Journal articles on the topic 'Micro b-open set'

In this paper, a new kind of set called a Micro b-open set in micro topological space is introduced. Basic properties of Micro b-open sets are analyzed and also this set used to introduce and study the new types of functions called Micro b-irresolute, Micro b-continuous and Micro Wb-continuous functions.

The purpose of this research is generalize the concepts of micro openset, so we study the relation this new concept open set micro-pre-open set, micro-semi-open set, micro open set, and micro openset and micro open continuity and micro irresolute continuity in micro topological space and study the relationship among them, many theorems were proved as characteristic of these type of continuity and some examples were introduced.

Virtual testing has lately gained widespread acceptance among scientists as a simple, accurate, and reproducible method to determine the mechanical properties of heterogeneous microstructures, early in the production process. As a result of the rapid expansion of the use of composites in aerospace design, virtual testing techniques are, in fact, deemed extremely useful to eliminate unnecessary tests and to reduce cost and time associated with generating allowables for lengthy lifing analyses of structures. Leveraging on a limited set of experimental data, a Progressive Failure Analysis can accurately predict the life and safety of a component/assembly, simply tapping on the physics of its micro-/macro- mechanics material properties, manufacturing processes, and service environments. The robust methodology is showcased using blind predictions of fatigue stiffness degradation and residual strength in tension and compression after fatigue compared with test data from Lockheed Martin Aeronautics and Air Force Research Laboratory). The multi-scale progressive failure analysis methodology in the GENOA software considers uncertainties and defects and evaluated the damage and fracture evolution of three IM7-977-3 laminated composite layups at room temperature. The onset and growth of composite damage was predicted and compared with X-ray CT. After blind predictions, recalibrations were performed with knowledge of the test data using the same set of inputs for all layups and simulations. Damage and fracture mechanism evolution/tracking throughout the cyclic loading is achieved by an integrated multi-scale progressive failure analysis extended FEM solution: (a) damage tracking predicts percentage contributing translaminar and interlaminar failure type, initiation, propagation, crack growth path, and observed shift in failure modes, and (b) fracture mechanics (VCCT, DCZM) predicts crack growth (Crack Tip Energy Release Rate vs. Crack Length), and delamination. The predictive methodology is verified using a building block validation strategy that uses: (a) composite material characterization and qualification (MCQ) software, and (b) the GENOA multi-scale progressive failure analysis fatigue life, stiffness degradation, and post-fatigue strength predictions for open-hole specimens under tension/compression at RTD. The unidirectional tension, compression, and in-plane shear lamina properties supplied by Lockheed Martin Aeronautics and the Air Force Research Laboratory (based on the D3039, D638, D3518 tests) were used by MCQ to reverse engineer effective fiber and matrix static and fatigue properties for the IM7-977-3 material system. The use of constituent properties identified the root cause problem for composite failure and enabled the detection of damage at the micro-scale of the material where damage is incepted. For all three case studies (namely, layups [0/45/90/−45]2s, [+60, 0, −60]3s, and [+30, +60, 90, −60, −30]2s), the blind predictions on the fatigue stiffness degradation and residual strength of the open-hole coupon in tension/compression under cyclic loading (with R = 0.1) at RTD were evaluated using a FE mesh (made of 2k shell elements), in which only one shell element, containing all plies, was employed through the thickness. The results of all analyses correlated very well with the tests, including the damage micro-graphs generated during the cyclic loading.

THE INFLUENCE OF MICRO-INFLUENCERS AND DIGITAL MARKETING ON PURCHASE DECISIONS OF TIKTOK SHOP CUSTOMERS IN BENGKULU CITY Andhes Tiani Putri, Meylaty F 12Faculty Of Economic Email: How to cite: ………………………….(fill in Editor) INFORMASI ARTIKEL ABSTRACT Article History: Accepted : ….. (fill in editor) Revised : ….. (fill in editor) Approved : ….. (fill in editor) This study aims to determine the influence of Micro-Influencers and Digital Marketing on Purchase Decisions of TikTok Shop customers in Bengkulu City, both partially and simultaneously. Using 120 respondents, the research analyzes the impact of these variables through multiple data analysis techniques, including instrument testing, classical assumption testing, multiple linear regression, and hypothesis testing. The results show that Micro-Influencers and Digital Marketing both have a significant impact on purchase decisions individually and collectively, highlighting their crucial role in consumer behavior on TikTok Shop. Keywords: 3-5 words or phrases represent the focus of writing. Written with letters Times New Roman 10 pt Italic. Pages: …… (fill in editor) This is an open access article under the CC–BY-SA license Keywords: Micro-Influencer, Digital Marketing, Purchase Decision INTRODUCTION In recent years, digital marketing has become one of the primary strategies businesses use to reach consumers. This trend has grown even further with the emergence of micro-influencers—individuals with a relatively small but highly engaged following on social media. Micro-influencers are considered more authentic and have a stronger personal connection with their audience, making them more impactful on consumer purchasing decisions compared to major celebrities. In this context, local businesses in Bengkulu, such as those on TikTok, leverage micro-influencers to increase their product exposure. By using platforms like Instagram or TikTok, they can reach a relevant audience more effectively and efficiently. Moreover, the concept of digital marketing has become a crucial topic in the modern era, aligning with the growing consumer awareness of social and environmental responsibility. Digital marketing aims to create long-term value for businesses while ensuring environmental sustainability. Businesses in Bengkulu, including those on TikTok, face the challenge of not only focusing on short-term profitability but also adopting business practices that support sustainable digital marketing. Through collaborations with micro-influencers who promote eco-friendly products or digital messages, local stores have the opportunity to build a more positive brand image and meet the demands of modern consumers who are increasingly concerned with digital marketing issues. LITERATURE REVIEW (Hasibuan, 2016) states that purchase decision is the process of selecting a product or service after evaluating various factors. Consumers consider aspects such as needs, desires, price, and the benefits offered by the product before making a decision. This decision results from a complex analysis, including comparisons between different options available in the market. (Japarianto, 2020) emphasizes that a purchase decision is the outcome of a consumer's evaluation of various product or service options to fulfill their needs. This process involves considering internal factors (such as attitudes, perceptions, and experiences) as well as external factors (such as promotions, pricing, or trends). The decision is made after consumers weigh all the available information. (Chaniago & Majid, 2023) state that micro-influencers are individuals with a following of between 1,000 and 100,000 on social media, known for their personal closeness to their audience. They are considered more trustworthy due to their authentic interactions and are seen as more relatable compared to influencers with larger followings. (Girsang, 2020) mentions that micro-influencers are individuals with a smaller but highly loyal follower base, particularly within specific communities or niches. They are often regarded as a trusted source of information by their followers, making them highly effective in marketing campaigns targeting specific market segments. (As-Syahri, 2024) states that digital marketing is the process of promoting products or services through digital media, including the internet and other electronic devices. As-Syahri emphasizes that digital marketing encompasses various strategies, such as SEO (Search Engine Optimization), social media marketing, email marketing, and digital advertising. This process allows companies to reach a broader audience in a more efficient and measurable way compared to traditional marketing. Additionally, digital marketing enables direct interaction between brands and consumers, creating a more personalized experience. (Triskamto, 2024) explains that digital marketing includes all forms of marketing conducted through digital platforms. This involves the use of websites, applications, and social media to deliver marketing messages to target audiences. Digital marketing offers advantages in data analysis, allowing marketers to track campaign performance in real-time, make adjustments, and optimize strategies based on collected data. Triskamto also highlights the importance of creating engaging and relevant content to capture consumers' attention in the competitive digital landscape. RESEARCH METHOD This chapter outlines the research methodology used in the study on the influence of micro-influencers and digital marketing on purchasing decisions. The research was conducted among consumers who have shopped on TikTok in Bengkulu, with initial observations made in October 2024. A descriptive quantitative approach was employed, comparing data collected through a questionnaire from a representative sample population. The target population consists of TikTok consumers, with a purposive sampling method used to select 120 respondents based on specific criteria. This ensures that the sample is suitable for the study's objectives. Data collection was carried out using a well-structured questionnaire designed to gather detailed information, allowing respondents to provide their insights without feeling pressured. The questionnaire utilized a Likert scale for responses, facilitating quantitative analysis. Data analysis techniques included instrument testing, where validity and reliability tests were conducted to confirm the effectiveness of the questionnaire. Validity was assessed through correlation thresholds, while reliability was evaluated using Cronbach's Alpha, with acceptable values set above 0.60. The classical assumption testing involved normality, multicollinearity, and heteroscedasticity tests to ensure the data met the necessary statistical assumptions. Finally, multiple linear regression analysis was employed to assess the impact of the independent variables (micro-influencer and digital marketing) on the dependent variable (purchasing decisions). Hypothesis testing included both partial (t-test) and simultaneous (F-test) evaluations to determine the influence of the independent variables on the dependent variable. This comprehensive methodology ensures the reliability and validity of the study's findings. RESEARCH RESULTS AND DISCUSSION Tables and Figures Chapter 4: Results and Discussion This chapter presents the results of the study, analyzing data collected from 120 respondents regarding the influence of micro-influencers and digital marketing on purchasing decisions. The findings are categorized into several sections, including demographic characteristics, instrument testing results, and key findings related to the research variables. General Overview of TikTok Shop TikTok Shop is an e-commerce feature integrated into the TikTok platform, allowing users to purchase products directly through videos and live streams. It promotes interaction between sellers and consumers, enhancing the shopping experience. Respondent Characteristics The demographic characteristics of the respondents are summarized in the following tables: Table 4.1: Respondent Characteristics by Gender Characteristic Number Percentage (%) Male 85 70.83 Female 35 29.17 Total 120 100% Table 4.2: Respondent Characteristics by Age Age Range Number Percentage (%) 17 - 29 years 80 53.72 30 - 40 years 30 37.03 > 40 years 10 9.25 Total 120 100% Instrument Testing Results The validity and reliability of the questionnaire were confirmed, ensuring accurate data collection. Table 4.3: Validity Test Results Variable Item Correlation Validity Micro-Influencer H1 .874 Valid Digital Marketing P1 .485 Valid Table 4.4: Reliability Test Results Variable Cronbach’s Alpha Reliability Micro-Influencer .900 Reliable Digital Marketing .620 Reliable Purchase Decision .681 Reliable Key Findings The results indicate that both micro-influencers and digital marketing significantly influence purchasing decisions on TikTok Shop. Statistical analysis revealed: Micro-influencer (X1) has a significant positive effect on purchasing decisions. Digital marketing (X2) also significantly impacts purchasing decisions. Table 4.5: Summary of Hypothesis Testing Variable t-value Significance (p) Micro-Influencer 1.246 < 0.05 Digital Marketing 4.586 < 0.05 The findings support the hypothesis that both micro-influencers and digital marketing are effective tools in influencing consumer behavior. The close relationship between micro-influencers and their audience enhances trust and credibility, which are crucial for driving purchasing decisions. Additionally, effective digital marketing strategies further amplify this effect, leading to increased sales and brand loyalty. This chapter highlights the significant role of micro-influencers and digital marketing in shaping consumer purchasing behavior, providing valuable insights for businesses aiming to optimize their marketing strategies on platforms like TikTok. CONCLUSION Conclusion Based on the research findings and discussions regarding the influence of micro-influencers and digital marketing on purchasing decisions at TikTok Shop, several conclusions can be drawn: Significant Influence of Micro-Influencers: The study demonstrates that micro-influencers have a significant positive impact on purchasing decisions. Their ability to establish a personal connection with their audience enhances their credibility, leading to increased consumer trust and influence over buying behavior. Impact of Digital Marketing: Digital marketing strategies also show a significant effect on purchasing decisions. The integration of various digital platforms allows for more effective engagement with consumers, which in turn boosts brand awareness and sales. Combined Effect: The simultaneous influence of both micro-influencers and digital marketing indicates that businesses can enhance their marketing effectiveness by leveraging both elements. The collaborative approach between these two factors significantly drives purchasing decisions among consumers. Recommendations Based on the conclusions drawn from the study, the following recommendations are proposed: Utilize Micro-Influencers: Businesses should consider partnering with micro-influencers who resonate with their target audience. The authenticity and relatability of micro-influencers can help in building trust and encouraging consumer purchases. Enhance Digital Marketing Strategies: Companies should invest in comprehensive digital marketing strategies that incorporate various channels, including social media, email marketing, and content marketing. Engaging and relevant content will further attract and retain consumers. Monitor Consumer Feedback: It is crucial for businesses to continuously monitor consumer feedback and adapt their strategies accordingly. Understanding consumer preferences and behaviors will aid in refining marketing efforts and improving overall effectiveness. Integrate Sustainability: As consumers become more environmentally conscious, integrating sustainability into marketing strategies can enhance brand image and attract a wider audience. Promoting products that align with sustainable practices can significantly influence purchasing decisions. Further Research: Future research should explore the long-term effects of micro-influencers and digital marketing on consumer behavior across different demographics and market segments. This could provide deeper insights into optimizing marketing strategies. This chapter emphasizes the essential role of micro-influencers and digital marketing in influencing purchasing decisions, offering practical recommendations for businesses seeking to enhance their marketing effectiveness in the digital age. REFERENCE Adireja, M. H., Barkah, C. S., & Novel, N. J. A. (2024). Implementasi Strategi Influencer Marketing untuk Membangun Brand Awareness di Industri Skincare Pria. Jurnal Indonesia : Manajemen Informatika Dan Komunikasi, 5(3), 2976–2983. https://doi.org/10.35870/jimik.v5i3.952 As-Syahri, H. (2024). Socius: Jurnal Penelitian Ilmu-Ilmu Sosial Peranan Influencer Marketing Sebagai Strategi Pemasaran Digital 5.0. 1. https://doi.org/10.5281/zenodo.12783992 Astuti, R. (2020). 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As the world embraces a technological revolution on how everyday devices are connected to the internet, users provide sensitive information using the internet which is broken down and distributed as packets throughout the network. Packet sniffers tap to these packets, capable of potentially compromising security and privacy of unsuspecting users. This study aims to put into the test some well-known Intrusion Detection Systems (IDS) and observe how they fare against popular packet-sniffing tools such as Wireshark and tcpdump. The varied sniffing methods and techniques from various sniffing tools will provide an evaluation of performance of the intrusion detection systems.[1]  Ansari, S., Rajeev, S. & Chandrashekar, H. Packet Sniffing: A brief introduction. Potentials IEEE, Vol. 21 (5), (2002), pp. 17-19.[2]  Chomsiri, T. Sniffing packets on LAN without ARP spoofing.  IEEE in Convergence and Hybrid Information Technology ICCIT'08. Third International Conference, Vol. 2 (1), (2008), pp. 472-477.[3]  Anh, N. & Shorey, R. Network sniffing tools for WLANs: merits and limitations. 2005 IEEE International Conference on Personal Wireless Communications, (2005).[4]  Hu, Q., Asghar, M. & Brownlee, N. Evaluating network intrusion detection systems for high-speed networks. Telecommunication Networks and Applications Conference (ITNAC) 2017 27th International, (2017), pp. 1-6.[5]  Guo, K., Lu, H. & Yu, R. Packet Capture and Protocol Analysis Based on Winpcap. 2016 International Conference on Robots & Intelligent System (ICRIS), (2016).[6]  Goyal, P. & Goyal, A. Comparative study of two most popular packet sniffing tools-Tcpdump and Wireshark. 2017 9th International Conference on Computational Intelligence and Communication Networks (CICN), (2017).[7]  Meghana, J., Subashri, T. & Vimal, K. A survey on ARP cache poisoning and techniques for detection and mitigation. Signal Processing Communication and Networking (ICSCN). 2017 Fourth International Conference, (2017), pp. 1-6.[8]  Arzhakov, A. & Silnov, D. Architecture of multithreaded network scanner. IEEE Micro/Nanotechnologies and Electron Devices (EDM) 2017 18th International Conference of Young specialists, (2017), pp. 43-45.[9]  Bhosale, D. & Mane, V. Comparative study and analysis of network intrusion detection tools. 2015 International Conference on Applied and Theoretical Computing and Communication Technology (iCATccT), (2015).[10] Tirumala, S., Sathu, H. & Sarrafzadeh, A. Free and open source intrusion detection systems. Machine Learning and Cybernetics (ICMLC) 2015 International Conference, (2015).[11] Albin, E. & Rowe, N. A realistic experimental comparison of the suricata and snort intrusion -detection systems. Advanced Information Networking and Applications Workshops (WAINA) 26th International Conference, (2012).[12] Africa, A., Mesina, A., Izon, J. & Quitevis, B. Development of a Novel Android Controlled USB File Transfer Hub. Journal of Telecommunication, Electronic and Computer Engineering, Vol. 9 (2-8), (2017), pp. 1-5.[13] SmartSniff. (2018). https://www.nirsoft.net/utils/smsniff.html.  [14] Wireshark. (2018).  https://www.wireshark.org/.[15] Windump. (2013). https://www.winpcap.org/windump/. [16] Dsniff, Dug Song. (2018). https://www.monkey.org/~dugsong/dsniff/.[17] Cain and Abel. (2018). http://www.oxid.it/cain.html. [18] Ettercap. (2018).  http://www.ettercap-project.org/ettercap/index.html. [19] Network grep. (2018).  http://ngrep.sourceforge.net/usage.html. [20] Nmap. (2018). https://nmap.org/. [21] Africa, A., Aguilar, J., Lim Jr., C., Pacheco, P. & Rodrin, S. Automated Aquaculture System that Regulates Ph, Temperature and Ammonia. 9th International Conference on Humanoid, Nanotechnology, Information Technology, Communication and Control, Environment, and Management (HNICEM), (2017).[22]S.Dhar. (2018). http://www.just.edu.jo/~tawalbeh/nyit/incs745/presentations/Sniffers.pdf.  [23] A. Africa, A Rough Set-Based Expert System for diagnosing information system communication networks. International Journal of Information and Communication Technology, Vol. 11 (4), (2017), pp. 496-512.[24]Africa, A., Bautista, S., Lardizabal, F., Patron, J. & Santos, A. Minimizing Passenger Congestion in Train Stations through Radio Frequency Identification (RFID) coupled with Database Monitoring System. ARPN Journal of Engineering and Applied Sciences, Vol. 12 (9), (2017), pp. 2863-2869.[25] Africa, A. & Cabatuan, M. A Rough Set Based Data Model for Breast Cancer Mammographic Mass Diagnostics. International Journal of Biomedical Engineering and Technology, Vol. 18 (4), (2015), pp. 359-369.[26] Africa, A. A Rough Set Based Solar Powered Flood Water Purification System with a Fuzzy Logic Model. ARPN Journal of Engineering and Applied Sciences, Vol. 12 (3), (2017), pp. 638-647.[27] Africa, A. A Mathematical Fuzzy Logic Control  Systems Model Using Rough Set Theory for Robot Applications. Journal of Telecommunication, Electronic and Computer Engineering, Vol. 9 (2-8), (2017), pp. 7-11.[28] Brucal, S., Africa, A. & Dadios, E. Female Voice Recognition using Artificial Neural Networks and MATLAB Voicebox Toolbox. Journal of Telecommunication, Electronic and Computer Engineering, Vol. 10 (1-4), (2018), pp. 133-138.[29] Africa, A. & Velasco, J. Development of a Urine Strip Analyzer using Artificial Neural Network using an Android Phone. ARPN Journal of Engineering and Applied Sciences, Vol. 12 (6), (2017), pp. 1706-1712.[30] Loresco, P. & Africa, A. ECG Print-out Features Extraction Using Spatial-Oriented Image Processing Techniques. Journal of Telecommunication, Electronic and Computer Engineering, Vol. 10 (1-5), (2018), pp. 15-20.[31] Snort. (2018). https://www.snort.org/. [32] Africa, A. A Logic Scoring of Preference Algorithm using ISO/IEC 25010:2011 for Open Source Web Applications Moodle and Wordpress. ARPN Journal of Engineering and Applied Sciences, Vol. 13 (15), (2018).[33]Suricata. (2018). https://suricata-ids.org/. [34] Gadde, S., Ganta, R., Gupta, A., Rao, R. & Rao, K. Securing Internet of Things (IoT) Using Honey Pots. International Journal of Engineering and Technology, Vol. 7 (2.7), (2018), pp. 820-824.

Satellite ocean-colour based models of size-fractionated primary production (PP) have been developed for the oceans on a global level. Uncertainties exist as to whether these models are accurate for temperate Shelf seas. In this paper, an existing ocean-colour based PP model is tuned using a large in situ database of size-fractionated measurements from the Celtic Sea and Western English Channel of chlorophyll-a (Chl a) and the photosynthetic parameters, the maximum photosynthetic rate ( P m B ) and light limited slope ( α B ). Estimates of size fractionated PP over an annual cycle in the UK shelf seas are compared with the original model that was parameterised using in situ data from the open ocean and a climatology of in situ PP from 2009 to 2015. The Shelf Sea model captured the seasonal patterns in size-fractionated PP for micro- and picophytoplankton, and generally performed better than the original open ocean model, except for nanophytoplankton PP which was over-estimated. The overestimation in PP is in part due to errors in the parameterisation of the biomass profile during summer, stratified conditions. Compared to the climatology of in situ data, the shelf sea model performed better when phytoplankton biomass was high, but overestimated PP at low Chl a.

Relevance. The verb constitutes a highly complex and multifaceted element of the Ukrainian language. It is a subject of intense scientific inquiry, yet it simultaneously evades complete resolution amidst a multitude of contradictions and open issues. Each time new micro-problem areas emerged, the findings were presented in the context of established or evolving grammatical science. However, the results were not always indisputable, given the plurality of linguistic thought and approaches. In particular, the issue of the verbal prefix, including its identification, theoretical qualification related to the very nature of prefixed verbs, and a multitude of related and pivotal problems concerning the verbal prefix as a morpheme variant, represents a significant challenge. The objective of this article is to present the results of analysis of the system of verb prefixes in the Ukrainian language, focusing on the ways in which they manifest semantic features of nuclearity and the potential role they play as a parameter of authorial speech. The extensive scope of the research problem has led to the following main tasks: a) to identify the principal features of the prefix morpheme; b) to put forward a philosophical and methodological approach to the process of cognitive activity in primary and secondary forms and the formation of relevant terminology; c) to base the analysis process on information about a specific verb prefix and the corresponding prefix verb name. The procedural denotation is attested by explanatory dictionaries, as well as quantitative indicators of the author's file; d) to substantiate the notion of «seminal prefix nucleation» and to identify the means of its use (on the examples of several productive verb prefixes). Results. The paper delineates the semantic core of productive verb prefixes and explores the potential for some of them to serve as a parameter of authorial speech. The article puts forth the notion that the dynamics of scientific advancement are occurring at a faster and more qualitative pace than the static, particularly in the realms of heredity and the continuity of the general worldview, aesthetic and scientific progress. This leads to rapid changes in scientific paradigms. Even the fundamental stereotypes in the selection of scientific direction, and thus the verbal «coding» of the dominant themes of research, are evolving in accordance with the prevailing trends. This article includes numerous observations regarding the vast assortment of terms that have been derived from the nomination and metalinguistic ordering of concepts that are directly or indirectly associated with the prefix, which plays a crucial and sometimes contentious role in morphemics. The primary objective of the study was to substantiate the hypothesis that the prefix plays a pivotal role in the formation of verb names in the Ukrainian language. The prefix «ви-», identified and analysed on the basis of various features of semantic nuclearity, has been shown to provide new guidelines and approaches not only to this prefix, but also to the possibility of using them to study other prefixes. Furthermore, the article considers the potential for identifying the semantic nuclearity of other productive verb prefixes as a possible parametric factor of authorial speech. The texts by M. Kotsiubynskyi and S. Andrukhovych are presented as objective factors of the author's speech parameter based on a set of quantitative indicators of the functioning of productive prefixes in verb forms representing different variants of the verb category «proceduralism».

<strong>Kozioł Maciej, Piech Piotr, Wokurka Wojciech, Maciejewski Ryszard. Advances in osteosynthesis - a basic overview of modern fixation materials. J</strong><strong>ournal of Education, Heal</strong><strong>th and Sport. 2022;12(7):416-428</strong><strong>. eISSN 2391-8306. DOI </strong><strong>http://dx.doi.org/10.12775/JEHS.2022.12.07.041</strong> <strong>https://apcz.umk.pl/JEHS/article/view/JEHS.2022.12.07.041</strong> <strong>https://zenodo.org/record/6815186</strong> &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; <strong>The journal has had 40 points in Ministry of Education and Science of Poland parametric evaluation. Annex to the announcement of the Minister of Education and Science of December 21, 2021. No. The journal has had 40 points in Ministry of Education and Science of Poland parametric evaluation. Annex to the announcement of the Minister of Education and Science of December 21, 2021. No. 32343.</strong> <strong>Has a Journal&#39;s Unique Identifier: 201159. Scientific disciplines assigned: Physical Culture Sciences (Field of Medical sciences and health sciences); Health Sciences (Field of Medical Sciences and Health Sciences).</strong> &nbsp; <strong>Punkty Ministerialne z 2019 - aktualny rok 40 punkt&oacute;w. Załącznik do komunikatu Ministra Edukacji i Nauki z dnia 21 grudnia 2021 r. Lp. 32343. Posiada Unikatowy Identyfikator Czasopisma: 201159.</strong> <strong>Przypisane dyscypliny naukowe: Nauki o kulturze fizycznej (Dziedzina nauk medycznych i nauk o zdrowiu); Nauki o zdrowiu (Dziedzina nauk medycznych i nauk o zdrowiu).</strong> &nbsp; <strong>&copy; The Authors 2022;</strong> <strong>This article is published with open access at Licensee Open Journal Systems of Nicolaus Copernicus University in Torun, Poland</strong> <strong>Open Access. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author (s) and source are credited. This is an open access article licensed under the terms of the Creative Commons Attribution Non commercial license Share alike.</strong> <strong>(http://creativecommons.org/licenses/by-nc-sa/4.0/) which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited.</strong> <strong>The authors declare that there is no conflict of interests regarding the publication of this paper.</strong> &nbsp; <strong>Received: 22.06.2022. Revised: 22.06.2022. Accepted: 10.07.2022.</strong> &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; <strong>Advances in osteosynthesis - a basic overview of modern fixation materials</strong> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Postępy w osteosyntezie &ndash; podstawowy przegląd nowoczesnych materiał&oacute;w zespalajacych &nbsp; <strong>Maciej Kozioł [1], Piotr Piech [1], Wojciech Wokurka [2], Ryszard Maciejewski [2]</strong> &nbsp; [1] Orthopedics and Traumatology Department, Medical Univeristy of Lublin, Poland [2] Human Anatomy Department, Medical Univeristy of Lublin, Poland &nbsp; <strong>Abstract.</strong> The dynamic development of trauma-orthopedic surgery and accompanying material technology has led in recent years to the need for close cooperation between researchers in these fields. In a short time, thanks to the cooperation of engineers and doctors, the general approach to the method of bone anastomosis has changed significantly. The need to optimize the effects of treatment, i.e. to quickly recovery, reduce the number of postoperative complications, reduce the number of reoperations, and reduce the costs of procedures and treatment used has resulted in the development of many new technologies that have set trends in modern traumatology. The widespread use of LCP (Locking compression plate) and locking screws, the development of polymers and biopolymers with a modified chemical structure, a significant improvement in the biocompatibility and cytocompatibility of the materials used, and the implementation of products with significant micro-roughness that improve osseointegration are the well-known and commonly used effects of this cooperation today . Materials science related to orthopedics is an extremely complex and multi-threaded field. Its continuous development requires a periodic summary of the results and development directions provided, which allows faster evaluation and interpretation by researchers. The purpose of the following work is to summarize the latest research on materials and methods used in osteosynthesis in a legible way for potential recipients of this information from various fields. &nbsp; <strong>Abstrakt. </strong>Dynamiczny rozw&oacute;j chirurgii urazowo-ortopedycznej i towarzyszącej jej technologii materiałowej doprowadził w ostatnich latach do potrzeby ścisłej wsp&oacute;łpracy pomiędzy badaczami tych dziedzin.&nbsp; W kr&oacute;tkim czasie dzięki wsp&oacute;łpracy inżynier&oacute;w i lekarzy istotnym zmianom uległo og&oacute;lne podejście do sposobu zespoleń kości. Potrzeba optymalizacji efekt&oacute;w leczenia tj. szybkiego przywr&oacute;cenia chorych do sprawności, ograniczenia liczby powikłań pooperacyjnych, zmniejszenia ilości reoperacji i redukcja koszt&oacute;w stosowanych procedur i leczenia wpłynęła na rozw&oacute;j wielu nowych technologii, kt&oacute;re wyznaczyły trendy w nowoczesnej traumatologii. Powszechne użycie płyt LCP (<em>Locking compression plate</em>) i wkręt&oacute;w blokowanych, rozw&oacute;j polimer&oacute;w i biopolimer&oacute;w o zmodyfikowanej strukturze chemicznej, znaczna poprawa biokompatybilności i cytokompatybilności stosowanych materiał&oacute;w oraz wdrożenie do użycia produkt&oacute;w o istotnej mikrochropowatości poprawiających osteointegrację to dobrze znane i powszechnie stosowane efekty wspomnianej wsp&oacute;łpracy już dzisiaj. Inżynieria materiałowa związana z ortopedią jest dziedziną niezwykle skomplikowaną i wielowątkową. Ciągły jej rozw&oacute;j wymaga okresowego podsumowania dostarczanych wynik&oacute;w i kierunk&oacute;w rozwoju co umożliwia ich szybszą ocenę i interpretację przez badaczy. Celem poniższej pracy jest podsumowanie najnowszych badań dotyczących materiał&oacute;w i metod używanych w osteosyntezie w spos&oacute;b czytelny dla potencjalnych odbiorc&oacute;w tych informacji z r&oacute;żnych dziedzin. &nbsp; <strong>Key words: </strong>materials in orthopedics, osteointegration, polymers, biopolymers, bio-absorbable materials. &nbsp; <strong>Słowa kluczowe: </strong>materiały w ortopedii, osteointegracja, polimery, biopolimery, materiały bio-wchłanialne.

Introduction. Today we observed of tendency to reduction of honey bee population in the world what according to honey gathering. For example, in Ukraine, according to statistical data for 7 inhabitants of Poltava region accounts for 1 bee familie, compared with that of 300 years ago, it reached within 3 bee families per citizen. Science and practice open many secrets according biology of bees, allowing bee efficient manage vital functions for humans. But despite the opening of beekeeping is the only farm animal that over 100 years has not been the intervention of human hands to create a new breeds of honeybees. Genetic intensify the search in the field of beekeeping conditions in Ukraine needs to initiate breeding program as planned waste zoning and aspects of reproduction of bees.&#x0D; The aime of research: determining the characteristics of four intrapedigrees types in the Carpathian bee breed using the methods of population and molecular genetics.&#x0D; Materials and Methods: Sampling was performed from top five lines: Sinevir, Rakhiv, Vuchkivskyy and Hoverla bee colonies were taken 10 bee worker. For molecular genetic analysis used 20 insects of each Carpathian bees breed type with observance of the principle of representativeness. DNA extraction performed from homogenate tissues using the standard commercial kit «DNA Sorb B», «Amplisense», this some modifications [1] during sample preparation. Reaction mix purification from bees wax leading this octane. The structure of the primers used for genotyping of bees and their code designations are: OPA-1(3’- CAG GCC CTT C -5’); OPA-4 (3’- AAT CGG GCT G -5’); B15 (3’- GGA GGG TGT T -5’); S1 (3’-AGC AGC AGC AGC AGC AGC C-5’). The program amplification of RAPD - primers: 1 cycle: 940 - 3min .; 2-35 cycle: 940 - 1 min., 360 - 30s., 720 - 1hv.36 cycle (final elongation): 720 - 10 min. The program amplification with primers S1: 1 cycle of 94 ° C - 4min 2 - 31 cycle: 57 ° C - 2 minutes; 72 ° C - 4min; 94 ° C - 1 minute, 32 cycle: 57 ° C - 3 minutes; 72 ° C - 7 minutes. Electrophoretic separation of amplified sections performed in 2% agarose gel in Tris borate buffer conditions. Size of amplification products control was carried out using molecular weight marker 1 kb - Ledder plus ( «Fermentas», Vilnius, Latvia). Processing of the profiles was performed in a standard computer program GELSTAT [4]. Genetic distances were calculated in terms of genetic similarity indices obtained GELSTAT program as follows: Dxy = - lnI&#x0D; Building a kladohramm performed according to the values of genetic distances TREE program and MEGA 4 [5; 6]. Statistical analysis of amplicon frequencies, heterozygosity, linage similarity, etc., performed by Fisher's algorithm [7].&#x0D; Results and discussion. Molecular genetic studies on four primers made it possible to analyze 95 DNA fragments of different lengths, matching the same number of anonymous genetic loci of the genome of bees. Apply primer in RAPD - 15 made it possible to identify 18 amplification products in a range of molecular sizes from 410 to 1000 b. p. It noted that the DNA fragment size 410 b.p. 100% met all the members of Carpathian bees breed and described one genetic monomorfic locus. DNA size band 445 b. p. elektrophoregramme was found in only 20% of bees Vuchkivskiy type in the absence of individuals in other populations. Statistical comparisons (Fisher's criterion) frequency distribution of DNA fragments obtained with primer in -15 revealed a significant number of types of identification markers intrapedigrees of Carpathian bees. The largest number of DNA - fragments set for Vuchkivsksy type whose size is reached within the following limits: 1000, 630, 580 and 485b.p. DNA fragment of 1000 b.p. general was absent in bees Rakhiv type and Synevir, and its frequency in the population of Representatives Hoverla was 0.600 (p &lt;0.001). As individuals, the members of such Synevir, observed no amplicon size 630 b. p. A fragment of a molecular weight of 710 b. p. observed with a frequency of 0.600 to 0.400 bees and type Rahiv, Goverla and 0,100 individuals in such Vuchkivskyy (p &lt;0,05; p &lt;0,01), respectively. Bees type Rahiv, Goverla and can be identified among other types of Carpathian breed presence significantly higher frequency amplification product whose size is 655, 515 b. p., 830 b. p, 530 b. p., respectively. Statistical analysis of the frequency distribution of the products of amplification bees four types derived from molecular genetic analysis of four primers in PCR was performed to identify the most characteristic identification of DNA fragments bees each of type [8]. Based on these characteristics were constructed genetic formula intrapedigrys types of Carpathian breed. According genetic formulas the highest number of specific DNA - fragments characterized by bees for types of Sinevir and Vuchkivskyy, that the overwhelming number of such markers has been found Sinevir system ISSR-S1 (four amplicons) and for the type of system was Vuchkivskyy informative method of RAPD B-15 primer (six DNA fragments). Type Rahiv different from the others by the presence of six DNA markers, such as bees of Hoverla characterized only four specific genetic loci.&#x0D; For the main parameters of population parameters highest level of genetic diversity characterized types Goverla and Rahiv because in terms of total heterozygosity significantly different from the type of bees from Sinevir and Vuchkivskyy values ​​of 0.362 and 0.354, respectively (p &lt;0,01; p &lt;0,001). Moreover, the type of bees Hoverla observed the largest number of polymorphic loci - 54.9%, with a minimum of meaning in a population of individuals Sinevir type, and the lowest value of intrapedigrys similarity (number of DNA fragments in the same study group) were observed for a sample of bees type Rakhiv (0.665, p &lt;0.001).&#x0D; Determining the genetic distances between breeds in genealogical structure can be used as a methodical approach predicting the effectiveness of a combination of lines and types for heterosis effect on purebred descendants basis.The maximum value of genetic distance algorithm M. Ney was established between the types Synevir and Rakhiv (0.435), slightly less than this value typical of the combination VUChK - Synevir (0.426) and VUChK - Rakhiv (0.423). The smallest genetic distance calculated between individuals and types Hoverla ‒ Vuchkivskyy, which indicates their high genetic relationship and the undesirability of crossing the representatives of these types together. Application of unweighted pair-group clustering based on the calculated distances made it possible to analyze the nature of the genetic relationships between intrapedigrys types of Carpathian breed in graphic terms.&#x0D; According dendrogram representatives Synevir types and Rahiv presented by individual branches, indicating their genetic identity. The Goverla and Vuchkivskyy type of bees and united in a common underklaster, due not only to the minimum calculated measure of genetic distance between data types, but confirmed the historical part of the establishment pedigree group Hoverla, based line mares are exactly the type Vuchkivskyy and lost insect genealogical group of Kolochavskiy type.&#x0D; Conclusion Determining genetic specificity linage types of Carpathian breed bees allowed to obtain the following results: 1) chosen for the study of molecular genetic markers is sufficiently informative for determining the unique, specific features of each breed group and the identification of any sample Carpathian bees with the opportunity to consider linage certain type; 2) received genetic formula of Carpathian bees linage types are proof of the impact of breeding activities and can form the basis the protection of intellectual property of their authors; 3) used molecular genetic markers may serve as a forecasting tool optimal compatibility of linage types to obtain heterosis effect in their offspring.&#x0D; The prospect of the research of this area is to select as a methodological tool for measuring genetic polymorphism Carpathian breed more accurate, reproducible and standardized markers, locus-specific micro satellite analysis, STR, research of structural genes single nucleotide polymorphism, SNP analysis, accumulation and formation the databases to assess the state of development, management and preservation unique of Carpathian bees gene pool.

The degradation of proton exchange membrane fuel cells (PEMFCs) stands as a critical determinant in evaluating the robustness of fuel cell systems, crucial for the commercial-scale transition to sustainable energy. For zero-emission vehicles, fuel cell stacks need to demonstrate lifespans exceeding 8,000 (light-duty vehicles) and 30,000 hours (heavy-duty vehicles)[1]. For achieving robust PEMFC systems at commercial scale, it is crucial to precisely understand the degradation pathways to effectively pinpoint and mitigate degradation issues. To achieve this, customized small-scale fuel cell fixtures are utilized for accelerated stress testing (AST) and X-ray computed tomography (XCT) in-situ and ex-situ visualization [2,3]. This approach enables comprehensive analysis of membrane electrode assembly (MEA) aging processes. Our group previously employed this approach to identify chemical, mechanical, and chemo-mechanical degradation mechanisms in commonly used perfluorosulfonic acid (PFSA) ionomer membranes[4,5]. In recent years, transitioning from PFSA membranes to hydrocarbon-based (HC) membranes in PEMFCs has gained significant attention. This shift eliminates fluorinated compounds typically found in PFSA membranes, aligning with sustainability goals, and reducing environmental impact. This transition signifies both technological advancement and innovation, driving forward the quest for efficient, cost-effective, and environmentally friendly energy solutions. Despite their promising characteristics, HC membranes are more susceptible to mechanical degradation than PFSA ones[6], presenting unique compatibility challenges due to their distinct chemical composition. Historically, PEMFC systems intentionally developed and optimized for PFSA may face performance issues and premature failure with HC membranes. This research therefore investigates the unique aspects of HC membrane degradation within a conventional fuel cell design with PFSA ionomer catalyst layers, using 4D in-situ XCT to understand chemo-mechanical degradation, enhancing fuel cell technology. Herein, ZEISS Xradia® 520 Versa micro XCT system was used to visualize samples. Repetitive scans were taken at room temperature in a dry state of the MEA. The first set of MEAs consisted of reinforced HC membranes (Pemion-PF1-HLF8-15-X) of 15 µm thickness, spray-coated with PFSA catalyst ink to form catalyst-coated membranes (CCMs), and assembled with gas diffusion layers (Freudenberg H14C15, 190 µm) featuring smooth and crack-free microporous layer. The AST protocol was custom-designed for chemo-mechanical membrane degradation and included open circuit voltage hold for chemical degradation and relative humidity cycling for mechanical degradation, which were applied consecutively. It has been demonstrated that MEAs undergo dimensional changes during wet-dry AST cycles due to alternating conditions of hydration and dehydration[5,7]. It is apparent from the degraded XCT image (Figures 1a-d) that cracks were initiated and propagated between the membrane and catalyst layers, with cracks prominently visible near agglomerated electrode particles. Differential swelling between the membrane and catalyst layer, compounded by varying rates of expansion or contraction, resulted in magnified mechanical stresses at the membrane-catalyst layer interfaces. The second set of cells consisted of the same reinforced HC membrane assembled with commercial gas diffusion electrodes (GDEs; 0.5 mg/cm² 60% Pt on Vulcan, Sigracet 22 BB, 215 µm). The existence of interfacial voids (Figure 1e) was observed in the pristine sample at both anode and cathode membrane-GDE interfaces, likely due to interfacial adhesion fatigue (IAF)[8]. Uneven compression and interfacial incompatibility during fuel cell assembly results in IAF. When these GDE based MEAs were subjected to wet-dry AST cycles, mechanical stress was induced at the membrane-GDE interfaces[9, 10]. The uneven mechanical stress can aggravate creep propagation in the membrane. Due to creep propagation, membrane thinning at the edges was evident from the XCT images, and resulted in electrode shorting, as shown in Figure 1(f and g). It is believed that constraints during creep failure hindered membrane's return to original thickness after swelling, causing permanent dimensional changes. The combined effects of cyclic dimensional changes and membrane thinning due to the creep mechanism contributed to performance degradation and reduced durability of GDE-based MEAs. Acknowledgements This work was supported by the Natural Sciences and Engineering Research Council of Canada, Ionomr Innovations Inc., Canada Foundation for Innovation, British Columbia Knowledge Development Fund, Pacific Economic Development Canada, and Canada Research Chairs. References C.S. Gittleman et al., Joule, 5 (2021) 1660–1677. Y. Singh et al., J Power Sources, 412 (2019) 224–237. D. Ramani et al., Electrochim Acta, 380 (2021) 138194. Y. Chen et al., J Power Sources, 520 (2022) 230674. Y. Chen et al., J Electrochem Soc, 170 (2023) 114526. S.H. Mirfarsi et al., Int J Hydrogen Energy, 47 (2022) 13460–13489. A. Sadeghi Alavijeh et al., J Power Sources, 427 (2019) 207–214. X. Huang et al., J Polym Sci B Polym Phys, 44 (2006) 2346–2357. V.A. Sethuraman et al., J Electrochem Soc, 155 (2008) B50. S.H. Mirfarsi et al., Int J Hydrogen Energy, 50 (2024) 1507–1522. Figure 1

The cystic fibrosis gene product cystic fibrosis transmembrane conductance regulator (CFTR) is a low conductance, cAMP-regulated Cl- channel. Removal of cytosolic ATP causes a cessation of cAMP-dependent kinase-phosphorylated CFTR channel activity that resumes upon ATP addition. (Anderson, M. P., H. A. Berger, D. R. Rich, R. J. Gregory, A. E. Smith, and M. J. Welsh. 1991. Cell. 67:775-784). The aim of this study was to quantify possible effects of ATP on CFTR gating. We analyzed multichannel records since only 1 of 64 patches contained a single channel. ATP increased the channel open probability (Po) as a simple Michaelis-Menten function of concentration; the effect was half maximal at 24 microM, reached a maximum of 0.44, and had a Hill coefficient of 1.13. Since the maximum Po was not 1, the simplest description of the effect of ATP on CFTR gating is the noncooperative three-state mechanism of del Castillo and Katz (1957. Proceedings of the Royal Society of London. B. 146:369-381). We analyzed current fluctuations to quantify possible changes in CFTR gating. The power density spectra appeared to contain a single Lorentzian in the range of 0.096-31 Hz. Analysis of the corner frequency (fc) of this Lorentzian revealed that ATP increased 2 pi fc as a Michaelis-Menten function with a Hill coefficient of 1.08, and it provided estimates of the ATP dissociation constant (44 tau open (154 ms), and the ATP-sensitive tau close [(185 ms) (44 microM/[ATP] + 1)]. These results suggest that the binding reaction is rapid compared to the opening and closing rates. Assuming that there is a single set of closed-to-open transitions, it is possible to verify the outcome of fluctuation analysis by comparing fluctuation-derived estimates of Po with measures of Po from current records. The two values were nearly identical. Thus, noise analysis provides a quantitative description of the effect of ATP on CFTR opening. The noncooperative three-state model should serve as a basis to understand possible alterations in CFTR gating resulting from regulators or point mutations.

The experiments described examine single channel currents recorded through Torpedo acetylcholine receptor channels stably expressed by a mouse fibroblast cell line. Closed-duration histograms were constructed from currents elicited by 0.5-300 microM acetylcholine (ACh). The concentration dependence of closed durations is well described by a four-state linear scheme with the addition of open-channel block by ACh. Analysis of closed durations measured at low concentrations gives estimates of the rate of opening of doubly liganded receptors, beta, the rate of dissociation of ACh from doubly liganded receptors, k-2, and the rate of channel closing, alpha. The rate of ACh dissociation from singly liganded receptors, k-1, is then deduced from closed-duration histograms obtained at intermediate ACh concentrations. With k-1, k-2 and beta determined, the rates of ACh association, k+1 and k+2, are estimated from fitting closed-duration histograms obtained over a range of high ACh concentrations. A complete set of rate constants is presented for three experimental conditions: (a) Ca2(+)-free extracellular solution containing 1 mM free Mg2+ at 22 degrees C, (b) Ca2(+)-free solution at 12 degrees C, and (c) extracellular Ca2+ and Mg2+, both at 0.5 mM, at 22 degrees C. For all three conditions the dissociation constant for the first agonist binding site is approximately 100-fold lower than that for the second site. The different affinities are due primarily to different dissociation rates. Both the association and dissociation rates depend strongly on temperature. At 22 degrees C ACh associates at diffusion-limited rates, whereas at 12 degrees C association is 30- to 60-fold slower. Also slowed at 12 degrees C are beta (4-fold), k-2 (3-fold), k-1 (25-fold), and alpha (15-fold). In contrast to the activation rate constants, those for ACh-induced block decrease only twofold between 22 and 12 degrees C. Changing from a Ca2(+)-free to a Ca2(+)-containing extracellular solution does not affect k+1 and k+2, but increases beta (twofold) and decreases k-2, k-1, and alpha (all twofold). Spectral analysis of single channel currents supports the parameter estimates obtained from fitting the open- and closed-duration histograms, and improves resolution of brief channel blockages produced by ACh.

Abstract This study investigates the impact of micro-credentials on educational administrators' professional growth and development. The study was guided by two research questions and one hypothesis. The study adopted the descriptive design. The study's population consists of 1589 teaching staff of the Rivers State University. A sample size of 477 teachers, representing 30% of the study population was determined using the simple random sampling technique. The instrument for data collection was a questionnaire named "Investigating the Impact of Micro-credentials on Educational Administrators' Professional Growth and Development Questionnaire (IIMEAPGDQ)&rdquo; respectively which was developed by the researcher. The questionnaire consisted of three sections. The questionnaire was validated through the researchers&rsquo; supervisor and other expert reviews and the reliability was established using Cronbach's Alpha (&alpha; = 0.75). The reliability and validity of the instruments used were also evaluated to ensure consistent and accurate measurement. Data was analyzed using descriptive statistics, such as mean, and standard deviation to answer the research questions while the inferential statistics of the Independent t-test were used to test the hypotheses at a 0.05 level of significance which means that any results with a p-value less than 0.05 were considered statistically significant, with the aid of SPSS Version 26.0. The findings revealed that micro-credentials positively impact educational administrators' professional growth and development. The study concluded that micro-credentials enhance administrators' professional competence, job performance, and career prospects. Based on the findings, it was recommended among others that institutions should prioritize the integration of micro-credentials into their professional development programs, leveraging their potential to enhance educational administrators' skills and expertise. <em>Keywords:</em><em> </em>Micro-credentials, Professional Growth, Educational Administrators, Development. <strong>Introduction</strong> In the rapidly evolving education sector, professional growth and development of educational administrators have become increasingly important to the success of academic institutions. As the demands on schools and educational systems continue to expand, there is a growing recognition of the need for innovative approaches by professional development to close the gap with these changes (Westfahl &amp; Wilkins, 2017). One such approach that has gained significant attention in recent years is using micro-credentials. Micro-credentials are also known as digital badges, nano degrees, micro-certifications, web badges, mini degrees and open badges in the form of compact and competency-based recognitions that verify an individual's skills, knowledge, or achievements in a specific area (Parsons et al., 2023). Micro-credentials are short, focused credentials designed to provide in-demand skills, know-how and experience. Unlike traditional degrees or certifications that often require extensive time commitments and cover broad areas of study, micro-credentials are typically focused on discrete skills or competencies and can be earned in a relatively short period. This flexibility and specificity make micro-credentials an attractive option for continuous professional development, particularly in fields like educational administration where the needed skills and knowledge are constantly evolving. The Nigerian educational system, like many others around the world, faces several challenges in ensuring the quality and effectiveness of its educational administrators. As such, Kwaghbo (2021) noted that effective leadership is essential for improving the quality of education and student outcomes. However, he further added that many administrators lack the necessary skills and knowledge to effectively manage the complex challenges facing modern schools. This gap in professional competence emphasizes the need for innovative and effective approaches to professional development for educational administrators in Nigeria. The concept of micro-credentials is relatively new in the Nigerian educational landscape, but it holds significant potential to address the professional development needs of educational administrators. No wonder, Oluwafemi (2019) argued that the present global yearning for education to gainful living and self-reliance indicates that there is an urgent need for effective and well-trained individuals who would be able to take care of themselves and contribute meaningfully and productively to the development of the society mentally, socially and economically. Micro-credentials compromise an invaluable opportunity to turn an ability you spent years developing into something traceable and proven (Flintberg, 2022). A credential provides information about the extra educational or professional steps someone has taken in search of constant improvement. If vocational, they will often be tagged with terminology related to skills and competencies. When finding qualified or dedicated professionals is increasingly challenging and time-consuming, micro-credentials can give individuals an edge and indicate to recruiters that they are committed to constant learning. For educational institutions, offering micro-credentials can be a means of commitment to celebrating all types of experiences and learning opportunities, valuing these moments and outcomes as part of a professional journey. Educational administrators therefore, can choose to pursue micro-credentials in specific areas where they need to develop their skills or knowledge, rather than being constrained by the one-size-fits-all approach of many traditional professional development programs. This aligns well with the principles of adult learning theory, which emphasizes the importance of self-directed and relevant learning experiences for adult learners. Moreover, micro-credentials can provide a more flexible and accessible means of professional development. In a country like Nigeria, where geographical and resource constraints can often limit access to traditional professional development opportunities, the digital nature of many micro-credential programs could potentially democratize access to high-quality professional learning. Olatunji and Adewumi (2021) noted that the integration of technology in teacher professional development in Nigeria has the potential to overcome many of the barriers associated with traditional face-to-face training programs. It is clear that micro-credentials are ultimately situated in the business models of companies and how they compete. For companies, whether private or public, that operate in a volatile and fast-changing environment, the commitment and the ability to continue learning are key aspects of customer quality and competitiveness. For employers, the implications are they have to organise work in ways that give employees opportunities to use their competencies in full and to further learn through work, as well as more structured learning. Giving people an opportunity to grow in their jobs and showcase their achievements is an excellent way to encourage personal and professional development and the DNA of customer centricity and innovation (Shapiro in Alangari, 2024).&nbsp; Moreover, micro-credentials can boost engagement and motivate people to transform their skills into shareable achievements, and for employers having insights into the competence base increases company agility. Micro-credentials can also highlight a learning path for a dedicated learner, being the first block of learning and a stacked credential in a shift to a new occupation or a step up the career ladder to a new job role. This can motivate and encourage the learner while helping the education institution nudge the learner to the next level, building stronger partnerships and having a visible role in professional communities and the labour market. Since micro-credentials are not about time dedicated to learning a craft or the amount of money invested into improving, this type of certificate is more inclusive and respectful of people&rsquo;s time. <strong>Statement of the Problem</strong> Educational administrators certainly have proven their creativity and flexibility in the past few years by growing skills in virtual instruction, connecting with students, and cooperating with colleagues as well as recognizing their growth. These allow them to participate in professional learning that is personalized, directly connected to their work and competency-based. Micro-credentials are closely tied to educational administrators' identifying their career paths to fulfil workforce demands suited to a knowledge-based economy, as well as provide exposure to the higher education environment for those individuals seeking a career change. However, the adoption and effectiveness of micro-credentials in the Nigerian context are not without challenges.&nbsp; The system is characterized by unequal access to technology and internet connectivity, which could limit the accessibility of digital micro-credential programs. There are arguments that for micro-credentials to be an effective tool for professional development in Nigeria, efforts must be made to address these infrastructural challenges and ensure equitable access to digital learning opportunities. Despite these challenges, the potential benefits of micro-credentials for the professional growth and development of educational administrators in Nigeria warrant further investigation. This study aims to explore the impact of micro-credentials on educational administrators' professional growth and development. The specific objectives are to examine the extent micro-credentials enhance educational administrators' professional growth and development as well as the perceived challenges of implementing micro-credentials in educational administration. <strong>Research Questions</strong> The study was guided by the following research questions: 1.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; To what extent do micro-credentials enhance educational administrators' professional growth and development? 2.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; What are the perceived challenges educational administrators face while undergoing micro-credentials? <strong>Hypotheses</strong> The following was formulated and statistically tested at a 0.05 level of significance: There is a significant difference between the mean rating on the extent to which micro-credentials enhance male and female educational administrators' professional growth and development. <strong>Social Cognitive Theory</strong> Social Cognitive Theory (SCT) started as the Social Learning Theory (SLT) in the 1960s by Albert Bandura. It developed into the SCT in 1986 and posits that learning occurs in a social context with a dynamic and reciprocal interaction of the person, environment, and behaviour (LaMorte, 2022). The unique feature of SCT is the emphasis on social influence and its emphasis on external and internal social reinforcement. SCT considers how individuals acquire and maintain behaviour, while also considering the social environment in which individuals perform the behaviour. The theory considers a person's past experiences, which factor into whether behavioural action will occur. These past experiences influence reinforcements, expectations, and expectancies, all of which shape whether a person will engage in a specific behaviour and the reasons why a person engages in that behaviour. The goal of SCT is to explain how people regulate their behaviour through control and reinforcement to achieve goal-directed behaviour that can be maintained over time (LaMorte, 2022). The SCT is built on five key principles. Observational learning (O'Leary, 2020), modelling influences behaviour, self-efficacy, and reinforcement, whether through rewards or punishments, shapes behaviour, while cognitive processes like attention, motivation, and memory mediate learning. In educational administration, SCT holds significant relevance. Teacher development programs can be designed to emphasize observation, modelling, and practice, while administrative training can focus on observational learning, self-efficacy, and reinforcement. SCT also helps administrators understand how to foster a positive school culture through modelling and reinforcement. <strong>Conceptual Clarification</strong> <strong>Micro-credentials </strong> Micro-credentials are innovation opportunities in higher education that allow administrators to acquire specific skills or knowledge to help advance their careers and also improve their profession. According to Ahsan et al. (2023), these skills or training are designed to provide more flexible learning pathways than traditional degrees which require less time to improve on specific competencies demanded by employers. Sharma et al. (2024) further added that micro-credentials help to bridge the gap between academic learning and university requirements by offering targeted career and skill development opportunities. These credentials are particularly valuable in today's rapidly evolving university requirements, where specific technical and professional competencies need frequent updating. Ghasia et al. (2019) emphasized the potential of micro-credentials in democratizing education, particularly in developing contexts like Tanzania. Their research reveals that micro-credentials can provide a more accessible pathway in education for administrators who are willing to advance in their professional growth and development. The value of micro-credentials lies in their ability to provide targeted, administrators-relevant skills while maintaining academic administrative rigour. Ahsan et al. (2023) noted that successful implementation requires careful consideration of market demands, pedagogical approaches, and technological infrastructure. Their systematic review suggests that micro-credentials are most effective when they align with both industry needs and academic standards. In the same vein, Sharma et al. (2024) argued that micro-credentials are becoming increasingly important in creating a more flexible and responsive higher education ecosystem. They suggest that blended learning approaches, combining online and face-to-face instruction, can enhance the effectiveness of micro-credential programs while improving academic administrators' growth and development. In Nigeria, micro-credentials in the university education system, offer the potential for administrators to respond more quickly to current contemporary issues facing university education through the requisition of significant skills for effective university administration and educational goal attainment. Halim et al. (2024) asserted that successful implementation requires a multi-stakeholder approach, involving educators, administrators, industry partners, and students in the design and delivery of micro-credential programs. The evidence from these studies suggests that micro-credentials are not merely a temporary trend but rather a fundamental shift in how administrators can certify learning to advance their professional growth and development.&nbsp; <strong>Micro-Credentials and Educational Administrators' Professional Growth and Development</strong> The emergence of micro-credentials has revolutionized higher education, transforming the way professionals like administrators acquire skills and advance their careers. In today's fast-paced, ever-evolving educational system, adaptability and agility are crucial. Micro-credentials create a focused, efficient, and flexible approach to learning, concentrating on specific skills or knowledge areas. The university system in Nigeria operates on a unique framework, where administrative promotions are heavily based on academic accomplishments. To ascend the administrative ladder, individuals must demonstrate exceptional scholarly competency, evident through publications in reputable journals and active participation in conferences. Academic contributions serve as a vital metric for evaluating an administrator's suitability for promotion. The quantity and quality of publications in reputable journals hold significant weight, as they feature an administrator's proficiency and commitment to advancing knowledge, growth and development in their field. Conference attendance and presentations are equally crucial, unveiling an administrator's ability to engage with peers, share research findings, and stay abreast of global educational trends. These scholarly pursuits not only enhance personal credibility but also reflect positively on the institution. In this context, administrators must strike a balance between their managerial responsibilities and academic pursuits. They must allocate time and resources to conduct research, author papers, and participate in conferences, all while ensuring the seamless operation of their departments. This system fosters a culture of scholarship and intellectual consistency within Nigerian universities, encouraging administrators to remain active contributors to their fields when they tie promotions to academic achievements, institutions incentivize excellence, driving administrators to excel as both managers and scholars. In essence, this approach enhances the overall quality of university administration, as leaders are equipped with the knowledge, expertise, and networks necessary to navigate complex academic landscapes effectively. They also offer enhanced career prospects, increased flexibility and accessibility, continuous learning and upskilling, and personalized education pathways. Additionally, micro-credentials complement traditional degree programmes where educational administrators must embrace this shift, leveraging micro-credentials to foster professional growth, enhance employability, and redefine the future of higher education. <strong>Challenges Educational Administrators Face while Undergoing Micro-Credentials</strong> Micro-credential which promotes administrators' professional growth and development is also faced with several challenges in terms of implementation. They include: <strong>1. Technological Integration Barriers: </strong>During the initial rollout of micro-credentials, educational administrators struggled significantly with integrating new digital systems into existing institutional infrastructure. Research by Dane (2024), asserted that institutions faced persistent challenges with their student information systems in tracking and recording micro-credential completions because most administrators had unexpected technical conflicts between their legacy systems and new micro-credential platforms, leading to manual workarounds that consumed significant staff time and resources. <strong>2. Professional Development Gaps: </strong>Educational administrators encountered substantial difficulties in preparing faculty for micro-credential delivery and assessment. According to Kıbaru F. (2018), administrators across multiple institutions face significant challenges in providing adequate training for faculty. Most critically, administrators struggled to help instructors transition from traditional assessment methods to competency-based evaluation systems required for micro-credentials, often resulting in inconsistent assessment practices and delayed program implementations. <strong>3. Promotion Compliance Issues: </strong>One of the most pressing challenges administrators faced was navigating accreditation requirements while implementing micro-credential programs. Van der Hijden and Martin (2023), several administrators grappled with maintaining compliance with existing accreditation standards while introducing micro-credentials. Sometimes as a result of significant delays in programme launches due to uncertainties about how micro-credentials align with traditional accreditation frameworks, particularly regarding credit hour equivalencies and learning outcome documentation. <strong>4. Budget Allocation Difficulties: </strong>Administrators encountered severe financial planning challenges during micro-credential implementation phases. Brown et al. (2022) asserted that administrators consistently underestimated academic publications, training and development costs during the early stages of micro-credential adoption. The study showed that unexpected expenses arose primarily from technology infrastructure upgrades, staff training, and marketing efforts, forcing many administrators to reallocate resources from other educational programs or seek additional funding sources mid-implementation. <strong>5. Stakeholder Resistance Management: </strong>Managing resistance from various stakeholders proved to be a significant challenge for administrators during micro-credential rollout. According to Aharonian and Schatz Oppenheimer (2024), administrators faced active resistance from multiple stakeholder groups in the institutions. Faculty members expressed concerns about academic rigour and workload increases, while department chairs worried about resource allocation and program cannibalization. Additionally, administrators struggled to address concerns from traditional degree program students who feared their credentials might be devalued by the introduction of micro-credentials. These challenges provide valuable insights into the complexities administrators face during micro-credential implementation.&nbsp; <strong>Review of Related Empirical Studies</strong> Oluwafemi (2019), examined the effects of programmed instruction, questioning and assignment teaching methods on the academic achievement of students in business studies in Oyo state, Nigeria. The study sought to determine the effect of programmed instruction, questioning and assignment teaching methods as well as gender on the academic achievement of students in Business Studies. Five research questions guided the study and five null hypotheses were tested at a 0.05 level of significance. Pretest, post-test non-equivalent group, and quasi-experimental research design were used for the study. The population of the study comprised all junior secondary II business studies students in Oyo state. A purposive sampling technique was used to comprise a sample of 201 students for the study. The instrument for data collection was a self-designed Business Studies Achievement Test (BSAT) which was validated by three experts. A reliability coefficient of 0.77 was obtained for the test items using Kuder-Richardson (KR20). Mean was used to analyze data relating to the research questions while analysis of covariance (ANCOVA) was used to test the hypotheses at a 0.05 level of significance. Findings revealed that students taught business studies using programmed instruction, questioning and assignment teaching methods performed better with higher post-test scores than those taught using conventional teaching methods. Students taught using the assignment method performed best with the highest post-test scores than those taught using programmed instruction and questioning methods. The three methods favoured both genders in the classroom but males benefitted more from programmed instruction while females benefitted more from the assignment teaching method. All the null hypotheses were rejected except the hypothesis on gender. Based on the findings of the study, it was concluded that the three teaching methods have the potential to improve students&rsquo; academic achievement in Business Studies. The study therefore recommended among others that business studies teachers should use programmed instruction, questioning and assignment teaching methods to enhance students' academic achievements in Business Studies. School administration and stakeholders in education should provide adequate training and resources needed to employ the three teaching methods for teaching business studies. Pirkkalainen et al. (2023), examined how might micro-credentials influence institutions and empower learners in higher education. A four-step Delphi study approach was used to explore how micro-credentials may shape higher education (HE) in the next 5&ndash;10 years. Educational experts undertook a consensus-building activity utilising workshops and surveys: (1) initial identification of enabling factors (i.e. drivers) and beneficial outcomes (i.e. impacts) of micro-credentials; (2) prioritisation based on importance; (3) identification of enabling factors considered essential for each beneficial outcome and (4) analysis of the extent to which micro-credentials might be accepted in HE, with participants reflecting on the importance of the previously identified enablers and outcomes for alternative scenarios. The findings of the study light on three alternative possible futures for micro-credentials. Expert consensus indicated that the potential of micro-credentials lies especially among educational institutions and the networks of institutions innovating beyond, and within, traditional study offerings and programmes. Future wide-scale adoption of micro-credentials was considered unpredictable, due to external factors at the ecosystem level, and beyond institutions&rsquo; strategies and control. The study concluded that, for the successful uptake of micro-credentials, the same benefits do not need to accrue for institutions and learners: a &lsquo;one-size-fits-all&rsquo; approach is not necessary or optimal. For the wider-scale influence of micro-credentials to be felt, there is a need for considerable international and national strategy development and implementation to overcome a variety of policy- and technology-related barriers that HEIs cannot influence or tackle on their own. Tee et al. (2024 conducted a study on marketing micro-credentials: understanding learners' engagement and willingness to pay more. A survey questionnaire was used to collect data from 354 respondents who are working adults living in the major economic states in Malaysia. Data analysis was performed using the analysis of a moment structures (AMOS) statistical software and SPSS (Statistical Package for the Social Sciences) PROCESS macro. The results show the significance of programme design factors (i.e., flexibility, system quality and content quality) in determining the learning experience. The learning experience is found as a mediator in the relationship between programme design factors learner engagement and WTPM. In addition, the moderation assessment confirms that enjoyment during learning strengthens the relationship between experience and behavioural responses. <strong>Methodology</strong> This study employed a descriptive research design to investigate the impact of micro-credentials on educational administrators' professional growth and development. The population consisted of 1589 teaching staff of Rivers State University. A sample size of 477 teachers, representing 30% of the population, was selected using simple random sampling. The data collection instrument was a questionnaire titled "Investigating the Impact of Micro-credentials on Educational Administrators' Professional Growth and Development Questionnaire (IIMEAPGDQ)". The questionnaire comprised three sections: Section A collected demographic data, while Section B addressed the research questions using a 4-point Likert scale, with options ranging from Very High Extent (VHE),&nbsp;High Extent (HE),&nbsp;Low Extent (LE) and Very Low Extent (VLE), assigned values of 4, 3, 2, and 1 respectively. To ensure validity, the questionnaire underwent expert reviews, including the researcher's supervisor. Reliability was established using Cronbach's Alpha, yielding a coefficient of 0.75.Data analysis involved descriptive statistics, specifically mean and standard deviation, to address the research questions. Inferential statistics, particularly the Independent t-test, tested hypotheses at a 0.05 significance level. Statistical Package for Social Sciences (SPSS) Version 26.0 facilitated data analysis. Results with a p-value less than 0.05 were deemed statistically significant. <strong>Data Presentation and Results</strong> A total of 477 copies of questionnaires were distributed among the teaching staff of Rivers State University and after two weeks, upon retrieval, 432 were valid representing 90.5% of the total population and were coded into the SPSS for the analysis. <strong>Answers to Research Questions</strong> <strong>Research Question 1: </strong>To what extent do micro-credentials enhance educational administrators' professional growth and development? Table 1: Showed Response Rate on Descriptive Statisticsmicro-credentials enhance educational administrators' professional growth and development &nbsp; Items N Mean Std. D. Remark &nbsp; 1. Micro-credentials enhance my professional growth and development as an educational administrators 432 2.862 .974 High Extent &nbsp; 2. My academic publication in reputable journals enhances my administrative promotion 432 2.781 1.001 High Extent &nbsp; 3. My work time allocated to research, authoring papers, and conference participation enhances my professional growth and development 432 3.015 1.192 High Extent &nbsp; 4. My tying promotions to academic achievements incentivize excellence among educational administrators. 432 3.162 1.102 High Extent &nbsp; 5. I pursue micro-credentials to enhance my skills and career prospects as an educational administrator 432 2.912 1.095 High Extent Grand Mean &nbsp; 2.94 &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; The analysis of the extent to which micro-credentials enhance educational administrators' professional growth and development yielded a grand mean of 2.94. This result indicates that educational administrators generally perceive micro-credentials as having a moderate to high impact on their professional growth and development. This implies that micro-credentials are viewed as a valuable tool for educational administrators' ongoing professional development. <strong>Research Question 2: </strong>What are the perceived challenges of implementing micro-credentials in educational administration? Table 2: Showed Response Rate on Descriptive Statistics on the perceived challenges of implementing micro-credentials in educational administration &nbsp; &nbsp; N Mean Std. D. Remark &nbsp; 6.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Technological Integration Barriers 432 3.126 .985 High Extent 7.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Professional Development Gaps 432 2.905 3.215 High Extent 8.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Promotion Compliance Issues 432 3.174 .994 High Extent 9.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Budget Allocation Difficulties 432 2.960 1.089 High Extent 10.&nbsp;&nbsp;&nbsp; Stakeholder Resistance Management 432 3.074 1.570 High Extent Grand Mean 432 3.04 &nbsp; High Extent &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; The analysis of the perceived challenges of implementing micro-credentials in educational administration revealed a grand mean of 3.04. This result indicates that educational administrators strongly agree that implementing micro-credentials poses significant challenges. This implies that the respondents overwhelmingly acknowledge the existence of substantial obstacles to successful micro-credential implementation. These challenges may encompass technological integration, professional development gaps, accreditation compliance issues, budget allocation difficulties, and stakeholder resistance. <strong>Test of Hypothesis</strong> <strong>Hypothesis 1: </strong>There is a significant difference between the mean rating on the extent to which micro-credentials enhance male and female educational administrators' professional growth and development. Summary of independent t-test analysis on the difference between the mean responses of male and female teaching staff on the extent to which micro-credentials enhance male and female educational administrators' professional growth and development. &nbsp; Students N <strong>x̅</strong> S.D <strong>Df</strong> <strong>t</strong> <strong>t_tab</strong> <strong>Sig.</strong> <strong>Decision</strong> Male teaching staff 304 24.67 1.96 475 6.41 1.96 0.00 Significant Female teaching staff 173 22.82 1.92 &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; The results of Hypothesis One reveal a statistically significant difference in the perceived impact of micro-credentials on professional growth and development between male and female educational administrators. The calculated t-value of 6.41 exceeds the critical t-value of 1.96, and the p-value of 0.00 is less than the level of significance of 0.05, leading to the rejection of the null hypothesis. This finding revealed that micro-credentials have a differing impact on the professional growth and development of male and female administrators, with gender playing a significant role in shaping their perceptions of micro-credentials effectiveness. The significant difference between the mean ratings of male and female teaching staff implies that institutions should consider gender-specific strategies to optimize the effectiveness of micro-credentials.&nbsp; <strong>DISCUSSION OF FINDINGS</strong> <strong>Impact of micro-credentials on professional growth and development&nbsp;</strong> The findings of this study reveal that micro-credentials have a positive impact on the professional growth and development of educational administrators. This result aligns with existing literature, which suggests that micro-credentials can empower learners and transform institutions in higher education (Pirkkalainen et al., 2023). Their finding added that flexible, and accessible learning opportunities and micro-credentials enable administrators to acquire new skills and knowledge, enhancing their professional competence. This, in turn, contributes to improved job performance, increased confidence, and enhanced career prospects. The study's results also emphasised the potential of micro-credentials to address specific professional development needs, bridging gaps in administrative expertise. As Pirkkalainen et al. (2023) noted, micro-credentials can facilitate institutional innovation, fostering a culture of continuous learning and professional growth. <strong>Conclusion</strong> The advent of micro-credentials has revolutionized professional development in higher education, offering a flexible, focused, and accessible approach to learning. As educational administrators navigate the challenges of their roles, micro-credentials have emerged as a vital tool for enhancing their professional growth and development. This study investigated the impact of micro-credentials on educational administrators' professional growth and development. There exists a positive impact of micro-credentials on educational administrators' professional growth and development. This implies that micro-credentials enhance administrators' professional competence, job performance, and career prospects. The flexibility, accessibility, and focus of micro-credentials make them an attractive option for administrators seeking to address specific professional development needs. <strong>Recommendations</strong> Based on the findings of the study, the following recommendations were made: &middot;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Institutions should prioritize the integration of micro-credentials into their professional development programs, leveraging their potential to enhance educational administrators' skills and expertise. This may involve providing accessible and flexible learning pathways, recognizing and rewarding micro-credential completion, and encouraging a culture of continuous learning. &middot;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Institutions should proactively address the significant challenges hindering micro-credential implementation by investing in technological infrastructure, providing targeted training and support, clarifying accreditation and compliance procedures, allocating sufficient resources, and fostering stakeholder engagement and buy-in. &nbsp; <strong>REFERENCES</strong> Aharonian, N., &amp; Schatz Oppenheimer, O. (2024). &lsquo;If you do not write, you dry up&rsquo;: Tensions in teacher educator research and academic writing.<em> Education Sciences, 14(9), 972.</em> Ahsan, K., Akbar, S., Kam, B., &amp; Abdulrahman, M. D. A. (2023). Implementation of micro-credentials in higher education: A systematic literature review.&nbsp;<em>Education and Information Technologies</em>,&nbsp;<em>28</em>(10), 13505-13540. Alangari, H. (2024). Transforming learning: The rise of micro-credentials in higher education. In <em>Digital Transformation in Higher Education, Part A</em> (pp. 83-100). Emerald Publishing Limited. Brown, M., McGreal, R., &amp; Peters, M. (2023). A strategic institutional response to micro-credentials: Key questions for educational leaders<em>. Journal of Interactive Media in Education, 3</em>(1), 45-57. Dane, Z. R. (2024). <em>Providing engaging and relevant professional learning opportunities: An evaluation of the perceived efficacy of micro-credentials &amp; digital badges upon k-12 educators in a large urban school district</em> (Doctoral dissertation, Northeastern University). Flintberg, B. (2022). <em>Digital micro-credentials for upskilling and reskilling in the vocational sector: A study on the potential use of a blockchain-based ICT system for micro-credentials in Sweden.</em> Unpublished Dissertation. University Press. Ghasia, M., Machumu, H., &amp; Smet, E. (2019). Micro-credentials in higher education institutions: An exploratory study of its place in Tanzania.&nbsp;<em>International Journal of Education and Development Using ICT</em>,&nbsp;<em>15</em>(1), 56-62. Halim, F. S. A., Luaran, J. E., &amp; Jill, L. S. S. (2024). Unravelling challenges of higher education institutions in implementing effective micro-credentials: A multi-stakeholder qualitative study.&nbsp;<em>Asian Journal of University Education</em>,&nbsp;<em>20</em>(1), 114-126. Kıbaru, F. (2018). Supporting faculty to face challenges in the design and delivery of quality courses in virtual learning environments. <em>Turkish Online Journal of Distance Education, 19</em>(4), 176-197. Kwaghbo, T. M. (2021). Organizing and managing business teacher education and training programs in higher educational institutions in Nigeria for quality business education delivery. <em>Nigerian Journal of Business Education, 8</em>(1), 51-59. LaMorte, W. W. (2022). <em>The social cognitive theory.</em> Boston University School of Public Health. https://sphweb.bumc.bu.edu/otlt/mph Liu, X., Peng, M. Y. P., Anser, M. K., Chong, W. L., &amp; Lin, B. (2020). Key teacher attitudes for sustainable development of student employability by social cognitive career theory: the mediating roles of self-efficacy and problem-based learning. <em>Frontiers in Psychology, 11</em>, 1945. Olatunji, T. I., &amp; Adewumi Adebisi, T. (2021). Comparative analysis of operational structures in single-and dual-mode distance learning institutions in Nigeria. <em>International Review of Research in Open and Distributed Learning, 22</em>(1), 59-77. O'Leary, M. (2020). <em>Classroom observation: A guide to the effective observation of teaching and learning.</em> Routledge. Oluwafemi, P. A. (2019). <em>Effects of programmed instruction, questioning and assignment teaching methods on academic achievement of students in business studies in Oyo State, Nigeria.</em> University Press. Parsons, D., Sparks, H., Vo, D., &amp; Singh, A. (2023). MOOCS and micro-credentials as launch pads to further education: Challenges and experiences. In <em>Massive Open Online Courses-Current Practice and Future Trends. IntechOpen.</em> Pirkkalainen, H., Sood, I., Padron Napoles, C., Kukkonen, A., &amp; Camilleri, A. (2023). How might micro-credentials influence institutions and empower learners in higher education? <em>Educational Research, 65</em>(1), 40-63. Sharma, H., Jain, V., Mogaji, E., &amp;Babbilid, A. S. (2024). Blended learning and augmented employability: a multi-stakeholder perspective of the micro-credentialing ecosystem in higher education.&nbsp;<em>International Journal of Educational Management</em>, 3(1), 78-86. Smith, M. J. O. (2021). <em>The impact of self-efficacy on personalized professional learning for google certification. </em>Widener University. Tee, P. K., Cham, T. H., Aw, E. C. X., Khudaykulov, A., &amp; Zhang, X. (2024). Marketing micro-credentials: understanding learners' engagement and willingness to pay more. <em>International Journal of Educational Management, 38</em>(4), 1001-1020. Van der Hijden, P., &amp; Martin, M. (2023). Short courses, micro-credentials, and flexible learning pathways: A blueprint for policy development and action. <em>International Institute for Educational Planning, </em>1-56. Wang, Y., Chaw, L. Y., Leong, C. M., Lim, Y. M., &amp; Barut, A. (2024). Massive open online courses learners' continuance intention: shaping a roadmap to micro-credentials. <em>International Journal of Educational Management, 38</em>(4), 978-1000. Westfahl, S. A., &amp; Wilkins, D. B. (2017). The leadership imperative: A collaborative approach to professional development in the global age of more for less.&nbsp;<em>Stan. L. Rev.</em>,&nbsp;<em>69</em>, 1667.

The sun bathes our planet with far more energy than humankind will possibly ever need (&gt; 8,000 times the current demand). Yet, sustainable energy provision is among the most pressing challenges faced today. In order to unlock the vast potential of clean solar energy, we need disruptive technologies capable of efficiently harvesting sunlight, while being deployable at unprecedented scales. Available commercial photovoltaics (PV) can hardly cope sustainably with the sheer scale of this challenge. Silicon solar panels are the major commercial PV, they are based on a very Earth-abundant element, but their fabrication is extremely energy intensive. Conversely, thin film solar panels based on CdTe and Cu(In,Ga)Se2 require far less energy to produce, but some of their constituent elements are quite rare on the Earth’s crust. Hence, in both cases, the short term economic and ecologic sustainabilities are dubious. Recently, an advanced PV concept, called microconcentrator PV [1], has been conceived, which is free from raw materials availability constraints and is based on sunlight absorbers requiring low energy to grow. To demonstrate microconcentrator PV at laboratory scale, research groups have been using optical projection lithography (OPL), generating arrays of Cu(In,Ga)Se2 circles with tens of micrometer diameter. However, OPL cannot be scaled credibly to terawatt deployment. Industrial uptake of microconcentrator PV is only possible with a technique that ensures both high semiconductor quality, and high throughput at capital expenditure comparable or lower than currently available PVs. Inspired by the research of the US partner Gary Friedman [2], the Italian PI D. Colombara has patented a disrupting microfabrication technique [3] that could be scaled economically to deploy terawatts of microconcentrator PV. Our intent in this project is to leverage our complementary know-how to empower young PV pioneers and establish a lasting cooperation in this new promising field. Acknowledgements This work was supported in part by the Italian Ministry of Foreign Affairs and International Cooperation, grant number US23GR17. REMAP has received funding from the European Commission PathFinder Open programme under grant agreement No. 101046909. Funded by the European Union. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or European Innovation Council and SME Executive Agency (EISMEA). Neither the European Union nor the granting authority can be held responsible for them. References [1] M. Alves, A. Pérez-Rodríguez, P. J. Dale, C. Domínguez, and S. Sadewasser, ‘Thin-film micro-concentrator solar cells’, J. Phys. Energy, vol. 2, 012001, 2019, doi: 10.1088/2515-7655/ab4289. [2] B. B. Yellen, G. Fridman, and G. Friedman, ‘Ferrofluid lithography’, Nanotechnology, vol. 15, S562–S565, 2004, doi: 10.1088/0957-4484/15/10/011. [3] D . Colombara, ‘Method for patterning a surface of a substrate’ 2019. Available: https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2020136057

<strong>Obuchowska Aleksandra, W&oacute;jcik Justyna, Standyło Arkadiusz, Obuchowska Karolina, Ozga Alicja, Kimber-Trojnar Żaneta, </strong> <strong>Leszczyńska-Gorzelak Bożena</strong><strong>. Assessment of students&#39; knowledge on iodine supplementation by women planning pregnancy. Journal of Education, Health and Sport. 2020;10(9):270‑277. eISSN 2391-8306. DOI http://dx.doi.org/10.12775/JEHS.2020.10.09.030</strong> <strong>https://apcz.umk.pl/czasopisma/index.php/JEHS/article/view/JEHS.2020.10.09.030</strong> <strong>https://zenodo.org/record/4024544</strong> &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; <strong>The journal has had 5 points in Ministry of Science and Higher Education parametric evaluation. &sect; 8. 2) and &sect; 12. 1. 2) 22.02.2019.</strong> <strong>&copy; The Authors 2020;</strong> <strong>This article is published with open access at Licensee Open Journal Systems of Nicolaus Copernicus University in Torun, Poland</strong> <strong>Open Access. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author (s) and source are credited. This is an open access article licensed under the terms of the Creative Commons Attribution Non commercial license Share alike.</strong> <strong>(http://creativecommons.org/licenses/by-nc-sa/4.0/) which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited.</strong> <strong>The authors declare that there is no conflict of interests regarding the publication of this paper.</strong> &nbsp; <strong>Received: 20.08.2020. Revised: 25.08.2020. Accepted: 11.09.2020.</strong> &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; <strong>Assessment of students&#39; knowledge on iodine supplementation by women planning pregnancy</strong> &nbsp; <strong>Aleksandra Obuchowska^1a, Justyna W&oacute;jcik^1b, Arkadiusz Standyło^1c, Karolina&nbsp;Obuchowska^1d, Alicja Ozga^1e, Żaneta Kimber-Trojnar^2f, </strong> <strong>Bożena Leszczyńska-Gorzelak^2g</strong> &nbsp; ^aaobuchowska12@gmail.com; https://orcid.org/0000-0003-0464-2695 ^bjustynawojcik455@gmail.com; https://orcid.org/0000-0001-7163-6784 ^c a.standylo@gmail.com; https://orcid.org/0000-0002-5154-4759 ^{d &shy;}karolinaobuchowska99@gmail.com; https://orcid.org/0000-0003-4519-8236 ^eaozga1@gmail.com; https://orcid.org/0000-0003-1291-905X ^fzkimber@poczta.onet.pl; https://orcid.org/0000-0001-7295-0409 ^gb.leszczynska@umlub.pl; https://orcid.org/0000-0002-0221-1982 &nbsp; ¹Student&#39;s Scientific Association at the Chair and Department of Perinatology and Obstetrics, Medical University of Lublin, Poland ²Chair and Department of Perinatology and Obstetrics, Medical University of Lublin, Poland &nbsp; <strong>Corresponding author: </strong> Aleksandra Obuchowska Student&#39;s Scientific Association at the Department of Perinatology and Obstetrics Jaczewskiego 8 Street 20-954 Lublin, Poland e-mail: aobuchowska12@gmail.com <strong>Abstract: </strong> <strong>Introduction:</strong> Iodine is essential for the synthesis of the thyroid hormones. Proper thyroid function in a pregnant woman depends on the accumulated reserves of iodine in the pre-pregnancy period. If they are sufficient, the production of thyroid hormones is adequate even in the case of increased demand. Adequate iodine status during pregnancy is crucial for maternal health and foetal growth. Severe iodine deficiency causes maternal and foetal hypothyroxinaemia, leading to irreversible brain damage, manifested by mental retardation and neurological disorders. The daily requirement for iodine for pregnant women has been determined by The World Health Organization (WHO) at 250 &micro;g. Most likely, the demand for it cannot be covered only by dietary iodine intake, so iodine should be supplemented in the form of tablets at a dose of 150-200 &micro;g daily. <strong>Purpose:</strong> The aim of the study is to assess students&#39; knowledge on iodine supplementation by women planning pregnancy and pregnant women. <strong>Material and method: </strong>Students&#39; knowledge regarding the importance of iodine supplementation was verified using anonymous Internet questionnaire. The obtained results were analysed and checked on the basis of scientific literature. <strong>Results: </strong>The percentage of students who think iodine supplementation should be recommended is 22.7%, and 9.7% believe it is not necessary. Only 8.8% of the respondents chose the correct recommended dose of iodine. Surprisingly, most of the respondents do not know the recommended iodine dose, as much as 71.7% of them. <strong>Conclusions: </strong>The proper supply of iodine has a decisive impact on the proper functioning of the body, and its deficiency has serious consequences, especially in the womb. The problem of iodine deficiency is serious. This is also confirmed by the results of our surveys. It is important to educate students on the recommendations for iodine supplementation in women of childbearing age. &nbsp; <strong>Key words: </strong>Iodine, Iodine deficiency disorder, pregnancy, supplementation among students, dietary supplements, foetal development

Asymmetric membrane currents and fluxes of Ca2+ release were determined in skeletal muscle fibers voltage clamped in a Vaseline-gap chamber. The conditioning pulse protocol 1 for suppressing Ca2+ release and the "hump" component of charge movement current (I gamma), described in the first paper of this series, was applied at different test pulse voltages. The amplitude of the current suppressed during the ON transient reached a maximum at slightly suprathreshold test voltages (-50 to -40 mV) and decayed at higher voltages. The component of charge movement current suppressed by 20 microM tetracaine also went through a maximum at low pulse voltages. This anomalous voltage dependence is thus a property of I gamma, defined by either the conditioning protocol or the tetracaine effect. A negative (inward-going) phase was often observed in the asymmetric current during the ON of depolarizing pulses. This inward phase was shown to be an intramembranous charge movement based on (a) its presence in the records of total membrane current, (b) its voltage dependence, with a maximum at slightly suprathreshold voltages, (c) its association with a "hump" in the asymmetric current, (d) its inhibition by interventions that reduce the "hump", (e) equality of ON and OFF areas in the records of asymmetric current presenting this inward phase, and (f) its kinetic relationship with the time derivative of Ca release flux. The nonmonotonic voltage dependence of the amplitude of the hump and the possibility of an inward phase of intramembranous charge movement are used as the main criteria in the quantitative testing of a specific model. According to this model, released Ca2+ binds to negatively charged sites on the myoplasmic face of the voltage sensor and increases the local transmembrane potential, thus driving additional charge movement (the hump). This model successfully predicts the anomalous voltage dependence and all the kinetic properties of I gamma described in the previous papers. It also accounts for the inward phase in total asymmetric current and in the current suppressed by protocol 1. According to this model, I gamma accompanies activating transitions at the same set of voltage sensors as I beta. Therefore it should open additional release channels, which in turn should cause more I gamma, providing a positive feedback mechanism in the regulation of calcium release.

ABSTRACT This study aimed to investigate the impact of Sharia Supervisory Board (SSB) on Islamic Social Responsibility (ISR) by Islamic Commercial Banks in Indonesia. Secondary data were collected from the annual reports of 14 Islamic Commercial Banks in Indonesia from 2010 to 2020, resulting in 125 firm-year data. A panel data regression was applied to analyze the data. The analysis results show that SSB age, cross-membership, and meeting frequency have a positive effect on ISR, whereas size has a negative effect. However, this analysis results did not manage to support the effect of SSB qualifications (Ph.D.) and expertise on ISR. The findings imply that SSB needs to increase the frequency of meetings to improve its supervisory function over Shariah banks' management, including in encouraging more comprehensive ISR disclosure. The contribution of this research is that research focuses on the characteristics of SSB on ISR by using Sharia Enterprise Theory (SET) to provide a better understanding of how these factors influence the implementation of ISR in the sharia context. Keywords: Sharia Supervisory Board Characteristics, Social Reporting, Islamic Bank, Indonesia ABSTRAK Tujuan dari penelitian ini adalah untuk menyelidiki dampak SSB terhadap Islamic Social Reporting (ISR) oleh Bank Umum Syariah di Indonesia. Data sekunder dikumpulkan dari laporan tahunan 14 Bank Umum Syariah di Indonesia sejak 2010 hingga 2020, menghasilkan 125 data firm-year. Regresi data panel diterapkan untuk menganalisis data. Hasil analisis menunjukkan bahwa usia SSB, lintas keanggotaan, dan frekuensi pertemuan berpengaruh positif pada ISR, sedangkan ukuran berpengaruh negatif. Namun hasil analisis ini tidak mendukung pengaruh kualifikasi (Ph.D.) dan keahlian SSB terhadap ISR. Temuan ini menyiratkan bahwa DPS perlu meningkatkan frekuensi pertemuan dalam rangka meningkatkan fungsi pengawasan terhadap manajemen bank syariah, termasuk dalam mmendorong pengungkapan ISR yang lebih komprehensif. kontribusi dalam penelitian ini yaitu Penelitian memfokuskan pada karakteristik SSB terhadap ISR dengan menggunakan Syariah Enterprise Theory (SET) untuk memberikan pemahaman yang lebih baik tentang bagaimana faktor-faktor tersebut mempengaruhi pelaksanaan ISR dalam konteks syariah. Kata Kunci: Karakteristik Dewan Pengawas Syariah, Pelaporan Sosial, Bank Syariah, Indonesia. REFERENCES Abdullah, W. A. W., Percy, M., &amp; Stewart, J. (2013). Shari’ah disclosures in Malaysian and Indonesian Islamic banks The Shari’ah governance system. Journal of Islamic Accounting and Business Research, 4(2), 100–131. doi:10.1108/JIABR-10-2012-0063 Adiertanto, C. P., &amp; Chariri, A. (2013). 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ABSTRAK Wakaf tunai merupakan salah satu instrumen keuangan syariah berbasis sosial yang penting untuk dikembangkan karena mudah digunakan sesuai dengan kebutuhan masyarakat yang ada, seperti untuk modal usaha mikro bagi petani. Mayoritas petani adalah masyarakat kelas menengah ke bawah yang menggantungkan mata pencahariannya dari hasil panen. Pandemi Covid-19 yang melanda Indonesia dan dunia membuat banyak keluarga mengalami kekurangan finansial, termasuk keluarga petani. Wakaf dapat membantu pembiayaan produksi para petani melalui program Wakaf Modal Usaha Mikro. Namun, masih terdapat beberapa kendala program ini sehingga program tersebut tidak berjalan secara maksimal. Penelitian ini bertujuan untuk menganalisis prioritas masalah dan solusi untuk mengoptimalkan peran wakaf modal usaha mikro bagi petani di Lamongan. Jenis penelitian ini adalah metode campuran dengan menggunakan pendekatan Analytic Network Process (ANP). Data diperoleh dari wawancara mendalam dengan tujuh pakar wakaf dan pertanian. Prioritas masalah adalah pada di nazhir terkait minimnya anggaran untuk pelayanan administrasi, sedangkan prioritas solusinya adalah menambah anggaran untuk pelayanan administrasi. Hasil penelitian dapat digunakan oleh Badan Wakaf Indonesia (BWI) bersama dengan Dinas Tanaman Pangan, Hortikultura, dan Perkebunan untuk mengembangkan wakaf di bidang pertanian. Kata Kunci: Wakaf Uang, Wakaf Produktif, Pemberdayaan Petani, Modal Usaha Mikro. ABSTRACT Cash waqf is one of the essential social-based Islamic financial instruments to be developed since it is easily used according to the existing community's needs, such as for micro-business capital for farmers. Most working as farmers are middle- to lower-class people who depend on their livelihoods from harvests. The Covid-19 pandemic that has hit Indonesia and the world has left many families experiencing financial shortages, including farming families. The Micro Business Capital Waqf program has been implemented in Lamongan. However, this program still encountered several problems that made the program not optimally conducted and could not be implemented in the broader community. This study aims to analyze the priority problems and solutions to optimize the role of micro-business capital waqf for farmers in Lamongan. This type of research was a mixed method using the Analytic Network Process (ANP) approach. Data were obtained from in-depth interviews with seven waqf and agriculture experts. The priority problem in Nazhir was related to the lack of budget for administrative services, while the priority solution was increasing the funding for administrative services. The research results can be used by the Indonesian Waqf Board (BWI) and the Department of Food Crops, Horticulture, and Plantations to develop waqf in agriculture. Keywords: Cash Waqf, Productive Waqf, Farmer Empowerment, Micro Business Capital. REFERENCES Ali, K. M., Yuliani, M., Mulatsih, S., &amp; Abdullah, Z. (2018). Aspek-aspek prioritas manajemen wakaf di Indonesia. Al Falah: Journal of Islamic Economics, 3(1), 1-26. DOI: http://dx.doi.org/10.29240/jie.v3i1.345 Amuda, Y. J. (2017). Commercialization of cash waqf in Nigeria: An analysis of its implementation. Doctoral Dissertation, Academy of Islamic Studies, University of Malaya Amuda, Y. J., Embi, A. C., &amp; Babatunde, O. H. (2014). An agricultural approach to the commercialization of cash waqf between Malaysia and Nigeria. Journal of Advanced Management Science, 2(4), 344–348. DOI: 10.12720/joams.2.4.344-348 Ascarya, &amp; Yumanita, D. (2018). Analisis rendahnya pengumpulan zakat di Indonesia dan alternatif solusinya. Working Paper Bank Indonesia, WP/9/2018 Faizin, Y. (2021). Micro business capital waqf for lamongan farmer Global Wakaf. (2020a). Global wakaf solusi sejatinya membangun kehidupan. Dipresentasikan dalam acara Indonesia Sharia Economic Festival (ISEF). Indonesia Sharia Economic Festival (ISEF). Retrieved from https://isef.co.id/wp-content/uploads/2020/11/5.-GLOBAL-WAKAF_ISEF_06102020.pdf Global Wakaf. (2020b). Panen raya bahagiakan petani binaan global wakaf. Retrieved from https://news.act.id/berita/panen-raya-bahagiakan-petani-binaan-global-wakaf Hamzani, A. (2015). Perkembangan hukum wakaf di Indonesia. Diya Media Group Hasan, S., &amp; Rajafi, A. (2018). Pengelolaan tanah wakaf masjid di kota manado. Aqlam: Journal of Islam and Plurality, 3(2). DOI: http://dx.doi.org/10.30984/ajip.v3i2.726 Huda, N., Rini, N., Mardoni, Y., Hudori, K., &amp; Anggraini, D. (2017). Problems, solutions, and strategies priority for waqf in Indonesia. Journal of Economic Cooperation &amp; Development, 38(1), 29-54. Hydra, M. (2020). An integrative model of waqf, sadaqah, and takaful for poverty alleviation through empowering women farmers in the rural Gambia. Journal of Islamic Finance, 5(3), 1–12. Indonesian Waqf Board. (2021). Gerakan nasional wakaf uang 2021. Retrieved from https://www.bwi.go.id/5806/2021/01/21/gerakan-nasional-wakaf-uang-2021/ Khamim. (2021). Micro business capital waqf for lamongan farmer Kurniawan, E., Iman, N., &amp; Santoso, A. (2021). Simas Waqfuna sebagai solusi pengelolaan wakaf untuk multi oraganisasi yang transparan (Simas Waqfuna). IKRA-ITH Informatika: Jurnal Komputer Dan Informatika, 5(3), 18–27. Lin, J. H., &amp; Yang, C. J. (2016). Applying analytic network process to the selection of construction projects. Open Journal of Social Sciences, 4(3), 41. DOI:10.4236/jss.2016.43007 Melinda, S., Qurrat, V. ., Yusida, E., Purnamasari, V., Seprillina, L., &amp; Hussain, N. (2021). The role of cash waqf as a source of micro business financing for strengthening the local economy: a case study in Gunung Kawi district, Malang regency. Review of Integrative Business and Economics Research, 10(1), 136–144. Ministry of Communication and Information Technology. (2022, October 7). Perkuat literasi wakaf secara berkelanjutan pemerintah libatkan forum jurnalis wakaf indonesia. Retrieved from kominfo.go.id: https://www.kominfo.go.id/content/detail/44786/perkuat-literasi-wakaf-secara-berkelanjutan-pemerintah-libatkan-forum-jurnalis-wakaf-indonesia/0/berita#:~:text=Badan%20Wakaf%20Indonesia%20(BWI)%20mencatat,2021%20senilai%20855%20miliar%20rupiah. Ministry of Religion of the Republic of Indonesia. (2013a). Panduan pengelolaan wakaf tunai. Retrieved from https://jatim.kemenag.go.id/file/file/panduanwakaf/ynqo1425024661.pdf Ministry of Religion of the Republic of Indonesia. (2013b). Pedoman pengelolaan dan perkembangan wakaf. Direktoral Jendral Bimbingan Masyarakat Islam. Direktorat Pemberdayaan Wakaf Muntaqo, F. (2015). Problematika dan prospek wakaf produktif di Indonesia. Al-Ahkam, 1(25), 83-108. DOI: 10.21580/ahkam.2015.1.25.195 Purba, H. J., Yusuf, E. S., &amp; Erwidodo. (2020). Dampak pandemi Covid-19 terhadap pertumbuhan ekonomi dan sektor pertanian. In Dampak Pandemi Covid-19: Perspektif Adaptasi dan Resiliensi Sosial Ekonomi Pertanian (pp. 23-46). Jakarta: IAARD PRESS Rahman, Y. (2021). Micro business capital waqf for Lamongan farmers Rizal, M. S. (2021). Manajemen wakaf tunai berbasis masjid di masa Covid-19 (studi pengelolaan dana wakaf sebagai modal usaha mikro jama’ah di Masjid Baiturrohman Madiun) [Institut Islam Negeri Ponorogo]. Tesis tidak dipublikasikan. Institut Islam Negeri Ponorogo. Rusydiana, A. &amp; Devi, A. (2013). Analytic network process: Pengantar teori dan aplikasi. Smart Publishing. Saptono, I. (2019). Wakaf untuk kesejahteraan ummat dalam konteks revolusi industry 4.0. Dipresentasikan dalam Indonesia Waqf Summit 2019. Sapuan, N. M., Rajadurai, J., Zeni, N. A. M., &amp; Hashim, S. L. M. (2018). Developing a holistic business model for an efficient waqf property in Malaysia. Global Business and Management Research, 10(3), 445. Sisyanto. (2017). Micro business capital waqf for Lamongan farmers Sulistiani, S. (2017). Perbaruan hukum wakaf di Indonesia. PT Refika Aditama Syahyuti., &amp; Aldillah, R. (2020). Upaya menekan dampak pandemi Covid-19 pada sumber daya manusia pertanian. In Dampak Pandemi Covid-19: Perspektif Adaptasi dan Resiliensi Sosial Ekonomi Pertanian (pp. 549-570). Jakarta: IAARD PRESS. Syahyuti., &amp; Elizabeth, R. (2020). Kebutuhan jaring pengaman sosial untuk petani pada masa pandemi Covid-19. In Dampak Pandemi Covid-19: Perspektif Adaptasi dan Resiliensi Sosial Ekonomi Pertanian (pp. 481-498). Jakarta: IAARD PRESS. Thanker, M. A. M. T., Amin, M. F., Thaker, H. M. T., Khaliq, A., &amp; Pitchay, A. A. (2020). Cash waqf model for micro enterprise's human capital development. ISRA International Journal of Islamic Finance, 13(1), 66-83. DOI: 10.1108/IJIF-08-2018-0091 Utomo, Y. (2021). Micro business capital waqf for Lamongan farmers Youneszadeh, H., Ardeshir, A., &amp; Sebt, M. (2017). Exploring critical success factors in urban housing projects using fuzzy analytic network process. Civil Engineering Journal, 3(11), 1048–1067. DOI: 10.28991/cej-030937 Yuli, S. B. C. (2015). Optimalisasi peran wakaf dalam pemberdayaan Usaha Mikro, Kecil dan Menengah (UMKM). Ekonomika-Bisnis, 6(1), 1–15. DOI: https://doi.org/10.22219/jibe.v6i1.2269 Yusof, M. A., Aziz, M. R. A., &amp; Johari, F. (2013). The relationship between level of income and willingness of muslim community to contribute for Islamic waqf bank. In 5th Islamic Economic System Conference (IECONS 2013), Berjaya Times Square Hotel, Kuala Lumpur on 4th-5th September. Retrieved from http://www.nuradli.com/iecons2013/4B-1.pdf

Abstract. 1. The conference The third conference on "Nonlinear VAriability in Geophysics: scaling and multifractal processes" (NVAG 3) was held in Cargese, Corsica, Sept. 10-17, 1993. NVAG3 was joint American Geophysical Union Chapman and European Geophysical Society Richardson Memorial conference, the first specialist conference jointly sponsored by the two organizations. It followed NVAG1 (Montreal, Aug. 1986), NVAG2 (Paris, June 1988; Schertzer and Lovejoy, 1991), five consecutive annual sessions at EGS general assemblies and two consecutive spring AGU meeting sessions. As with the other conferences and workshops mentioned above, the aim was to develop confrontation between theories and experiments on scaling/multifractal behaviour of geophysical fields. Subjects covered included climate, clouds, earthquakes, atmospheric and ocean dynamics, tectonics, precipitation, hydrology, the solar cycle and volcanoes. Areas of focus included new methods of data analysis (especially those used for the reliable estimation of multifractal and scaling exponents), as well as their application to rapidly growing data bases from in situ networks and remote sensing. The corresponding modelling, prediction and estimation techniques were also emphasized as were the current debates about stochastic and deterministic dynamics, fractal geometry and multifractals, self-organized criticality and multifractal fields, each of which was the subject of a specific general discussion. The conference started with a one day short course of multifractals featuring four lectures on a) Fundamentals of multifractals: dimension, codimensions, codimension formalism, b) Multifractal estimation techniques: (PDMS, DTM), c) Numerical simulations, Generalized Scale Invariance analysis, d) Advanced multifractals, singular statistics, phase transitions, self-organized criticality and Lie cascades (given by D. Schertzer and S. Lovejoy, detailed course notes were sent to participants shortly after the conference). This was followed by five days with 8 oral sessions and one poster session. Overall, there were 65 papers involving 74 authors. In general, the main topics covered are reflected in this special issue: geophysical turbulence, clouds and climate, hydrology and solid earth geophysics. In addition to AGU and EGS, the conference was supported by the International Science Foundation, the Centre Nationale de Recherche Scientifique, Meteo-France, the Department of Energy (US), the Commission of European Communities (DG XII), the Comite National Francais pour le Programme Hydrologique International, the Ministere de l'Enseignement Superieur et de la Recherche (France). We thank P. Hubert, Y. Kagan, Ph. Ladoy, A. Lazarev, S.S. Moiseev, R. Pierrehumbert, F. Schmitt and Y. Tessier, for help with the organization of the conference. However special thanks goes to A. Richter and the EGS office, B. Weaver and the AGU without whom this would have been impossible. We also thank the Institut d' Etudes Scientifiques de Cargese whose beautiful site was much appreciated, as well as the Bar des Amis whose ambiance stimulated so many discussions. 2. Tribute to L.F. Richardson With NVAG3, the European geophysical community paid tribute to Lewis Fry Richardson (1881-1953) on the 40th anniversary of his death. Richardson was one of the founding fathers of the idea of scaling and fractality, and his life reflects the European geophysical community and its history in many ways. Although many of Richardson's numerous, outstanding scientific contributions to geophysics have been recognized, perhaps his main contribution concerning the importance of scaling and cascades has still not received the attention it deserves. Richardson was the first not only to suggest numerical integration of the equations of motion of the atmosphere, but also to attempt to do so by hand, during the First World War. This work, as well as a presentation of a broad vision of future developments in the field, appeared in his famous, pioneering book "Weather prediction by numerical processes" (1922). As a consequence of his atmospheric studies, the nondimensional number associated with fluid convective stability has been called the "Richardson number". In addition, his book presents a study of the limitations of numerical integration of these equations, it was in this book that - through a celebrated poem - that the suggestion that turbulent cascades were the fundamental driving mechanism of the atmosphere was first made. In these cascades, large eddies break up into smaller eddies in a manner which involves no characteristic scales, all the way from the planetary scale down to the viscous scale. This led to the Richardson law of turbulent diffusion (1926) and tot he suggestion that particles trajectories might not be describable by smooth curves, but that such trajectories might instead require highly convoluted curves such as the Peano or Weierstrass (fractal) curves for their description. As a founder of the cascade and scaling theories of atmospheric dynamics, he more or less anticipated the Kolmogorov law (1941). He also used scaling ideas to invent the "Richardson dividers method" of successively increasing the resolution of fractal curves and tested out the method on geographical boundaries (as part of his wartime studies). In the latter work he anticipated recent efforts to study scale invariance in rivers and topography. His complex life typifies some of the hardships that the European scientific community has had to face. His educational career is unusual: he received a B.A. degree in physics, mathematics, chemistry, biology and zoology at Cambridge University, and he finally obtained his Ph.D. in mathematical psychology at the age of 47 from the University of London. As a conscientious objector he was compelled to quit the United Kingdom Meteorological Office in 1920 when the latter was militarized by integration into the Air Ministry. He subsequently became the head of a physics department and the principal of a college. In 1940, he retired to do research on war, which was published posthumously in book form (Richardson, 1963). This latter work is testimony to the trauma caused by the two World Wars and which led some scientists including Richardson to use their skills in rational attempts to eradicate the source of conflict. Unfortunately, this remains an open field of research. 3. The contributions in this special issue Perhaps the area of geophysics where scaling ideas have the longest history, and where they have made the largest impact in the last few years, is turbulence. The paper by Tsinober is an example where geometric fractal ideas are used to deduce corrections to standard dimensional analysis results for turbulence. Based on local spontaneous breaking of isotropy of turbulent flows, the fractal notion is used in order to deduce diffusion laws (anomalous with respect to the Richardson law). It is argued that his law is ubiquitous from the atmospheric boundary layer to the stratosphere. The asymptotic intermittency exponent i hypothesized to be not only finite but to be determined by the angular momentum flux. Schmitt et al., Chigirinskaya et al. and Lazarev et al. apply statistical multifractal notions to atmospheric turbulence. In the former, the formal analogy between multifractals and thermodynamics is exploited, in particular to confirm theoretical predictions that sample-size dependent multifractal phase transitions occur. While this quantitatively explains the behavior of the most extreme turbulent events, it suggests that - contrary to the type of multifractals most commonly discussed in the literature which are bounded - more violent (unbounded) multifractals are indeed present in the atmospheric wind field. Chigirinskaya et al. use a tropical rather than mid-latitude set to study the extreme fluctuations form yet another angle: That of coherent structures, which, in the multifractal framework, are identified with singularities of various orders. The existence of a critical order of singularity which distinguishes violent "self-organized critical structures" was theoretically predicted ten years ago; here it is directly estimated. The second of this two part series (Lazarev et al.) investigates yet another aspect of tropical atmospheric dynamics: the strong multiscaling anisotropy. Beyond the determination of universal multifractal indices and critical singularities in the vertical, this enables a comparison to be made with Chigirinskaya et al.'s horizontal results, requiring an extension of the unified scaling model of atmospheric dynamics. Other approaches to the problem of geophysical turbulence are followed in the papers by Pavlos et al., Vassiliadis et al., Voros et al. All of them share a common assumption that a very small number of degrees of freedom (deterministic chaos) might be sufficient for characterizing/modelling the systems under consideration. Pavlos et al. consider the magnetospheric response to solar wind, showing that scaling occurs both in real space (using spectra), and also in phase space; the latter being characterized by a correlation dimension. The paper by Vassiliadis et al. follows on directly by investigating the phase space properties of power-law filtered and rectified gaussian noise; the results further quantify how low phase space correlation dimensions can occur even with very large number of degrees of freedom (stochastic) processes. Voros et al. analyze time series of geomagnetic storms and magnetosphere pulsations, also estimating their correlation dimensions and Lyapounov exponents taking special care of the stability of the estimates. They discriminate low dimensional events from others, which are for instance attributed to incoherent waves. While clouds and climate were the subject of several talks at the conference (including several contributions on multifractal clouds), Cahalan's contribution is the only one in this special issue. Addressing the fundamental problem of the relationship of horizontal cloud heterogeneity and the related radiation fields, he first summarizes some recent numerical results showing that even for comparatively thin clouds that fractal heterogeneity will significantly reduce the albedo. The model used for the distribution of cloud liquid water is the monofractal "bounded cascade" model, whose properties are also outlined. The paper by Falkovich addresses another problem concerning the general circulation: the nonlinear interaction of waves. By assuming the existence of a peak (i.e. scale break) at the inertial oscillation frequency, it is argued that due to remarkable cancellations, the interactions between long inertio-gravity waves and Rossby waves are anomalously weak, producing a "wave condensate" of large amplitude so that wave breaking with front creation can occur. Kagan et al., Eneva and Hooge et al. consider fractal and multifractal behaviour in seismic events. Eneva estimates multifractal exponents of the density of micro-earthquakes induced by mining activity. The effects of sample limitations are discussed, especially in order to distinguish between genuine from spurious multifractal behaviour. With the help of an analysis of the CALNET catalogue, Hooge et al. points out, that the origin of the celebrated Gutenberg-Richter law could be related to a non-classical Self-Organized Criticality generated by a first order phase transition in a multifractal earthquake process. They also analyze multifractal seismic fields which are obtained by raising earthquake amplitudes to various powers and summing them on a grid. In contrast, Kagan, analyzing several earthquake catalogues discussed the various laws associated with earthquakes. Giving theoretical and empirical arguments, he proposes an additive (monofractal) model of earthquake stress, emphasizing the relevance of (asymmetric) stable Cauchy probability distributions to describe earthquake stress distributions. This would yield a linear model for self-organized critical earthquakes. References: Kolmogorov, A.N.: Local structure of turbulence in an incompressible liquid for very large Reynolds number, Proc. Acad. Sci. URSS Geochem. Sect., 30, 299-303, 1941. Perrin, J.: Les Atomes, NRF-Gallimard, Paris, 1913. Richardson, L.F.: Weather prediction by numerical process. Cambridge Univ. Press 1922 (republished by Dover, 1965). Richardson, L.F.: Atmospheric diffusion on a distance neighbour graph. Proc. Roy. of London A110, 709-737, 1923. Richardson, L.F.: The problem of contiguity: an appendix of deadly quarrels. General Systems Yearbook, 6, 139-187, 1963. Schertzer, D., Lovejoy, S.: Nonlinear Variability in Geophysics, Kluwer, 252 pp, 1991.

<strong>ABSTRACT</strong> The biomining can happen in basalt rocks from ridges which contains around 25% Mn in the form of different minerals and oxides due to deep sea hydrothermal activity. The prime goal of this experiment was to demonstrate Mn mobilization from natural Mn minerals and oxides using striping voltammetry from basalt near deep-sea hydrothermal vents (DSHVs) by a potential bacterial isolate R6 which was isolated from this environment. Natural basalt sample was collected from the carls berg ridge during ABP-36 cruise and was characterized by scanning electron microscopy (SEM) and X-ray diffractometry. Bacterial isolation was done in laboratory by spread plate method using 100uM Mn amended NA media plates. Isolated bacteria R6 (Accsession No. LK934696) and basalt sample were used in a laboratory batch experiment. The isolate R6 (identified as marine Bacteria <em>Imtechella halotolerans sp.</em>) and natural basalt rock were placed in 100% seawater in the presence and absence of an organic carbon supplement as 0.01% glucose (analogous abiotic and chemical controls systems were also included). This laboratory batch experiment was incubated in the dark at 28 &plusmn; 2 &deg;C for 6 months and cell bio mass, pH, Eh and concentrations of mobilized Mn ions were measured over time.&nbsp; The presence of the bacteria induced the release of Mn from the basalt relative to the controls, especially with the addition of the organic carbon supplement. Bacteria was able to draw significant mobilization rate 27985.91 and 4797.37 &mu;g g^-1 d^-1 with and without glucose added in biological experiment part when compared with abiotic and chemical controls.&nbsp; Bacterial colonies on the basalt fragments surfaces were examined by SEM which shows evidence of extrapolysaccherides secretion and mineral precipitation. The results of this study suggest that chemoorganotrophic bacteria are involved in the cycling of Mn mobilization in basalt near DSHVs. <strong>Keywords</strong>: bacteria, basalt, mobilization, hydrothermal vent, marine, mineral <strong>REFERENCES</strong> APHA, AWWA, WEF. 2005. Oxidation-Reduction Potential (ORP). In: Standard Methods for the Examination of Water and Wastewater. Eaton AD, Clesceri LS, Rice EW, Greenberg AE (eds) Washington, DC, pp. 2-75 to 2-79. Amy, P.S., Morita, R.Y., 1983. Starvation survival patterns of sixteen freshly isolated open-ocean bacteria. Appl. Environ. Microbiol. 45, 1109-1115. 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Roh, Y., Liu, S.V., Li, G., Huang, H., Phelps, T.J., Zhou, J., 2002. Isolation and characterization of metal-reducing <em>Thermoanaerobacter </em>strains from deep subsurface environments of the Piceance basin, Colorado. Appl. Environ. Microbiol. 68, 6013-6020. Roy, P., Balaram, V., Kumar, A., Satyanarayanan, M., Rao, T.G., 2007. New REE and trace element data on two kimberlitic reference materials by ICP-MS. Geostand. Geoanal. Res. 31, 261-273 Russin, P., Ehrlich, H.L., 1995. Developments in microbial leaching: mechanisms of manganese solubilisation. Adv. Biochem. Eng/Biotechnol. 52, 1-26. Shehata, T.E., Marr, A.G., 1971. Effect of nutrient concentration on the growth of <em>Escherichia coli. </em>J. Bacteriol. 107,210-216. Sujith, P.P., Khedekar, V.D., Girish, A.P., Loka Bharathi, P.A., 2010. Immobilization of nickel by bacterial isolates from the Indian ridge system and the chemical nature of the accumulated metal. Geomicrobiol. J. 27, 424-434. Sujith P.P, Mourya B.S, Krishanmurthi S., Meena R.M.&nbsp; and Loka Bharathi P.A^*, 2014. Mobilization of manganese by basalt associated Mn(II)-oxidizing bacteria from the Indian Ridge System. Chemosphere. 95, 486-495. Seyfried WE Jr, Mottl MJ. 1995. Geologic setting and chemistry of deep-sea hydrothermal vents. In: Karl DM, editor. The Microbiology of Deep-Sea Hydrothermal Vents, Boca Raton, FL: CRC Press. pp. 1&ndash;34. Thamdrup, B., 2000. Microbial manganese and iron reduction in aquatic sediments. Adv. Microb. Ecol. 16, 41-84. Torrella, F., Morita, R.Y., 1981. Microcultural study of bacterial size changes and microcolony formation by heterotrophic bacteria in seawater. Appl. Environ. Microbiol. 41, 518-527. Torseth IH, Furnes H, Tumyr O. 1991.Atextural and chemical study of Icelandic palagonite of varied composition and its bearing on the mechanism of the glass-palagonite transformation. Geochim Cosmochim Acta 55:731&ndash;749. Torseth IH, Furnes H, Heldal M. 1992. 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Introduction: Currently, the only disease-modifying therapy for degenerative mitral stenosis (DMS) due to mitral annular calcification (MAC) is open surgical valve repair or replacement, but it is technically challenging, and patients with DMS are often at elevated risk of surgical complications. As such, there is an unmet need for less-invasive strategies to treat DMS. Given the ability of shock-wave grade therapeutic ultrasound to disrupt calcifications, we set out to determine whether therapeutic ultrasound may be a possible treatment strategy for DMS. Hypothesis: This study aimed to evaluate whether ex vivo histotripsy can produce internal disruption of calcified nodules in the mitral annulus, a precursor to improving valve function in DMS. Methods: All experiments were performed on a formalin-fixed human cadaveric heart with severe MAC (Figure 1 A). Histotripsy cavitation was directed at calcified nodules utilizing a sonic concepts HIFUPlex Focused 128 element,1.1MHz array therapy transducer (Figure 1 E). B-mode ultrasound imaging was used to guide therapy. Transducer settings were PRF: 60 Hz, Duty cycle: 8x10^-4, transmit frequency: 1.1 MHz, HIFU Voltage: 60 V, resulting in an estimated pressure output of 34.6 MPa_pk. Results: A total of three histotripsy experiments were performed on the primary dominant calcium nodule. Visual assessment of treatment X-ray microtomography (Micro-CT) revealed qualitative fracturing of the calcified nodule from baseline (Figure 1 B), after the first treatment (Figure 1 C), and after three treatments (Figure 1 D). Gross visual inspection and Micro-CT demonstrated no changes to the surrounding fibrous annulus. Conclusions: In a feasibility study involving a fixed cadaveric heart, histotripsy therapeutic ultrasound demonstrated the potential to internally disrupt a MAC nodule without injuring the mitral annulus. Further studies are needed to address the ability of histotripsy to improve valve function in DMS.

The purpose of this study were (1) Identify the factors that impede business development of micro, small and medium enterprises in East Java, (2) identify strategies flagship relevant for micro, small and medium enterprises in East Java, (3) Develop a strategy formulation the development of micro, small and medium enterprises in East Java as the alleviation of poverty. by using the SWOT method, it can be concluded that (1) the factors that impede business development of micro, small and medium enterprises in East Java is the low marketing,Jurnal Teknik Industri HEURISTIC, vol 12, no 2, Oktober 2015, halaman 142-156 ISSN: 1693-8232143promotion is still lacking, innovation competitor products quickly, the condition of the competitors are very strict, government policy to reduce public subsidies, high inflation, weak niai rupiah against the $ US, demands a quality product at a price that is increasingly Competitive, the decline of the country's economy, political situation in the country is not stable, (2) Strategy flagship relevant to micro, small and medium enterprises in East Java Growth strategy, showed no growth and product diversification efforts, micro, small and medium enterprises, Stability strategy, shows the strategy adopted without changing the strategic direction set. (3) The formulation of the development strategy of micro, small and medium enterprises in East Java as the fight against poverty is (a) Creating opportunities for new markets, for example, Open gallery to accommodate everyone who wants to learn about batik, designs, colors and patterns are new , Producing cotton batik arise, fabric doby, woven fabrics batik, fabric paris (semi ciffon), silk, production of herbal medicine in the form of sachets, Forms/ways of marketing, micro, small and medium enterprises : exhibitions, internet, stand at the site of micro, small, and intermediate, product marketing process: www. petismadura.com through on line, by telephone, came to the site of micro, small and medium enterprises, (b) Measures craftsmen of micro, small and medium enterprises to face competition, for example, cotton batik patterned producing contemporary craftsmen who work in Tanjungbumi invited back to the village in Sampang, designs, colors and patterns are new, it needs creative freedom batik, and according to standard batik, development capital, collecting batik former, For medicinal products: produce products with packaging per ounce, for product paste: improved quality, competitive prices, CSR for young, expanding the image to the public, To handicraft: should innovation models, especially the products of bags and wallets, information from the Internet, (c) Efforts craftsmen that demand the products increases, for example, promotion of one to the other, for batik products: create a new motive, the exhibition, plus employee and machine, plus shops, for the product chips fish : without chemicals, for products skull cap: rent for wholesale, product diversification is koko, exhibits 10 times per year.Keywords: SWOT, micro small and medium enterprise, poverty alleviation

Abstract Introduction Endomyocardial biopsy (EMB) is the gold standard for diagnosis of several cardiac diseases, yet its use is limited by low diagnostic yield and significant complication risks. The size of the current devices allows only limited steering to different parts of the ventricle walls. In transplant monitoring, repeated biopsies with the current devices can cause scarring that makes it increasingly difficult to obtain adequate samples. We hypothesised that several of the shortcomings of EMB can be avoided with a smaller and more steerable device. Further, we hypothesised that the novel sampling procedure could be coupled to a low-input molecular analysis method, such as RNA-sequencing (RNA-seq), to provide molecular characterisation of the tissue without the need of large biopsy samples. Purpose To develop an EMB device with significantly smaller dimensions, for future use in diagnostics and research investigations. Specific aims were to test feasibility and safety of the procedure, as well as the quality of the generated molecular data. Methods 65 “micro biopsy” (micro-EMB) device prototypes were designed and evaluated in-house. The prototypes were evaluated either in an ex-vivo simulator or in acute non-survival pig experiments (n=23). Once the final device design was reached, an in vivo trial was set up using six naive Yorkshire farm pigs. Micro-EMB, conventional EMB, skeletal muscle and blood samples were collected for RNA-seq characterisation and comparison. In half of the animals (n=3), micro-EMB was the only intervention in order to prioritise safety evaluations. The animals were monitored for one week. Results The final device design has an outer diameter (OD) of 0.4 mm, compared to a conventional 11 mm device (in the opened position), Fig 1A. The device can be directed to different parts of the myocardium in both ventricles. In the in vivo evaluation in swine, 81% of the biopsy attempts (n=157) were successful. High quality RNA-seq data was generated from 91% of the sequenced heart micro-biopsy samples (n=32). The gene expression signatures of samples taken with the novel device were comparable with samples taken with a conventional device, Fig 1B. No major complications were detected either during periprocedural monitoring or during the follow-up. The tissue mark after micro-biopsy was markedly smaller than after conventional endomyocardial biopsy. A) Bioptome dimensions. B) RNA-seq data. Conclusions Our preliminary data suggest that the novel submillimeter biopsy device, coupled with RNA-seq, provides a feasible method to obtain molecular data from the myocardium. The method is less traumatic and has a higher flexibility compared to conventional methods, enabling safer and more specific sampling from different parts of the myocardium. In the long term, the procedure could open unprecedented diagnostic and research possibilities. Future studies should be directed to establish the capabilities of the novel method in a relevant disease model. Acknowledgement/Funding Family Erling Persson Foundation. The Söderberg foundations. KID (Karolinska Institutet). The 4D project. Stockholm county council. Astra Zeneca.

Digital nomads (DNs) are hyper-mobile, location-independent workers whose practices blur traditional boundaries between labour, leisure, home and travel. They rely on digital tools to work and on computer-mediated communication to share knowledge and resources. Their resource-sharing culture is vital for self-efficacy and self-actualisation — two fundamental values that define the DN identity. Community identity is a constant social-semiotic construct mutually determined by (micro) interactions and the (macro) influence of collectively shared meanings and symbols. However, most of our understanding of community identity comes from structural and synchronic properties that often assume identity “exists” as an entity, separate from underlying collective dynamics. In this paper, we approach community identity diachronically, by introducing a quantitative typology that projects conversational timelines on two dimensions relevant to understanding the process of community identity construction: (a) the temporal orientation to the community core (or peripheral) conversation topics, and (b) interaction pattern anomalies. We cast three years of r/digitalnomad threads as a set of conversation topics, describing the interaction dynamics on these topics using the proposed typology. The central questions asked by this paper are whether there was a pre-pandemic stable community expression, and if so, how the COVID-19 pandemic may have perturbed it. Since lockdowns and travel restrictions impinged on fundamental DN values, the nature of the topics and interaction patterns that characterise the r/digitalnomad subreddit could have changed its character. We found a stable pre-pandemic balanced expression of core and peripheral conversation topics with regular interaction patterns. This identity expression was perturbed temporarily in the middle of the lockdown period when the community shifted focus to interactions about visa issues. As many countries began to re-open their borders around May 2021, a record-breaking number of interactions disrupted identity expression more profoundly. First, we observed constant interaction anomalies. Second, the community orientation revealed multi-factorial emergent issues, most of which revolved around conversations about what it means to be a DN, resource sharing and restrictions. We hypothesise that an influx of outsiders may have caused a clash of social norms and triggered a transformation of the DN identity that was still ongoing at the end of the studied period, in December 2021.

To develop higher efficiency and lower emission gasoline engines, ultra-lean burning under high Reynolds number conditions is desired. It is believed that enhancement of tumble flow in the engine cylinder is effective for increase of turbulent intensity, resulting in improvement of characteristics of flame propagation and ignition under a strong discharge, while the enhancement of tumble flow might cause heat loss from the wall. Investigations of characteristics of turbulence and distributions of wall and gas temperature in engine cylinders are still challenging due to transient and high pressure, and due to cycle-to-cycle variations. In the previous study (Jainski et al., 2013), a micro particle image velocimetry (micro PIV) measurements were conducted in an engine cylinder at up to 1100 rpm and the characteristics of velocity boundary layer around a cylinder head were investigated. The study has shown that the log-law does not properly present the measured velocity distributions near the wall. In our previous study (Shimura et al., 2018), a micro PIV was conducted in a motored engine cylinder to investigate velocity boundary layer characteristics near piston top before the top dead center (TDC) at a constant engine speed of 2000 rpm to deepen understanding characteristics of velocity boundary layer in engine cylinder with tumble flow. The velocity boundary layer was well fitted to the Blasius theory at 30 CAD before TDC and deviated from the theory at 15 CAD before TDC. However, the obtained data was two components of velocity in the measurement plane, which means that effects of magnitude of velocity were not clear in the previous measurement. In this study, stereoscopic micro PIV was conducted to elucidate the effects of magnitude and direction of velocity on the characteristics of velocity boundary layer near the piston top in the tumble enhanced SI IC optical engine. The tumble enhanced SI IC optical engine used in the previous study (Shimura et al., 2018; Matsuda et al., 2019) was used also in this study. The bore is 75 mm and the stroke is 112.5 mm. Length of the connecting rod is 250 mm. The engine has two intake valves of the diameter 29 mm and two exhaust valves of the diameter 25 mm. The compression ratio is 13.0. The optical access is achieved through the quartz glass cylinder. A tumble enhancing intake port is used for the sake of improvement of ignition and flame propagation. The engine speed can be set up to 2000 rpm at the maximum. The overall flow fields taken by a preliminary PIV experiment can be seen in the literatures (Shimura et al., 2018; Matsuda et al., 2019). The laser beams for PIV are from two Nd:YAG lasers (LOTIS, LS-2131, 150 mJ/pulse, 532 nm) are led to the same optical axis by a mirror and a polarizing beam splitter. The laser beam is formed into laser sheet of 180 µm thickness by laser sheet forming optics and led into the engine cylinder. The scattering light was collected by long distance microscope lenses (Quester, SZM100) and imaged onto CCD cameras (Princeton Technology, ES4020) in the stereoscopic alignment with 18 degrees. To compensate for the difference in the focal length caused by the quartz engine liner, a cylindrical lens of 1000 mm focal length was placed in front of each long distance microscope lens. SiO2 of 1 µm mean diameter was used for tracer particles. The micro PIV was operated at about 6.6 Hz to be synchronized with engine speed. The time separation of the successive particle images was 1.5 µs. The field of view of the micro PIV was 3.5 mm × 3.5 mm on the piston top including central axis of the cylinder. Here, x and y coordinates are set to the direction from the exhaust to the intake valves and the direction from the piston to the pent roof, respectively. z coordinates is perpendicular to x and y axes, and the orizin of the coordinates is set at the center of the piston top. The spatial resolution of PIV, which is defined by the size of interrogation region, is 108.8 µm × 54.4 µm. Vector spacing is 54.4 µm × 27.2 µm. The first vector position is about 27.2 µm away from the wall. The measurements were conducted at 345 CAD. The engine was motored at 2000 rpm and operated for three intake valve open timings of -30 CAD. The operation condition of the engine tested contain strong cycle-to-cycle variations, which results in the large root-mean-square values of velocity fluctuation near the center of the piston top (Shimura et al., 2018). To evaluate flow characteristics in the cycle-to-cycle variations, conditional averaging based on magnitude of fluid velocity is used in this study. Figure 1(a) shows a histogram of the magnitude of combined velocity of u and w. The magnitude of velocity can be considered as momentum of fluid because few fluctuation of density is considered and temperature boundary layer is enough thin compared to the velocity boundary layer. The large variations in the momentum can be observed in Fig. 1(a). The large variations are considered to be caused by the variations of tumble core locations. The fraction of the large momentum Here, the momentum are classified into C1 to C4 based on fractions (C1: 54.4%, C2: 19.5%, C3: 19.5%, C4: 6.6%). Figure 1(b) and (c) shows mean velocity distribution classified into C1 and C4 in Fig. 1(a). The distribution is fitted to the log-law velocity profile of developed wall turbulence. The mean velocity profile for C1 shows large discrepancy from that of general turbulent boundary layers, while that for C4 show relatively close to that of general turbulent boundary layer. C2 and C3, which are not shown here, have trend between the C1 and C4 profiles. These results show that the velocity profiles which can be assumed to be the developed turbulent boudary layer in the targeted condition is less than half of cycles, which means partial applicability of conventional CFD models for prediction of boundary layer of the engine condition.

Abstract Background Multidrug resistant (MDR) Gram-negative bacteria, including metallo-β-lactamase (MBL) producers, pose significant treatment challenges. This study investigated efficacy and safety of aztreonam-avibactam (ATM-AVI) in the treatment of complicated intra-abdominal infection (cIAI) or hospital-acquired)/ventilator-associated pneumonia (HAP/VAP) due to Gram-negative bacteria, including MBL-producing MDR pathogens, with limited or no treatment options. Methods REVISIT was a phase 3, prospective, randomized, multicenter, open-label, central assessor-blinded study in hospitalized adults. Patients were randomized 2:1 to ATM-AVI (± metronidazole [MTZ]; cIAI patients only) or meropenem (MER) ± colistin (COL) for 5–14 (cIAI) or 7–14 (HAP/VAP) days. Clinical cure at the test-of-cure (TOC) visit in the intent-to-treat (ITT) and clinically evaluable (CE) analysis sets were the primary efficacy endpoints. Key secondary endpoints included microbiological responses at TOC, 28-day mortality, and safety. No formal hypothesis testing was planned. Results In total, 422 patients were randomized (ATM-AVI ± MTZ, n=282; MER ± COL, n=140). Adjudicated clinical cure rates at TOC are shown in Table 1. Favorable microbiological response rates at TOC (micro-ITT analysis set) were 75.7% for ATM-AVI ± MTZ and 73.9% for MER ± COL; 28-day all-cause mortality rates for ATM-AVI ± MTZ and MER ± COL were 1.9% (4/208) vs 2.9% (3/104), and 10.8% (8/74) vs 19.4% (7/36) in cIAI and HAP/VAP, respectively. Adverse events (AEs) are summarized in Table 2. There were no treatment-related serious AEs in the ATM-AVI group. Conclusion ATM-AVI (± MTZ) was effective in treating patients with cIAI and HAP/VAP, displaying similar efficacy to MER ± COL. ATM-AVI was generally well tolerated. These data support potential use of ATM-AVI for the treatment of serious infections caused by susceptible Gram-negative bacteria. Further analyses will focus on MBL-producing pathogens. Trial registration. NCT03329092. Study sponsored by Pfizer. ATM-AVI is jointly developed with AbbVie, also supported by the United States Biomedical Advanced Research and Development Authority (BARDA) and the European Innovative Medicines Initiative (IMI), under the COMBACTE-CARE consortium. Disclosures Yehuda Carmeli, MD, Merck: Advisor/Consultant|Merck: Grant/Research Support|Merck: Honoraria|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Pfizer: Honoraria|Qpex: Advisor/Consultant|Qpex: Grant/Research Support|Qpex: Honoraria|Roche: Advisor/Consultant|Roche: Grant/Research Support|Roche: Honoraria Georgios L. Daikos, PhD, MSD: Honoraria|Pfizer: Advisor/Consultant|Pfizer: Honoraria|Viatris: Honoraria Rosa-María Jiménez Rodríguez, MD, PhD, Abex: Honoraria|B. Braun: Honoraria|Johnson &amp; Johnson: Honoraria Halley Rogers, MPH, Pfizer: Ownership Interest Michele Wible, MS, Pfizer: Ownership Interest Francis Arhin, PhD, Pfizer: Ownership Interest Joanne Leaney, PhD, Pfizer: Ownership Interest Rienk Pypstra, MD, MBA, Pfizer: Stocks/Bonds Joseph Chow, MD, Pfizer: Ownership Interest

In this work, we present a means of controlling the cross-stream trajectory of a compound drop under the combined action of a transverse electric field and an oscillatory axial pressure gradient in a microfluidic channel. We bring out the decisive role of the flow pulsation in delaying the drop's attainment of a steady-state transverse position. With an enhancement in the frequency of oscillation, both the axial and transverse movement of the drop may be preferentially attenuated, with its dynamic traversal occurring in a locus offset to the central axis as precisely set in by the action of transverse electric forcing, to bring in exclusive controllability on the drop trajectory along with its eventual position of settlement. In addition, we observe that a leaky dielectric compound drop having the electrical permittivity ratio of the inner-to-outer droplet phase surpassing their respective electrical conductivity ratio is not only capable of selectively altering the direction of the resulting drop trajectory from towards the channel-centerline to away from the same, but at the same time facilitates a precise settling of the same at an intermediate transverse location by harnessing the interplay of electrical and hydrodynamic shear. We further identify the key dimensionless parameters along with their desirable ranges accountable for the directional switching of the drop trajectory with high specificity. These findings open up novel perspectives of controllable maneuvering of the double emulsion system in a confined microenvironment bearing decisive implications in engineering and biology.

Babies and toddlers are amassing huge followings on social media, achieving microcelebrity status, and raking in five figure sums. In East Asia, many of these lucrative “micro­-microcelebrities” rise to fame by inheriting exposure and proximate microcelebrification from their social media Influencer mothers. Through self-branding techniques, Influencer mothers’ portrayals of their young’ children’s lives “as lived” are the canvas on which (baby) products and services are marketed to readers as “advertorials”. In turning to investigate this budding phenomenon, I draw on ethnographic case studies in Singapore to outline the career trajectory of these young children (under 4yo) including their social media presence, branding strategies, and engagement with their followers. The chapter closes with a brief discussion on some ethical considerations of such young children’s labour in the social media age.Influencer MothersTheresa Senft first coined the term “microcelebrity” in her work Camgirls as a burgeoning online trend, wherein people attempt to gain popularity by employing digital media technologies, such as videos, blogs, and social media. She describes microcelebrities as “non-actors as performers” whose narratives take place “without overt manipulation”, and who are “more ‘real’ than television personalities with ‘perfect hair, perfect friends and perfect lives’” (Senft 16), foregrounding their active response to their communities in the ways that maintain open channels of feedback on social media to engage with their following.Influencers – a vernacular industry term albeit inspired by Katz &amp; Lazarsfeld’s notion of “personal influence” that predates Internet culture – are one type of microcelebrity; they are everyday, ordinary Internet users who accumulate a relatively large following on blogs and social media through the textual and visual narration of their personal lives and lifestyles, engage with their following in “digital” and “physical” spaces, and monetize their following by integrating “advertorials” into their blog or social media posts and making physical appearances at events. A pastiche of “advertisement” and “editorial”, advertorials in the Influencer industry are highly personalized, opinion-laden promotions of products/services that Influencers personally experience and endorse for a fee. Influencers in Singapore often brand themselves as having “relatability”, or the ability to persuade their followers to identify with them (Abidin). They do so by make consciously visible the backstage (Goffman) of the usually “inaccessible”, “personal”, and “private” aspects of mundane, everyday life to curate personae that feel “authentic” to fans (Marwick 114), and more accessible than traditional celebrity (Senft 16).Historically, the Influencer industry in Singapore can be traced back to the early beginnings of the “blogshop” industry from the mid-2000s and the “commercial blogging” industry. Influencers are predominantly young women, and market products and services from diverse industries, although the most popular have been fashion, beauty, F&amp;B, travel, and electronics. Most prominent Influencers are contracted to management agencies who broker deals in exchange for commission and assist in the production of their vlogs. Since then, the industry has grown, matured, and expanded so rapidly that Influencers developed emergent models of advertorials, with the earliest cohorts moving into different life stages and monetizing several other aspects of their personal lives such as the “micro-microcelebrity” of their young children. What this paper provides is an important analysis of the genesis and normative practices of micro-microcelebrity commerce in Singapore from its earliest years, and future research trajectories in this field.Micro-Microcelebrity and Proximate MicrocelebrificationI define micro-microcelebrities as the children of Influencers who have themselves become proximate microcelebrities, having derived exposure and fame from their prominent Influencer mothers, usually through a more prolific, deliberate, and commercial form of what Blum-Ross defines as “sharenting”: the act of parents sharing images and stores about their children in digital spaces such as social networking sites and blogs. Marwick (116-117), drawing from Rojek’s work on types of celebrity – distinguishes between two types of microcelebrity: “ascribed microcelebrity” where the online personality is made recognizable through the “production of celebrity media” such as paparazzi shots and user-produced online memes, or “achieved microcelebrity” where users engage in “self-presentation strateg[ies]”, such as fostering the illusion of intimacy with fans, maintaining a persona, and selective disclosure about oneself.Micro-microcelebrities lie somewhere between the two: In a process I term “proximate microcelebrification”, micro-microcelebrities themselves inherit celebrity through the preemptive and continuous exposure from their Influencer mothers, many beginning even during the pre-birth pregnancy stages in the form of ultrasound scans, as a form of “achieved microcelebrity”. Influencer mothers whose “presentational strategies” (cf. Marshall, “Promotion” 45) are successful enough (as will be addressed later) gain traction among followers, who in turn further popularize the micro-microcelebrity by setting up fan accounts, tribute sites, and gossip forums through which fame is heightened in a feedback loop as a model of “ascribed microcelebrity”.Here, however, I refrain from conceptualizing these young stars as “micro-Influencers” for unlike Influencers, these children do not yet curate their self-presentation to command the attention of followers, but instead are used, framed, and appropriated by their mothers for advertorials. In other words, Influencer mothers “curate [micro-microcelebrities’] identities into being” (Leaver, “Birth”). Following this, many aspects of their micro-microcelebrities become rapidly commodified and commercialized, with advertisers clamoring to endorse anything from maternity hospital stays to nappy cream.Although children of mommybloggers have the prospect to become micro-microcelebrities, both groups are conceptually distinct. Friedman (200-201) argues that among mommybloggers arose a tension between those who adopt “the raw authenticity of nonmonetized blogging”, documenting the “unglamorous minutiae” of their daily lives and a “more authentic view of motherhood” and those who use mommyblogs “primarily as a source of extra income rather than as a site for memoir”, focusing on “parent-centered products” (cf. Mom Bloggers Club).In contrast, micro-microcelebrities and their digital presence are deliberately commercial, framed and staged by Influencer mothers in order to maximize their advertorial potential, and are often postured to market even non-baby/parenting products such as fast food and vehicles (see later). Because of the overt commerce, it is unclear if micro-microcelebrity displays constitute “intimate surveillance”, an “almost always well-intentioned surveillance of young people by parents” (Leaver, “Born” 4). Furthermore, children are generally peripheral to mommybloggers whose own parenting narratives take precedence as a way to connect with fellow mothers, while micro-microcelebrities are the primary feature whose everyday lives and digital presence enrapture followers.MethodologyThe analysis presented is informed by my original fieldwork with 125 Influencers and related actors among whom I conducted a mixture of physical and digital personal interviews, participant observation, web archaeology, and archival research between December 2011 and October 2014. However, the material presented here is based on my digital participant observation of publicly accessible and intentionally-public digital presence of the first four highly successful micro-microcelebrities in Singapore: “Baby Dash” (b.2013) is the son of Influencer xiaxue, “#HeYurou” (b.2011) is the niece of Influencer bongqiuqiu, “#BabyElroyE” (b.2014) is the son of Influencer ohsofickle, and “@MereGoRound” (b.2015) is the daughter of Influencer bongqiuqiu.The microcelebrity/social media handles of these children take different forms, following the platform on which their parent/aunt has exposed them on the most. Baby Dash appears in all of xiaxue’s digital platforms under a variety of over 30 indexical, ironic, or humourous hashtags (Leaver, “Birth”) including “#pointylipped”, #pineappledash”, and “#面包脸” (trans. “bread face”); “#HeYurou” appears on bongqiuqiu’s Instagram and Twitter; “#BabyElroyE” appears on ohsofickle’s Instagram and blog, and is the central figure of his mother’s new YouTube channel; and “@MereGoRound” appears on all of bongqiuqiu’s digital platforms but also has her own Instagram account and dedicated YouTube channel. The images reproduced here are screenshot from Influencer mothers’ highly public social media: xiaxue, bongqiuqiu, and ohsofickle boast 593k, 277k, and 124k followers on Instagram and 263k, 41k, and 17k followers on Twitter respectively at the time of writing.Anticipation and Digital EstatesIn an exclusive front-pager (Figure 1) on the day of his induced birth, it was announced that Baby Dash had already received up to SGD25,000 worth of endorsement deals brokered by his Influencer mother, xiaxue. As the first micro-microcelebrity in his cohort (his mother was among the pioneer Influencers), Baby Dash’s Caesarean section was even filmed and posted on xiaxue’s YouTube channel in three parts (Figure 2). xiaxue had announced her pregnancy on her blog while in her second trimester, following which she consistently posted mirror selfies of her baby bump.Figure 1 &amp; 2, screenshot April 2013 from ‹instagram.com/xiaxue›In her successful attempt at generating anticipation, the “bump” itself seemed to garner its own following on Twitter and Instagram, with many followers discussing how the Influencer dressed “it”, and how “it” was evolving over the weeks. One follower even compiled a collage of xiaxue’s “bump” chronologically and gifted it to the Influencer as an art image via Twitter on the day she delivered Baby Dash (Figure 3 &amp; 4). Followers also frequently speculated and bantered about how her baby would look, and mused about how much they were going to adore him. Figure 3 &amp; 4, screenshot March 2013 from ‹twitter.com/xiaxue› While Lupton (42) has conceptualized the sharing of images that precede birth as a “rite of passage”, Influencer mothers who publish sonograms deliberately do so in order to claim digital estates for their to-be micro-microcelebrities in the form of “reserved” social media handles, blog URLs, and unique hashtags for self-branding. For instance, at the 3-month mark of her pregnancy, Influencer bongqiuqiu debuted her baby’s dedicated hashtag, “#MereGoRound” in a birth announcement on her on Instagram account. Shortly after, she started an Instagram account, “@MereGoRound”, for her baby, who amassed over 5.5k followers prior to her birth. Figure 5 &amp; 6, screenshot March 2015 from instagram.com/meregoround and instagram.com/bongqiuqiuThe debut picture features a heavily pregnant belly shot of bongqiuqiu (Figure 5), creating much anticipation for the arrival of a new micro-microcelebrity: in the six months leading up to her birth, various family, friends, and fans shared Instagram images of their gifts and welcome party for @MereGoRound, and followers shared congratulations and fan art on the dedicated Instagram hashtag. During this time, bongqiuqiu also frequently updated followers on her pregnancy progress, not without advertising her (presumably sponsored) gynecologist and hospital stay in her pregnancy diaries (Figure 6) – like Baby Dash, even as a foetus @MereGoRound was accumulating advertorials. Presently at six months old, @MereGoRound boasts almost 40k followers on Instagram on which embedded in the narrative of her growth are sponsored products and services from various advertisers.Non-Baby-Related AdvertorialsPrior to her pregnancy, Influencer bongqiuqiu hopped onto the micro-microcelebrity bandwagon in the wake of Baby Dash’s birth, by using her niece “#HeYurou” in her advertorials. Many Influencers attempt to naturalize their advertorials by composing their post as if recounting a family event. With reference to a child, parent, or partner, they may muse or quip about a product being used or an experience being shared in a bid to mask the distinction between their personal and commercial material. bongqiuqiu frequently posted personal, non-sponsored images engaging in daily mundane activities under the dedicated hashtag “#HeYurou”.However, this was occasionally interspersed with pictures of her niece holding on to various products including storybooks (Figure 8) and shopping bags (Figure 9). At first glance, this might have seemed like any mundane daily update the Influencer often posts. However, a close inspection reveals the caption bearing sponsor hashtags, tags, and campaign information. For instance, one Instagram post shows #HeYurou casually holding on to and staring at a burger in KFC wrapping (Figure 7), but when read in tandem with bongqiuqiu’s other KFC-related posts published over a span of a few months, it becomes clear that #HeYurou was in fact advertising for KFC. Figure 7, 8, 9, screenshot December 2014 from ‹instagram.com/bongqiuqiu›Elsewhere, Baby Dash was incorporated into xiaxue’s car sponsorship with over 20 large decals of one of his viral photos – dubbed “pineapple Dash” among followers – plastered all over her vehicle (Figure 10). Followers who spot the car in public are encouraged to photograph and upload the image using its dedicated hashtag, “#xiaxuecar” as part of the Influencer’s car sponsorship – an engagement scarcely related to her young child. Since then, xiaxue has speculated producing offshoots of “pineapple Dash” products including smartphone casings. Figure 10, screenshot December 2014 from ‹instagram.com/xiaxue›Follower EngagementSponsors regularly organize fan meet-and-greets headlined by micro-microcelebrities in order to attract potential customers. Photo opportunities and the chance to see Baby Dash “in the flesh” frequently front press and promotional material of marketing campaigns. Elsewhere on social media, several Baby Dash fan and tribute accounts have also emerged on Instagram, reposting images and related media of the micro-microcelebrity with overt adoration, no doubt encouraged by xiaxue, who began crowdsourcing captions for Baby Dash’s photos.Influencer ohsofickle postures #BabyElroyE’s follower engagement in a more subtle way. In her YouTube channel that debut in the month of her baby’s birth, ohsofickle produces video diaries of being a young, single, mother who is raising a child (Figure 11). In each episode, #BabyElroyE is the main feature whose daily activities are documented, and while there is some advertising embedded, ohsofickle’s approach on YouTube is much less overt than others as it features much more non-monetized personal content (Figure 12). Her blog serves as a backchannel to her vlogs, in which she recounts her struggles with motherhood and explicitly solicits the advice of mothers. However, owing to her young age (she became an Influencer at 17 and gave birth at 24), many of her followers are teenagers and young women who respond to her solicitations by gushing over #BabyElroyE’s images on Instagram. Figure 11 &amp; 12, screenshot September 2015 from ‹instagram.com/ohsofickle›PrivacyAs noted by Holloway et al. (23), children like micro-microcelebrities will be among the first cohorts to inherit “digital profiles” of their “whole lifetime” as a “work in progress”, from parents who habitually underestimate or discount the privacy and long term effects of publicizing information about their children at the time of posting. This matters in a climate where social media platforms can amend privacy policies without user consent (23), and is even more pressing for micro-microcelebrities whose followers store, republish, and recirculate information in fan networks, resulting in digital footprints with persistence, replicability, scalability, searchability (boyd), and extended longevity in public circulation which can be attributed back to the children indefinitely (Leaver, “Ends”).Despite minimum age restrictions and recent concerns with “digital kidnapping” where users steal images of other young children to be re-posted as their own (Whigham), some social media platforms rarely police the proliferation of accounts set up by parents on behalf of their underage children prominently displaying their legal names and life histories, citing differing jurisdictions in various countries (Facebook; Instagram), while others claim to disable accounts if users report an “incorrect birth date” (cf. Google for YouTube). In Singapore, the Media Development Authority (MDA) which governs all print and digital media has no firm regulations for this but suggests that the age of consent is 16 judging by their recommendation to parents with children aged below 16 to subscribe to Internet filtering services (Media Development Authority, “Regulatory” 1). Moreover, current initiatives have been focused on how parents can impart digital literacy to their children (Media Development Authority, “Empowered”; Media Literacy Council) as opposed to educating parents about the digital footprints they may be unwittingly leaving about their children.The digital lives of micro-microcelebrities pose new layers of concern given their publicness and deliberate publicity, specifically hinged on making visible the usually inaccessible, private aspects of everyday life (Marshall, “Persona” 5).Scholars note that celebrities are individuals for whom speculation of their private lives takes precedence over their actual public role or career (Geraghty 100-101; Turner 8). However, the personae of Influencers and their young children are shaped by ambiguously blurring the boundaries of privacy and publicness in order to bait followers’ attention, such that privacy and publicness are defined by being broadcast, circulated, and publicized (Warner 414). In other words, the publicness of micro-microcelebrities is premised on the extent of the intentional publicity rather than simply being in the public domain (Marwick 223-231, emphasis mine).Among Influencers privacy concerns have aroused awareness but not action – Baby Dash’s Influencer mother admitted in a national radio interview that he has received a death threat via Instagram but feels that her child is unlikely to be actually attacked (Channel News Asia) – because privacy is a commodity that is manipulated and performed to advance their micro-microcelebrities’ careers. As pioneer micro-microcelebrities are all under 2-years-old at present, future research warrants investigating “child-centred definitions” (Third et al.) of the transition in which they come of age, grow an awareness of their digital presence, respond to their Influencer mothers’ actions, and potentially take over their accounts.Young LabourThe Ministry of Manpower (MOM) in Singapore, which regulates the employment of children and young persons, states that children under the age of 13 may not legally work in non-industrial or industrial settings (Ministry of Manpower). However, the same document later ambiguously states underaged children who do work can only do so under strict work limits (Ministry of Manpower). Elsewhere (Chan), it is noted that national labour statistics have thus far only focused on those above the age of 15, thus neglecting a true reflection of underaged labour in Singapore. This is despite the prominence of micro-microcelebrities who are put in front of (video) cameras to build social media content. Additionally, the work of micro-microcelebrities on digital platforms has not yet been formally recognized as labour, and is not regulated by any authority including Influencer management firms, clients, the MDA, and the MOM. Brief snippets from my ethnographic fieldwork with Influencer management agencies in Singapore similarly reveal that micro-microcelebrities’ labour engagements and control of their earnings are entirely at their parents’ discretion.As models and actors, micro-microcelebrities are one form of entertainment workers who if between the ages of 15 days and 18 years in the state of California are required to obtain an Entertainment Work Permit to be gainfully employed, adhering to strict work, schooling, and rest hour quotas (Department of Industrial Relations). Furthermore, the Californian Coogan Law affirms that earnings by these minors are their own property and not their parents’, although they are not old enough to legally control their finances and rely on the state to govern their earnings with a legal guardian (Screen Actors Guild). However, this similarly excludes underaged children and micro-microcelebrities engaged in creative digital ecologies. Future research should look into safeguards and instruments among young child entertainers, especially for micro-micrcocelebrities’ among whom commercial work and personal documentation is not always distinct, and are in fact deliberately intertwined in order to better engage with followers for relatabilityGrowing Up BrandedIn the wake of moral panics over excessive surveillance technologies, children’s safety on the Internet, and data retention concerns, micro-microcelebrities and their Influencer mothers stand out for their deliberately personal and overtly commercial approach towards self-documenting, self-presenting, and self-publicizing from the moment of conception. As these debut micro-microcelebrities grow older and inherit digital publics, personae, and careers, future research should focus on the transition of their ownership, engagement, and reactions to a branded childhood in which babies were postured for an initimate public.ReferencesAbidin, Crystal. “Communicative Intimacies: Influencers and Perceived Interconnectedness.” Ada: A Journal of Gender, New Media, &amp; Technology. Forthcoming, Nov 2015.Aiello, Marianne. “Mommy Blog Banner Ads Get Results.” Healthcare Marketing Advisor 17 Nov. 2010. HealthLeaders Media. 16 Aug. 2015 ‹http://healthleadersmedia.com/content/MAR-259215/Mommy-Blog-Banner-Ads-Get-Results›.Azzarone, Stephanie. “When Consumers Report: Mommy Blogging Your Way to Success.” Playthings 18 Feb. 2009. 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Authors: Chen Weijin, Zheng Yue &amp; Wang Biao ### Abstract In this protocol we present the typical procedures of conducting computational experiments on the domain structure of low dimensional ferroelectrics using a personal computer (PC). Based on an example of the phase-field model (PFM) for ferroelectric nanoplatelet, a computational experiment consisting of physical modeling, programming, simulation, result analysis and post processing, will be illustrated. Our physical model appropriately takes into account the effects of boundary conditions, inhomogeneous elastic and electrostatic fields, ambient temperature, size and shape of the low dimensional ferroelectrics, which makes it convenient to conduct a series of virtual experiments, e.g., applying external mechanical or electric field to affect the domain structure of nanostructures. ### Introduction Low dimensional ferroelectrics, i.e., ferroelectrics with extreme geometrical confinement along some dimensions, are under active investigations for their important roles in device miniaturization and functional device development. (1,2) People are particularly concerned about the regular responses of domain structures in low dimensional ferroelectrics to the application of external fields. Nevertheless, to synthesize ferroelectric nanostructures of regular size and shape is still a difficult issue at present, not to say the experimental probe and control of nanoscale ferroelectric domain structures. A natural way to come out the predicament is to conduct computational experiments. With appropriate modeling and simulating the physical processes, computational experiments can provide us convincing results to test the correctness of theory, interpret the results of hands-on experiments, and more importantly, to predict new regularity and phenomenon. Especially for situations where hands-on experiments are not feasible or quite expensive, conducting computational experiments is an attractive alternative. This is why recently computational physics has achieved great advance and becomes an important and individual subject of physics other than just an assistant tool of theoretical physics. A complete computational experiment consists of several parts, including physical modeling, programming, simulation, result analysis and post processing. Physical processes are represented through numerically solving the governing equations, which depend on the theoretical description of the processes. Accordingly, different simulation methods could be adopted. For ferroelectrics, a quantity of simulation methods has been developed, including thermodynamic ones like phase-field model (PFM) (3,4), atomistic level simulations, e.g., molecular dynamic simulation (MD) (5) and effective Hamiltonian simulation (EH) (6), and first-principle calculations (FPC) (7), etc. Basing on an example of phase-field model for ferroelectric nanoplatelet, in this protocol we present the procedures of conducting computational experiments on low dimensional ferroelectrics using a personal computer (PC). The detail of the phase-field model and derivation of finite element method to solve the electromechanical fields can be found in the associated publication. ### Equipment 1. A personal computer (PC) with a typical operating system (Windows/Linux). - C language compiler (the program is assumed written in C language), e.g., Visual C++ (http://www.microsoft.com) for Windows systems, or GNU Compiler Collection (http://gcc.gnu.org) for Linux systems. - MATLAB (http://www.mathworks.com) (version 7.1 or above). ### Procedure **I. Physical modeling of low dimensional ferroelectrics** 1. Determine your model system. You should determine the dimensionality of the system (i.e., 0-D nanoplatelet 1-D nanowire and 2-D nanofilm), material, and electric and mechanical boundary conditions, which are recommended to be written as optional tags in your program. In this demo, we consider a PbTiO3 ferroelectric nanoplatelet under surface traction and open-circuit condition as shown in Figure 1. - Construct the system’s free energy. In general, the free energy of a ferroelectric is a functional of polarization, and should incorporate the effects of polarization inhomogeneity, elastic and electric fields. It can be written as a sum of the bulk free energy (including a Landau-type potential, gradient energy, elastic energy and electric energy) and surface free energy. - Tip: The construction of free energy is very important in phase-field model of ferroelectrics. The governing equation, i.e., the Time Dependent Ginzburg–Landau (TDGL) equation, describes that the evolution of domain structure toward its equilibrium is driven by the decrease of the system’s free energy. - Caution: The free energy should be generally constructed under the thermodynamic framework. - Design your experiment. In our case, the experiment is to apply mechanical loads to the nanoplatelet to affect its domain structure. **II. Programming** The flow diagram of phase-field simulation at a given condition (e.g., fixed temperature, open-circuited and mechanical load) is as shown in Figure 2. Followings are the main functional parts of the corresponding program. 1. Initialization. In this part of program, the parameters and variables needed for simulation would be defined and initialized. It is recommended to write this part in a way that it can either use the default values of the parameters and variables or read them from input files. - Tip: For numerical accuracy and convenience, it is recommended to make the parameters and variables dimensionless so that their values fall into a suitable range. - Grid division of the system. This process would generate some tables, from which you can easily find out the information of the nodes and elements, such as its coordinates, adjacent nodes and elements. In our case, we make the following tables, - Tnode——Each row stores a node’s number, coordinates and adjacent elements, - Tnodes——Each row stores a node’s number and its adjacent nodes, - Telement——Each row stores an element’s number, coordinates and its nodes. - Tip: These tables can largely simplify the calculation of element stiffness matrices, element node displacement/potential vectors, and the assemblage of global stiffness matrices and displacement/potential vectors. - Calculation of element stiffness matrices and the assemblage of global stiffness matrices. In most cases, the stiffness matrices (including elastic and electric) can be considered unchanged during the process, therefore the calculation only need to be done by once. - Tip: According to the boundary conditions, the computation size and accuracy requirement, the electric and elastic fields can be solved by different methods, such as conjugate gradient method (CGM), finite-element method (FEM), and fast Fourier transformation (FFT). For our case of a ferroelectric nanoplatelet with a moderate size, finite-element method is suitable to solve the electric and elastic fields. - Calculation of the elastic field. Calculate the element node displacement vectors, and assemble them into the global node displacement vector. Solve the equation [Ku]{U}={Fu}. This should be done at each step of polarization evolution. - Calculation of the electric field. Calculate the element node potential vectors, and assemble them into the global node potential vector. Solve the equation [Kφ]{Ф}={Fφ}. This should be done at each step of polarization evolution. - Tip: Iterative methods, e.g. the Gauss-Seidel iteration method, would be suitable to equations [Ku]{U}={Fu} and [Kφ]{Ф}={Fφ}. - Calculation of the evolution force and polarization field at next step. Simple explicit difference methods or semi-implicit Fourier-spectral algorithms8 can be used to solve the governing equation, i.e., the TDGL equation. - Error analysis. Calculate the error between the polarization field at this step and next step. If the error is small enough, end program; otherwise, repeat steps 3-6. - Input and output. In our case, the input includes a parameter file storing the value of parameters and a configuration file storing the initial polarization field. The output includes a log file recording the monitoring information during the simulation and files storing the polarization field at selected steps. **III. Simulation** We would like to simulate how the mechanical load affects the formation of domain structure in the nanoplatelet. 1. Prepare a set of parameter files. In our case, the parameter files differ only in the value of surface traction. - For each parameter files, compile and run the program. - Tip: You can also make some modifications on the original program, so that the whole simulation can be fulfilled by running the program by once. **IV. Result analysis &amp; Post processing** At this stage, we assume that there is an output file storing the polarization field, e.g., p_final.txt. The file stores the node’s number, its coordinates and the polarization components. Now we will show how to use MATLAB (http://www.mathworks.com) to visualize the domain structures. 1. Start MATLAB. - Import file p_final.txt into the Workspace as shown in Figure 3. - Run the following commands to obtain a vector plot the domain structure as shown in Figure 4a, - quiver3(p_final(:,3),p_final(:,2),p_final(:,4),p_final(:,6),p_final(:,5),p_final(:,7)); axis equal; axis off; - Run the following commands to add a color plot of the polarization magnitude to Figure 4a as shown in Figure 4b, - meshing=[10 3 30]; - nx=meshing(1)+1; - ny=meshing(2)+1; - nz=meshing(3)+1; - m=0; - for i=1:nx - for j=1:ny - for k=1:nz - m=m+1; - P(i,j,k)= (p_final(m,5)*p_final(m,5)+p_final(m,6)*p_final(m,6)+p_final(m,7)*p_final(m,7))^0.5; - end - end - end - Xslice=[0,ny-1];Yslice=[0,nx-1];Zslice=[0,nz-1]; - hold on;h =slice(x,y,z,P,Xslice,Yslice,Zslice); - set(h,’LineStyle’,’none’); - Change the colormap to your preference. For example, run the following commands to as shown in Figure 4c, - color_round=[1 0 1];color_floor=[0 1 0]; - n_color=64; - for i=1:n_color - map(i,1)=color_round(1)+(i-1)*(color_floor(1)-color_round(1))/(n_color-1); - map(i,2)=color_round(2)+(i-1)*(color_floor(2)-color_round(2))/(n_color-1); - map(i,3)=color_round(3)+(i-1)*(color_floor(3)-color_round(3))/(n_color-1); - end - colormap(map); ### Timing In our case of a 10×3×30 meshing nanoplatelet at a given surface traction and open-circuited condition, the simulation time of polarization evolution from random toward equilibrium ranges from tens of minutes to several hours on a PC (3.00 GHz, 1.75GB internal memory). In general, the time of a simulation would strongly depend on the size of system, the algorithms, the required calculation accuracy, the computer performance and the simulated process. When phase transition happens, the simulation time would be much longer. ### Troubleshooting I. How can I make sure whether the results are right? 1. Repeat some solved problems with your own program. - Compare the results of FEM with those of other methods, such as finite difference method. - Analyze whether the results are physical. II. If the polarization field does converge to a suitable range: 1. Check whether time step is too large. - Check the other parameters. - Check whether the elastic and electric field are calculated correctly. Tip: Write the program based a former one, and make sure the former program is correct. III. If the calculation of the elastic/electric field does not converge: 1. Check the correctness of the tables that used to generate the stiffness matrices and force vectors. - Check whether you have appropriately handled the boundary conditions. - Try other algorithms. ### Anticipated Results Results of the experiment are presented in Figure 5. The results indicate that mechanical loads can affect the nucleation and balance of the vortices in the nanoplatelet, and lead to various equilibrium vortex domain structures. ### References 1. Scott, J. F. Nanoferroelectrics: statics and dynamics. *J. Phys.: Condens. Matter* 18, R361 (2006). - Rørvik, P. M., Grande, T. &amp; Einarsrud, M.-A. One-dimensional nanostructures of ferroelectric perovskites. *Adv. Mater*. 23, 4007-4034 (2011). - Chen, L. Q. Phase-Field Method of Phase Transitions/Domain Structures in Ferroelectric Thin Films: A Review. *J. Am. Ceram. Soc*. 91, 1835–1844 (2008). - Chen, W. J, Zheng, Y. &amp; Wang, B. Phase field simulations of stress controlling the vortex domain structures in ferroelectric nanosheets. *Appl. Phys. Lett*. 100, 062901 (2012). - Phillpot, S. R., Sinnott, S. B. &amp; Asthagiri, Atomic-Level Simulation of Ferroelectricity in Oxides: Current Status and Opportunities. *A. Annu. Rev. Mater. Res*. 37, 239 (2007). - Zhong, W., Vanderbilt, D &amp; Rabe, K. M. First-principles theory of ferroelectric phase transitions for perovskites: The case of BaTiO3. *Phys. Rev. B* 52, 6301 (1995). - Shimada, T., Umeno, Y. &amp; Kitamura, T. Ab initio study of stress-induced domain switching in PbTiO3. *Phys. Rev. B* 77, 094105 (2008). - Chen, L.Q., Shen J. Applications of semi-implicit Fourier-spectral method to phase field equations. *Computer Physics Communications* 108, 147 (1998). ### Acknowledgements Helpful discussions with Dr. D. C. Ma and Dr. S. P. Lin are gratefully acknowledged. The authors also acknowledge the financial support of the National Natural Science Foundation of China (NSFC) (Nos. 10902128, 11232015, 50802026, 10972239). Y. Z. also thanks support by the Fundamental Research Funds for the Central Universities to Micro&Nan;; Physics and Mechanics Research Laboratory, New Century Excellent Talents in University, Research Fund for the Doctoral Program of Higher Education, Fok Ying Tung Foundation, Natural Science Funds for Distinguished Young Scholar of Guangdong Province of China, and Educational Commission of Guangdong Province of China. ### Figures **Figure 1 : Scheme of the model system**. [Download Figure 1](http://www.nature.com/protocolexchange/system/uploads/2333/original/Figure1.tif?1352989520) *A ferroelectric nanoplatelet under surface traction and open-circuited condition*. **Figure 2: Flow diagram of phase-field simulation**. [Download Figure 2](http://www.nature.com/protocolexchange/system/uploads/2334/original/Figure2.tif?1352942087) *The polarization evolution is simulated at a given condition, e.g., T=300K, open-circuited and zero mechanical load. Finite element methods are used to solve the elastic and electric fields*. **Figure 3: Scheme of importing file p_final.txt into MATLAB and illustration of the data.** [Download Figure 3](http://www.nature.com/protocolexchange/system/uploads/2335/original/Figure3.tif?1352942149) *Each row of the p_final matrix stores a node number, node coordinates and polarization components*. **Figure 4: Some result of visualizing the domain structure of a nanoplatelet by using MATLAB**. [Download Figure 4](http://www.nature.com/protocolexchange/system/uploads/2336/original/Figure4.tif?1352942239) *(a) A vector plot the domain structure using quiver3() function. (b) Adding a color plot of the polarization magnitude using slice() function. (c) A plot after changing the colormap to your preference*. **Figure 5: Anticipated results**. [Download Figure 5](http://www.nature.com/protocolexchange/system/uploads/2339/original/Figure5.tif?1352987121) *Snapshots of the domain morphologies during the polarization evolution for a nanoplatelet under different surface traction but with the same initial random perturbation at T=300K.* ### Associated Publications **Vortex Domain Structure in Ferroelectric Nanoplatelets and Control of its Transformation by Mechanical Load**. W. J. Chen, Yue Zheng, and Biao Wang. *Scientific Reports* 2 () 12/11/2012 [doi:10.1038/srep00796](http://dx.doi.org/10.1038/srep00796) ### Author information **Chen Weijin, Zheng Yue &amp; Wang Biao**, State Key Laboratory of Optoelectronic Materials and Technologies, Sun Yat-sen University, Guangzhou 510275, China. School of Physics and Engineering, Sun Yat-sen University, Guangzhou 510275, China. Correspondence to: Zheng Yue (zhengy35@mail.sysu.edu.cn) Wang Biao (wangbiao@mail.sysu.edu.cn) *Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2519) (2012) doi:10.1038/protex.2012.054. Originally published online 16 November 2012*.

Authors: David Blum, Samuel LaBarge, The Reproducibility Project: Cancer Biology†* ### Abstract The [Reproducibility Project: Cancer Biology](https://osf.io/e81xl/wiki/home/) seeks to address growing concerns about reproducibility in scientific research by conducting replications of 50 papers in the field of cancer biology published between 2010 and 2012. This Registered Report describes the proposed replication plan of key experiments from “Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion” by Straussman and colleagues, published in Nature in 2012 (Straussman et al., 2012).The key experiments being replicated in this study are from Figures 2A, C and D (and Supplemental Figure 11) and Figure 4C (and Supplemental Figure 19) (Straussman et al., 2012). Figure 2 demonstrates resistance to drug sensitivity conferred by co-culture with some stromal cell lines, and identifies the secreted factor responsible as HGF. In Figure 4, Straussman and colleagues show that blocking the HGF receptor MET abrogates HGF’s rescue of drug sensitivity. The Reproducibility Project: Cancer Biology is a collaboration between the [Center for Open Science](http://centerforopenscience.org/) and [Science Exchange](https://www.scienceexchange.com/), and the results of the replications will be published by eLife. ### Introduction Resistance to oncoprotein-targeted chemotherapy is a common occurrence during cancer treatment and identifying the mechanisms of resistance is important in improving treatment options. Specifically, BRAF-mutant melanomas, which show an initial response to RAF inhibitors, usually become resistant to the therapy (Nickoloff and Vande Woude, 2012). The identification of stroma-mediated resistance in BRAF-mutant melanomas, through the secretion of hepatocyte growth factor (HGF), therefore indicates a potential therapeutic strategy through combination treatment of RAF inhibitors and inhibition of the HGF activated pathway (Straussman et al., 2012). This report is the first to identify paracrine HGF as a potential mechanism for the development of drug resistance (Ghiso and Giordano, 2013; Glaire et al., 2012). In Figure 2A of their paper, Straussman and colleagues tested the effect of fibroblast-conditioned medium on the proliferation of BRAF-mutant melanoma cells grown in the presence of the BRAF inhibitor PLX4720. Using a cell proliferation assay, they reported that fibroblast-conditioned medium rescued BRAF-mutant melanoma cells from PLX4720 sensitivity, which indicated that a secreted factor was involved. This was a key finding demonstrating that the stromal environment of the tumor cells could mediate their response to drug treatment. This experiment will be replicated in Protocol 3. Straussman and colleagues went on to identify the secreted factor responsible for acquired drug resistance as HGF. In Figure 2C, they demonstrate that treating melanoma cell lines with PLX4720 in combination with increasing concentrations of exogenous HGF increased proliferation as compared to treatment with drug alone. This finding showed a similar effect to treatment with conditioned media from stromal cells that secrete HGF (see Figure 2A) and supports the hypothesis that HGF is the growth factor responsible for rescuing melanoma cells from drug sensitivity. This experiment will be replicated in Protocol 4. Straussman and colleagues demonstrated that the HGF-mediated rescue of melanoma cells from drug sensitivity was mediated through HGF’s cognate receptor tyrosine kinase MET by treating melanoma cell lines co-cultured with stromal cell lines in the presence of PLX4720 with the MET inhibitor crizotinib, as shown in Figure 2D and Supplemental Figure 11. Treatment with crizotinib reduced the increase in proliferation due to co-culture with an HGF-secreting stromal cell line. This experiment provided further support for the hypothesis that HGF was responsible for rescue from drug sensitivity, and also provided evidence that that rescue was MET dependent. This experiment is replicated in Protocol 5. Lastly, Straussman and colleagues reported sustained activation of both ERK and AKT in HGF-treated melanoma cells during BRAF inhibition, and to a lesser extent MEK inhibition, as shown in Figure 4C and Supplemental Figure 19 by Western blot. This confirms activation of pro-survival pathways in response to HGF treatment even in the presence of PLX4720. These experiments are replicated in Protocol 6. To date, a direct replication has been reported; Lezcano and colleagues (Lezcano et al., 2014) published a replication of Figure 3 of Straussman et al Nature 2013 wherein Straussman and colleagues evaluated HGF expression in patient-derived primary melanoma samples and observed a negative correlation between expression of HGF and response to therapy (Straussman et al., 2012). While Lezcano and colleagues’ replication also detected the presence of HGF in human melanoma tumor cells and stromal cells with increased expression at disease progression, they did not identify a statistically significant correlation between HGF expression and clinical outcome (Lezcano et al., 2014). While both of the studies come to different conclusions about the association of stromal HGF and clinical outcome, the 95% confidence intervals of the standardized measure of the effect (cohen’s d) for each study substantially overlap. A study published around the same time as the work of Straussman and colleagues supports the negative association between HGF and clinical response to RAF inhibitor treatments through an analysis of HGF levels in patient plasma samples (Wilson et al., 2012). In other systems, additional labs have observed a similar role for HGF in acquired drug resistance. Caenepeel and colleagues reported that HGF rescued melanoma cell lines, notably SK-MEL-5, from BRAF or MEK inhibition using vemurafenib (an analogue of PLX4720) or PD0325901, respectively, and the rescue was attenuated by MET inhibition (Caenepeel et al., 2013). Nakagawa and colleagues observed that tumor-secreted (not stromal secreted) HGF could induce resistance to the VEGFR inhibitor lenvatinib, and that this resistance could be overcome by co-treatment with golvatinib, a MET inhibitor (Nakagawa et al., 2014). Etnyre and colleagues reported that c-MET and BRAF inhibitors had synergistic inhibitory effects when exposed in combination to melanoma cell lines (Etnyre et al., 2013). Casbas-Hernandez and colleagues co-cultured MCF10 cells with immortalized mammoplasty-derived fibroblasts, and observed a correlation between the levels of fibroblast-secreted HGF and the differentiation of the MCF10 cells towards a ductal carcinoma phenotype. They also observed a correlation between HGF expression and the more invasive basal-like tumors as opposed to the less invasive luminal tumors (Casbas-Hernandez et al., 2013). HGF is also being evaluated as a potential biomarker to indicate potential treatment choices (Penuel et al., 2013; Xie et al., 2013). ### Materials and Methods Unless otherwise noted, all protocol information was derived from the original paper, references from the original paper, or information obtained directly from the authors. **Protocol 1: Determining the range of detection of the replicating lab’s plate reader** This is a general protocol that determines the range of detection of the plate reader. Because the plate reader in use by the replicating lab is different than the plate reader used in the original study, we are determining what the range of detection is for the replicating lab’s plate reader. Sampling: - SK-MEL-5 - 8,000 cells/well x 4 replicates - 4,000 cells/well x 4 replicates - 2,000 cells/well x 4 replicates - 1,000 cells/well x 4 replicates - 500 cells/well x 4 replicates -250 cells/well x 4 replicates - 125 cells/well x 4 replicates - 62.5 cells/well x 4 replicates - 31.25 cells/well x 4 replicates - The experiment is done a total of once. Materials and Reagents: - Reagents that are different from ones originally used are noted with an asterisk (*).  Procedure: 1. Seed 4 wells of a 384-well black plate with 8,000 cells/well all the way to 31.25 cells/well (serial 1:2 dilutions) with pLex-TRC206 SK-MEL-5 cells in 60 µl per well using phenol-red free medium using an automated workstation. Note: All cells will be sent for mycoplasma testing and STR profiling. Note: Ensure at least 85% of SK-MEL-5 cells are GFP-positive before start of experiment. Cells can be enriched using FACS or puromycin (0.5-2 µg/ml), however do not grow cells under antibiotic selection on a regular basis. - a. Total wells seeded = 36 - b. Medium for assay: phenol-red free DMEM supplemented with 1 mM sodium pyruvate, 10% FBS, and 1X Pen-Strep-Glut. - c. Fill wells with 60 µl/well of clear media in at least 2 rows and 2 columns around wells that are being included in the experiment. - The next day after seeding, read GFP fluorescence (Synergy HT Microplate Reader). - Subtract the average reading from media-only wells from the wells with cells. Deliverables: - Data to be collected: - Raw GFP fluorescence readings. - Graph of GFP fluorescence readings vs cell number. Confirmatory analysis plan: - Statistical Analysis: - Coefficient of determination of data values. Known differences from the original study: - Synergy HT Microplate Reader used instead of Molecular Devices SpectraMax M5e Microplate Reader – both can detect GFP fluorescence and the Synergy HT Microplate Reader will be evaluated for sensitivity of detection (Protocol 1) and to determine if the gradient is similar to the original study (≤ 5%) (Protocol 2). Provisions for quality control: This protocol will ensure that the replicating lab’s plate reader is comparable to the original lab’s plate reader. - A lab from the Science Exchange network with extensive experience in conducting cell viability assays will perform these experiments. - All cells will be sent for STR profiling to confirm identity and mycoplasma testing to confirm lack of mycoplasma contamination. - SK-MEL-5 cells will be confirmed to have at least 85% of the cells GFP-positive before the start of experiment. **Protocol 2: Determining the detection variability of the replicating lab’s plate reader** This is a general protocol that determines the variability in detection of the plate reader. Because the plate reader in use by the replicating lab is different than the plate reader used in the original study, we are determining what the variability of detection is for the replicating lab’s plate reader. Sampling: - SK-MEL-5: - 2,000 cells/well x 384 replicates - Experiment will be done a total of once. Materials and Reagents: - Reagents that are different from ones originally used are noted with an asterisk (*)  Procedure: 1. Seed all wells of a 384-well black plate with 2,000 pLex-TRC206 SK-MEL-5 cells (provided by authors) in 60 µl per well using phenol-red free medium using an automated workstation. - Note: All cells will be sent for mycoplasma testing and STR profiling. - Note: Ensure at least 85% of SK-MEL-5 cells are GFP-positive before start of experiment. Cells can be enriched using FACS or antibiotics, however do not grow cells under antibiotic selection on a regular basis. - a. Medium for assay: phenol-red free DMEM supplemented with 1 mM sodium pyruvate, 10% FBS, and 1X Pen-Strep-Glut. - b. Fill wells with 60 µl/well of clear media in at least 2 rows and 2 columns around wells that are being included in the experiment. - The next day after seeding, read GFP fluorescence (Synergy HT Microplate Reader). - a. Subtract the average reading from media-only wells from the wells with cells. Deliverables: - Data to be collected: - Raw GFP fluorescence readings. - Difference of each individual well and the average reading across the plate. Confirmatory analysis plan: - Statistical Analysis: - Standard deviation of data values. Known differences from the original study: - Synergy HT Microplate Reader used instead of Molecular Devices SpectraMax M5e Microplate Reader – both can detect GFP fluorescence and the Synergy HT Microplate Reader will be evaluated for sensitivity of detection (Protocol 1) and to determine if the gradient is similar to the original study (≤ 5%) (Protocol 2). Provisions for quality control: This protocol will ensure that the replicating lab’s plate reader is comparable to the original lab’s plate reader. - A lab from the Science Exchange network with extensive experience in conducting cell viability assays will perform these experiments. - All cells will be sent for STR profiling to confirm identity and mycoplasma testing to confirm lack of mycoplasma contamination. - SK-MEL-5 cells will be confirmed to have at least 85% of the cells GFP-positive before the start of experiment. **Protocol 3: Co-culture proliferation assay** This protocol outlines how to culture melanoma cell lines with conditioned medium from three stromal cell lines with or without the RAF inhibitor PLX4720 to analyze cell proliferation rates, as is described in Figure 2A. Sampling: - Experiment to be repeated a total of 4 times for a minimum power of 81%. - See Power Calculations section for details - Each experiment has six conditions to be run in quadruplicate per experiment: - SK-MEL-5 untreated control [additional control] - SK-MEL-5 vehicle (DMSO) control - SK-MEL-5 treated with 2 µM PLX4720 and with unconditioned medium - SK-MEL-5 treated with 2 µM PLX4720 and with conditioned medium from CCD-1090Sk cells that do not secrete HGF - SK-MEL-5 treated with 2 µM PLX4720 and with conditioned medium from PC60163A1 cells that do secrete HGF - SK-MEL-5 treated with 2 µM PLX4720 and with conditioned medium from LL 86 cells that do secrete HGF Materials and Reagents: - Reagents that are different from ones originally used are noted with an asterisk (*)  Procedure: 1. Prepare Pre-Conditioned Medium (PCM); fresh PCM must be prepared the same day it is used in the treatment of SK-MEL-5 cells; this step is repeated three times to ensure fresh PCM is available on the needed day: - a. Three days before the PCM is needed, seed 3 x 10 cm tissue culture plates with 0.5x10e6 LL 86 cells each, 3 x 10 cm tissue culture plates with 1x10e6 PC60163A1 cells each, and 3 x 10 cm tissue culture plates with 2x10e6 CCD-1090Sk cells each (9 plates total) in 10 ml of phenol-red free medium each and grow for 3 days. - b. 3 days after seeding, collect medium from each cell line using the plate closest to 80-90% confluent. - i. 75-95% confluency can be used. - c. Filter through 0.45 µm syringe filter with a 10 ml syringe and dilute filtered PCM 1:1 in fresh phenol-red free medium. Total volume = 20 ml. - i. Use same day. - ii. Do not dilute for day 0 of treatment (these wells will already have 20 µl of media in them). - On day 0, seed 120 wells of a 384-well black plate with 1,900 pLex-TRC206 SK-MEL-5 cells in 20 µl per well using phenol-red free medium using an automated workstation. - Note: - All cells will be sent for mycoplasma testing and STR profiling. - Ensure at least 85% of SK-MEL-5 cells are GFP-positive before start of experiment. Cells can be enriched using FACS or antibiotics, however do not grow cells under antibiotic selection on a regular basis. - Do not exceed a rate of 5-10 µl/sec and do not let the tip end closer than 1mm to the well bottom. - a. Fill wells with 50 µl/well of media in at least 2 rows and 2 columns around wells that are being included in the experiment. - i. Medium for assay: phenol-red free DMEM supplemented with 1 mM sodium pyruvate, 10% FBS, and 1X Pen-Strep-Glut. - b. To wells in step a, add 20 µl of fresh undiluted PCM from appropriate stromal cells generated as described in step 1 (see Sampling section for Cohorts) or phenol-red free medium alone (Cohort 1). - On day 1 after seeding, read GFP fluorescence (Synergy HT Microplate Reader). - a. Subtract the average reading from media-only wells from the wells with cells. - After reading GFP fluorescence, refresh media and add drug using an automated workstation. - a. Change medium for each cohort to 40 µl fresh diluted PCM from appropriate stromal cell lines generated as described in step 1 or phenol-red free medium alone. - b. Within each cohort, add 10 µl of 5X PLX4720, DMSO dilution or 10 µl phenol-red free medium to each appropriate well to bring the final volume per well up to 50 µl. - i. 5X PLX4720: Make up stocks of 10mM PLX4720 in DMSO, then dilute 1:1000 in media to make up 10µM PLX4720. This is a 5X stock. - ii. DMSO dilution: Dilute 1 µl DMSO with 999 µl media. Add 10 µl of this mix to DMSO wells. - __1. These dilutions in media prevent toxicity from excess DMSO. - On day 4 after seeding, read GFP fluorescence. - a. Subtract the average reading from media-only wells from the wells with cells. - After reading GFP fluorescence, change medium in appropriate wells to 40 µl fresh diluted PCM from appropriate stromal cell lines generated as described in step 1 or phenol-red free medium alone using an automated workstation. - a. Add 10 µl of 5X PLX4720, DMSO dilution or 10 µl phenol-red free medium to each appropriate well to bring the final volume per well up to 50 µl. - i. 5X PLX4720: Make up stocks of 10 mM PLX4720 in DMSO, then dilute 1:1000 in media to make up 10 µM PLX4720. This is a 5X stock. - ii. DMSO dilution: Dilute 1 µl DMSO with 999 µl media. Add 10 µl of this mix to DMSO wells. - On day 7 after seeding, read GFP fluorescence and document bright-field and GFP images (BD, Pathway 435 Bioimager). - a. Subtract the average reading from media-only wells from the wells with cells. - Data analysis: - a. Remove background fluorescence by subtracting the average reading from media-only wells from the wells with cells for each plate reading. - b. Subtract the readings of day 1 from the other plates (day 4 and day 7) for the same wells. - c. Average the quadruplicates. - d. Calculate the effect of PLX4720 in the presence or absence of conditioned media by normalizing the number of cells after 7 days of treatment (as measured by GFP fluorescence) to the number of cells present in the SK-MEL-5 vehicle control condition. - Repeat experiment independently three additional times. Deliverables: - Data to be collected: - Raw GFP fluorescence readings from days 1, 4 and 7. - Normalized fluorescence proliferation data. - Fluorescent and bright field micrographs of cells from day 7. - Bar chart of relative proliferation as a % of untreated control for all conditions. (Use data from Day 7 - Day 1 background) (Compare to Figure 2A) - A semi-logarithmic graph of proliferation (log) vs time (linear) over 3 time points after seeding. Confirmatory Analysis Plan: - Statistical Analysis of the Replication Data: - One-way ANOVA comparing the proliferation of PLX4720-treated cells cultured with unconditioned medium, CCD-1090Sk conditioned medium, LL 86 conditioned medium, or PC60163A1 conditioned medium. - Planned comparisons with the Bonferroni correction: - unconditioned medium to PC60163A1 conditioned medium - unconditioned medium to LL 86 conditioned medium - CCD-1090Sk to PC60163A1 conditioned medium - CCD-1090Sk to LL 86 conditioned medium - Meta-analysis of original and replication attempt effect sizes: - Compare the effect sizes of the original data to the replication data and use a meta-analytic approach to combine the original and replication effects, which will be presented as a forest plot. Known differences from original study: - The replication will only use one of the three melanoma cell lines used by the original authors, the SK-MEL-5 cell line. The replication will exclude SK-MEL-28 and G-361 cells. - The replication will include an additional control, untreated SK-MEL-5 cells in addition to the vehicle (DMSO) treated SK-MEL-5 cells used in the original study. - A Synergy HT Microplate Reader will be used instead of a Molecular Devices SpectraMax M5e Microplate Reader – both can detect GFP fluorescence and the Synergy HT Microplate Reader will be evaluated for range of detection (Protocol 1) and detection variability (Protocol 2) - A BD Pathway 435 Bioimager used instead of a Zeiss Axio Observer.Z1 – both are fluorescence microscopes with high-throughput screening capabilities. - The replicating lab does not have a ViCell XR cell viability counter, and thus will seed a larger number of cells per well (1,900 instead of 1,700 cells/well). Provisions for quality control: All data obtained from the experiment - raw data, data analysis, control data and quality control data - will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (https://osf.io/p4lzc/). - A lab from the Science Exchange network with extensive experience in conducting cell viability assays will perform these experiments. - All cells will be sent for STR profiling to confirm identity and mycoplasma testing to confirm lack of mycoplasma contamination. - SK-MEL-5 cells will be confirmed to have at least 85% of the cells GFP-positive before the start of experiment. **Protocol 4: Recombinant HGF proliferation assay** This protocol assesses changes in proliferation when melanoma cells are treated with the RAF inhibitor PLK4720 with or without HGF, as is described in Figure 2C. The cells are also treated with a MEK inhibitor, PD184352. Sampling: - Experiment to be repeated a total of 3 times for a final power of 99%. - See Power Calculations section for details - Each experiment has 12 conditions to be done in quadruplicate per experiment: - SK-MEL-5 untreated control [additional control] - SK-MEL-5 vehicle (DMSO) control - SK-MEL-5 2 µM PLX4720 + 0 ng/ml HGF - SK-MEL-5 2 µM PLX4720 + 6.25 ng/ml HGF - SK-MEL-5 2 µM PLX4720 + 12.5 ng/ml HGF - SK-MEL-5 2 µM PLX4720 + 25 ng/ml HGF - SK-MEL-5 2 µM PLX4720 + 50 ng/ml HGF - SK-MEL-5 1 µM PD184352 + 0 ng/ml HGF - SK-MEL-5 1 µM PD184352 + 6.25 ng/ml HGF - SK-MEL-5 1 µM PD184352 + 12.5 ng/ml HGF - SK-MEL-5 1 µM PD184352 + 25 ng/ml HGF - SK-MEL-5 1 µM PD184352 + 50 ng/ml HGF Materials and Reagents: - Reagents that are different from the ones originally used are noted with a *.  Procedure: 1. On day 0, seed 48 wells of a 384 well clear-bottom plate with 2,800 pLex-TRC206 SK-MEL-5 cells in 40 µl of phenol-red free medium each using an automated workstation. - Note: - All cells will be sent for mycoplasma testing and STR profiling. - Ensure at least 85% of SK-MEL-5 cells are GFP-positive before start of experiment. Cells can be enriched using FACS or antibiotics, however do not grow cells under antibiotic selection on a regular basis. - Do not exceed a rate of 5-10 µl/sec and do not let the tip end closer than 1 mm to the well bottom. - a. Fill wells with 60 µl/well of clear media in at least 2 rows and 2 columns around wells that are being included in the experiment. - b. Medium of all cell lines for assay: phenol-red free DMEM supplemented with 1 mM sodium pyruvate, 10% FBS, and 1X Pen-Strep-Glut. - On day 1 after seeding, read GFP fluorescence (Synergy HT Microplate Reader). - a. Subtract the average reading from media-only wells from the wells with cells. - After reading GFP fluorescence, add to the appropriate wells; 10 µl 6X HGF or phenol-red free medium alone. Then add to the appropriate wells the following; 10 µl 6X PLX4720, 10 µl 6X PD184352, 10 µl DMSO dilution, or 10 µl phenol-red free medium alone. - a. 6X HGF: Make up stocks of 100 ug/ml HGF, then dilute accordingly to make 6X working concentrations of each required HGF dilution. - b. 6X PLX4720: Make up stocks of 12 mM PLX4720 in DMSO, then dilute 1:1000 in media to make up 12 uM PLX4720 for use at 6X for the assay to avoid excessive DMSO toxicity. - c. 6X PD184352: Make up stocks of 6 mM PD184352 in DMSO, then dilute 1:1000 in media to make up 6 uM PD184352 for use at 6X for the assay to avoid excessive DMSO toxicity. - d. DMSO dilution: Dilute 1 µl DMSO with 999 µl media. Add 10 µl of this mix to DMSO dilution wells. a. These media dilutions are to avoid toxicity from excessive DMSO. - On day 4 after seeding, read GFP fluorescence. - a. Subtract the average reading from media-only wells from the wells with cells. - After reading GFP fluorescence, change medium in all wells to 40 µl fresh phenol-red free medium using an automated workstation. Then add to the appropriate wells; 10 µl 6X HGF or phenol-red free medium alone. Then add to the appropriate wells the following; 10 µl 6X PLX4720, 10 µl 6X PD184352, 10 µl DMSO dilution, or 10 µl phenol-red free medium alone. - a. 6X HGF: Make up stocks of 100 ug/ml HGF, then dilute accordingly to make 6X working concentrations of each required HGF dilution. - b. 6X PLX4720: Make up stocks of 12 mM PLX4720 in DMSO, then dilute 1:1000 in media to make up 12 uM PLX4720 for use at 6X for the assay to avoid excessive DMSO toxicity. - c. 6X PD184352: Make up stocks of 6 mM PD184352 in DMSO, then dilute 1:1000 in media to make up 6 uM PD184352 for use at 6X for the assay to avoid excessive DMSO toxicity. - d. DMSO dilution: Dilute 1 µl DMSO with 999 µl media. Add 10 µl of this mix to DMSO dilution wells. - a. These media dilutions are to avoid toxicity from excessive DMSO. - On day 7 after seeding, read GFP fluorescence and document bright-field and GFP images (BD, Pathway 435 Bioimager). - a. Subtract the average reading from media-only wells from the wells with cells. - Data analysis: - a. Remove background fluorescence by subtracting the average reading from media-only wells from the wells with cells for each plate reading. - b. Subtract the readings of day 1 from the other plates (day 4 and day 7) for the same wells. - c. Average the quadruplicates. - d. Calculate the effect of PLX4720 and PD184352 in the presence or absence of HGF by normalizing the number of cells after 7 days of treatment (as measured by GFP fluorescence) to the number of cells present in the SK-MEL-5 vehicle control condition. - Repeat experiment independently two additional times. Deliverables: - Data to be collected: - Raw GFP fluorescence readings from days 1, 4 and 7. - Normalized fluorescence proliferation data. - Fluorescent and bright field micrographs of cells from day 7. - Bar chart of relative proliferation as a % of untreated control for all conditions. (Use data from Day 7 - Day 1 background) (Compare to Figure 2C) - A semi-logarithmic graph of proliferation (log) vs time (linear) over 3 time points after seeding. Confirmatory Analysis Plan: - Statistical Analysis: - Compare the proliferation rate of PLX4720 treated cells treated with 0, 6.25, 12.5, 25, or 50 ng/ml HGF. Also compare each HGF cohort to the proliferation rate of vehicle treated and untreated cells. - One-way ANOVA - Compare the proliferation rate of PD184352 treated cells treated with 0, 6.25, 12.5, 25, or 50 ng/ml HGF. Also compare each HGF cohort to the proliferation rate of vehicle treated and untreated cells. - One-way ANOVA - Meta-analysis of original and replication attempt effect sizes: - Compare the effect sizes of the original data to the replication data use a meta-analytic approach to combine the original and replication effects, which will be presented as a forest plot. Known differences from original study: - The replication will only use one of the three melanoma cell lines used by the original authors, the SK-MEL-5 cell line. The replication will exclude SK-MEL-28 and G-361 cells. - The replication will include an additional control, untreated SK-MEL-5 cells in addition to the vehicle (DMSO) treated SK-MEL-5 cells used in the original study. - A Synergy HT Microplate Reader used instead of a Molecular Devices SpectraMax M5e Microplate Reader – both can detect GFP fluorescence and the Synergy HT Microplate Reader will be evaluated for range of detection (Protocol 1) and detection variability (Protocol 2) - A BD Pathway 435 Bioimager used instead of a Zeiss Axio Observer.Z1 – both are fluorescence microscopes with high-throughput screening capabilities. - The replicating lab does not have a ViCell XR cell viability counter, and thus will seed a larger number of cells per well (2,800 instead of 2,500 cells/well). Provisions for quality control: All data obtained from the experiment - raw data, data analysis, control data and quality control data - will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (https://osf.io/p4lzc/). - A lab from the Science Exchange network with extensive experience in conducting cell viability assays will perform these experiments. - All cells will be sent for STR profiling to confirm identity and mycoplasma testing to confirm lack of mycoplasma contamination. - SK-MEL-5 cells will be confirmed to have at least 85% of the cells GFP-positive before the start of experiment. **Protocol 5: Inhibitor proliferation assay** This experiment confirms that the rescue from drug sensitivity is due to HGF signaling by co-treating cells with crizotinib, an inhibitor of MET, the receptor tyrosine kinase for HGF, as seen in Figure 2D and Supplemental Figure 11. Sampling: - Run the experiment six times in total for a minimum power of 80%. - See Power Calculations section for details - Each experiment has 10 cohorts: - Each cohort consists of - SK-MEL-5 cells alone - SK-MEL-5 co-cultured with LL86 cells - SK-MEL-5 co-cultured with CCD-1090Sk cells - __Each condition will be run in quadruplicate. - The cohorts are treated with the following drugs: - Cohort 1: no drug treatment [additional control] - Cohort 2: Treated with vehicle (DMSO) control - Cohort 3: Treated with 0.2 µM crizotinib and vehicle - Cohort 4: Treated with 0.2 µM PHA-665752 and vehicle [additional control] - Cohort 5: Treated with 2 µM PLX4720 and vehicle - Cohort 6: Treated with 2 µM PLX4720 and 0.2 µM crizotinib - Cohort 7: Treated with 2 µM PLX4720 and 0.2 µM PHA-665752 [additional] - Cohort 8: Treated with 1 µM PD184352 and vehicle - Cohort 9: Treated with 1 µM PD184352 and 0.2 µM crizotinib - Cohort 10: Treated with 1 µM PD184352 and 0.2 µM PHA-665752 [additional control] Materials and Reagents: - Reagents that are different from ones originally used are noted with an asterisk (*)  Procedure: 1. On day 0, seed 40 wells of a 384 well clear-bottom plate with 1,900 LL86 stromal cells in 20 µl phenol-red free media, seed 40 wells with 1,900 CCD-1090Sk stromal cells in 20 µl media, and seed 40 wells with phenol-red free medium alone using an automated workstation. - Note: - All cells will be sent for mycoplasma testing and STR profiling. - Ensure at least 85% of SK-MEL-5 cells are GFP-positive before start of experiment. Cells can be enriched using FACS or antibiotics, however do not grow cells under antibiotic selection on a regular basis. - Do not exceed a rate of 5-10 µl/sec and do not let the tip end closer than 1 mm to the well bottom - a. Total wells seeded: 120 - b. Fill wells with 60 µl/well of clear media in at least 2 rows and 2 columns around wells that are being included in the experiment. - c. Medium of all cell lines for assay: phenol-red free DMEM supplemented with 1 mM sodium pyruvate, 10% FBS, and 1X Pen-Strep-Glut. - In wells from Step 1, seed 1,900 pLex-TRC206 SK-MEL-5 cells in 20 µl phenol-red free medium per well using an automated workstation. - On day 1 after seeding, read GFP fluorescence (Synergy HT Microplate Reader). a. Subtract the average reading from media-only wells from the wells with cells. - Add appropriate drugs to each well (final volume = 60 µl). - a. Formulation of drug stock solutions: - i. 6X PLX4720: Make up stocks of 12 mM PLX4720 in DMSO, then dilute 1:1000 in media to make up 12 µM PLX4720 for use at 6X for the assay to avoid excessive DMSO toxicity. - ii. 6X PD184352: Make up stocks of 6 mM PD184352 in DMSO, then dilute 1:1000 in media to make up 6 µM PD184352 for use at 6X for the assay to avoid excessive DMSO toxicity. - iii. 6X crizotinib: Make up stocks of 1.2 mM crizotinib in DMSO, then dilute 1:1000 in media to make up 1.2 µM PD184352 for use at 6X for the assay to avoid excessive DMSO toxicity. - iv. 6X PHA-665752: Make up stocks of 1.2 mM PHA-665752 in DMSO, then dilute 1:1000 in media to make up 1.2 µM PD184352 for use at 6X for the assay to avoid excessive DMSO toxicity. - v. DMSO dilution: Dilute DMSO 1:1000 in medium to avoid excessive DMSO toxicity. - b. Cohort 1: Add 20 µl phenol-red free medium - c. Cohort 2: Add 10 µl DMSO dilution and 10 µl medium - d. Cohort 3: Add 10 µl 6X crizotinib and 10 µl medium - e. Cohort 4: Add 10 µl 6X PHA-665752 and 10 µl medium - f. Cohort 5: Add 10 µl 6X PLX4720 and 10 µl medium - g. Cohort 6: Add 10 µl 6X PLX4720 and 10 µl 6X crizotinib - h. Cohort 7: Add 10 µl 6X PLX4720 and 10 µl 6X PHA-665752 - i. Cohort 8: Add 10 µl 6X PD184352 and 10 µl medium - j. Cohort 9: Add 10 µl 6X PD184352 and 10 µl 6X crizotinib - k. Cohort 10: Add 10 µl 6X PD184352 and 10 µl 6X PHA-665752 - On day 4 after seeding, read GFP fluorescence. - a. Subtract the average reading from media-only wells from the wells with cells. - Change medium in relevant wells to 40 µl fresh media, then add appropriate drugs as per Step 4 using an automated workstation. - On day 7 after seeding, read GFP fluorescence and document bright-field and GFP images (BD, Pathway 435 Bioimager). - a. Subtract the average reading from media-only wells from the wells with cells. - Data analysis: - a. Remove background fluorescence by subtracting the average reading from media-only wells from the wells with cells for each plate reading. - b. Subtract the readings of day 1 from the other plates (day 4 and day 7) for the same wells. - c. Average the quadruplicates. - d. Calculate the effect of PLX4720, PD184352, Crizotinib, PHA-665752, PLX4720 + Crizotinib, PLX4720 + PHA665752, PD184352 + Crizotinib, PD184352 + PHA665752, DMSO, or untreated in the presence or absence of stromal cells by normalizing the number of cells after 7 days of treatment (as measured by GFP fluoresence) to the number of cells present in the vehicle control treated SK-MEL-5 cells alone condition. - Repeat experiment independently five additional times. Deliverables - Data to be collected: - Raw GFP fluorescence readings from days 1, 4 and 7. - Normalized fluorescence proliferation data. - Fluorescent and bright field micrographs of cells from day 7. - Bar chart of relative proliferation as a % of untreated control for all conditions. (Use data from Day 7 - Day 1 background) (Compare to Figure SF11) - A semi-logarithmic graph of proliferation (log) vs time (linear) over 3 time points after seeding. Confirmatory analysis plan: - Statistical Analysis of replication data: - Three-way ANOVA comparing the proliferation of vehicle-treated, PLX4720-treated, or PD184352-treated cells also treated with vehicle, crizotinib, or PHA-665752 cultured with or without stromal cells followed by: - Two-way ANOVA comparing the proliferation of vehicle-treated cells treated with vehicle, crizotinib, or PHA-665752 cultured with or without stromal cells. - Two-way ANOVA comparing the proliferation of PLX4720-treated cells treated with vehicle, crizotinib, or PHA-665752 cultured with or without stromal cells. - Planned comparisons with the Bonferroni correction: - Vehicle-treated LL 86 cells compared to vehicle-treated no stromal cells - Vehicle-treated LL 86 cells compared to vehicle-treated CCD-1090Sk cells - Vehicle-treated LL 86 cells compared to crizotinib-treated LL 86 cells - Vehicle-treated LL 86 cells compared to PHA-665752-treated LL 86 cells - Two-way ANOVA comparing the proliferation of PD184352-treated cells treated with vehicle, crizotinib, or PHA-665752 cultured with or without stromal cells. - Planned comparisons with the Bonferroni correction: -__Vehicle-treated LL 86 cells compared to crizotinib-treated LL 86 cells -__Vehicle-treated LL 86 cells compared to PHA-665752-treated LL 86 cells - Meta-analysis of original and replication attempt effect sizes: - Compare the effect sizes of the original data to the replication data use a meta-analytic approach to combine the original and replication effects, which will be presented as a forest plot. Known differences from original study: - Supplemental Figure 11 tests co-culture of SK-MEL-5 cells with 9 stromal cell lines. We have chosen LL86 cells, which showed the largest rescue of proliferation, and CCD-1090Sk cells, which showed the least rescue. - Additional controls added by the replication team: - Treatment with PHA-665752 - In addition to inhibiting MET, crizotinib also targets ALK, ROS1 and RON. In order to confirm that the effects of crizotinib are due to targeting of MET, we will also use a more selective MET inhibitor, PHA-665752 (Cui, 2014; Parikh et al., 2014). - The replication will include an additional control, untreated SK-MEL-5 cells in addition to the vehicle (DMSO) treated SK-MEL-5 cells used in the original study. - A Synergy HT Microplate Reader used instead of a Molecular Devices SpectraMax M5e Microplate Reader - Both can detect GFP fluorescence and the Synergy HT Microplate Reader will be evaluated for range of detection (Protocol 1) and detection variability (Protocol 2) - A BD Pathway 435 Bioimager used instead of a Zeiss Axio Observer.Z1 - Both are fluorescence microscopes with high-throughput screening capabilities. - The replicating lab does not have a ViCell XR cell viability counter, and thus will seed a larger number of cells per well (1,900 instead of 1,700 cells/well). Provisions for quality control: All data obtained from the experiment - raw data, data analysis, control data and quality control data - will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (https://osf.io/p4lzc/). - A lab from the Science Exchange network with extensive experience in conducting cell viability assays will perform these experiments. - All cells will be sent for STR profiling to confirm identity and mycoplasma testing to confirm lack of mycoplasma contamination. - SK-MEL-5 cells will be confirmed to have at least 85% of the cells GFP-positive before the start of experiment. **Protocol 6: Inhibitor western blot assay of ERK and AKT signaling** This experiment assesses the protein levels of various activated downstream pathway signaling component proteins in the presence or absence of HGF and drugs, as seen in Figure 4C and Supplemental Figure 19. Sampling: - Repeat the experiment six times in total for a minimum power of 85%. - See Power Calculations section for details - Each experiment contains seven conditions: - SK-MEL-5 cells treated with: - untreated [additional control] - vehicle (DMSO) control - 2 µM PD184352 - 2 µM PLX4720 - 25 ng/ml HGF + vehicle - 25 ng/ml HGF + 2µM PD184352 - 25 ng/ml HGF + 2µM PLX4720 Materials and Reagents: - Reagents that are different from ones originally used are noted with an asterisk (*)   Procedure: 1. On day 0, plate 5x10e5 pLex-TRC206 SK-MEL-5 cells in 2 ml media per well for a total of 7 wells across 2 x 6-well plates. - Note: - All cells will be sent for mycoplasma testing and STR profiling. - Ensure at least 85% of SK-MEL-5 cells are GFP-positive before start of experiment. Cells can be enriched using FACS or antibiotics, however do not grow cells under antibiotic selection on a regular basis. - a. Medium of all cell lines for assay: DMEM supplemented with 10% FBS and 1X Pen-Strep. - On day 1 add the appropriate additives to each well. - a. Formulation of stock solutions: Note: These dilutions are to avoid toxicity from excessive DMSO. - i. 1000X HGF: Make a stock of 25 µg/ml HGF. - ii. 1000X PLX4720: Make a stock of 20 mM PLX4720 in DMSO, then dilute 1:10 in media to make a 2 mM PLX4720 working solution. - iii. 1000X PD184352: Make a stock of 20 mM PD184352 in DMSO, then dilute 1:10 in media to make a 2 mM working solution. - iv. DMSO dilution: Dilute DMSO 1:10 in medium. - b. For media only: add 2 µl media - c. For DMSO: Add 2 µl DMSO dilution - d. For 2 µM PD184352: Add 2 µl 1000X PD184352 - e. For 2 µM PLX4720: Add 2 µl 1000X PLX4720 - f. For 25 ng/ml HGF + DMSO: Add 2 µl 1000X HGF and 2 µl DMSO dilution - g. For 25 ng/ml HGF + 2 µM PD184352: Add 2 µl 1000X HGF and 2 µl 100X PD184352 - h. For 25 ng/ml HGF + 2 µM PLX4720: Add 2 µl 1000X HGF and 2 µl 100X PLX4720. - 24 hr after drug treatment, prepare cells for lysis. - a. Quickly wash cells with ice-cold PBS and remove excess PBS. - b. Add 0.5 ml or less of ice-cold lysis buffer to wells on ice. - i. Lysis Buffer: 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 1mg/ml NaF and one pellet per 10 ml each of PhosSTOP Phosphatase Inhibitor and Complete Mini Protease Inhibitor. - c. Scrape cells off dish with cell scraper. - d. Collect cells in a 1.5 ml centrifuge tube on ice. - e. Incubate on ice for 30 min with periodic vortexing. - f. Spin down at 4˚C and remove supernatant into separate tube. - Determine protein concentration by using the DC Protein Assay Kit II following manufacturer’s instructions. - Mix 50 µg total cell lysate with NuPAGE Sample Reducing Agent and run on two 4-12% Bis-Tris gels with a protein molecular weight marker at 120 V. - Transfer onto membrane using replicating lab’s transfer protocol. - After the transfer, stain the membrane with Ponceau to visualize transferred protein. Image membrane, then wash out the Ponceau stain. [additional quality control step] - Wet membrane with PBS for 5 min, then block membranes in Odyssey blocking buffer (LI-COR, 927-40000) following manufacturer’s instructions. - Probe membrane with the following primary antibodies diluted in Odyssey blocking buffer at 4°C with gentle shaking, overnight. - a. mouse anti-c-Met (Cell Signaling, 3148); 1:1000; 145kDa - b. rabbit anti-pMet Tyr 1349 (Cell Signaling, 3133); 1:1000; 145kDa - c. mouse anti-AKT (Cell Signaling, 2920); 1:2000; 60kDa - d. rabbit anti-pAKT (Cell Signaling, 4060); 1:2000; 60kDa - e. mouse anti-MEK (Cell Signaling, 4694); 1:1000; 45kDa - f. rabbit anti-pMEK (Cell Signaling, 9154); 1:1000; 45kDa - g. mouse anti-ERK (Santa Cruz, 135900); 1:200; 44,42kDa - h. rabbit anti-pERK (Cell Signaling, 4370); 1:2000; 44,42kDa - i. rabbit anti-GAPDH (Cell Signaling, 2118); 1:1000; 37kDa - i. Loading control - j. Note: multiple gels will need to be run to probe for this many proteins. Do not strip between probing with different phospho antibodies, just wash membrane well (4 x 10 min PBS-T) and then add next antibody. Suggest grouping as follows: - i. Gel 1: Probe pAKT [rabbit 60kDa], then pMEK [rabbit 45kDa], then AKT [mouse 60kDa], then MEK [mouse 45kDa], then GAPDH [rabbit 37kDa]. - ii. Gel 2: Probe pMet Tyr 1349 [rabbit 145kDa], then pERK [rabbit 44,42kDa], then c-Met [mouse 145kDa], then ERK [mouse 44,42kDa], then GAPDH [rabbit 37kDa]. - Wash membranes in PBS + 0.1% Tween 20 4 x 5 min. - Detect primary antibodies with anti-rabbit or anti-mouse IRDye secondary antibodies (LICOR) diluted in Odyssey blocking buffer for 30-60 min protected from light following manufacturer’s instructions. - Wash membranes in PBS + 0.1% Tween 20 4 x 5 min. - Rinse membrane with PBS to remove residual Tween 20. - Detect near infrared fluoresence with the Odyssey Infrared Imaging System. - Quantify signal intensity with Odyssey Application Software. - a. For each antibody subtract background intensity from values and then divide by the GAPDH loading control. - b. Calculate the effect of PLX4720, PD184352, or vehicle in the presence or absence of HGF by normalizing the band intensities (after background and loading correction) to the band intensity of the SK-MEL-5 vehicle control condition. - Repeat experiment independently five additional times. Deliverables: - Data to be collected: - Odyssey images of probed membranes (full images with ladder) - Raw and quantifed signal intensities normalized for GAPDH loading and total pan-protein levels. - Bar graphs of normalized mean signal intensities (Compare to Figure S19) Confirmatory Analysis plan: - Statistical Analysis of replication data: - Two-way ANOVA comparing the relative phopho-AKT band intensities of cells treated with vehicle, PLX4720, or PD184352 in the presence or absence of HGF. - Planned comparisons with the Bonferroni correction: - PLX4720-treated cells in the absence of HGF compared to PLX4720-treated cells in the presence of HGF - Two-way ANOVA comparing the relative phopho-ERK band intensities of cells treated with vehicle, PLX4720, or PD184352 in the presence or absence of HGF. - Planned comparisons with the Bonferroni correction: - PLX4720-treated cells in the absence of HGF compared to PLX4720-treated cells in the presence of HGF - Two-way ANOVA comparing the relative phopho-MET (Tyr1349) band intensities of cells treated with vehicle, PLX4720, or PD184352 in the presence or absence of HGF. - Planned comparisons with the Bonferroni correction: - Cells treated in the absence of HGF and treated with vehicle, PLX4720, or PD184352 compared to cells treated in the presence of HGF and treated with vehicle, PLX4720, or PD184352 - Meta-analysis of original and replication attempt effect sizes: - Compare the effect sizes of the original data to the replication data and use a meta-analytic approach to combine the original and replication effects, which will be presented as a forest plot. Known differences from original study: - Provider lab transfer protocol used instead of iBlot Gel Transfer Device (Invitrogen, IB1001) using Program 4 – both are capable of transferring protein efficiently, and to determine completeness of the transfer, the gel may be stained (Step 8). - The replication will include an additional control, untreated SK-MEL-5 cells in addition to the vehicle (DMSO) treated SK-MEL-5 cells used in the original study. - The replication will not include the pMet Tyr1234/5, RAF1, and pRAF1 antibodies included in the original study. Provisions for quality control: All data obtained from the experiment - raw data, data analysis, control data and quality control data - will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (https://osf.io/p4lzc/). - A lab from the Science Exchange network with extensive experience in conducting cell viability assays and performing Western blots will perform these experiments. - All cells will be sent for STR profiling to confirm identity and mycoplasma testing to confirm lack of mycoplasma contamination. - SK-MEL-5 cells will be confirmed to have at least 85% of the cells GFP-positive before the start of experiment. ### Power Calculations All calculations are determined in order to reach at least 80% power. **Protocol 1** No power calculations required. **Protocol 2** No power calculations required. **Protocol 3** Summary of original data: Note: Original data values were shared by authors.  - Standard deviation was calculated using formula SD = SEM*(SQRT n) Test family - ANOVA: Fixed effects, omnibus, one-way, alpha error = 0.05 - Power calculations were performed from effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). - ANOVA F statistic calculated with Graphpad Prism 6.0 - Partial η2 calculated from (Lakens, 2013) Power calculations for replication:  Test family - two tailed t-test; difference between two independent means, Bonferroni’s correction: alpha error = 0.0125. - Calculations were performed from effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). Power Calculations for replication:  **Protocol 4** Summary of original data: Note: Original data values were shared by authors.  Test family: - ANOVA: Fixed effects, omnibus, one way, alpha error = 0.05 - Power calculations were performed from effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). - ANOVA F statistic calculated with Graphpad Prism 6.0 - Partial η2 calculated from (Lakens, 2013)  Summary of original data: Note: Original data values were shared by authors.  Test family: - ANOVA: Fixed effects, omnibus, one way, alpha error = 0.05 - Power calculations were performed from effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). - ANOVA F statistic calculated with Graphpad Prism 6.0 - Partial η2 calculated from (Lakens, 2013) Power Calculations for replication:  **Protocol 5** Summary of original data: Note: numbers were shared by original authors.  Test family: - 3-way ANOVA Between subjects: Fixed effects, special, main effects and interactions, alpha error = 0.05 - Power calculations were performed from effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). - ANOVA F statistic calculated with R software 3.1.1 (R Core Team, 2014) - Partial η2 calculated from (Lakens, 2013) Power Calculations for replication:  Test family: - 2-way ANOVA Between subjects: Fixed effects, special, main effects and interactions, alpha error = 0.05 - Power calculations were performed from effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). - ANOVA F statistic calculated with Graphpad Prism 6.0 - Partial η2 calculated from (Lakens, 2013) Power Calculations for replication (BRAF/MEK inhibitor):  Test family: - two tailed *t*-test; difference between two independent means, Bonferroni’s correction: alpha error = 0.0125. - Power calculations were performed for effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). Power Calculations for replication (PLX4720 group):  Test family: - two tailed *t*-test; difference between two independent means, Bonferroni’s correction: alpha error = 0.025. - Power calculations were performed for effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). Power Calculations for replication (PD184352 group):  **Protocol 6** Summary of original data: Note: numbers were estimated from bar chart in Supplemental Figure S19.  Test family: - 2-way ANOVA Between subjects: Fixed effects, special, main effects and interactions, alpha error = 0.05 - Power calculations were performed from effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). - ANOVA F statistic calculated with Graphpad Prism 6.0 - Partial η2 calculated from (Lakens, 2013) Power Calculations for replication:  Test family - two tailed *t*-test; difference between two independent means, Bonferroni’s correction: alpha error = 0.05. - Note: Calculations were performed for effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). Power calculations for replication (pAKT group):  Note: HGF/PD184352 compared to vehicle/PD184352 is not included as the number of needed samples is too large Test family - two tailed *t*-test; difference between two independent means, Bonferroni’s correction: alpha error = 0.05. - Note: Calculations were performed for effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). Power calculations for replication (pERK group):  Note: HGF/PD184352 compared to vehicle/PD184352 is not included as the number of needed samples is too large. Test family - two tailed *t*-test; difference between two independent means, Bonferroni’s correction: alpha error = 0.05. - Note: Calculations were performed for effects reported in original study using G*Power software (version 3.1.7) (Faul et al., 2007). Power calculations for replication (pMET(Tyr1349) group):  ### Acknowledgements The Reproducibility Project: Cancer Biology core team would like to thank the original authors, in particular Ravid Straussman and Michal Barzily-Rokni, for generously sharing critical information and reagents to ensure the fidelity and quality of this replication attempt. We would also like to thank the following companies for generously donating reagents to the Reproducbility Project: Cancer Biology; BioLegend, Charles River Laboratories, Corning Incorporated, DDC Medical, EMD Millipore, Harlan Laboratories, LI-COR Biosciences, Mirus Bio, Novus Biologicals, and Sigma-Aldrich. ### References 1. Caenepeel, S, Cajulis, E, Kendall, R, Coxon, A, and Hughes, P. Targeting HGF-mediated resistance to vemurafenib in V600E BRAF mutant melanoma cell lines. In: *Proceedings of the 104th Annual Meeting of the American Association for Cancer Research*, Washington, DC. 2013. Cancer Res 73: Abstract. doi: 10.1158/1538-7445.AM2013-3405. - Casbas-Hernandez, P, Arcy, MD, Roman-Perez, E, Brauer, HA, McNaughton, K, Miller, SM, Chhetri, RK, Oldenburg, AL, Fleming, JM, Amos, KD, Makowski, L, and Troester, MA. 2013. Role of HGF in epithelial-stromal cell interactions during progression from benign breast disease to ductal carcinoma in situ. *Breast Cancer Research* 15:R82. doi: 10.1186/bcr3476. - Cui, JJ. 2014. Targeting Receptor Tyrosine Kinase MET in Cancer: Small Molecule Inhibitors and Clinical Progress. *J. Med. Chem*. 57:4427-53. doi: 10.1021/jm401427c. - Faul, F, Erdfelder, E, Lang, AG, and Buchner, A. 2007. G* Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences. *Behavior research methods* 39:175-91. - Lezcano, C, Lee, C-W, Larson, AR, Menzies, AM, Kefford, RF, Thompson, JF, Mihm, MC, Ogino, S, Long, GV, Scolyer, RA, and Murphy, GF. 2014. Evaluation of stromal HGF immunoreactivity as a biomarker for melanoma response to RAF inhibitors. 1-10. doi: 10.1038/modpathol.2013.226. - Nakagawa, T, Matsushima, T, Kawano, S, Nakazawa, Y, Kato, Y, Adachi, Y, Abe, T, Semba, T, Yokoi, A, Matsui, J, Tsuruoka, A, and Funahashi, Y. 2014. Lenvatinib in combination with golvatinib overcomes hepatocyte growth factor pathway-induced resistance to vascular endothelial growth factor receptor inhibitor. *Cancer Sci* 105:723-30. doi: 10.1111/cas.12409. - Nickoloff, BJ, and Vande Woude, G. 2012. Hepatocyte growth factor in the neighborhood reverses resistance to BRAF inhibitor in melanoma. *Pigment Cell Melanoma Res* 25:758-61. doi: 10.1111/pcmr.12020. - Parikh, R, Wang, P, Beumer, J, Chu, E, and Appleman, L. 2014. The potential roles of hepatocyte growth factor (HGF)-MET pathway inhibitors in cancer treatment. OTT 969. doi: 10.2147/OTT.S40241. - Penuel, E, Li, C, Parab, V, Burton, L, Cowan, KJ, Merchant, M, Yauch, RL, Patel, P, Peterson, A, Hampton, GM, Lackner, MR, and Hegde, PS. 2013. HGF as a Circulating Biomarker of Onartuzumab Treatment in Patients with Advanced Solid Tumors. *Molecular Cancer Therapeutics* 12:1122-30. doi: 10.1158/1535-7163.MCT-13-0015. - Straussman, R, Morikawa, T, Shee, K, Barzily-Rokni, M, Qian, ZR, Du, J, Davis, A, Mongare, MM, Gould, J, Frederick, DT, Cooper, ZA, Chapman, PB, Solit, DB, Ribas, A, Lo, RS, Flaherty, KT, Ogino, S, Wargo, JA, and Golub, TR. 2012. Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion. *Nature* 487:500-04. doi: 10.1038/nature11183. - Wilson, TR, Fridlyand, J, Yan, Y, Penuel, E, Burton, L, Chan, E, Peng, J, Lin, E, Wang, Y, Sosman, J, Ribas, A, Li, J, Moffat, J, Sutherlin, DP, Koeppen, H, Merchant, M, Neve, R, and Settleman, J. 2012. Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors. *Nature* 487:505-09. doi: 10.1038/nature11249. - Xie, Q, Su, Y, Dykema, K, Johnson, J, Koeman, J, De Giorgi, V, Huang, A, Schlegel, R, Essenburg, C, Kang, L, Iwaya, K, Seki, S, Khoo, SK, Zhang, B, Buonaguro, F, Marincola, FM, Furge, K, Vande Woude, GF, and Shinomiya, N. 2013. Overexpression of HGF Promotes HBV-Induced Hepatocellular Carcinoma Progression and Is an Effective Indicator for Met-Targeting Therapy. *Genes &amp; Cancer* 4:247-60. doi: 10.1177/1947601913501075. ### Author Information † The RP:CB core team consists of Elizabeth Iorns (Science Exchange, Palo Alto, California), William Gunn (Mendeley, London, United Kingdom), Fraser Tan (Science Exchange, Palo Alto, California), Joelle Lomax (Science Exchange, Palo Alto, California), and Timothy Errington (Center for Open Science, Charlottesville, Virginia). David Blum, Bioexpression and Fermentation Facility, University of Georgia, Athens, Georgia Samuel LaBarge, City of Hope, Duarte, California Correspondence to Fraser Tan, fraser@scienceexchange.com. Competing Interests: We disclose that EI, FT, and JL are employed by and hold shares in Science Exchange Inc. The experiments presented in this manuscript will be conducted by EG at the Monoclonal Antibody Core Facility, which is a Science Exchange lab. No other authors disclose conflicts of interest related to this manuscript. Funding: The Reproducibility Project: Cancer Biology is funded by the Laura and John Arnold Foundation, provided to the Center for Open Science in collaboration with Science Exchange. The funder had no role in study design or the decision to submit the work for publication.

Authors: Yuki Goto, Takayuki Katoh &amp; Hiroaki Suga ### Abstract Genetic code reprogramming is a method for the reassignment of arbitrary codons from proteinogenic amino acids to non-proteinogenic ones, and thus specific sequences of non-standard peptides can be ribosomally expressed according to their mRNA templates. We have developed a protocol that facilitates the genetic code reprogramming using flexizymes integrated with a custom-made in-vitro translation apparatus, referred to as the flexible in-vitro translation (FIT) system. Flexizymes are flexible tRNA acylation ribozymes that enable the preparation of a diverse array of non-proteinogenic acyl-tRNAs. These acyl-tRNAs read vacant codons created in the FIT system, yielding the desired non-standard peptides with diverse exotic structures, such as N-methyl amino acids, D-amino acids and physiologically stable macrocyclic scaffolds. We here describe a protocol for the preparation of DNA templates for flexizymes, tRNAs, microhelix RNA, and mRNAs that are used for genetic code reprogramming with flexizymes. ### Reagents 1. Tris(hydroxymethyl)aminomethane (Tris), nuclease and protease tested (Nacalai tesque, cat. no. 35434-34) - Hydrochloric acid (35%) (Nacalai tesque, cat. no. 18321-05) ! CAUTION Corrosive; irritant. Wear gloves and protecting glasses. - Potassium chloride (Nacalai tesque, cat. no. 28514-75) - Triton X-100 (Nacalai tesque, cat. no. 35501-15) - Magnesium chloride hexahydrate (Nacalai tesque, cat. no. 20909-55) - 100 mM dATP solution (Nippon gene, custom made) - 00 mM dCTP solution (Nippon gene, custom made) - 100 mM dGTP solution (Nippon gene, custom made) - 100 mM dTTP solution (Nippon gene, custom made) - Oligodeoxyribonucleotides (Operon) - Agarose for 50–800 bp fragment (Nacalai tesque, cat. no. 01147-96) - Ethidium bromide solution (10 mg/ml) (Nacalai tesque, cat. no. 14631-94) ! CAUTION Mutagen; irritant. Wear gloves. - Acetic Acid (Kanto chemical, cat. no. 01021-00) ! CAUTION Corrosive; irritant. - 0.5 mol/l-EDTA solution (pH 8.0) (Nacalai tesque, cat. no. 14347-21) - Phenol, saturated with TE buffer (Nacalai tesque, cat. no. 26829-96) ! CAUTION Toxic; corrosive, irritant. - Chloroform (Nacalai tesque, cat. no. 08402-55) ! CAUTION Irritant. - 3-Methyl-1-butanol (isoamyl alcohol) (Nacalai tesque, cat. no. 02715-45) ! CAUTION Flammable. - Ethanol (Nacalai tesque, cat. no. 14713-95) ! CAUTION Flammable. - Sodium chloride (Nacalai tesque, cat. no. 31320-05) - Dithiothreitol, nuclease tested (Nacalai tesque, cat. no. 14128-04) - Spermidine (Nacalai tesque, cat. no. 32108-04) ! CAUTION Corrosive. - Adenosine 5’-triphosphate disodium salt n-hydrate (Wako Pure Chemical Industries, cat. no. 133-00725) - CTP sodium salt (JENA Bioscience, cat. no. NU-1011) - GTP sodium salt (JENA Bioscience, cat. no. NU-1012) - UTP sodium salt (JENA Bioscience, cat. no. NU-1013) - Potassium hydroxide (Nacalai tesque, cat. no. 28616-45) ! CAUTION Corrosive; irritant. Wear gloves and protecting glasses. - Manganese (II) chloride tetrahydrate (Wako Pure Chemical Industries, cat. no. 133-00725) - RQ1 RNase-free DNase (1 U/μl) (Promega, cat. no. M610A) - 2-Propanol (Nacalai tesque, cat. no. 29112-05) ! CAUTION Flammable. - 3’,3”,5’,5”-tetrabromophenolsulfonphthalein sodium salt (BPB) (Sigma, cat. no. B-8026) - 40% (w/v)-Acrylamide/Bis mixed solution (19:1), nuclease and protease tested (Nacalai tesque, cat. no. 00857-55) ! CAUTION May cause nervous damage; cancer suspect agent. Wear gloves. - Urea (Nacalai tesque, cat. no. 35905-64) - Ammonium peroxodisulfate (Wako Pure Chemical Industries, cat. no. 016-08021) ! CAUTION Strong oxidizer; irritant. - N, N, N’, N’-Tetramethylethylenediamine (TEMED) (Nacalai tesque, cat. no. 33401-72) ! CAUTION Flammable; irritant. - Boric acid (Nacalai tesque, cat. no. 05215-05) **REAGENT SETUP** 1. RNase-free water Water generated by an ultrapure water system. - 10× PCR buffer 500 mM KCl, 100 mM Tris-HCl (pH 9.0), and 1% (v/v) Triton X-100. For preparation of 50 ml solution, mix 12.5 ml of 2 M KCl, 5 ml of 1 M Tris-HCl (pH9.0), and 0.5 ml of Triton X-100, then add water up to 50 ml. Store it in aliquots at -20°C (stable for years). - 5 mM dNTPs Mix 1 ml of 100 mM dATP, 1 ml of 100 mM dCTP, 1 ml of 100 mM dGTP, and 1 ml of 100 mM dTTP, with 16 ml of water. Store it in aliquots at -20°C (stable for at least a year). - Taq DNA polymerase Heterogeneously express Taq DNA polymerase in E. coli and purify it according to the previously reported way (1). - 50× TAE Mix 247 g of Tris-base, 57 ml of acetic acid, 100 ml of 0.5 M EDTA, then add water up to 1 L. - 3% Agarose gel containing ethidium bromide Weigh out 3 g of agarose in 300-ml conical flask and add 1x TAE up to 100 ml. Microwave the flask for about 2 min to melt the agarose completely. After cooling down to about 70°C, add 5 μl of 10 mg/ml ethidium bromide and mix well. Then, pour the gel slowly into the gel maker and insert the comb. Leave to set for about 1 h and remove the comb. Can be stored in 1x TAE at room temperature for several months. - Phenol/chloroform/isoamyl alcohol solution (25:24:1) Mix 50 ml of TE-saturated phenol, 48 ml of chloroform, and 2 ml of isoamyl alcohol. Can be stored at 4°C for at least a year. - Chloroform/isoamyl alcohol solution (24:1) Mix 96 ml of chloroform and 4 ml of isoamyl alcohol. Can be stored at room temperature for at least a year. - 70% Ethanol Mix 12 ml of water and 28 ml of ethanol. Can be stored at room temperature for at least six months. - 10× T7 buffer 400 mM Tris-HCl (pH 8.0), 10 mM spermidine, and 0.1% (v/v) Triton-X. For preparation of 10 ml solution, add 0.485 g of Tris, 50 µl of 2 M spermidine, and 10 µl of Triton X-100 to 6 ml of RNase-free water. Adjust the pH of solution to 8.0 by adding 1 M HCl, and then add water up to 10 ml. Store it in aliquots at -20°C (stable for years). - 25 mM NTPs First, make 100 mM stock solution of each NTP (ATP, CTP, GTP, and UTP). Dissolve 600 mg of each NTP in 10 ml of RNase-free water. Dilute a small portion of the solution with 100 mM MOPS-KOH buffer (pH 7.0), and measure the absorbance to determine the concentration (the molar absorption coefficients at pH 7.0 for ATP, CTP, GTP, and UTP are 15,400 at 259 nm, 9,000 at 271 nm, 13,700 at 253 nm, and 10,000 at 262 nm, respectively). Adjust the concentration to 100 mM by adding RNase-free water. Then, mix equal volume of 100 mM ATP, CTP, GTP, and UTP to make 25 mM NTPs solution. Store it in aliquots at -20°C (stable for years). - T7 RNA polymerase Express His6-tagged T7 RNA polymerase in E. coli and purify it according to standard methods. - Acrylamide gel solution (8, 12, or 20%) Mix 14.4 g of urea, 4 ml of 5× TBE, and proper amount of 40% acrylamide/bisacrylamide (19/1) solution (8, 12, and 20 ml to make 8, 12, and 20% gel, respectively), and add RNase-free water up to 40 ml. Mix gently until urea dissolves completely. Add 400 µl of 10% APS and 30 µl of TEMED to the solution right before pouring to a slab-gel equipment. - 2× RNA loading buffer 8 M urea, 2 mM EDTA, 2 mM Tris, and 0.004% BPB. For preparation of 50 ml solution, mix 24 g of urea, 372 mg of EDTA·2Na·2H2O, 12 mg of Tris, and 250 μl of 2% BPB, then add water up to 50 mL. pH of the resulting solution should be around 7.5. Can be stored at room temperature for at least a year. - 5× TBE Mix 53.91 g of Tris, 27.51 g of boric acid, and 20 ml of 0.5 M EDTA (pH 8.0), then add water up to 1 L. Can be stored at room temperature for at three months. - 1× TBE Mix 800 ml of water and 200 ml of 5× TBE. Can be stored at room temperature for at least six months. ### Equipment 1. Ultrapure water system (Sartorius, arium 611UV) - Vortex mixer (TM-2000, IWAKI) - Thermal cycler (Techne, PC-312) - Microwave oven (SHARP, RE-T11) - Submarine gel electrophoresis equipment (Advance, Mupid-ex) - Refrigerated micro centrifuge (TOMY, MX-305) - Air incubator (Isuzu seisakusho, FR-115S) - Slab-gel electrophoresis equipment (BIO CRAFT, BE-140) - Power supply for electrophoresis (Bio-rad, PowerPac Basic) - TLC plate 20 cm×20 cm Silica gel 60 F254 (Merck, cat. no. 1.05715.0009) - UV hand lamp (260 nm) (COSMO BIO, cat. no. CSL-4C) - 0.45 μm Syringe driven filter unit (Millipore, Millex-LH, cat. no. SLLH H25NK) - UV-Vis spectrometer (GE Healthcare, Nanovue) **EQUIPMENT SETUP** 1. PCR thermal cycler Program 1: 95°C for 1 min; 5 cycles of 50°C for 1 min and 72°C for 1min. Program 2: 95°C for 1 min; 12 cycles of 95°C for 40 s, 50°C for 40 s, and 72°C for 40 s. Program 3: 95°C for 1 min; 5 cycles of 95°C for 40 s, 50°C for 40 s, and 72°C for 40 s. - Polyacrylamide gel electrophoresis Assemble gel plates (16 cm × 16 cm) to make 2 mm-thick gel. Pour acrylamide gel solution into the gel plates. Stand it at room temperature until the gel solidifies. Use 1× TBE as a running buffer. ### Procedure **Preparation of DNA template for flexizymes** ●TIMING 3 h Extension - 1 Prepare a master mix solution for the following extension and PCR reactions on ice by mixing 120 μl of 10× PCR buffer, 12 μl of 250 mM MgCl2, 60 μl of 5 mM dNTPs, and 9 μl of Taq DNA polymerase with 1000 μl of RNase-free water. Keep the master mix solution on ice. - 2 Mix 0.5 μl of 200 μM Fx-F primer and 0.5 μl of 200 μM a reverse primer in a 200-μl PCR tube with 100 μl of the master mix solution. For the preparation of eFx dFx, and aFx, use eFx-R1, dFx-R1, and aFx-R1, respectively, as a reverse primer. - 3 Set the sample in a PCR thermal cycler and carry out the extension reaction with Program 1. - PAUSE POINT The extension product may be stable at -20°C for years. PCR - 4 Mix 5 μl of the extension product (without any purification) with 2.5 μl of 200 μM T7-F primer, 2.5 μl of 200 μM a reverse primer, and 1000 μl of the master mix solution. Aliquot the resulting solution into five 200-μl PCR tubes. For the preparation of eFx, dFx, and aFx, use eFx-R2, dFx-R2, and aFx-R2, respectively, as a reverse primer. Divide the resulting solution to 5 aliquots in 200-μl PCR tubes. - 5 Set the tubes in a PCR thermal cycler and run it with Program 2. - 6 Check the amplified DNA by agarose gel electrophoresis. If the band corresponding to the objective band is faint, run 2–3 additional PCR cycles. Purification of the PCR product - 7 Combine the DNA solutions and add 1 ml of phenol/chloroform/isoamyl alcohol solution to the resulting sample. Shake the tube intensely. - 8 Centrifuge the sample at 15,000×g for 5 min at 25°C. - 9 Recover the water layer and mix it with 1 ml of chloroform/isoamyl alcohol solution. Shake the tube intensely. - 10 Centrifuge the sample at 15,000×g for 5 min at 25°C. - 11 Recover the water layer and add 100 μl of 3 M NaCl and 2.2 ml of ethanol. Mix the sample well. - 12 Centrifuge the sample at 15,000×g for 15 min at 25°C. - 13 Remove the supernatant and add 500 μl of 70% ethanol to the tube. - 14 Centrifuge the sample at 15,000×g for 15 min at 25°C. - 15 Remove the supernatant completely. Open the tube lid and cover it with tissues, then dry the DNA at room temperature for 10 min. - 16 Add 100 μl of RNase-free water and resuspend the DNA pellet. - **PAUSE POINT** The DNA solution can be stored at -20°C for years. **Preparation of DNA template for tRNAs** ●TIMING 4 h Extension - 17 Prepare a master mix solution for the following extension and PCR reactions on ice by mixing 132 μl of 10× PCR buffer, 13.2 μl of 250 mM MgCl2, 66 μl of 5 mM dNTPs, and 9.9 μl of Taq DNA polymerase with 1100 μl of RNase-free water. Keep the master mix solution on ice. - 18 Mix 0.5 μl of 20 μM a forward primer and 0.5 μl of 20 μM a reverse primer in a 200-μl PCR tube with 10 μl of the master mix solution. - **CRITICAL STEP** For the preparation of tRNA^Asn-E2 XXX, use Asn-E2-F and Asn-E2-XXX-R1 as a forward and reverse primer, respectively. For the preparation of tRNA^fMetE CAU, use fMetE-F and fMetE-R1 as a forward and reverse primer, respectively. - 19 Set the sample in a PCR thermal cycler and carry out the extension reaction with Program 1. 1st PCR - 20 Mix 10 μl of the extension product (without any purification) with 0.5 μl of 200 μM T7-F primer, 0.5 μl of 200 μM a reverse primer, and 190 μl of the master mix solution in a 200-μl PCR tube. - **CRITICAL STEP** For the preparation of tRNAAsn-E2XXX and tRNAfMetECAU, use Asn-E2-R2 and fMetE-R2, respectively, as a reverse primer. - 21 Set the tubes in a PCR thermal cycler and run it with Program 3. 2nd PCR - 22 Mix 5 μl of the PCR product (without any purification) with 2.5 μl of 200 μM T7-F primer, 2.5 μl of 200 μM a reverse primer, and 1000 μl of the master mix solution. Aliquot the resulting solution into five 200-μl PCR tubes. Divide the resulting solution to 5 aliquots in 200-μl PCR tubes. - **CRITICAL STEP** For the preparation of tRNA^Asn-E2 XXX and tRNA^fMetE CAU, use Asn-E2-R3 and fMetE-R3, respectively, as a reverse primer. - 23 Set the tubes in a PCR thermal cycler and run it with Program 2. - 24 Check the amplified DNA by agarose gel electrophoresis. If the band corresponding to the objective band is faint, run 2–3 additional PCR cycles. Purification of the PCR product - 25 Combine the DNA solutions and add 1 ml of phenol/chloroform/isoamyl alcohol solution to the resulting sample. Shake the tube intensely. - 26 Centrifuge the sample at 15,000×g for 5 min at 25°C. - 27 Recover the water layer and mix it with 1 ml of chloroform/isoamyl alcohol solution. Shake the tube intensely. - 28 Centrifuge the sample at 15,000×g for 5 min at 25°C. - 29 Recover the water layer and add 100 μl of 3 M NaCl and 2.2 ml of ethanol. Mix the sample well. - 30 Centrifuge the sample at 15,000×g for 15 min at 25°C. - 31 Remove the supernatant and add 500 μl of 70% ethanol to the tube. - 32 Centrifuge the sample at 15,000×g for 15 min at 25°C. - 33 Remove the supernatant completely. Open the tube lid and cover it with tissues, then dry the DNA at room temperature for 10 min. - 34 Add 100 μl of RNase-free water and resuspend the DNA pellet. - **PAUSE POINT** The DNA solution can be stored at -20°C for years. **Preparation of DNA template for microhelix RNA** ●TIMING 10 min Annealing of oligonucleotides - 35 Mix 1.25 μl of 200 μM mihx-F primer, 1.25 μl of 200 μM mihx-R primer, 20 μl of 50 mM KCl, and 2.5 μl of 10× T7 buffer. - 36 Heat the sample at 95°C for 3 min, then slowly cool it at room temperature over 5 min. - 37 Add 75 μl of water to the sample, and directly use the resulting solution as a DNA template solution for in vitro transcription reaction. **Synthesis of RNAs by in vitro transcription** ●TIMING 7h - 38 Prepare DNA template for in vitro transcription according to the methods described above. - **CRITICAL STEP** All the following steps should be performed in an RNase-free manner. Use RNase-free tubes, pipettes, pipette tips, and water. Wear gloves at all times. - 39 Prepare in vitro transcription reaction mixture. This step can be performed using option A or option B depending on RNA molecules to be made. - A. Preparation of flexizymes. - Mix 100 μl of 10× T7 buffer, 100 μl of 100 mM DTT, 120 μl of 250 mM MgCl2, 200 μl of 25 mM NTPs, 15 μl of 2 M KOH, 100 μl of DNA template, and 20 μl of T7 RNA polymerase with 345 μl of RNase-free water. - B. Preparation of tRNAs and microhelix RNA. - Mix 100 μl of 10× T7 buffer, 100 μl of 100 mM DTT, 90 μl of 250 mM MgCl2, 150 μl of 25 mM NTPs, 11.25 μl of 2 M KOH, 50 μl of 100 mM GMP, 100 μl of DNA template, and 20 μl of T7 RNA polymerase with 303 μl of RNase-free water. 40 Incubate the transcription reaction mixture in an air incubator at 37°C for 5 hours. Ｗhite precipitations of inorganic pyrophosphate occurs if transcription reaction occurs. ? TROUBLESHOOTING - 41 Add 20 μl of 100 mM MnCl2 and 4 μl of DNase I to the reaction mixture. Incubate the solution at 37°C for additional 30 min. - 42 Add 75 μl of 500 mM EDTA (pH 8.0), 100 μl of 3 M NaCl, and 1 ml of isopropanol. Mix the sample well and stand it at room temperature for 5 min. - 43 Centrifuge the sample at 15,000×g for 5 min at 25°C. - 44 Remove the supernatant completely. Open the tube lid and cover it with tissues, then dry the RNA at room temperature for 10 min. - **PAUSE POINT** The RNA pellet can be stored at -20°C for at least a week. **Purification of RNAs by polyacrylamide gel electrophoresis** ●TIMING 6h - 45 Add 100 μl of RNase-free water and resuspend the RNA pellet. Mix the RNA solution with 100 μl of 2× RNA loading buffer. - 46 Incubate the sample on a heat block at 95°C for 1 min. - 47 Apply the resulting sample onto a denaturing polyacrylamide gel and run it. (Use 12% polyacrylamide gel with 250V for 1 hour to purify flexizymes. Use 8% polyacrylamide gel with 250 V for 1 hour to purify tRNAs. Use 20% polyacrylamide gel with 280 V for 3 hour to purify microhelix RNA.) - 48 Remove the gel from gel plates and put it on a TLC plate containing a fluorescent indicator covered with a plastic wrap. Cover the gel with another plastic wrap. - 49 Visualize RNA band by irradiating with 260 nm UV lamp in a dark room. Trace the pattern of RNA band with a marker. - ! CAUTION Wear UV-protecting glasses to protect your eyes from harmful UV light. - **CRITICAL STEP** Mark the band promptly. Irradiation of UV at a short range for a long time may cause RNA damage. ? TROUBLESHOOTING - 50 Cut the gel by a razor along the mark. Crush the gel pieces containing RNA finely in a 50 ml tube. - **CRITICAL STEP** Recovery yield of RNA can be improved by well breaking the gel into a paste. - 51 Add 3 ml of 0.3 M NaCl to the resulting gel paste, then shake the tube at room temperature for 1 hour. - 52 Centrifuge the sample at 15,000×g for 5 min at 25°C. - 53 Recover the supernatant carefully. - 54 Add 2 ml of 0.3 M NaCl to the gel pellet, then shake the tube at room temperature for additional 1 hour. - 55 Centrifuge the sample at 15,000×g for 5 min at 25°C. - 56 Recover the supernatant carefully. - 57 Repeat steps 54–56 one more time. - 58 Combine all supernatants, and filter it by a 0.45 μm syringe filter. - 59 Add 2-fold volume of ethanol to the resulting RNA solution. Mix the sample well. - 60 Centrifuge the sample at 15,000×g for 15 min at 25°C. - 61 Remove the supernatant and add 1000 μl of 70% ethanol to the tube and wash the pellet. - 62 Centrifuge the sample at 15,000×g for 3 min at 25°C. - 63 Remove the supernatant completely. Open the tube lid and cover it with tissues, then dry the RNA at room temperature for 10 min. - 64 Add 50 μl of RNase-free water and resuspend the RNA pellet. - 65 Determine the concentration of RNA by a UV spectrometer (The length of tRNAs, aFx, dFx, eFx, and microhelix RNA are 76, 47, 46, 45, and 22 mer, respectively). - 66 Dilute the RNA solution with RNase-free water to make a 250 μM stock solution of flexizyme. - **PAUSE POINT** The RNA solution (flexizymes, tRNAs, and microhelix RNA) are stored at -20°C for at least two years. - ANTICIPATED RESULTS 15,000¬–45,000 pmol of RNA can be obtained from a 1 ml-scale transcription reaction. These amounts of RNAs allow us to carry out 60–180 translation reactions. **Preparation of DNA templates for translation** ●TIMING 6 h Extension - 67 Design primers required for synthesis of the objective DNA template (Fig. S2). The forward primers (mDNA-F1 and mDNA-F2) contain a region from the T7 promoter to start codon, while the reverse primers (mDNA-R1, mDNA-R2 and mDNA-R3) mainly consist of a peptide coding region that can be variable. Alteration of the sequences of the reverse primers yields various template DNAs that code different peptide sequences. - **CRITICAL STEP** See “EXPERIMENTAL DESIGN” section of the main text for the required sequence elements in DNA template for FIT system. - 68 Prepare extension reaction mixture (50 μl). Mix 5 μl of 10× PCR buffer, 0.5 μl of 250 mM MgCl2, 2.5 μl of 5 mM dNTPs, 2.5 μl of 10 μM mDNA-F1 primer, 2.5 μl of 10 μM mDNA-R1 primer, 0.5 μl of Taq DNA polymerase, and 36.5 μl of RNase-free water in a 200-μl PCR tube. - 69 Set the sample tube in a thermal cycler and perform the extension reaction with program 1. 1st PCR - 70 Prepare the 1st PCR mixture (50 μl). Mix 5 μl of 10× PCR buffer, 0.5 μl of 250 mM MgCl2, 2.5 μl of 5 mM dNTPs, 2.5 μl of 10 μM mDNA-F2 primer, 2.5 μl of 10 μM mDNA-R2 primer, 0.5 μl of Taq DNA polymerase, 1.25 μl of the extension product from step 69, and 35.25 μl of RNase- free water in a 200-μl PCR tube. - 71 Set the sample tube in a thermal cycler and run the PCR using the program 3. 2nd PCR - 72 Prepare the 2nd PCR mixture (200 μl). Mix 20 μl of 10× PCR buffer, 2 μl of 250 mM MgCl2, 10 μl of 5 mM dNTPs, 1 μl of 100 μM mDNA-F2 primer, 1 μl of 100 μM mDNA-R3 primer, 2 μl of Taq DNA polymerase, 5 μl of the 1st PCR product from step 71, and 159 μl of RNase- free water in a 200-μl PCR tubes. - 73 Set the sample tube in a thermal cycler and run the PCR using the program 2. - **PAUSE POINT** The PCR product is stable for at least 1 year at -20 °C. - 74 Analyze the product of the PCR by 3% agarose gel electrophoresis and ethidium bromide staining. The reaction should yield a ~110 bp product after the 2nd PCR. Purification of the PCR product - 75 Add 200 μl of phenol/chloroform/isoamyl alcohol solution. Shake the tube vigorously by a vortex mixer. - 76 Centrifuge the tube at 15,000x g for 5 min at 25°C. - 77 Recover the water layer in a new tube and add 200 μl of chloroform/isoamyl alcohol. Shake the tube vigorously by vortexing. - 78 Centrifuge the tube at 15,000×g for 5 min at 25°C. - 79 Recover the water layer. Add 20 μl of 3M NaCl and 440 μl of ethanol. Mix the solution well. - 80 Centrifuge the tubes at 15,000×g for 15 min at 25°C. - 81 Remove the supernatant and add 200 μl of 70% ethanol. - 82 Centrifuge the tubes at 15,000×g for 5 min at 25°C. - 83 Remove the supernatant. Dry the DNA pellet at room temperature for 10 min. - 84 Resuspend the DNA pellet in 20 μl of RNase-free water. - **PAUSE POINT** The purified PCR product can be kept frozen at -20 °C for years. ### Timing - Steps 1–16, Preparation of DNA template for flexizymes: 3h - Steps 17–34, Preparation of DNA template for tRNAs: 4h - Steps 35–37, Preparation of DNA template for microhelix RNA: 10 min - Steps 38–44, Synthesis of RNAs by in vitro transcription: 7 h - Steps 45–66, Purification of RNAs by polyacrylamide gel electrophoresis: 6 h - Steps 67–84, Preparation of DNA templates for translation: 6 h ### Troubleshooting - No or little precipitation in Step 40. - **Possible reason**: Improper pH of the reaction mixture. - **Solution**: Acidity of NTPs solution may depend on lots of reagents. Adjust the amount of 2 M KOH to make the final pH value of reaction mixture around 8.0. - No or little observed band corresponding to the objective RNA in Step 49. - **Possible reason**: Failure in transcription reaction. - **Solution**: Ensure that all reagents used for transcription are correct. Check the pH of reaction mixture as described above. - Smear bands in Step 49. - **Possible reason**: Contamination of RNase. - **Solution**: Pay attention not to contaminate reagents and reaction mixture by RNase. Wear gloves when performing experiments. Prepare all reagents freshly when RNase contamination is suspected. ### References 1. Pluthero, F.G. Rapid purification of high-activity Taq DNA polymerase. *Nucleic Acids Res*. 21, 4850-4851 (1993). ### Acknowledgements We thank Mr. Yusuke Yamagishi for the discussion and proof-reading. We thank Dr. Hiroshi Murakami for the contributions to the development of the methods presented in this study. We also thank Dr. Patrick C. Reid for proofreading. This work was supported by grants of Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (S) (16101007), Specially Promoted Research (21000005), a research and development projects of the Industrial Science and Technology Program in the New Energy and Industrial Technology Development Organization (NEDO), and the World Class University project of the MEST and the NRF (R31-2008-000-10103-0) to H.S., grants of Japan Society for the Promotion of Science Grants-in-Aid for Young Scientists (B) (22750145) to Y.G., and (B)(22710210) to T.K.. ### Figures **Figure 1: Cloverleaf structure of tRNAfMetECAU and tRNAAsnE2NNN used in this study**.  *tRNAfMetECAU and tRNAAsnE2NNN are engineered initiator and elongator tRNAs from tRNAfMetCAU and tRNAAsnGUU, respectively. Arrows indicate the sites of mutations. Red characters indicate the nucleotides in their original natural tRNA molecules (tRNAfMetCAU or tRNAAsnGUU). The “nnNNNnn” sequence in tRNAAsnE2NNN indicates anticodon triplet (NNN) and anticodon codon loop sequence (nn___nn), which should be appropriately chosen for the objective reprogrammed codon. For example, GGA, GUG, and GCG anticodon triplet with CU___AA, UU___AA, and CU___GA loop sequence could be preferentially used in order to maximize the decoding efficiency and fidelity of UCC, CAC, and CGC codons, respectively*. **Figure 2: Preparation of the DNA template used for the translation reaction by 3-step PCR**. [Download Figure 2](http://www.nature.com/protocolexchange/system/uploads/1717/original/FigureS2.tif?1296732440) *Green arrows indicate the regions made by the extension reaction. Blue and red arrows show the regions amplified by PCR. Peptide coding region can be designed variably by using the corresponding reverse primers*. **Table 1: Primer sequences used in this protocol**.  - *aXXX denotes the anticodon sequence of tRNA to be made*. - *bYYY corresponds to the complementary triplet of XXX. yy___yy corresponds to the complementary sequence of anticodon loop, xx___xx*. ### Author information **Yuki Goto, Takayuki Katoh &amp; Hiroaki Suga**, Department of Chemistry, Graduate School of Science, The University of Tokyo Correspondence to: Hiroaki Suga (hsuga@chem.s.u-tokyo.ac.jp) *Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2020) (2011) doi:10.1038/protex.2011.209. Originally published online 12 May 2011*.

<strong>Piotrowska Paulina, W&oacute;jcik Justyna, Obuchowska Aleksandra, Ozga Alicja, Kimber-Trojnar Żaneta, Leszczyńska‑Gorzelak&nbsp;Bożena.</strong><strong> Folic acid supplementation among students. </strong><strong>Journal of Education, Health and Sport. 2019;9(9):4</strong><strong>92</strong><strong>-</strong><strong>498</strong><strong>. eISSN 2391-8306. DOI</strong><strong> </strong><strong>http://dx.doi.org/10.5281/zenodo.3416015</strong> <strong>http://ojs.ukw.edu.pl/index.php/johs/article/view/74</strong><strong>60</strong> &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; <strong>The journal has had 5 points in Ministry of Science and Higher Education parametric evaluation. &sect; 8. 2) and &sect; 12. 1. 2) 22.02.2019.</strong> &nbsp; <strong>&copy; The Authors 2019;</strong> <strong>This article is published with open access at Licensee Open Journal Systems of Kazimierz Wielki University in Bydgoszcz, Poland</strong> <strong>Open Access. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author (s) and source are credited. This is an open access article licensed u</strong><strong>nder the terms of the Creative Commons Attribution Non commercial license Share alike.</strong> <strong>(http://creativecommons.org/licenses/by-nc-sa/4.0/) which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited.</strong> <strong>The authors declare that there is no conflict of interests regarding the publication of this paper.</strong> <strong>Received: 25.08.2019. Revised: 30.08.2019. Accepted: 14.09.2019.</strong> &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; <strong>Folic acid supplementation among students</strong> &nbsp; <strong>Paulina Piotrowska</strong>^{<strong>1a</strong>}<strong>, Justyna W&oacute;jcik</strong>^{<strong>1b</strong>}<strong>, Aleksandra Obuchowska</strong>^{<strong>1c</strong>}<strong>, Alicja Ozga</strong>^{<strong>1d</strong>}<strong>, Żaneta Kimber-Trojnar</strong>^{<strong>2e</strong>}<strong>, Bożena Leszczyńska-Gorzelak</strong>^{<strong>2f </strong>} &nbsp; ¹Student&#39;s Scientific Association at the Chair and Department of Obstetrics and Perinatology, Medical University of Lublin, Poland ²Chair and Department of Obstetrics and Perinatology, Medical University of Lublin, Poland &nbsp; ^apaulina.piotrowska222@gmail.com ^bjustynawojcik455@gmail.com ^caobuchowska12@gmail.com ^daozga1@gmail.com ^ezkimber@poczta.onet.pl ^fb.leszczynska@umlub.pl &nbsp; &nbsp; <strong>Corresponding author:</strong> Paulina Piotrowska Student&#39;s Scientific Association at the Chair and Department of Obstetrics and Perinatology Jaczewskiego 8 Street 20-954 Lublin, Poland e-mail: paulina.piotrowska222@gmail.com &nbsp; &nbsp; <strong>Abstract:</strong> <strong>Introduction: </strong>Pregnancy is a period in which the supply of vitamins, micro- and macroelements is essential for the proper growth of the fetus. One of the most important substances, which is believed to have a considerable influence on the proper fetus growth is folic acid. Folic acid (i.e. vitamin B9) plays a significant role in rapidly dividing cells and lack of this vitamin causes serious fetal defects - for example neural tube anomalies. Supplementation is important not only during pregnancy but also during the period of pregnancy planning. <strong>Methods: </strong>Students&#39; knowledge regarding the importance of folic acid supplementation was verified using anonymous Internet questionnaire. The obtained results were analyzed using statistical methods and checked on the basis of scientific literature. <strong>Results: </strong>The percentage of students who supply folic acid is 18,1% and only 9,5% do this regularly. The most common cause of supply is due to the fact that that folate can be found in vitamin kits students usually take. Part of respondents claim that they plan the pregnancy and want the fetus to develop correctly. Other causes of vitamin B9 intake is prevention of arteriosclerosis, vascular system diseases, anemia, lung, esophagus and uterus cancer. <strong>Conclusion: </strong>Outcomes showed that students are not fully aware of the importance of vitamin B9 during pregnancy and there is a great need to share knowledge about folic acid supplementation among people in procreative age. &nbsp; <strong>Key words: </strong>folic acid, pregnancy, supplementation among students, dietary supplements

Photo by Adhy Savala on Unsplash ABSTRACT During a crisis, when healthcare capacity becomes overwhelmed and cannot meet regular standards of patient care, crisis standards of care are invoked to distribute scarce hospital space, staff, and supplies. When transitioning between conventional standards of care and crisis standards, hospitals may have to manage resources under scarcity constraints in an intermediate phase defined as the contingency phase. While much attention has been paid to the ethics of crisis standard of care protocols, contingency measures were more widely implemented, though little exists within the literature on the ethics of contingency measures or a clearly explicated contingency standard of care. This paper addresses three ethical issues with the current contingency response to COVID-19: the lack of formalization, the risks of using short-term solutions for prolonged contingency shortages, and the danger of exacerbating health disparities through hospital-level resource allocation. To mitigate these ethical issues, I offer recommendations for reimagining resource allocation during contingency standards of care. INTRODUCTION When transitioning between conventional standards of care and crisis standards, or in situations where shortages do not immediately threaten care delivery, hospitals may have to manage scarce resources in an intermediate phase, known as the “contingency” phase.[1] While much attention has been paid to the ethics of crisis standards, less literature covers the ethics of contingency measures or a clearly explicated contingency standard of care. Many states and hospital systems do not have contingency standards of care to dictate allocation absent an event triggering crisis standards. Crisis standards of care, used when healthcare capacity becomes overwhelmed and cannot meet regular standards of patient care, reflect ethical priorities relevant in times of shortage or other emergencies. These priorities include saving the most lives, the stewardship of scarce resources, and justice relating to equitable resource distribution.[2] Crisis standards of care delineate specialized allocation protocols and triage decision-making bodies at the institutional or state levels. Crisis standards of care require formal activation at the state level, and in the absence of clear triggers or governmental willingness to use them, hospitals may adopt informal strategies to manage allocation in the form of contingency measures. The contingency phase is defined by two simultaneous goals: prevent or stall crisis-level scarcity by managing limited resources and providing patient care that is functionally equivalent to usual care.[3] In other words, allocate scarce resources with no significant health consequences to patients. However, this is an unrealistic expectation: meeting a patient’s medical needs and allocating resources on the basis of scarcity instead of medical indications can be at odds, creating ethical tension. This paper addresses three ethical issues with the current contingency response stemming from this tension: the lack of formalization, the risk of using short-term solutions for prolonged contingency shortages, and the danger of exacerbating health disparities through hospital-level resource allocation. To mitigate these ethical issues, I offer recommendations for reimagining resource allocation during contingency standards of care. l. Lack of Formalization One shortcoming of current contingency measures is that they fail to meet the same level of procedural detail and clarity as crisis standards. The early COVID-19 surges in Italy and France demonstrated the pitfalls of bedside allocation in the absence of procedural guidance. The acute scarcity of critical care resources forced doctors in these countries to make allocation decisions at the bedside, which often resulted in de facto age-based allocation as well as experiences of moral distress and shame among providers.[4] In France, medical allocation guidelines and statistics were never released to the public, raising concerns over the role of transparency in implementing crisis standards and triage guidelines and causing the public to question the trustworthiness of provider triage.[5] Though many states in the US have crisis standards of care that can be implemented in the case of a large-scale triage event, these measures vary widely. A 2020 review of 31 crisis standards of care in the US found that only 18 contained strong “ethical grounding,” 28 used “evidence-based clinical processes and operations,” 21 included “ongoing community and provider engagement, education, and communication,” and 16 had “clear indicators, triggers, and lines of responsibility.”[6] The need for standardization, public transparency, and guidelines for crisis standards of care to prevent bedside allocation has been widely recognized. However, these issues remain unresolved by public policy or legislative efforts during the contingency period before (or after) crisis standards apply. A recent public health study that observed triage team members in a high-fidelity triage simulation highlighted the challenges of making equitable frontline allocation decisions.[7] In the simulation, participants nudged patient priority status up or down depending on what they subjectively identified as morally relevant factors. Through the simulation, participants reported difficulty separating implicit biases about patient characteristics from their clinical judgment. In the absence of formal institutional or regional guidelines for allocation during contingency-level shortages, there are few to no procedural safeguards against biased, ad hoc, and non-transparent rationing. Without formalized or standardized contingency allocation guidance, providers are left to make bedside allocation decisions that are susceptible to individual biases and patterns of unintended discrimination. An example of this susceptibility is seen when hospitals allow patients who no longer benefit from ICU resources to continue occupying ICU beds. This is based on a first-come-first-served (FCFS) approach to bed allocation. FCFS is often a default for patient intake, which led to disparities in care access during the early COVID-19 pandemic. Media reports of hospitals with “plenty of space” being unwilling to accept patients from overwhelmed, lower-income hospitals illustrate that the FCFS default advantages those who could show up first to a particular hospital: often privileged, well-funded healthcare systems that were inaccessible to low-income communities.[8] FCFS is blind to several morally relevant factors, including the likelihood of survival to discharge, reciprocity (i.e. prioritizing healthcare workers), and varying degrees of access to healthcare. Therefore, it inappropriately privileges those in proximity to healthcare systems or with social connections enabling greater initial access to care.[9] During crisis standards of care, excessive mortality that would result from FCFS is mitigated through formalized system-wide triage protocols based on current patient health status and potential benefit from resources. Crisis and contingency standards may provide liability coverage for providers who reallocate critical care beds away from those who no longer benefit during periods of scarcity. This liability coverage shifts bed allocation away from an FCFS model, but only if the policy is well-defined, clearly established, and known to providers. Without a formal system to guide the process or transition from the usual method of allocation to the contingency period, contingency decisions about who gets a scarce resource may continue to operate on an implicit FCFS basis, even when approaching crisis levels of scarcity. Additionally, these decisions will fall unsustainably on individual providers or transfer center workers, leading to moral distress on the frontlines when hospitals are already strained. Lessons from the crisis and contingency responses during COVID-19 can improve future contingency responses. There are multiple ways of achieving equity during contingency allocation, ranging from hospital-level to state-level policy changes. State-wide policies and interventions to facilitate resource-sharing can relieve some of the scarcity burdens that hospitals may face during the contingency period. For example, moving ICU patients to lower levels of care once they have sufficiently recovered is a challenge for doctors, who often call other hospitals to find open beds. In these situations, providers who do not move patients who no longer benefit from ICU beds unknowingly reinforce the FCFS system in which those who arrive first keep the scarce beds, while those who arrive later or wait for one are disadvantaged by having limited access to them. State-wide patient transfer centers, often facilitated by state public health departments, present an alternative by balancing patient needs and bed distribution more equitably and efficiently than individual physicians do, as demonstrated following COVID-19 surges in hospitalization.[10] These centers aid not only in allocating open tertiary care beds, but also in identifying open beds at lower levels of care and assisting physicians with transferring out patients who can be safely downgraded and no longer benefit from tertiary care resources. However, the simplest solution is to encourage the creation of ethics guidance or protocols for contingency allocation at the hospital level. In hospitals, institutional ethics guidance can help providers navigate difficult decisions and conversations with patients. When providers face time-sensitive allocation decisions, like the allocation of open ICU beds, the guidance would be a useful tool for making transparent, principled, and ethically justified allocation decisions in real-time to mitigate the risk of ad hoc or implicit rationing. ll. Unsuited for Prolonged Resource Shortages Secondly, neither contingency nor crisis standards are currently designed to respond to prolonged strains on the healthcare system. Since the start of the pandemic, a prolonged period of staffing shortages began and is projected to persist.[11] However, both crisis and contingency standards assume that the system will eventually return to conventional standards of care. For example, as a contingency or crisis standard, many hospitals deferred elective surgeries to preserve limited resources for emergency and life-saving procedures. Massachusetts, for instance, issued a public health emergency order that required hospitals to defer 50 percent of all non-essential and non-urgent (elective) surgeries. This order demonstrates the use of this contingency measure in response to prolonged staffing and bed shortages.[12] However, the deferral of elective procedures can result in adverse long-term community health consequences. Medical conditions typically addressed through elective surgery, such as joint replacement surgeries for osteoarthritis patients, may worsen if delayed. This can result in greater numbers of acute emergencies, the need for more complex surgical procedures later, increased reliance on pain medications, and longer recovery times.[13] Without a greater understanding of long-term complications in community health, existing contingency strategies, such as the deferral of elective surgeries, may be unsuitable for prolonged shortages. This becomes a greater threat to patient safety when contingency measures inappropriately take the place of crisis standards, risking the long-term implementation of emergency measures designed for temporary use. Although some state emergency planning documents identify indicators and triggers for activating contingency and crisis operations,[14] this transition is not always clear in action. For example, New York did not implement crisis standards of care during the early COVID-19 pandemic despite being one of the hardest-hit cities in the US.[15] Other states, including California, Texas, and Florida, did not activate crisis standards of care, leaving hospitals to implement informal contingency measures that ultimately required allocation strategies very similar or identical to many crisis standards of care protocols.[16] Due to the hesitance to activate crisis standards, ad hoc contingency measures and bedside decision-making prevailed over formal triage protocols. If contingency measures are not set forth in objective documents and are inappropriately used in the place of crisis standards, these short-term measures may result in an unfair or non-transparent distribution of scarce resources. When shortages in space, staff, or supplies jeopardize the ability to provide necessary care for critically ill patients under a conventional standard of care, failures to activate crisis standards risk the inappropriate use of ad hoc contingency measures in their place. With clear contingency standards of care, the duration of an ad hoc approach could be limited. Crisis standards are defined and activated at the regional or state-wide level, but outside of hospital-specific resource limitations, there are generally no standardized indications or triggers for transitioning into and out of contingency measures. Leaving contingency needs to individual hospitals may seem beneficial but defining the contingency period at the hospital level and the crisis period at the state or regional level blurs the line about when it is appropriate for decision makers to activate crisis standards, risking delayed activation or failure to activate them at all. Therefore, it is important that state policies implement automatic triggers for activation that clearly delineate between contingency and crisis responses.[17] Automatic triggers based on validated metrics like remaining available resources can inform the appropriate decision makers about when they must activate crisis standards. These triggers should be transparent to the public, validated, and updated over time with evolving data. These automatic triggers would prevent confusion, inconsistent guidelines, and inequitable contingency allocation at the hands of distressed providers when crisis standards are needed. Defining when to begin crisis standards could help limit the length of the contingency period. This would protect against the inappropriate application of contingency measures to crisis-level scarcity and prolonged shortages that they could not sustainably ameliorate. lll. Potential to Exacerbate Health Disparities Inconsistencies in contingency allocation open the door to disparities in care and unequal distribution of scarcity burdens among different communities based on their location or health needs. This is a concern because it is unclear whether contingency measures can meet their goal of achieving functionally equivalent patient outcomes when resource allocation must be balanced with patient-centered care.[18] The care under contingency standards is meant to be functionally equivalent to regular care. The definition assumes (or may wrongly suggest) that any contingency strategy in place to avoid critical scarcity has no significant impact on patient outcomes. While functional equivalence is attainable, there is currently little research into which contingency measures achieve functionally equivalent outcomes and which patient groups may be disproportionately affected by harmful resource allocation strategies. Although the transition from contingency standards to crisis standards is defined by the inability to provide functionally equivalent care, the difference in practice may merely be a distinction between visible, immediate sacrifices to patient well-being during crises and less-obvious, long-term decrements in community health due to protracted contingency care alterations. Two common contingency measures are cause for concern over disparate patient outcomes and the attainability of functional equivalence. First, restricting emergency room visits by the patient’s degree of need has worrying consequences. In late 2021 and early 2022, hospitals in Massachusetts faced widespread staffing shortages, leading to an emergency order that restricted emergency visits to emergency needs.[19] While this order is a reasonable method of allocating limited staff in the emergency department during severe shortages, it is doubtful that the outcomes of this restriction were equivalent to usual care. Health issues that are soon-to-be emergencies are filtered out until they worsen, resulting in patients overflowing to urgent care clinics or presenting to ERs with more severe forms of sicknesses later on. Given the empirical evidence demonstrating ER treatment and admission disparities that disadvantage Black and Hispanic patients, such a measure would only exacerbate these disparities by further limiting access to needed care.[20] Second, altered staffing ratios, which stretch a limited number of providers to meet patient needs during a staffing shortage, are another concerning yet common contingency measure. Staffing allocation is often viewed similarly to the allocation of space and medical equipment, such that contingency alterations to staffing operations may not seem like they significantly jeopardize patient care quality and outwardly appear functionally equivalent.[21] However, lower ratios of qualified nurses are associated with poor outcomes such as higher inpatient mortality[22] and lower survival rates of in-hospital cardiac arrest for Black patients.[23] These examples highlight the strong potential for contingency measures to amplify social health disparities, particularly when adopted over a prolonged time frame. Lowered standards of care in crisis allocation disproportionately impact racial and ethnic minorities.[24] For example, crisis standards of care used clinical scoring systems that were not developed or validated for crisis triage to prioritize access to life-saving treatments during the COVID-19 pandemic. This practice actively gives rise to racial health disparities and discrimination against disabled patients.[25] Not only were the standards inequitable in practice, but they varied widely from state to state and sometimes even from hospital to hospital, creating disparities across and within geographic regions.[26] If contingency measures are similarly implemented across hospitals or hospital departments without standardization or advance planning to ensure equitable outcomes, it is likely that the burden of a lower standard of care will fall primarily on disadvantaged patient groups and racial minorities. However, standardization alone may be insufficient. Other factors like varying levels of details on patients’ charts between hospitals could produce unfair outcomes if used to determine patient admission or transfer priority, even if the criteria for admissions and transfers are consistent. Thus, ongoing monitoring for unintended patterns of disparity must accompany standardization to ensure that blind spots in the allocation process are identified and corrected. Bioethics has long been preoccupied with the micro-allocation of limited resources within hospitals instead of confronting the structural inequities that underlie broader scarcity and patient needs. The traditional dilemma of allocating limited hospital resources among a certain number of patients overlooks questions about how other resources have already been allocated, which patients were present at the hospital in the first place, where hospitals have (and have not) been built, and whether previous allocation strategies created bias in the broader distribution of resources. Therefore, to achieve fairness, bioethicists must pay attention to aspects of the broader distribution of resources, such as social determinants of health and the allocation of preventative resources at the public health level. One strategy for measuring and addressing these disparities is the Area Deprivation Index (ADI). The ADI quantifies the effects of race, class, and socioeconomic background by geographic region for use in public health research and the prioritization of resources.[27] It has shown promise in identifying geographic regions in need of targeted community health efforts for diabetes management based on electronic patient health records.[28] The ADI and similar tools would be useful in proactively deciding how to allocate public health resources when hospitals are strained. Moreover, through using population health and resource data, public health organizations may forecast contingency shortages allowing for the adoption of early measures to mitigate health disparities that might otherwise be amplified from hospital-level contingency allocation decisions. CONCLUSION Meeting community health needs during periods of contingency scarcity, both before and after crisis standards of care apply, will require contingency standards of care rather than a bedside ad hoc distribution of scarce resources. While it is not inherently ethically unjustifiable for hospitals to adopt measures that may lower the standard of care during contingency standards, the necessity of these measures requires that bioethicists consider how equity, transparency, and the overall aim of functional equivalence can best be achieved under conditions of scarcity. The long-term health consequences of existing contingency measures, the potential for ad hoc and inconsistent allocation of scarce resources, and the need for consensus about when it becomes appropriate to make the formal transition to crisis standards of care demand further consideration. Because contingency measures will likely amplify existing disparities as crisis standards have, hospital-level management of scarcity is inadequate. Public health measures should be adopted in parallel to anticipate and manage health needs at the community or state level when resources are strained. - [1] Altevogt, B. M., Stroud, C., Hanson, S. L., Hanfling, D., &amp; Gostin, L. O. (2009). Guidance for Establishing Crisis Standards of Care for Use in Disaster Situations: A Letter Report. The National Academies Press. https://doi.org/10.17226/12749 [2] Emanuel, E. J., Persad, G., Upshur, R., Thome, B., Parker, M., Glickman, A., Zhang, C., Boyle, C., Smith, M., &amp; Phillips, J. P. (2020). Fair Allocation of Scarce Medical Resources in the Time of Covid-19. New England Journal of Medicine, 382(21), 2049–2055. https://doi.org/10.1056/NEJMsb2005114 [3] Alfandre, D., Sharpe, V. A., Geppert, C., Foglia, M. B., Berkowitz, K., Chanko, B., &amp; Schonfeld, T. (2021). Between Usual and Crisis Phases of a Public Health Emergency: The Mediating Role of Contingency Measures. The American Journal of Bioethics, 21(8), 4–16. https://doi.org/10.1080/15265161.2021.1925778 [4] Rosenbaum, L. (2020). Facing Covid-19 in Italy—Ethics, Logistics, and Therapeutics on the Epidemic’s Front Line. New England Journal of Medicine, 382(20), 1873–1875. https://doi.org/10.1056/NEJMp2005492 [5] Orfali, K. (2020). What Triage Issues Reveal: Ethics in the COVID-19 Pandemic in Italy and France. Journal of Bioethical Inquiry, 17(4), 675–679. https://doi.org/10.1007/s11673-020-10059-y [6] Romney, D., Fox, H., Carlson, S., Bachmann, D., O’Mathuna, D., &amp; Kman, N. (2020). Allocation of Scarce Resources in a Pandemic: A Systematic Review of US State Crisis Standards of Care Documents. Disaster Medicine and Public Health Preparedness, 14(5), 677–683. https://doi.org/10.1017/dmp.2020.101 [7] Butler, C. R., Webster, L. B., Diekema, D. S., Gray, M. M., Sakata, V. L., Tonelli, M. R., &amp; Vranas, K. C. (2022). Perspectives of Triage Team Members Participating in Statewide Triage Simulations for Scarce Resource Allocation During the COVID-19 Pandemic in Washington State. JAMA Network Open, 5(4), e227639. https://doi.org/10.1001/jamanetworkopen.2022.7639 [8] Dwyer, J. (2020, May 14). One Hospital Was Besieged by the Virus. Nearby Was ‘Plenty of Space.’—The New York Times. The New York Times. https://www.nytimes.com/2020/05/14/nyregion/coronavirus-ny-hospitals.html [9] Persad, G., Wertheimer, A., &amp; Emanuel, E. J. (2009). Principles for allocation of scarce medical interventions. Lancet (London, England), 373(9661), 423–431. https://doi.org/10.1016/S0140-6736(09)60137-9 [10] Mitchell, S. H., Rigler, J., &amp; Baum, K. (2022). Regional Transfer Coordination and Hospital Load Balancing During COVID-19 Surges. JAMA Health Forum, 3(2), e215048. https://doi.org/10.1001/jamahealthforum.2021.5048 [11] ASPE. (2022, May 3). Impact of the COVID-19 Pandemic on the Hospital and Outpatient Clinician Workforce: Challenges and Policy Responses. ASPE. https://aspe.hhs.gov/reports/covid-19-health-care-workforce [12] Executive Office of Health and Human Services. (2021). Baker-Polito Administration Provides COVID-19 Update on Mask Advisory, Hospital Support | Mass.gov. https://www.mass.gov/news/baker-polito-administration-provides-covid-19-update-on-mask-advisory-hospital-support [13] The Lancet Rheumatology. (2021). Too long to wait: The impact of COVID-19 on elective surgery. The Lancet Rheumatology, 3(2), e83. https://doi.org/10.1016/S2665-9913(21)00001-1 [14] For an example of transition planning between crisis and contingency standards, see Minnesota Department of Health. (2021). Ethical Framework for Transitions Between Conventional, Contingency, and Crisis Conditions in Pervasive or Catastrophic Public Health Events with Medical Surge Implications (Minnesota Crisis Standards of Care). https://www.health.state.mn.us/communities/ep/surge/crisis/framework_transitions.pdf [15] Powell, T., &amp; Chuang, E. (2020). COVID in NYC: What We Could Do Better. The American Journal of Bioethics, 20(7), 62–66. https://doi.org/10.1080/15265161.2020.1764146 [16] Persoff, J., &amp; Wynia, M. K. (2021). Ethically Navigating the Murky Waters of “Contingency Standards of Care.” The American Journal of Bioethics, 21(8), 20–21. https://doi.org/10.1080/15265161.2021.1939810 [17] Board on Health Sciences Policy &amp; Institute of Medicine. (2013). Indicators and Triggers. In Crisis Standards of Care: A Toolkit for Indicators and Triggers. National Academies Press (US). http://www.ncbi.nlm.nih.gov/books/NBK202381/ [18] Frith, L., Draper, H., Fovargue, S., Baines, P., Redhead, C., &amp; Chiumento, A. (2021). Neither ‘Crisis Light’ nor ‘Business as Usual’: Considering the Distinctive Ethical Issues Raised by the Contingency and Reset Phases of a Pandemic. The American Journal of Bioethics, 21(8), 34–37. https://doi.org/10.1080/15265161.2021.1940363 [19] Rosseau, M. (2022, January 14). New emergency orders issued to help understaffed Mass. Hospitals. Boston.Com. https://www.boston.com/news/coronavirus/2022/01/14/new-emergency-orders-issued-to-help-understaffed-mass-hospitals/ [20] Zhang, X., Carabello, M., Hill, T., Bell, S. A., Stephenson, R., &amp; Mahajan, P. (2020). Trends of Racial/Ethnic Differences in Emergency Department Care Outcomes Among Adults in the United States From 2005 to 2016. Frontiers in Medicine, 7. https://www.frontiersin.org/articles/10.3389/fmed.2020.00300 [21] Hick, J. L., Hanfling, D., &amp; Wynia, M. (2022). Hospital Planning for Contingency and Crisis Conditions: Crisis Standards of Care Lessons from COVID-19. The Joint Commission Journal on Quality and Patient Safety. https://doi.org/10.1016/j.jcjq.2022.02.003 [22] Musy, S. N., Endrich, O., Leichtle, A. B., Griffiths, P., Nakas, C. T., &amp; Simon, M. (2021). The association between nurse staffing and inpatient mortality: A shift-level retrospective longitudinal study. International Journal of Nursing Studies, 120, 103950. https://doi.org/10.1016/j.ijnurstu.2021.103950 [23] Brooks Carthon, M., Brom, H., McHugh, M., Sloane, D. M., Berg, R., Merchant, R., Girotra, S., &amp; Aiken, L. H. (2021). Better Nurse Staffing Is Associated With Survival for Black Patients and Diminishes Racial Disparities in Survival After In-Hospital Cardiac Arrests. Medical Care, 59(2), 169–176. https://doi.org/10.1097/MLR.0000000000001464 [24] Annas, G. J., &amp; Crosby, S. S. (2021). Standard Racism: Trying to Use “Crisis Standards of Care” in the COVID-19 Pandemic. The American Journal of Bioethics, 21(8), 1–3. https://doi.org/10.1080/15265161.2021.1941424 [25] Wynia, M. K., &amp; Sottile, P. D. (2020). Ethical Triage Demands a Better Triage Survivability Score. The American Journal of Bioethics, 20(7), 75–77. https://doi.org/10.1080/15265161.2020.1779412 [26] Fink, S. (2020). Ethical Dilemmas in Covid-19 Medical Care: Is a Problematic Triage Protocol Better or Worse than No Protocol at All? The American Journal of Bioethics, 20(7), 1–5. https://doi.org/10.1080/15265161.2020.1788663 [27] Knighton, A. J., Savitz, L., Belnap, T., Stephenson, B., &amp; VanDerslice, J. (2016). Introduction of an Area Deprivation Index Measuring Patient Socioeconomic Status in an Integrated Health System: Implications for Population Health. EGEMS (Washington, DC), 4(3), 1238. https://doi.org/10.13063/2327-9214.1238 [28] Kurani, S. S., Lampman, M. A., Funni, S. A., Giblon, R. E., Inselman, J. W., Shah, N. D., Allen, S., Rushlow, D., &amp; McCoy, R. G. (2021). Association Between Area-Level Socioeconomic Deprivation and Diabetes Care Quality in US Primary Care Practices. JAMA Network Open, 4(12), e2138438. https://doi.org/10.1001/jamanetworkopen.2021.38438

<strong>W&oacute;jcik Justyna, Piotrowska Paulina</strong><strong>, Obuchowska Aleksandra, Ozga Alicja, Zych Anna, Kimber-Trojnar Żaneta, Leszczyńska-Gorzelak Bożena. </strong><strong>Restoring the patency of fallopian tubes using the transcervical selective hysterosalpingography methods - a clinical case analysis</strong><strong>. Journal of Education, Health and Sport. 2019;9(9):415-423. eISNN 2391-8306. DOI </strong><strong>http://dx.doi.org/10.5281/zenodo.3408517</strong> <strong>http://ojs.ukw.edu.pl/index.php/johs/article/view/7444</strong> &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; <strong>The journal has had 5 points in Ministry of Science and Higher Education parametric evaluation. &sect; 8. 2) and &sect; 12. 1. 2) 22.02.2019.</strong> <strong>&copy; The Authors 2019;</strong> <strong>This article is published with open access at Licensee Open Journal Systems of Kazimierz Wielki University in Bydgoszcz, Poland</strong> <strong>Open Access. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author (s) and source are credited. This is an open access article licensed under the terms of the Creative Commons Attribution Non commercial license Share alike.</strong> <strong>(http://creativecommons.org/licenses/by-nc-sa/4.0/) which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited.</strong> &nbsp; <strong>The authors declare that there is no conflict of interests regarding the publication of this paper.</strong> &nbsp; <strong>Received: 01.09.2019. Revised: 14.09.2019. Accepted: 15.09.2019.</strong> &nbsp; &nbsp; &nbsp; <strong>Restoring the patency of fallopian tubes using the transcervical selective hysterosalpingography methods - a clinical case analysis</strong> &nbsp; &nbsp; <strong>Justyna W&oacute;jcik</strong>^{<strong>1a</strong>}<strong>, Paulina </strong><strong>Piotrowska</strong>^{<strong>1b</strong>}<strong>, Aleksandra Obuchowska</strong>^{<strong>1c</strong>}<strong>, Alicja Ozga</strong>^{<strong>1d</strong>}<strong>, Anna&nbsp;Zych</strong>^{<strong>1e</strong>}<strong>, Żaneta Kimber-Trojnar</strong>^{<strong>2f</strong>}<strong>, Bożena Leszczyńska-Gorzelak</strong>^{<strong>2g</strong>} &nbsp; &nbsp; ¹Student&#39;s Scientific Association at the Chair and Department of Obstetrics and Perinatology, Medical University of Lublin, Poland ²Chair and Department of Obstetrics and Perinatology, Medical University of Lublin, Poland &nbsp; ^ajustynawojcik455@gmail.com; ^bpaulina.piotrowska222@gmail.com; ^caobuchowska12@gmail.com; ^daozga1@gmail.com; ^eanna.zych93@gmail.com; ^fzkimber@poczta.onet.pl; ^gb.leszczynska@umlub.pl; &nbsp; <strong>Corresponding author:</strong> Justyna W&oacute;jcik Student&#39;s Scientific Association at the Chair and Department of Obstetrics and Perinatology Jaczewskiego 8 Street 20-954 Lublin, Poland e-mail: justynawojcik455@gmail.com &nbsp; &nbsp; <strong>Abstract</strong> <strong>Introduction: </strong> Infertility caused by tubal occlusion is a relatively frequent affliction among women of child-bearing age. A common cause of tubal occlusion are mucous plugs accumulated inside the fallopian tubes, which results in blocking them. Selective hysterosalpingography (SHSG) is a method of removing the obstruction located in a proximal section of the fallopian tube. The most serious complication developed from this procedure is a fallopian tube perforation, however, it affects less than 1% of patients who undergo the treatment. The clinical success of recanalization of the fallopian tubes is estimated at about 85-88% and the frequency of successful pregnancies after the treatment is about 27%. <strong>Aim: </strong>To show the usage and effectiveness of SHSG in diagnosis and treatment in the obstruction of proximal section of fallopian tube. <strong>Case report: </strong>The patient, 33 years old, diagnosed with primary infertility, qualified to the SHSG at Independent Public Clinical Hospital No. 4 in Lublin. The first stage of SHSG was the insertion of a catheter into the uterine cavity and introduction of a contrast agent to the uterine cavity. The fallopian tubes did not release the contrast into the peritoneal cavity. After increasing the contrast, delivery pressure agent obtained the free flow of the contrast agent into the peritoneal cavity through the right fallopian tube. The uterine outlet of the left fallopian tube was selectively catheterized and a micro tool was inserted into the left fallopian tube. As the result, both fallopian tubes were unblocked. <strong>Summary</strong>: SHSG is a non-invasive and safe method of removing the obstruction located in the proximal section of the fallopian tube. Women subjected to SHSG can return to their normal activities in a few hours after treatment. &nbsp; <strong>Key words</strong>: female infertility, transcervical fallopian tube recanalization, selective hysterosalpingography, proximal tubal occlusion.

<strong>ABSTRACT</strong> Microbes are ubiquitous throughout the seawater and underlying sediment, which covers the vast region of the earth surface. Strains of different genera under the phylum <em>Actinobacteria</em> are ubiquitous all-over different environments of the earth surfaces. A well-studied aerobic genus, <em>Brevibacterium</em>, under the phylum also found to be omnipresent in both marine and terrestrial environments, ranging from coastal to open ocean. Which raise question, whether marine strains are phylogenetically distinct from others or not, and what are the special genomic adaptation marine strains acquire. To examine these, core and pan genome was studied for all the pure culture-based genomes of different strains under <em>Brevibacterium</em> genus. Both, 325 core genes-based as well as gene presence-absence matrix-based i.e. the contribution pattern of each genome to form the pan genome) phylogenetic analysis reveals that marine strains are distributed in polyphyletic clade but share adjacent position in phylograms, suggesting that special marine adaptation affects the genome evolution for isolates living in marine habitat. Starting from considering all the available genomes, different geographical province-specific (Atlantic Ocean, Indian Ocean and Pacific Ocean) genomic datasets were created, following core and pan genome analysis was performed for all the dataset. Which reveals that for marine adaptation 1638 number of core genes are essential, as these genes incorporated in all the genomes of marine strains under the genus <em>Brevibacterium</em>. Core genome expansion for genomes of Indian Ocean isolates and Arabian Sea oxygen minimum zone (OMZ) sediment&rsquo;s isolates were further studied in details, which depicts gene-encoding proteins involved in cell size adjustment, DNA repair machinery, heavy metal resistance, ions transport and other functions, becomes integrated into core genome and found to be pivotal genetic features for living nearby oceanic territory of OMZ. This marine province-specific core genome expansion study shows that strains despite belonging to different species level taxonomic group, possess ecologically significant core genes. A possible relation found to be existed in between seabed expansion, which creates new marine provinces in million years of time scale, and geographical province-specific microbial core genome evolution. &nbsp; <strong>INTRODUCTION</strong> Strains of the phylum <em>Actinobacteria</em> are ubiquitous in most of the natural environments, regardless of the environmental harshness, their habitat ranges from fresh to saline waterbodies and sediment systems of the both, hot springs, and soil of arid deserts (Ward and Bora 2006; Valverde et al. 2012; Mohammadipanah and Wink 2016; Neuenschwander et al. 2018; Deng et al. 2020). The genus <em>Brevibacterium</em>, can be regarded as typical under the phylum, as strains under it follows the similar habitability range (Chen et al. 2016; Jung et al. 2018; Belov et al. 2019; Pei et al. 2020; Zhao et al. 2023). Strains under the genus are generally obligately aerobic, gram positive, non-spore former (Collins 2006). Global ocean and its sediment system is the largest interconnected microbial habitat (Hoshino et al. 2020). The vast oceanic territory is generally divided into four major provinces, Pacific Ocean, Atlantic Ocean, Indian Ocean and Antarctic/Southern Ocean. Different physical and geochemical, characteristics of an environment are the driving force for the selection of only microorganisms possessing adaptive physiological traits necessary to sustain <em>in situ</em> (Dmitrijeva et al. 2024). In a particular environmental niche, ecological selection pressures affect the genome evolution through different mechanisms of genome alteration, such as spontaneous mutation and gain/loss of gene&rsquo;s functions; among these mechanisms horizontal transfer of genes encoding key survival features, between taxonomically diverse individuals is a highly effective way of genomic adaptation (Arnold et al. 2022). Strains of the <em>Brevibacterium</em>, are ubiquitous in the different geographic regions of the global marine territory. They are reported to be isolated from water/sediment sample of different regions of Pacific Ocean (Lee 2008, Pei et al. 2021), Atlantic Ocean (Pei et al. 2021), Indian Ocean (Bhadra et al. 2008; Chen et al. 2016). The aim of the current study is to examine how marine habitat-specific genome evolution takes place for strains of the <em>Brevibacterium</em> genus, which is distributed in both coastal to open ocean environment. Core and pan genome-based phylogenetic analysis were performed for all the pure culture strains under the genus to examine mono- or poly-phyletic origin of marine strains and relatedness between them. Core and pan genome functional analysis were done for all the pure culture isolate&rsquo;s genome, available under the genus <em>Brevibacterium</em> to reveal the actual size of the core and pan genome. How core genome expansion occurs for adaptation in marine habitat was examined (genes those added in the core genome only when genomes of marine strains considered). Further, core and pan genome analysis was done for different subsets of genomes obtained from isolates living in different marine provinces, which would reveal how core genome structure changes in accordance with the changes in marine provinces. Genomic adaptation to Indian ocean province was focused in detail along with special genomic features necessary to live in Arabian Sea OMZ sediment. Result of marine province specific genomic group/subset analysis was used to evaluate how distinct geographical territories shape core genome structure and functions of different strains of <em>Brevibacterium</em>. Furthermore, genomic plasticity of the marine isolates was analyzed by recently incorporated genes through horizontal gene transfer, in each of the marine isolate&rsquo;s genome. The study will add new understandings on how genome divergence is shaped by niche differentiation which is resulted from different geophysical conditions of earth. &nbsp; &nbsp; <strong>MATERIALS AND METHOD</strong> <strong>Genome of pure culture isolates available on GenBank</strong> 138 number of genome assemblies is available in GenBank database of National Center for Biotechnology Information (NCBI), under the genus <em>Brevibacterium</em> (as on March 2024). These only include all the genome assemblies under the genus which, derived from pure culture isolates, having proper annotation as well as proper taxonomic status (followed categorization of NCBI) and retrieved on March, 2024. The 138 genome assemblies include 25 complete genomes and 33 genomes from &lsquo;type material&rsquo; (under the genus 37 species have validly published). It was found that these strains were isolated from diverse habitats ranging from milk products, human infections, soil, freshwater or marine sediment systems. Genomes (represented by single complete circular chromosome) of <em>Brevibacterium</em> sp. BDJS002 and <em>Brevibacterium</em> sp. JSBI002 strains, isolated from Arabian Sea OMZ sediment system (Sarkar et al. 2024) were also included within 138 genome assemblies. TYGS (type strain genome server, DSMZ) was used to determine genome based taxonomic position whenever needed for the strains those have not species level taxonomic status (Meier-Kolthoff JP and G&ouml;ker 2019). &nbsp; <strong>Grouping of <em>Brevibacterium</em> strains on the basis of marine provinces</strong> Six hierarchical genome groups were formed concerning the niches from which pure culture of different <em>Brevibacterium</em> strains reported to be isolated i.e. on the basis of the characteristics/geographical region of the habitat from which 138 strains were isolated. All_Genomes dataset include all the 138 genomes of respective strains, found to be isolated from diverse environmental habitats (Supplementary Table S1). Marine_Genomes dataset include all the genome of strains those were isolated from seawater or marine sediment (genomes those were found to be obtained from strains present in seafood, not included, as geographic location of the seafood from which strain was isolated may not the actual geographic location of the habitat where strain naturally occurs). Atlantic_Ocean_Genomes dataset was created which includes three genomes of respective strains, isolated from water or sediment system of Atlantic Ocean. Pacific_Ocean_Genomes dataset was created including the four genomes those were isolated from water or sediment sample of Pacific Ocean territories. Indian_Ocean_Genomes dataset was created including all the six genomes isolated from strains living in geographic territories of Indian Ocean. Whereas, Arabian_Sea_Genomes dataset include genomes obtained from two pure culture strains isolated from OMZ sediment of Arabian Sea. &nbsp; <strong>Core and pan genome analysis and phylogeny</strong> Bacterial pan genome analysis pipeline (BPGA, version 1.3; Chaudhari et al. 2016) was used to determine pan and core genome of six hierarchical habitat-based group of strains distributed under the genus <em>Brevibacterium</em>. For orthologous clustering of genes, USEARCH algorithm (Edgar 2010) was used and 50% identity value was used as cutoff for clustering all the genes present in all the genomes of a given dataset. Six groups of genome assemblies were separately used to determine pan and core genome of respective group of strains. Pan genome describes distribution of all genes of a given dataset, in different orthologous gene families; pan genome includes core genes (genes present in each genome of a dataset), accessory genes (genes present in two or more genomes of a dataset) and unique genes (genes present in specific genome but not in others). Non-redundant reference sequences (RefSeq) of core, accessory and unique genes of any of the genomic dataset was extracted through BPGA script. After orthologous clustering of the all genes found in all genomes of a dataset, pan and core genome curve was plotted. Pan genome curve depict number of new gene families added after addition of each genome. Core genome curve illustrates how addition of genomes reduces the number of different gene families present throughout the genomes of each dataset. Using BPGA scripts 20 number of random permutations were carried out for addition of each genome and considering median values of number of all distinct gene families and number of commonly shared gene families of a given dataset to determine pan and core genome of that dataset to removing any biases. Power regression model was used for pan genome data and exponential model was used for core genome data for plotting pan and core genome curve and to determine whether pan genome is closed or open (Chaudhari et al. 2016). All the genes of a genome, those contain atypical G+C content (if G+C contents of a gene deviate from the average G+C content of the genome by more than two times of the standard deviation) was determined by atypical GC content analysis script of BPGA (Chaudhari et al. 2016). This atypical GC content of a gene indicate possible inclusion within the respective genome through horizontal gene transfer. Phylogenetic analysis was performed by considering both core genome and pan genome of all the 138 strains. For core genome based phylogenetic analysis, core genes were used for multiple sequences alignments by MUSCLE software (Edgar 2004). For pan genome based phylogenetic analysis, gene presence and absence based binary pan-matrix was considered for all the 138 genomes. Similarity and dissimilarity in contribution of genes to pan genome (orthologous gene clusters) was used to calculate gene matrix. Phylogenetic tree by neighbor-joining approach was created for both core genome and pan genome and Bio-Phylo-v2.0.1 was used for drawing phylogenetic tree (Vos et al. 2011). &nbsp; <strong>Annotation of core, accessory and unique genes</strong> Web-based utilities of eggNOG-mapper was used to functional identification of core, accessory and unique genes of a genomic dataset when required (Cantalapiedra et al. 2021). For this eggNOG 5 database was searched for a given query using search filters Minimum hit e-value = 0.001, Minimum hit bit-score = 60, Percentage identity = 40, Minimum % of query coverage = 20 and Minimum % of subject coverage = 20.<strong>&nbsp;</strong> <strong>&nbsp;</strong> <strong>&nbsp;</strong> <strong>RESULTS</strong> <strong>Range of habitability and genomic characteristics of pure cultured strains</strong> Genome assembly data was retrieved from the GenBank database for all pure-culture strains under <em>Brevibacterium</em> genus (138 strains), reported to be isolated from diverse environmental habitat such as, human infection, dairy product, soil, freshwater and marine sediment (Table 1; Supplementary Table S1). Among these 138 genome assemblies, 13 were found to be isolated from strains lining in marine environment and having proper geographical area from which sample of the inoculum obtained. These 13 marine strains were spread over three marine provinces; three were isolated from Atlantic Ocean, six were isolated from Indian Ocean (two from Arabian Sea OMZ sediment) and four were isolated from Pacific Ocean (Figure 1). Among these 13 strains 9 strains were taxonomically classified up to species level taxon; genome of remaining four were analyzed by TYGS. All the four were found to belong new species as none of these show &gt; 70% dDDH value with genomes of any type strain of <em>Brevibacterium</em> (Table 1). Genome assembly size of all 138 genomes of All_Genomes dataset, ranges from 2.3 Mb to 4.7 Mb having CheckM determined completeness 75.5% to 98.7%; whereas, G+C percentage ranging from 58% to 73%. Genome assembly size for 13 genomes of Marine_Genomes dataset ranges from 3.7 Mb to 4.5 Mb; for these genomes&rsquo; completeness ranges from 88.3% to 97.7% and G+C percentage ranges from 63% to 66% (Figure 2; Table 1; Supplementary Table S1). &nbsp; <strong>Overall core and pan genome structure and phylogeny</strong> Orthologous clustering of total gene repertoire of 138 genomes, obtained from all pure culture strains under the genus <em>Brevibacterium</em>, resulted in 325 number of non-redundant RefSeq genes shared by all the strains and regarded as core genome (Supplementary Table S2). Along with, 27914 number of non-redundant RefSeq genes can be obtained, within which all the genomic repertoire of 138 genomes were distributed, constituting the pan genome. To determine whether pan genome is open or closed, pan genome data was fitted on power regression model [on the equation f(x) = a . x ^ b where the value of a, x and b found to be as follows: a = 3674.6, x = 138 and b = 0.4] it was revealed that the pan genome is still open (as the value of b &le; 1) i.e. gene from new orthologous families is still being added in the gene pool of <em>Brevibacterium</em> strains (Figure 3). Phylogenetic tree derived by multiple sequence alignment of the 325 core genes, resulted in a phylogram (Figure 4) where different species level clusters existed on different node. 12 out of the 13 marine strains situated adjacent position on the phylogenetic tree. While only <em>Brevibacterium</em> sp. BDJS002 positioned distantly (Figure 4). Pan genome-based phylogeny was analyzed, considering each genome&rsquo;s contribution in the pangenome structure, through gene presence-absence data matrix (Figure 5). Here also similar distribution of marine strains observed. Pan genome constitute the non-redundant gene set of core, accessory (genes available in two or more genome) and unique genes (genes only available in a particular genome under consideration) determined through orthologous clustering. Highest percentage of (16.9%) non-redundant core genes were distributed in the J (Translation, ribosomal structure and biogenesis) COG category. Most percentage of non-redundant accessory genes were distributed in K (Transcription) COG category. Highest number of non-redundant unique genes for all the 138 genomes were found to be distributed in also the K (Transcription) COG category. For both accessory and unique genes distribution 2^nd highest distribution was found to be in E (Amino acid transport and metabolism) COG category (R COG category was not taking under consideration for this ranking as this category includes only genes those are predicted to be involved in general function). Along with this, core genes were found to be distributed with values more than 5% in M (Cell wall/membrane/envelope biogenesis), O (Post-translational modification, protein turnover, and chaperones), L (Replication, recombination and repair), C (Energy production and conversion), E (Amino acid transport and metabolism), H (Coenzyme transport and metabolism) and I (Lipid transport and metabolism) COG categories. Whereas, more than 5% accessory genes found to distributed in L (Replication, recombination and repair), G (Carbohydrate transport and metabolism), and P (Inorganic ion transport and metabolism) COG categories. More than 5% unique genes were distributed in G (Carbohydrate transport and metabolism), I (Lipid transport and metabolism), Q (Secondary metabolites biosynthesis, transport, and catabolism) and P (Inorganic ion transport and metabolism) COG categories individually (Figure 6A; Supplementary Table S3). &nbsp; <strong>Core genome expansion for marine habitat adaptation</strong> After orthologous clustering of total gene repertoire of 13 marine strains of <em>Brevibacterium</em>, 1963 non-redundant genes were found to be shared by all genomes i.e. representing the core genome size for the marine strains (Supplementary Table S4). The overall pan genome for these marine strains represented by 7788 number of non-redundant genes. After fitting pan genome data on power regression model [f(x) = a . x ^ b where the value of a, x and b found to be as follows: a = 3456.8, x = 13 and b = 0.3], it was found that it is still open in nature. Different COG categories found to be enriched in core, accessory and unique gene sets of the pan genome. Highest percentage (13.5%) of core genes were distributed in E (Amino acid transport and metabolism) COG category. Most percentages of accessory genes (13.8% and 13.1%) were found to be distributed in K (Transcription) and E COG categories respectively. For unique genes distribution in COG categories, the K (Transcription) category was found to be the highest. More than 5% core, accessory and unique genes were distributed in K (Transcription), C (Energy production and conversion), G (Carbohydrate transport and metabolism), E (Amino acid transport and metabolism) and P (Inorganic ion transport and metabolism) COG categories individually Figure 6B; Supplementary Table S3). After annotating these all 1963 number of core genes against KEGG database it was found that Membrane transport, Signal transduction (under major KEGG category - Environmental Information Processing), Folding, sorting and degradation, Replication and repair, Translation (under major KEGG category - Genetic Information Processing), Amino acid metabolism, Carbohydrate metabolism, Energy metabolism, Nucleotide metabolism (under major KEGG category - Metabolisms) KEGG sub-category become enriched in core genome of marine strains (Supplementary Table S5). Core genome that enriches for marine adaptation must be the results of subtraction of 325 non-redundant core genes of all the strains, from non-redundant core genes of marine strains. Thus (1963-325) = 1638 number of core genes might be essential for marine adaptation. When annotated on KEGG database, the 1638 number of core genes found to be distributed in all the COG categories (Supplementary Table S6). COG category-wise important genes and functional description of their encoded proteins include, ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category D (cell cycle control, cell division, chromosome partitioning), genes (<em>ftsK</em>, <em>ftsX</em>, <em>ftsW</em>, <em>parA</em>, <em>scpB</em>, <em>sepF</em> and <em>smc</em>) encoding proteins involved in cell division and chromosome segregation ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category M (cell wall/membrane/envelope biogenesis), genes encoding proteins involved in, first step of hexosamine metabolism (<em>glmS</em>), cell wall formation (<em>mraY</em>, <em>murB</em>, <em>murD</em>, <em>murG</em>, <em>murI</em>), catalyzes the interconversion of L-alanine and D- alanine (<em>alr</em>), mechanosensitive ion channel (<em>mscS</em>), regulation of osmotic pressure changes within the cell (<em>mscL</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category O (post-translational modification, protein turnover, and chaperones), genes encoding proteins involved in quality control of integral membrane protein (<em>ftsH</em>), proper folding of polypeptides in stressed condition (<em>groL</em>), different chaperones (<em>clpP, clpX</em>), response to hyperosmotic and heat shock by preventing the aggregation of stress-denatured proteins (<em>grpE</em>), redoxin (<em>resA</em>), thioredoxin (<em>trxA</em>), glutaredoxin (<em>nrdH</em>), heat shock protein (<em>hsp18</em>), degradation of misfolded proteins (<em>clpP</em>), glycoprotease (<em>yeaZ</em>), cytochrome oxidase assembly (<em>ctaA, ctaB</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category T (signal transduction mechanisms), genes encoding proteins involved in, ammonia assimilation (<em>glnE</em>), mediator of the stringent response that coordinates a variety of cellular activities in response to changes in nutritional abundance (<em>relA</em>), sensor kinase (<em>citA</em>), tyrosine kinase (<em>pknA</em>), transcriptional regulatory protein (<em>cseB</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category U (intracellular trafficking, secretion, and vesicular transport), genes encoding, part of the Sec protein translocase complex (<em>secA</em>, <em>secD</em>, <em>secE</em>), type II/IV secretion system protein (<em>cpaF</em>), proteins involved in targeting and insertion of nascent membrane proteins into the cytoplasmic membrane (<em>ffh</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category V (defense mechanisms) genes encoding, uncharacterized beta-lactamase and proteins from AcrB/AcrD/AcrF family ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category J (translation, ribosomal structure and biogenesis), gene encoding ribosomal proteins (<em>rpsA</em> to <em>rpsD</em>, <em>rpsF</em> to <em>rpsH</em>, <em>rpsJ</em>, <em>rpsK</em>, <em>rpsM</em> to <em>rpsT</em>, <em>rplA</em>, <em>rplC</em>, <em>rplD</em>, <em>rplF</em>, <em>rplI</em> to <em>rplQ</em>, <em>rplS</em>, <em>rplU</em> to <em>rplX</em>) and translational termination proteins (prfA to prfC) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category K (transcription), genes coding, primary sigma factor responsible for sigmoidal growth (<em>sigA</em>), uncharacterized bacterial transcriptional regulators, regulators of arginine biosynthesis genes (<em>argR</em>), regulatory protein (<em>hspR</em>), cold shock protein (<em>scoF4</em>), proteins needed for assembly of RNA polymerase (<em>rpoZ</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category L (replication, recombination and repair), gene encoding, DNA alkylation repair enzyme (<em>alkB</em>, <em>alkD</em>), DNA polymerase and associated proteins (<em>dnaA</em>, <em>dnaB</em>, <em>dnaE2</em>, <em>dnaG</em>, <em>dnaN</em>, <em>dnaQ</em>, <em>dnaX</em>, <em>polA</em>), recombination related proteins (<em>recB</em>, <em>recF</em>, <em>recG</em>, <em>recN</em>, <em>recQ</em>, <em>recQ</em>, <em>recR</em>), DNA damage recognition and processing protein machhinary (<em>uvrA</em>, <em>uvrD2</em>, <em>uvrC</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category C (energy production and conversion), genes encoding, component of the pyruvate dehydrogenase (<em>aceA</em>, <em>aceE</em>), regulators of arginine biosynthesis genes (<em>argR</em>), different subunit of ATP synthase (<em>atpA</em>, <em>atpB</em>, <em>atpC</em>, <em>atpE</em>, <em>atpF</em>, <em>atpG</em>), Sodium: dicarboxylate symporter (<em>gltT</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category G (carbohydrate transport and metabolism), genes encoding, phosphoglucomutase/phosphomannomutase (<em>manB</em>), phosphomannose isomerase type I (<em>manA</em>), glycosyl hydrolases (<em>otsB</em>), glycosyltransferase (<em>otsA</em>), phosphofructokinase (<em>pfkA</em>), bacterial extracellular solute-binding protein (<em>smoE</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category E (carbohydrate transport and metabolism), gene encoding, arginine synthase and lyase family proteins (<em>argB</em>, <em>argC</em>, <em>argE</em>, <em>argF</em>, <em>argG</em>, <em>argH</em>, <em>argJ</em>), proteins involved in shikimate compound related metabolism (<em>aroA</em>, <em>aroB</em>, <em>aroE</em>, <em>aroK</em>, <em>aroQ</em>), proteins responsible for the synthesis of L- tryptophan (<em>trpA</em>, <em>trpB</em>, <em>trpC</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category F (nucleotide transport and metabolism), genes encoding, adenosine/AMP deaminase (add), carbamoyl-phosphate synthase (<em>carA</em>, <em>carB</em>), orotidine 5'-phosphate decarboxylase/HUMPS family (<em>pyrF</em>), enzyme that catalyzes the reversible phosphorylation of UMP to UDP (<em>pyrH</em>), CO dehydrogenase (<em>xdhA</em>), aldehyde oxidase and xanthine dehydrogenase (<em>xdhB</em>), enzyme that catalyzes the synthesis of GMP from XMP (<em>guaA</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category H (coenzyme transport and metabolism), gene encoding proteins that, catalyzes the phosphorylation of the 3'-hydroxyl group of dephosphocoenzyme A to form coenzyme A (<em>coaE</em>), reversibly transfers an adenylyl group from ATP to 4'- phosphopantetheine (<em>coaD</em>), catalyzes the NADPH-dependent reduction of glutamyl- tRNA (Glu) to glutamate 1-semialdehyde (<em>hemA</em>), have different enzymatic function (<em>nadA</em> to <em>nadE</em>, <em>thiD</em> to <em>thiG</em>, <em>thiL</em>, <em>thiM</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category I (lipid transport and metabolism), gene encoding different transferases (<em>accD1</em>, <em>acpS</em>, <em>acpP</em>), AMP-binding enzyme (<em>fadD</em>), thiolase (<em>fadI</em>), acyl transferase (<em>fabD</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category P (inorganic ion transport and metabolism), gene encoding, catalase (<em>katA</em>), bifunctional enzyme with both catalase and broad- spectrum peroxidase activity (<em>katG</em>), sodium/hydrogen exchanger family protein (<em>kefB</em>), NADH-Ubiquinone oxidoreductase complex I (<em>mrpA</em>/<em>mrpB</em>), NADH-ubiquinone/plastoquinone oxidoreductase (<em>mrpC</em>), Proton-conducting membrane transporter (<em>mrpD</em>), Na+/H+ ion antiporter subunit (<em>mrpE</em>, <em>mrpG</em>), binding-protein-dependent transport system inner membrane component (<em>oppB</em>), N-terminal TM domain of oligopeptide transport permease C (<em>oppC</em>), ABC transporter permease (<em>oppC4</em>), proteins that destroys radicals which are normally produced within the cells and which are toxic to biological systems (<em>sodA</em>) &nbsp; <strong>Marine province-specific core genome expansion</strong> Core and pan genome analysis for each of the three marine province-specific genomic dataset reveals that, core genome was represented by 2572, 2376 and 2243 number of non-redundant core genes for Atlantic_Ocean_Genomes, Pacific_Ocean_Genomes and Indian_Ocean_Genomes (Supplementary Tables S7, S8 and S9). Pan genome was represented by 4711, 5780 and 5462 number of non-redundant genes. Each of the pan genome was found open when pan genome data was fitted on above mentioned power regression model. From the above result, it was revealed that core genome of strains isolated from Atlantic Ocean, Indian Ocean and Pacific Ocean get expanded by 609, 413 and 280 number of core genes (Figure 7A). Annotation of these genes with eggNOG 5 database revealed that these core genes distributed over most of the COG categories (Figure 7B; Supplementary Table S10). COG category-wise important genes and functional description, which were added in the core genome of <em>Brevibacterium</em> strains living in Indian Ocean province, and their encoded proteins include, ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category M, gene encoding proteins involved in, cell wall formation, catalyzes the final step in the synthesis of UDP-N-acetylmuramoyl-pentapeptide (<em>murF</em>), catalyzes the conversion of 1-hydroxy-2-methyl-2-(E)- butenyl 4-diphosphate (HMBPP) into a mixture of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (<em>ispH</em>), UDP-N-acetylglucosamine 2-epimerase (<em>wecB</em>), CoA-binding domain (<em>capD</em>), belongs to the DapA family (<em>dapA_1</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category T, gene encoding proteins involved in, response regulator which acts during transcriptional antitermination (<em>pdtaR</em>), forkhead associated domain ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category U, gene encoding proteins involved in membrane protein translocation (<em>secF</em>, <em>secG</em>), Tfp pilus assembly protein FimV, preprotein translocase, involved in the tonB-independent uptake of proteins ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category J, gene encoding proteins involved in mRNA degradation which catalyzes the phosphorolysis of single-stranded polyribonucleotides processively in the 3'- to 5'-direction (<em>pnp</em>), one of the primary rRNA binding proteins required for association of the 30S and 50S subunits to form the 70S ribosome (<em>rplB</em>), binds directly to 23S rRNA and is necessary for the in vitro assembly process of the 50S ribosomal subunit (<em>rplT</em>), Interacts with and stabilizes bases of the 16S rRNA that are involved in tRNA selection (<em>rpsL</em>), ribosomal protein S9/S16 (<em>rpsI</em>). ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category K, gene encoding proteins, putative response regulator, deoxyribose operon repressor, proteins that participates in transcription elongation, termination and antitermination (<em>nusG</em>), protein involved in negative regulation of DNA-templated transcription initiation, proteins involved in transcription antitermination (<em>nusB</em>), addiction module antidote protein (<em>higA</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category L, gene encoding proteins, that releases the supercoiling and torsional tension of DNA (<em>topA</em>), topoisomerase II (<em>gyrB2</em>), type III restriction enzyme, res subunit (<em>sdrA</em>), DNA polymerase III delta subunit (<em>holB</em>), with LigD forms a non-homologous end joining DNA repair enzyme, which repairs dsDNA breaks with reduced fidelity (<em>ku</em>), involved in DNA repair and RecF pathway recombination (<em>recO</em>), 3'-to-5' exoribonuclease (<em>orn</em>), the RuvA-RuvB complex in the presence of ATP renatures cruciform structure in supercoiled DNA with palindromic sequence (<em>ruvA</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category C, gene encoding proteins, 2-oxoglutarate dehydrogenase, prokaryotic cytochrome b561, acetyl-CoA hydrolase/transferase C-terminal domain (<em>cat1</em>), produces ATP from ADP in the presence of a proton gradient across the membrane (<em>atpD</em>), succinyl-CoA synthetase functions in the citric acid cycle (<em>sucC</em>), belongs to the cytochrome c oxidase bacterial subunit CtaF family (<em>ctaF</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category G, gene encoding proteins, alpha-L-arabinofuranosidase, GntP family permease (gntT), glycoside hydrolases, catalyzes the reversible conversion of 2-phosphoglycerate into phosphoenolpyruvate (<em>eno</em>), glucosamine-6-phosphate isomerases/6-phosphogluconolactonase (<em>nagB</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category E, gene encoding proteins involved in, class-II DAHP synthetase family (<em>aroG</em>), type I 3-dehydroquinase (<em>aroD</em>), rifampin ADP-ribosyl transferase (<em>arr</em>), phosphoribosyl-ATP pyrophosphohydrolase (<em>hisE</em>), IGPS catalyzes the conversion of PRFAR and glutamine to IGP, AICAR and glutamate (<em>hisH</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category F, gene encoding proteins that catalyzes the ATP-dependent amination of UTP to CTP with either L-glutamine or ammonia as the source of nitrogen (<em>pyrG</em>), catalyzes the conversion of dihydroorotate to orotate with quinone as electron acceptor (<em>pyrD</em>), plays an important role in the de novo pathway of purine nucleotide biosynthesis (<em>purA</em>), catalyzes a salvage reaction resulting in the formation of AMP (<em>apt</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category H, gene encoding protein that catalyzes two steps in the biosynthesis of coenzyme A (<em>coaBC</em>), catalyzes the conversion of dethiobiotin to biotin (<em>bioB</em>), catalyzes the NADPH-dependent reduction of ketopantoate into pantoic acid (<em>apbA</em>), catalyzes the ferrous insertion into protoporphyrin IX (<em>hemH</em>), catalyzes the synthesis of beta-nicotinate D- ribonucleotide from nicotinate and 5-phospho-D-ribose 1-phosphate at the expense of ATP (<em>pncB</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category I, gene encoding, acyltransferase, short chain fatty acid transporter (<em>atoE</em>), synthesis of isoprenoid compounds (<em>ispF</em> to <em>ispH</em>), squalene/phytoene synthase (<em>crtB</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category P, gene encoding putative citrate transporter, ammonium transporter (<em>amt</em>), formate/nitrite transporter (<em>focA</em>), CorA-like Mg2+ transporter protein (<em>corA</em>), heme oxygenase (<em>hmuO</em>) The two <em>Brevibacterium</em> strains of Arabian Sea OMZ isolates share additional 208 number of core genes in addition to the shared core genome of six isolates from Indian Ocean region. The 208 genes are distributed in most of the COG categories (Supplementary Tables S11 and S12). COG category-wise important genes and functional description of their encoded proteins include, ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category M, gene encoding proteins for, bacterial sugar transferase (<em>epsL</em>), DegT/DnrJ/EryC1/StrS aminotransferase (<em>epsN</em>), putative lysyltransferase, putative epimerase ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category O, gene encoding proteins for, C-terminal four TMM region of protein-O-mannosyltransferase (<em>pmt</em>), putative ADP-ribosylglycohydrolase, putative XdhC Rossmann domain ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category T, gene encoding proteins for, 5TM C-terminal transporter carbon starvation CstA (<em>cstA</em>), forkhead associated domain (<em>garA</em>), low molecular weight phosphotyrosine protein phosphatase, low molecular weight phosphatase family (<em>arsC</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category U, gene encoding, putative signal peptidase, putative oxidoreductase ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category J, gene encoding amidase (<em>amiD</em>), putative acetyltransferases, putative N-acetylases of ribosomal proteins, proteins polyketide cyclase ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category K, gene encoding proteins involved in, trisaccharide binding, ParB-like nuclease domain (<em>parB</em>), putative GCN5-related N-acetyltransferase, iron dependent repressor, arsenical resistance operon repressor (<em>cmtR</em>), cold shock protein domain (<em>scoF4</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category L, gene encoding, DNA polymerase involved in damage-induced mutagenesis and translesion synthesis (<em>dnaE2</em>), sigma-70 factor, transposase, integrase core domain, helix-turn-helix domain of resolvase, excinuclease ABC activity ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category L, gene encoding, xanthine dehydrogenase, Na⁺/H⁺ antiporter (<em>nhaC</em>), Belongs to the class-I pyridine nucleotide-disulfide oxidoreductase family (<em>merA</em>), malate/L-lactate dehydrogenase ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category G, gene encoding phosphodiester glycosidase, glyceraldehyde 3-phosphate dehydrogenase, trehalose-phosphatase, NmrA family protein ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category E, gene encoding proteins involved in thiamine pyrophosphate, central domain (<em>mdlC</em>), pyridoxal-dependent decarboxylase, catalyzes the transfer of a methyl group from 5- methyltetrahydrofolate to homocysteine resulting in methionine formation (<em>metE</em>), belongs to the carbamate kinase family (<em>arcC</em>), hydroxymethylglutaryl-CoA lyase (<em>hmgL</em>), ATPases associated with a variety of cellular activities (<em>gluA</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category H, gene encoding 6-pyruvoyl tetrahydropterin synthase (<em>ptpS</em>) ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category I, gene encoding proteins belongs to the HMG-CoA reductase family, acyl-CoA dehydrogenase, C-terminal domain (<em>gcdH</em>), putative oxidoreductase ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Under COG category P, gene encoding, anion-transporting ATPase (<em>arsA</em>), sodium bile acid symporter family (<em>arsB</em>), flavin-binding monooxygenase-like (<em>arsO</em>), ATPases associated with a variety of cellular activities (<em>feuA</em>), FecCD transport family (<em>feuC</em>), periplasmic binding protein (<em>feuS</em>), chromate transporter (<em>chrA</em>), Na⁺/H⁺ antiporter that extrudes sodium in exchange for external protons (<em>nhaA</em>) &nbsp; <strong>Actively evolving genome of marine isolates</strong> Genes for which G+C content deviate from the average G+C content of respected genome by more than two times of the standard deviation, was identified by BPGA scripts (Chaudhari et al. 2016). These are the genes which may be integrated in the respective genome through different horizontal gene transfer mechanism. When 13 genomes of marine strains of <em>Brevibacterium</em> was analyzed for genes having atypical G+C content it was found that all the 13 genomes contain unique genes with atypical G+C content. When these atypical G+C content bearing genes for all the 13 marine isolates, were annotated against KEGG database by eggNOG mapper it was found that they fall under different orthologous groups (eggNOG_OGs; Supplementary Table S13). ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; In the genome of <em>Brevibacterium atlanticum</em> WO024, out of 33 unique genes having atypical G+C content, 4 genes were annotated up to orthologous group of Bacteria, 24 genes were annotated in orthologous group of Actinobacteria (while 1 from family Brevibacteriaceae), 3 genes were annotated in orthologous group pf Proteobacteria, 2 genes were annotated in orthologous group of Firmicutes. The function of these genes includes transcription regulation (<em>carD</em>, <em>ybcM</em>), putative sugar transporter, proteins involved in mercury resistance, type I restriction modification DNA specificity domain (<em>hsdM</em>). ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; In the genome of <em>Brevibacterium pigmentatum</em> YB235, out of 14 unique genes having atypical G+C content, 1 gene was annotated up to orthologous group of Bacteria, 11 genes were annotated in orthologous group of Actinobacteria (while 1 from family Brevibacteriaceae), 3 genes were annotated in orthologous group pf Proteobacteria, 1 gene were annotated in orthologous group of Cyanobacteria. The function of these genes includes response regulator, adenine specific DNA methylase, type III restriction enzyme, res subunit, ATPase, P-type transporting (<em>cybH</em>). ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; In the genome of<em> Brevibacterium oceani</em> WW007, out of 39 unique genes having atypical G+C content, 4 genes were annotated up to orthologous group of Bacteria, 32 genes were annotated in orthologous group of Actinobacteria (while 1 from family Brevibacteriaceae), 1 gene was annotated in orthologous group pf Proteobacteria, 2 genes were annotated in orthologous group of Firmicutes. The function of these genes includes transcriptional regulator, ABC transporter, bacterial extracellular solute-binding protein, sugar transporter, ABC-type nitrate sulfonate bicarbonate transport systems periplasmic components. ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; In the genome of<em> Brevibacterium</em> sp. BDJS002, out of 29 unique genes having atypical G+C content, 4 genes were annotated up to orthologous group of Bacteria, 24 genes were annotated in orthologous group of Actinobacteria (while 9 genes from family Brevibacteriaceae), 1 gene was annotated in orthologous group of Bacteroidetes. The function of these genes includes restriction endonuclease, death-on-curing family protein, resolvase, cytochrome c biogenesis protein transmembrane region, region found in RelA/SpoT proteins (<em>ywaC</em>). ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; In the genome of<em> Brevibacterium </em>sp. JSBI002, out of 21 unique genes having atypical G+C content, 19 genes were annotated in orthologous group of Actinobacteria (while no genes from family Brevibacteriaceae), 1 gene was annotated in orthologous group pf Proteobacteria, 1 gene was annotated in orthologous group of Firmicutes. The function of these genes includes glycosyltransferase, endonuclease, transposase, putative ATP-dependent Lon protease, belongs to the peptidase S8, bacterial toxin 35. ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; In the genome of<em> Brevibacterium oceani </em>BBH7, out of 23 unique genes having atypical G+C content, 4 genes were annotated up to orthologous group of Viruses, 2 genes were annotated up to orthologous group of Bacteria, 15 genes were annotated in orthologous group of Actinobacteria (while no genes from family Brevibacteriaceae), 1 gene was annotated in orthologous group of Firmicutes, 1 gene was annotated in orthologous group of Bacteroidetes. The function of these genes includes transcriptional regulator, DNA alkylation repair, endonuclease, viral recombinase domain. ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; In the genome of<em> Brevibacterium sediminis</em> COD27, out of 31 unique genes having atypical G+C content, 2 genes were annotated up to orthologous group of Bacteria, 22 genes were annotated in orthologous group of Actinobacteria (while 6 from family Brevibacteriaceae), 4 genes were annotated in orthologous group pf Proteobacteria, 1 gene was annotated in orthologous group of Bacteroidetes, 2 genes were annotated in orthologous group of Firmicutes. The function of these genes includes, amidinotransferase, transposase, nucleotidyl transferase AbiEii toxin, arsenite transmembrane transporter, Na⁺/H⁺ antiporter. ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; In the genome of<em> Brevibacterium limosum</em> o2, out of 34 unique genes having atypical G+C content, 4 genes were annotated up to orthologous group of Bacteria, 30 genes were annotated in orthologous group of Actinobacteria (while 3 from family Brevibacteriaceae). The function of these genes includes different glycosyl transferases (family 1, 2, 4), sigma-70 region 2, sugar transporter. ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; In the genome of<em> Brevibacterium marinum</em> DSM 18964, out of 30 unique genes having atypical G+C content, 6 genes were annotated up to orthologous group of Bacteria, 23 genes were annotated in orthologous group of Actinobacteria (while 1 from family Brevibacteriaceae), 1 gene were annotated in orthologous group of Firmicutes. The function of these genes includes transposases, protein possibly involved in utilization of glycolate and propanediol, different hydrolases activity. ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; In the genome of<em> Brevibacterium</em> sp. CCUG 69071, out of 30 unique genes having atypical G+C content, 1 gene was annotated in the orthologous group of Viruses, 28 genes were annotated in orthologous group of Actinobacteria (while 1 from family Brevibacteriaceae), 1 gene was annotated in orthologous group of Firmicutes. The function of these genes includes glycosyltransferase, transcriptional regulator, endonucleases, recombinase. ̵&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; In the genome of<em> Brevibacterium</em> sp. Marine, out of 37 unique genes having atypical G+C content, 3 genes were annotated up to orthologous group of Bacteria, 1 gene were annotated up to orthologous group of Archaea, 26 genes were annotated in orthologous group of Actinobacteria (while 5 from family Brevibacteriaceae), 4 genes were annotated in orthologous group pf Proteobacteria, 3 genes were annotated in orthologous group of Firmicutes. The function of these genes includes glycosyl transferases, aromatic amino acid lyase, reverse transcriptase, sodium solute symporter, DNA mismatch endonuclease (<em>vsr</em>). In the genome of<em> Brevibacterium sediminis</em> FXJ8.269 and <em>Brevibacterium sediminis</em> CGMCC 1.15472 have no unique genes. &nbsp; &nbsp; <strong>DISCUSSIONS</strong> Microbial metabolism in marine gets influenced by <em>in situ</em> physico-chemical features of the habitat, diversity of metabolisms differs in coastal seawater-sediment system from open ocean site&rsquo;s seawater-sediment system (D&rsquo;Hondt et al. 2009; D&rsquo;Hondt et al. 2015; Sarkar et al. 2022). Seawater and underlying sediment of a particular marine environment functions as environmental continuum, microbial metabolism taking place within a sediment system get influenced by its overlying seawater, which is the only way for nutrient supply (Marcus and Boero, 1998). Depending on the physico-chemical features such as sedimentation rate, bottom water oxygen availability, abundance of oxidized/reduced metallic ions different metabolic stratum existed in the marine sedimentary environment (Kallmeyer et al. 2012; D&rsquo;Hondt et al. 2015;&nbsp;Torres-Beltr&aacute;n et al. 2021). Though microbial community changes from coastal region to open ocean sites, aerobic strains of proteobacteria and actinobacteria found to be ubiquitous in distribution (Batzke et al. 2007; Bhattacharya et al. 2020, Sarkar et al. 2024) throughout the global marine habitat. Despite the extreme environmental condition in different marine provinces, wide distribution of aerobic bacteria throughout the marine system raises question about their adaptation. Genomic adaptation regarding microbial life in marine habitat was studied for the strains of an aerobic genus, <em>Brevibacterium</em>, revealing its marine origin and expanded core genome structure for adapting different marine provinces. &nbsp; <strong>Polyphyletic genome evolution for marine strains</strong> G+C content percentages and genomic sizes are distributed in narrow range when only marine strains were taken under consideration (Figures 2A and 2B; Supplementary Table S1) in comparison to all the 138 strains. This indicates, structurally genomes of the marine strains not fall apart. After orthologous clustering of all the genes of 138 genomes, 325 non-redundant genes were identified representing core genome. Major percentages of 325 non-redundant core genes were assigned to the translation, ribosomal structure and biogenesis metabolism indicating their important and central role in surviving/growing microbial life. But as all the strains under the genus is Gram-positive and have capabilities to adapt in diverse environmental habitat significant number of genes concerning cell wall formation, replication, energy production and conversion, amino acid transport and metabolism becomes conserved (Supplementary Table S2). The pangenome is still open indicating the adaptive possibilities of strains in adverse environmental condition. When these 325 core genes were used to multiple sequence alignment following phylogenetic tree was drawn by neighbor-joining approach, it was found that marine strains occupy adjacent position on phylogram (Figure 4). The same observation also noticed when gene presence-absence based phylogenetic tree was drawn with same approach, based on how each of the genome contribute in the pan genome (Figure 5). Only strain that fall distant in position was <em>Brevibacterium</em> sp. BDJS002. Polyphyletic close phylogenetic relationship between marine strains supported by both core and pan genome reveals that the marine strains were not diverged recently rather evolved through continuous adaptation in marine habitat. &nbsp; <strong>Genomics of nutrient uptake, osmotic pressure regulation and resistance mechanisms to genotoxicity of sediment, enriched in marine strains</strong> Number of core gene increased, when genome of marine strains was concerned, in comparison to the core genes for 138 genomes. When genome analysis was performed for 13 marine isolates of <em>Brevibacterium</em>, 1963 number of core genes represents the core genome. Highest percentages of core genes found to be distributed in, amino acid transport and metabolism COG category, signifying the importance of these genes in marine adaptation. Different strains of <em>Brevibacterium</em>, follow aerobic chemoorganoheterotrophic mode of nutrition (Bhadra et al. 2008; Lee 2008; Chen et al. 2016; Pei et al. 2021), uptake and utilization of readily available mono/oligomer of structural and functional cell component, from the environment is an adaptive strategy in energy limited environments (Stanley et al. 1987; Coffin et al. 1989). Marine waterbody and its sediment system are the active areas, where organic compounds remineralization takes place, resulting bioavailable amino acids and short peptide chains, in water column and underneath sediment (Fernandes et al. 2014; Choi et al. 2022). Amino acid is available in aquatic environments in simple monomer, short peptide chains and proteins (Coffin et al. 1989). &alpha;- and &beta;- amino acids along with their derivatives often used by bacteria as compatible solutes for osmoadaptation. Uptake of readily available amino acids from the surroundings and incorporating within the biomass is one of the adaptive features of marine adaptation to economically funneling available energy as microbes living in this habitat have to withstand the pressure of water column and high salinity (Stanley et al. 1987; Coffin et al. 1989). Along with the amino acid transport and metabolism, core genome of marine strains includes high percentages of core genes from carbohydrate transport and metabolism, and energy production and conversion COG category. Genes of both of these COG categories are help in proper energy budgeting of the microbial cell, by utilizing readily available compounds available in the surroundings. Polymeric carbohydrates and sugars also often used by microorganism as osmolyte to cope up with the higher osmotic pressure (Yancey 2020), thus transporter of them found to be adaptive strategy for marine habitat. Furthermore, for marine adaptation, enhancement of the core genes observed in the COG category concerning inorganic ion transport and metabolism. Exchange of inorganic ions with the surrounding environment is one of the important adaptations for marine habitat along with the transport of compatible solutes (Wood et al. 2001; Welsh 2020). Furthermore, for marine adaptation, addition of core genes observed in along the most of the COG categories (Figure 6B; Supplementary Table S6). Additional core genes found to be added in core genome for marine adaptation include mechanosensitive ion channel, proteins involved in proper folding of polypeptides in stressed condition, repairing machinery for DNA damage, membrane channel responsible for solute homeostasis of bacterial cytoplasm. In marine sediment different mixtures of chemical and organic compounds causes damage to the dsDNA of the cell (Kammann et al. 2000), to overcome this genotoxic potential of sediment the damage repairing machinery found to be an important adaptive strategy for strains under <em>Brevibacterium</em>. &nbsp; <strong>Marine province-specific genome evolution shed light on oceanic habitat evolution</strong> Strains of <em>Brevibacterium</em>, living in seawater-sediment system of the three major marine provinces i.e. Atlantic Ocean, Indian Ocean and Pacific Ocean found to bear three different sized core genomes consisting of core genes (Figure 7A). Core gene distribution in different COG categories for these three-province specific dataset not show a larger difference (Figure 7B) but in details they differ. Genomes from each of the marine province share additional number of core genes between themselves, along with the core genes those were observed for marine adaptation. Though interconnected through water body sediment system of individual oceanic region got isolated due its immensity. This indicate that marine adaptation is not an easy ubiquitous metabolic strategy for strains under the genus <em>Brevibacterium</em>, rather special genome-guided metabolisms need to be acquired. Isolated environment of each of the marine province affect the genome evolution for strains of <em>Brevibacterium</em>, revealing marine strains are not recently introduced microorganism rather evolved through divergence and evolution of marine habitat through different geological and plate tectonics. For the adaptation in Indian Ocean province, core genome expansion includes genes having function in cell division regulation and determination of diameter of peptidoglycan layer. Different proteins involved in releasing supercoiling of DNA, non-homologous end joining DNA repair machinery, different transcriptional regulators and inorganic ion transporter protein are integrated in the core genome of the strains of Indian Ocean territory (Supplementary Table S10). The Indian Ocean harbor Arabian Sea OMZ and Bay of Bengal OMZ, the isolation site of strains of <em>Brevibacterium</em> were nearby regions of these OMZs. For adaptation of aerobic bacteria in stressed environment (O₂ limitation, nutrient availability, heavy metal enriched, variation of in situ temperature and higher salinity) cell size adjustment, DNA repair machinery, transcriptional regulation at different stages, is found to be important in different bacteria (Dai and Zhu 2018; Dupuy et al. 2019; Mathivanan et al. 2021; Pedraza-Reyes et al. 2024). The expanded core genome of two Arabian Sea isolates included genes concerning metabolism under carbon starvation, encoding arsenic resistant machinery, sodium ion and proton transporter and cold shock proteins (Supplementary Table S12). Adaptation in environments with changing nutrient availability, heavy metal abundance, wide temperature fluctuation and high osmotic pressure these metabolisms found to be important for survivability (Schultz and Matin 1991; Rosen et al. 1999; Zhang and Gross 2021). When genomes of any of the three marine provinces taken under consideration and largest closed circular genome of that dataset used as subject, on which other genomes of the same dataset mapped using BLAST algorithm (E-value cutoff was set to 0.0001), similar pattern in mapped-unmapped portion found, which further provides in between genomic relatedness (Figures 8, 9 and 10) of three marine provinces. The two genomes of pure culture isolates <em>Brevibacterium</em> sp. BDJS002 and <em>Brevibacterium</em> sp. JSBI002 living in sediment of Arabian Sea OMZ, fall apart in both core genome- and pan genome-based phylogeny. Both of them show maximum digital DNA-DNA hybridization value (dDDH) with different type strains (Table 1); but when only genomes from Arabian Sea, were concerned these two strains share 2584 number of non-redundant core genes (core genome size also increases from Indian_Ocean_Genomes dataset), indicating marine habitat play pivotal role in shaping genome evolution through horizontal gene transfer to acquire niche specific metabolic potential. With decreasing vastness of marine province, a greater number of genes included in the core genome regardless of their species level taxonomic identity. Searching for recently introduced genes within the genomes of marine isolates, through horizontal gene transfer mechanism in genome specific unique gene set, reveals high genomic plasticity as genes from all the major lineages i.e. Proteobacteria, Firmicutes, Bacteroidetes found to be included in the genome, encoding different glycosyl transferases, endonucleases, and ion channels. Furthermore, the genes of viral origin also observed to be incorporated in the genome of the marine isolates (Supplementary Table S13). The core and pan genome-based analysis showed that by colonizing a geographical region, different strains of <em>Brevibacterium</em> share significant number of core genes in their genome which bear marine geographical province-specific metabolic functions (Figure 7A). It may also indicate that diversification of marine strains of <em>Brevibacterium</em> takes place along with the evolution and spreading of marine habitat due to plate tectonics. As marine habitat especially oceanic sediment system is a stable environment in respect of flowing waterbody or terrestrial habitat and imposes additional environmental pressure for intruders, this may help to preserve evolutionary lineage of genomic traits. Genomics with larger taxonomically diverse dataset have future research scope for study the pattern of marine habitat evolution and underlying genome evolution. &nbsp; <strong>SUPPLEMENTARY DATA</strong> Supplementary information and data are available online in the form of a Word file named Supplimentary_Information.docx, and an Excel file named Supplimentary_Dataset.xlsx. &nbsp; &nbsp; &nbsp; <strong>DATA AVAILABILITY</strong> GenBank accession numbers for the genomes under the study was presented in Supplementary Table 1. &nbsp; <strong>AUTHOR CONTRIBUTIONS</strong> J.S. conceived the study, designed the bioinformatical experiments, interpreted the results, and wrote the paper. &nbsp; <strong>FUNDING</strong> The study did not utilize any fund. <strong>&nbsp;</strong> <strong>COMPETING INTEREST</strong> The authors declare no competing interest. <strong>ETHICAL APPROVAL</strong> Not applicable for this study &nbsp; <strong>&nbsp;</strong> <strong>REFERENCES</strong> Arnold BJ, Huang IT, Hanage WP (2022) Horizontal gene transfer and adaptive evolution in bacteria. 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Comp Biochem Physiol A Mol Integr Physiol 130:437-460. https://doi.org/10.1016/S1095-6433(01)00442-1 Yancey PH (2020) Compatible and counteracting solutes. In&nbsp;Cellular and molecular physiology of cell volume regulation.&nbsp;CRC press 81-109. Zhang Y, Gross CA (2021) Cold Shock Response in Bacteria. Annu Rev Genet 55:377-400. https://doi.org/10.1146/annurev-genet-071819-031654 Zhao J, Shakir Y, Deng Y et al (2023) Use of modified ichip for the cultivation of thermo-tolerant microorganisms from the hot spring. BMC Microbiol 23:56. https://doi.org/10.1186/s12866-023-02803-2 &nbsp; &nbsp; &nbsp; <strong>FIGURE LEGENDS</strong> <strong>Figure 1. </strong>Approximate geographical location from which sampling was reported to be done for isolation of 13 marine pure culture strains of <em>Brevibacterium</em>. White dots were assigned for stains those having proper geographical location from which isolation inoculum was obtained; while yellow dots were assigned for strains those having only reported major geographical provinces (i.e. Atlantic Ocean, Indian Ocean and Pacific Ocean) from which isolation inoculum was obtained. <strong>Figure 2. </strong>(<strong>A</strong>) Distribution of G+C percentages for all the 138 genomes of pure culture strains of <em>Brevibacterium</em>. (<strong>B</strong>) Genomic size distribution for all the 138 genomes of pure culture strains of <em>Brevibacterium</em>. In both of the plots red-filled shape denote genomic data derived from marine strains. <strong>Figure 3. </strong>Core and pan genome plot of 138 genomes obtained from pure culture strains of <em>Brevibacterium</em>, representing one-by-one addition of the genome and changes in core and pan genome structure. <strong>Figure 4. </strong>325 core genes based phylogenetic tree for the 138 strains of <em>Brevibacterium</em>, created by neighbor-joining approach. Strains name written in red indicates pure culture isolates obtained from marine seawater/sediment and having proper geographical location of isolation site. <strong>Figure 5.</strong> Pan genome based phylogenetic tree for the 138 strains of <em>Brevibacterium</em>, created by neighbor-joining approach. Gene presence and absence based binary pan-matrix was considered for depicting relation between all the 138 genomes. Similarity and dissimilarity in contribution of genes to pan genome (orthologous gene clusters) was used to calculate gene matrix. Strains name written in red indicates pure culture isolates obtained from marine seawater/sediment and having proper geographical location of isolation site. <strong>Figure 6. </strong>(<strong>A</strong>) Distribution of core, accessory and unique genes of 138 genomes into different COG categories (<strong>B</strong>) Distribution of core, accessory and unique genes of 13 genomes of marine isolates, into different COG categories <strong>Figure 7. </strong>(<strong>A</strong>) Schematic diagram of core genome size for all the 13 marine strains of <em>Brevibacterium</em> and variation between core genome size for strains living in different marine provinces. (<strong>B</strong>) Distribution of core genes of strains living in Atlantic, Indian and Pacific Ocean provinces, into different COG categories <strong>Figure 8. </strong>Circular genome-genome mapping visualization for all the three genomes of Atlantic_Ocean_Genomes dataset. The inner to outer colored-ring represents, CDSs of <em>Brevibacterium atlanticum</em> WO024, mapped region of <em>Brevibacterium oceani</em> WW007 on <em>Brevibacterium atlanticum</em> WO024, mapped region of <em>Brevibacterium pigmentatum</em> YB235 on <em>Brevibacterium atlanticum</em> WO024. BLAST algorithm was used for mapping where E-value cutoff was set to 0.0001. <strong>Figure 9. </strong>Circular genome-genome mapping visualization for all the six genomes of Indian_Ocean_Genomes dataset. The inner to outer colored-ring represents, CDSs of <em>Brevibacterium </em>sp. BDJS002, mapped region of <em>Brevibacterium </em>sp. JSBI002<em> </em>on <em>Brevibacterium </em>sp. BDJS002, mapped region of <em>Brevibacterium sediminis </em>COD27 on <em>Brevibacterium </em>sp. BDJS002, mapped region of <em>Brevibacterium sediminis </em>FXJ8.269<em> </em>on <em>Brevibacterium </em>sp. BDJS002, mapped region of <em>Brevibacterium sediminis </em>CGMCC 1.15472 on <em>Brevibacterium </em>sp. BDJS002, mapped region of <em>Brevibacterium oceani </em>BBH7<em> </em>on <em>Brevibacterium </em>sp. BDJS002. BLAST algorithm was used for mapping where E-value cutoff was set to 0.0001. <strong>Figure 10. </strong>Circular genome-genome mapping visualization for all the four genomes of Pacific_Ocean_Genomes dataset. The inner to outer colored-ring represents, CDSs of <em>Brevibacterium limosum </em>o2, mapped region of <em>Brevibacterium marinum </em>DSM 18964 on <em>Brevibacterium limosum </em>o2, mapped region of <em>Brevibacterium </em>sp. Marine on <em>Brevibacterium limosum </em>o2, mapped region of <em>Brevibacterium </em>sp. CCUG 69071<em> </em>on <em>Brevibacterium limosum </em>o2. BLAST algorithm was used for mapping where E-value cutoff was set to 0.0001. &nbsp;

&#x0D; &#x0D; &#x0D; According to Oscar Wilde, the problem with socialism was that it took up too many evenings. Wilde’s aphorism alludes to a major issue that bedevils all attempts to influence the public sphere: the fact that public activities encroach unduly on citizens’ valuable time. In the 21st century, the dilemma of how to deal with “too many evenings” is one that many citizen journalists face as they give their own time to public pursuits. This paper will look at the development of the public citizen and what it means to be a citizen journalist with reference to some of the writer’s own experiences in the field. The paper will conclude with an examination of future possibilities. While large media companies change their change their focus from traditional news values, citizen journalism can play a stronger role in public life as long as it grasps some of the opportunities that are available. There are substantial compensations available to citizen journalists for the problems presented by Wilde’s evenings. The quote from Wilde is borrowed from Albert Hirschman’s Shifting Involvements, which among other things, is an examination of the disappointments of public action. Hirschman noted how it was a common experience for beginners who engage in public action to find that takes up more time than expected (96). As public activity encroaches not only on time devoted to private consumption but also on to the time devoted to the production of income, it can become a costly pursuit which may cause a sharp reaction against the “practice of citizenship” (Hirschman 97). Yet the more stimuli about politics people receive, the greater the likelihood is they will participate in politics and the greater the depth of their participation (Milbrath &amp; Goel 35). People with a positive attraction to politics are more likely to receive stimuli about politics and participate more (Milbrath &amp; Goel 36). Active citizenship, it seems, has its own feedback loops. An active citizenry is not a new idea. The concepts of citizen and citizenship emerged from the sophisticated polity established in the Greek city states about 2,500 years ago. The status of a citizen signified that the individual had the right to full membership of, and participation in, an independent political society (Batrouney &amp; Goldlust 24). In later eras that society could be defined as a kingdom, an empire, or a nation state. The conditions for a bourgeois public sphere were created in the 13th century as capitalists in European city states created a traffic in commodities and news (Habermas 15). A true public sphere emerged in the 17th century with the rise of the English coffee houses and French salons where people had the freedom to express opinions regardless of their social status (Habermas 36). In 1848, France held the first election under universal direct suffrage (for males) and the contemporary slogan was that “universal suffrage closes the era of revolutions” (Hirschman 113). Out of this heady optimism, the late 19th century ushered in the era of the “informed citizen” as voting changed from a social and public duty to a private right – a civic obligation enforceable only by private conscience (Schudson). These concepts live on in the modern idea that the model voter is considered to be a citizen vested with the ability to understand the consequences of his or her choice (Menand 1). The internet is a new knowledge space which offers an alternative reading of the citizen. In Pierre Lévy’s vision of cyberculture, identity is no longer a function of belonging, it is “distributed and nomadic” (Ross &amp; Nightingale 149). The Internet has diffused widely and is increasingly central to everyday life as a place where people go to get information (Dutton 10). Journalism initially prospered on an information scarcity factor however the technology of the Internet has created an information rich society (Tapsall &amp; Varley 18). But research suggests that online discussions do not promote consensus, are short-lived with little impact and end up turning into “dialogues of the deaf” (Nguyen 148). The easy online publishing environment is a fertile ground for rumours, hoaxes and cheating games to circulate which risk turning the public sphere into a chaotic and anarchic space (Nguyen 148). The stereotypical blogger is pejoratively dismissed as “pajama-clad” (Papandrea 516) connoting a sense of disrespect for the proper transmission of ideas. Nevertheless the Internet offers powerful tools for collaboration that is opening up many everyday institutions to greater social accountability (Dutton 3). Recent research by the 2007 Digital Futures project shows 65 percent of respondents consider the Internet “to be a very important or extremely important source of information” (Cowden 76). By 2006, Roy Morgan was reporting that three million Australians were visiting online news site each month (Cowden.76). Crikey.com.au, Australia’s first online-only news outlet, has become a significant independent player in the Australia mediascape claiming over 5,000 subscribers by 2005 with three times as many non-paying “squatters” reading its daily email (Devine 50). Online Opinion has a similar number of subscribers and was receiving 750,000 page views a month by 2005 (National Forum). Both Crikey.com.au and Online Opinion have made moves towards public journalism in an attempt to provide ordinary people access to the public sphere. As professional journalists lose their connection with the public, bloggers are able to fill the public journalism niche (Simons, Content Makers 208). At their best, blogs can offer a “more broad-based, democratic involvement of citizens in the issues that matter to them” (Bruns 7). The research of University of North Carolina journalism professor Philip Meyer showed that cities and towns with public journalism-oriented newspapers led to a better educated local public (Simons, Content Makers 211). Meyer’s idea of good public journalism has six defining elements: a) the need to define a community’s sense of itself b) devotion of time to issues that demand community attention c) devotion of depth to the issues d) more attention to the middle ground e) a preference for substance over tactics and f) encouraging reciprocal understanding (Meyer 1). The objective of public journalism is to foster a greater sense of connection between the community and the media. It can mean journalists using ordinary people as sources and also ordinary people acting as journalists. Jay Rosen proposed a new model based on journalism as conversation (Simons, Content Makers 209). He believes the technology has now overtaken the public journalism movement (Simons, Content Makers 213). His own experiments at pro-am Internet open at assignment.net have had mixed results. His conclusion was that it wasn’t easy for people working voluntarily on the Internet to report on big stories together nor had they “unlocked” the secret of successful pro-am methods (Rosen). Nevertheless, the people formerly known as the audience, as Rosen called them, have seized the agenda. The barriers to entry into journalism have disappeared. Blogging has made Web publishing easy and the social networks are even more user friendly. The problem today is not getting published but finding an audience. And as the audience fragments, the issue will become finding a niche. One such niche is local political activism. The 2007 Australian federal election saw many online sites actively promoting citizen journalism. Most prominent was Youdecide2007 at Queensland University of Technology, funded by the Australian Research Council (ARC) in partnership with SBS, Online Opinion and the Brisbane Institute. Site co-editor Graham Young said the site’s aim was to use citizen journalists to report on their own electorates to fill the gap left by fewer journalists on the ground, especially in less populated areas (Young). While the site’s stated aim was to provide a forum for a seat-by-seat coverage and provide “a new perspective on national politics” (Youdecide2007), the end result was significantly skewed by the fact that the professional editorial team was based in Brisbane. Youdecide2007 published 96 articles in its news archive of which 59 could be identified as having a state-based focus. Figure 1 shows 62.7% of these state-based stories were about Queensland. Figure 1: Youdecide2007 news stories identifiable by state (note: national stories are omitted from this table): State Total no. of stories %age Qld 37 62.7 NSW 8 13.6 Vic 6 10.2 WA 3 5.1 Tas 2 3.4 ACT 2 3.4 SA 1 1.6 Modern election campaigns are characterised by a complex and increasingly fragmented news environment and the new media are rapidly adding another layer of complexity to the mix (Norris et al. 11-12). The slick management of national campaigns are is counter-productive to useful citizen journalism. According to Matthew Clayfield from the citizen journalism site electionTracker.net, “there are very few open events which ordinary people could cover in a way that could be described as citizen journalism” (qtd. in Hills 2007). Similar to other systems, the Australian campaign communication empowers the political leaders and media owners at the expense of ordinary party members and citizens (Warhurst 135). However the slick modern national “on message” campaign has not totally replaced old-style local activity. Although the national campaign has superimposed upon the local one and displaced it from the focus of attention, local candidates must still communicate their party policies in the electorate (Warhurst 113). Citizen journalists are ideally placed to harness this local communication. A grassroots approach is encapsulated in the words of Dan Gillmor who said “every reporter should realise that, collectively, the readers know more than they do about what they write about” (qtd. in Quinn &amp; Quinn-Allan 66). With this in mind, I set out my own stall in citizen journalism for the 2007 Australian federal election with two personal goals: to interview all my local federal Lower House candidates and to attend as many public election meetings as possible. As a result, I wrote 19 election articles in the two months prior to the election. This consisted of 9 news items, 6 candidate interviews and 4 reports of public meetings. All the local candidates except one agreed to be interviewed. The local Liberal candidate refused to be interviewed despite repeated requests. There was no reason offered, just a continual ignoring of requests. Liberal candidates were also noticeably absent from most candidate forums I attended. This pattern of non-communicative behaviour was observed elsewhere (Bartlett, Wilson). I tried to turn this to my advantage by turning their refusal to talk into a story itself. For those that were prepared to talk, I set the expectation that the entire interview would be on the record and would be edited and published on my blog site. As a result, all candidates asked for a list of questions in advance which I supplied. Because politicians devote considerable energy and financial resources to ensure the information they impart to citizens has an appropriate ‘spin’ on it, (Negrine 10) I reserved the right to ask follow-up questions on any of their answers that required clarification. For the interviews themselves, I followed the advice of Spradley’s principle by starting with a conscious attitude of near-total ignorance, not writing the story in advance, and attempting to be descriptive, incisive, investigative and critical (Alia 100). After I posted the results of the interview, I sent a link to each of the respondents offering them a chance to clarify or correct any inaccuracies in the interview statements. Defamation skirts the boundary between free speech and reputation (Pearson 159) and a good working knowledge of the way defamation law affects journalists (citizen or otherwise) is crucial, particularly in dealing with public figures. This was an important consideration for some of the lesser known candidates as Google searches on their names brought my articles up within the top 20 results for each of the Democrat, Green and Liberal Democratic Party candidates I interviewed. None of the public meetings I attended were covered in the mainstream media. These meetings are the type of news Jan Schaffer of University of Maryland’s J-Lab saw as an ecological niche for citizen journalists to “create opportunities for citizens to get informed and inform others about micro-news that falls under the radar of news organisations who don’t have the resources” (Schaffer in Glaser). As Mark Bahnisch points out, Brisbane had three daily newspapers and a daily state based 7.30 Report twenty years ago which contrasts with the situation now where there’s no effective state parliamentary press gallery and little coverage of local politics at all (“State of Political Blogging”). Brisbane’s situation is not unique and the gaps are there to be exploited by new players. While the high cost of market entry renders the “central square” of the public sphere inaccessible to new players (Curran 128) the ease of Web access has given the citizen journalists the chance to roam its back alleys. However even if they fill the voids left by departing news organisations, there will still be a large hole in the mediascape. No one will be doing the hardhitting investigative journalism. This gritty work requires great resources and often years of time. The final product of investigative journalism is often complicated to read, unentertaining and inconclusive (Bower in Negrine 13). Margaret Simons says that journalism is a skill that involves the ability to find things out. She says the challenge of the future will be to marry the strengths of the newsroom and the dirty work of investigative journalism with the power of the conversation of blogs (“Politics and the Internet”). One possibility is raised by the Danish project Scoop. They offer financial support to individual journalists who have good ideas for investigative journalism. Founded by the Danish Association for Investigative Journalism and funded by the Danish Foreign Ministry, Scoop supports media projects across the world with the only proviso being that a journalist has to have an agreement with an editor to publish the resulting story (ABC Media Report). But even without financial support, citizens have the ability to perform rudimentary investigative journalism. The primary tool of investigative journalism is the interview (McIlwane &amp; Bowman 260). While an interview can be arranged by anyone with access to a telephone or e-mail, it should not be underestimated how difficult a skill interviewing is. According to American journalist John Brady, the science of journalistic interviewing aims to gain two things, trust and information (Brady in White 75). In the interviews I did with politicians during the federal election, I found that getting past the “spin” of the party line to get genuine information was the toughest part of the task. There is also a considerable amount of information in the public domain which is rarely explored by reporters (Negrine 23). Knowing how to make use of this information will become a critical success factor for citizen journalists. Corporate journalists use databases such as Lexis/Nexis and Factiva to gain background information, a facility unavailable to most citizen journalists unless they are either have access through a learning institution or are prepared to pay a premium for the information. While large corporate vendors supply highly specialised information, amateurs can play a greater role in the creation and transmission of local news. According to G. Stuart Adam, journalism contains four basic elements: reporting, judging, a public voice and the here and now (13). Citizen journalism is capable of meeting all four criteria. The likelihood is that the future of communications will belong to the centralised corporations on one hand and the unsupervised amateur on the other (Bird 36). Whether the motive to continue is payment or empowerment, the challenge for citizen journalists is to advance beyond the initial success of tactical actions towards the establishment as a serious political and media alternative (Bruns 19). Nguyen et al.’s uses and gratification research project suggests there is a still a long way to go in Australia. While they found widespread diffusion of online news, the vast majority of users (78%) were still getting their news from newspaper Websites (Nguyen et al. 13). The research corroborates Mark Bahnisch’s view that “most Australians have not heard of blogs and only a tiny minority reads them (quoted in Simons, Content Makers 219). The Australian blogosphere still waits for its defining Swiftboat incident or Rathergate to announce its arrival. But Bahnisch doesn’t necessarily believe this is a good evolutionary strategy anyway. Here it is becoming more a conversation than a platform “with its own niche and its own value” (Bahnisch, “This Is Not America”). As far as my own experiments go, the citizen journalism reports I wrote gave me no financial reward but plenty of other compensations that made the experience richly rewarding. It was important to bring otherwise neglected ideas, stories and personalities into the public domain and the reports helped me make valuable connections with public-minded members of my local community. They were also useful practice to hone interview techniques and political writing skills. Finally the exercise raised my own public profile as several of my entries were picked up or hyperlinked by other citizen journalism sites and blogs. Some day, and probably soon, a model will be worked out which will make citizen journalism a worthwhile economic endeavour. In the meantime, we rely on active citizens of the blogosphere to give their evenings freely for the betterment of the public sphere. References ABC Media Report. “Scoop.” 2008. 17 Feb. 2008 http://www.abc.net.au/rn/mediareport/stories/2008/2151204.htm#transcript&gt;. Adam, G. Notes towards a Definition of Journalism: Understanding an Old Craft as an Art Form. St Petersburg, Fl.: Poynter Institute, 1993. Alia, V. “The Rashomon Principle: The Journalist as Ethnographer.” In V. Alia, B. Brennan, and B. Hoffmaster (eds.), Deadlines and Diversity: Journalism Ethics in a Changing World. Halifax: Fernwood Publishing, 1996. Bahnisch, M. “This Is Not America.” newmatilda.com 2007. 17 Feb. 2008 http://www.newmatilda.com/2007/10/04/not-america&gt;. Bahnisch, M. “The State of Political Blogging.” Larvatus Prodeo 2007. 17 Feb. 2008 http://larvatusprodeo.net/2007/09/30/the-state-of-political-blogging/&gt;. Bartlett, A. “Leaders Debate.” The Bartlett Diaries 2007. 19 Feb. 2008 http://andrewbartlett.com/blog/?p=1767&gt;. Batrouney, T., and J. Goldlust. Unravelling Identity: Immigrants, Identity and Citizenship in Australia. Melbourne: Common Ground, 2005. Bird, R. “News in the Global Village.” The End of the News. Toronto: Irwin Publishing, 2005. Bruns, A. “News Blogs and Citizen Journalism: New Directions for e-Journalism.” In K. Prasad (ed.), E-Journalism: New Directions in Electronic News Media. New Delhi: BR Publishing, 2008. 2 Feb. 2008 http://snurb.info/files/News%20Blogs%20and%20Citizen%20Journalism.pdf&gt;. Cowden, G. “Online News: Patterns, Participation and Personalisation.” Australian Journalism Review 29.1 (July 2007). Curran, J. “Rethinking Media and Democracy.” In J. Curran and M. Gurevitch (eds.), Mass Media and Society. 3rd ed. London: Arnold, 2000. Devine, F. “Curse of the Blog.” Quadrant 49.3 (Mar. 2005). Dutton, W. Through the Network (of Networks) – The Fifth Estate. Oxford Internet Institute, 2007. 6 April 2007 http://people.oii.ox.ac.uk/dutton/wp-content/uploads/2007/10/ 5th-estate-lecture-text.pdf&gt;. Glaser, M. “The New Voices: Hyperlocal Citizen’s Media Sites Want You (to Write!).” Online Journalism Review 2004. 16 Feb. 2008 http://ojr.org/ojr/glaser/1098833871.php&gt;. Habermas, J. The Structural Transformation of the Public Sphere: An Inquiry into a Category of Bourgeois Society. Cambridge: Polity Press, 1989 [1962]. Hills, R. “Citizen Journos Turning Inwards.” The Age 18 Nov. 2007. 17 Feb. 2008 http://www.theage.com.au/news/federal-election-2007-news/citizen-journos- turning-inwards/2007/11/17/1194767024688.html&gt;. Hirschman, A, Shifting Involvements: Private Interest and Public Action. Princeton, NJ: Princeton UP, 1982. Hunter, C. “The Internet and the Public Sphere: Revitalization or Decay?” Virginia Journal of Communication 12 (2000): 93-127. Killenberg, G., and R. Dardenne. “Instruction in News Reporting as Community Focused Journalism.” Journalism &amp; Mass Communication Educator 52.1 (Spring 1997). McIlwane, S., and L. Bowman. “Interviewing Techniques.” In S. Tanner (ed.), Journalism: Investigation and Research. Sydney: Longman, 2002. Menand, L. “The Unpolitical Animal: How Political Science Understands Voters.” The New Yorker 30 Aug. 2004. 17 Feb. 2008 http://www.newyorker.com/archive/2004/08/30/040830crat_atlarge&gt;. Meyer, P. Public Journalism and the Problem of Objectivity. 1995. 16 Feb. 2008 http://www.unc.edu/%7Epmeyer/ire95pj.htm&gt;. Milbrath, L., and M. Goel. Political Participation: How and Why Do People Get Involved in Politics? Chicago: Rand McNally M, 1975. National Forum. “Annual Report 2005.” 6 April 2008 http://www.onlineopinion.com.au/documents/reports/ annual_report_to_agm_2005.pdf&gt;. Negrine, R. The Communication of Politics. London: Sage, 1996. Nguyen, A. “Journalism in the Wake of Participatory Publishing.” Australian Journalism Review 28.1 (July 2006). Nguyen, A., E. Ferrier, M. Western, and S. McKay. “Online News in Australia: Patterns of Use and Gratification.” Australian Studies in Journalism 15 (2005). Norris, P., J. Curtice, D. Sanders, M. Scammell, and H. Setemko. On Message: Communicating the Campaign. London: Sage, 1999. Papandrea, M. “Citizen Journalism and the Reporter’s Privilege.” Minnesota Law Review 91 (2007). Pearson, M. The Journalist’s Guide to Media Law. 2nd ed. Sydney: Allen &amp; Unwin, 2004. Quinn, S., and D. Quinn-Allan. “User-Generated Content and the Changing News Cycle.” Australian Journalism Review 28.1 (July 2006). Rosen, J. “Assignment Zero: Can Crowds Create Fiction, Architecture and Photography?” Wired 2007. 6 April 2008 http://www.wired.com/techbiz/media/news/2007/07/assignment_zero_all&gt;. Ross, K., and V. Nightingale. Media Audiences: New Perspectives. Maidenhead, Berkshire: Open UP, 2003. Schaffer, J. “Citizens Media: Has It Reached a Tipping Point.” Nieman Reports 59.4 (Winter 2005). Schudson, M. Good Citizens and Bad History: Today’s Political Ideals in Historical Perspective. 1999. 17 Feb. 2008 http://www.mtsu.edu/~seig/paper_m_schudson.html&gt;. Simons, M. The Content Makers. Melbourne: Penguin, 2007. Simons, M. “Politics and the Internet.” Keynote speech at the Brisbane Writers’ Festival, 14 Sep. 2007. Tapsall, S., and C. Varley (eds.). Journalism: Theory in Practice. South Melbourne: Oxford UP, 2001. Warhurst, J. “Campaign Communications in Australia.” In F. Fletcher (ed.), Media, Elections and Democracy, Toronto: Dundurn Press, 1991. White, S. Reporting in Australia. 2nd ed. Melbourne: MacMillan, 2005. Wilson, J. “Who’s Afraid of the Big Bad Electorate.” Youdecide2007 2007. 19 Feb. 2008 http://www.youdecide2007.org/content/view/283/101/&gt;. Young, G. “Citizen Journalism.” Presentation at the Australian Blogging Conference, 28 Sep. 2007. &#x0D; &#x0D; &#x0D; &#x0D; Citation reference for this article&#x0D; &#x0D; MLA Style&#x0D; Barry, Derek. "Wilde’s Evenings: The Rewards of Citizen Journalism." M/C Journal 10.6/11.1 (2008). echo date('d M. Y'); ?&gt; &lt;http://journal.media-culture.org.au/0804/09-barry.php&gt;. APA Style&#x0D; Barry, D. (Apr. 2008) "Wilde’s Evenings: The Rewards of Citizen Journalism," M/C Journal, 10(6)/11(1). Retrieved echo date('d M. Y'); ?&gt; from &lt;http://journal.media-culture.org.au/0804/09-barry.php&gt;. &#x0D;

According to Oscar Wilde, the problem with socialism was that it took up too many evenings. Wilde’s aphorism alludes to a major issue that bedevils all attempts to influence the public sphere: the fact that public activities encroach unduly on citizens’ valuable time. In the 21st century, the dilemma of how to deal with “too many evenings” is one that many citizen journalists face as they give their own time to public pursuits. This paper will look at the development of the public citizen and what it means to be a citizen journalist with reference to some of the writer’s own experiences in the field. The paper will conclude with an examination of future possibilities. While large media companies change their change their focus from traditional news values, citizen journalism can play a stronger role in public life as long as it grasps some of the opportunities that are available. There are substantial compensations available to citizen journalists for the problems presented by Wilde’s evenings. The quote from Wilde is borrowed from Albert Hirschman’s Shifting Involvements, which among other things, is an examination of the disappointments of public action. Hirschman noted how it was a common experience for beginners who engage in public action to find that takes up more time than expected (96). As public activity encroaches not only on time devoted to private consumption but also on to the time devoted to the production of income, it can become a costly pursuit which may cause a sharp reaction against the “practice of citizenship” (Hirschman 97). Yet the more stimuli about politics people receive, the greater the likelihood is they will participate in politics and the greater the depth of their participation (Milbrath &amp; Goel 35). People with a positive attraction to politics are more likely to receive stimuli about politics and participate more (Milbrath &amp; Goel 36). Active citizenship, it seems, has its own feedback loops. An active citizenry is not a new idea. The concepts of citizen and citizenship emerged from the sophisticated polity established in the Greek city states about 2,500 years ago. The status of a citizen signified that the individual had the right to full membership of, and participation in, an independent political society (Batrouney &amp; Goldlust 24). In later eras that society could be defined as a kingdom, an empire, or a nation state. The conditions for a bourgeois public sphere were created in the 13th century as capitalists in European city states created a traffic in commodities and news (Habermas 15). A true public sphere emerged in the 17th century with the rise of the English coffee houses and French salons where people had the freedom to express opinions regardless of their social status (Habermas 36). In 1848, France held the first election under universal direct suffrage (for males) and the contemporary slogan was that “universal suffrage closes the era of revolutions” (Hirschman 113). Out of this heady optimism, the late 19th century ushered in the era of the “informed citizen” as voting changed from a social and public duty to a private right – a civic obligation enforceable only by private conscience (Schudson). These concepts live on in the modern idea that the model voter is considered to be a citizen vested with the ability to understand the consequences of his or her choice (Menand 1). The internet is a new knowledge space which offers an alternative reading of the citizen. In Pierre Lévy’s vision of cyberculture, identity is no longer a function of belonging, it is “distributed and nomadic” (Ross &amp; Nightingale 149). The Internet has diffused widely and is increasingly central to everyday life as a place where people go to get information (Dutton 10). Journalism initially prospered on an information scarcity factor however the technology of the Internet has created an information rich society (Tapsall &amp; Varley 18). But research suggests that online discussions do not promote consensus, are short-lived with little impact and end up turning into “dialogues of the deaf” (Nguyen 148). The easy online publishing environment is a fertile ground for rumours, hoaxes and cheating games to circulate which risk turning the public sphere into a chaotic and anarchic space (Nguyen 148). The stereotypical blogger is pejoratively dismissed as “pajama-clad” (Papandrea 516) connoting a sense of disrespect for the proper transmission of ideas. Nevertheless the Internet offers powerful tools for collaboration that is opening up many everyday institutions to greater social accountability (Dutton 3). Recent research by the 2007 Digital Futures project shows 65 percent of respondents consider the Internet “to be a very important or extremely important source of information” (Cowden 76). By 2006, Roy Morgan was reporting that three million Australians were visiting online news site each month (Cowden.76). Crikey.com.au, Australia’s first online-only news outlet, has become a significant independent player in the Australia mediascape claiming over 5,000 subscribers by 2005 with three times as many non-paying “squatters” reading its daily email (Devine 50). Online Opinion has a similar number of subscribers and was receiving 750,000 page views a month by 2005 (National Forum). Both Crikey.com.au and Online Opinion have made moves towards public journalism in an attempt to provide ordinary people access to the public sphere. As professional journalists lose their connection with the public, bloggers are able to fill the public journalism niche (Simons, Content Makers 208). At their best, blogs can offer a “more broad-based, democratic involvement of citizens in the issues that matter to them” (Bruns 7). The research of University of North Carolina journalism professor Philip Meyer showed that cities and towns with public journalism-oriented newspapers led to a better educated local public (Simons, Content Makers 211). Meyer’s idea of good public journalism has six defining elements: a) the need to define a community’s sense of itself b) devotion of time to issues that demand community attention c) devotion of depth to the issues d) more attention to the middle ground e) a preference for substance over tactics and f) encouraging reciprocal understanding (Meyer 1). The objective of public journalism is to foster a greater sense of connection between the community and the media. It can mean journalists using ordinary people as sources and also ordinary people acting as journalists. Jay Rosen proposed a new model based on journalism as conversation (Simons, Content Makers 209). He believes the technology has now overtaken the public journalism movement (Simons, Content Makers 213). His own experiments at pro-am Internet open at assignment.net have had mixed results. His conclusion was that it wasn’t easy for people working voluntarily on the Internet to report on big stories together nor had they “unlocked” the secret of successful pro-am methods (Rosen). Nevertheless, the people formerly known as the audience, as Rosen called them, have seized the agenda. The barriers to entry into journalism have disappeared. Blogging has made Web publishing easy and the social networks are even more user friendly. The problem today is not getting published but finding an audience. And as the audience fragments, the issue will become finding a niche. One such niche is local political activism. The 2007 Australian federal election saw many online sites actively promoting citizen journalism. Most prominent was Youdecide2007 at Queensland University of Technology, funded by the Australian Research Council (ARC) in partnership with SBS, Online Opinion and the Brisbane Institute. Site co-editor Graham Young said the site’s aim was to use citizen journalists to report on their own electorates to fill the gap left by fewer journalists on the ground, especially in less populated areas (Young). While the site’s stated aim was to provide a forum for a seat-by-seat coverage and provide “a new perspective on national politics” (Youdecide2007), the end result was significantly skewed by the fact that the professional editorial team was based in Brisbane. Youdecide2007 published 96 articles in its news archive of which 59 could be identified as having a state-based focus. Figure 1 shows 62.7% of these state-based stories were about Queensland. Figure 1: Youdecide2007 news stories identifiable by state (note: national stories are omitted from this table): State Total no. of stories %age Qld 37 62.7 NSW 8 13.6 Vic 6 10.2 WA 3 5.1 Tas 2 3.4 ACT 2 3.4 SA 1 1.6 Modern election campaigns are characterised by a complex and increasingly fragmented news environment and the new media are rapidly adding another layer of complexity to the mix (Norris et al. 11-12). The slick management of national campaigns are is counter-productive to useful citizen journalism. According to Matthew Clayfield from the citizen journalism site electionTracker.net, “there are very few open events which ordinary people could cover in a way that could be described as citizen journalism” (qtd. in Hills 2007). Similar to other systems, the Australian campaign communication empowers the political leaders and media owners at the expense of ordinary party members and citizens (Warhurst 135). However the slick modern national “on message” campaign has not totally replaced old-style local activity. Although the national campaign has superimposed upon the local one and displaced it from the focus of attention, local candidates must still communicate their party policies in the electorate (Warhurst 113). Citizen journalists are ideally placed to harness this local communication. A grassroots approach is encapsulated in the words of Dan Gillmor who said “every reporter should realise that, collectively, the readers know more than they do about what they write about” (qtd. in Quinn &amp; Quinn-Allan 66). With this in mind, I set out my own stall in citizen journalism for the 2007 Australian federal election with two personal goals: to interview all my local federal Lower House candidates and to attend as many public election meetings as possible. As a result, I wrote 19 election articles in the two months prior to the election. This consisted of 9 news items, 6 candidate interviews and 4 reports of public meetings. All the local candidates except one agreed to be interviewed. The local Liberal candidate refused to be interviewed despite repeated requests. There was no reason offered, just a continual ignoring of requests. Liberal candidates were also noticeably absent from most candidate forums I attended. This pattern of non-communicative behaviour was observed elsewhere (Bartlett, Wilson). I tried to turn this to my advantage by turning their refusal to talk into a story itself. For those that were prepared to talk, I set the expectation that the entire interview would be on the record and would be edited and published on my blog site. As a result, all candidates asked for a list of questions in advance which I supplied. Because politicians devote considerable energy and financial resources to ensure the information they impart to citizens has an appropriate ‘spin’ on it, (Negrine 10) I reserved the right to ask follow-up questions on any of their answers that required clarification. For the interviews themselves, I followed the advice of Spradley’s principle by starting with a conscious attitude of near-total ignorance, not writing the story in advance, and attempting to be descriptive, incisive, investigative and critical (Alia 100). After I posted the results of the interview, I sent a link to each of the respondents offering them a chance to clarify or correct any inaccuracies in the interview statements. Defamation skirts the boundary between free speech and reputation (Pearson 159) and a good working knowledge of the way defamation law affects journalists (citizen or otherwise) is crucial, particularly in dealing with public figures. This was an important consideration for some of the lesser known candidates as Google searches on their names brought my articles up within the top 20 results for each of the Democrat, Green and Liberal Democratic Party candidates I interviewed. None of the public meetings I attended were covered in the mainstream media. These meetings are the type of news Jan Schaffer of University of Maryland’s J-Lab saw as an ecological niche for citizen journalists to “create opportunities for citizens to get informed and inform others about micro-news that falls under the radar of news organisations who don’t have the resources” (Schaffer in Glaser). As Mark Bahnisch points out, Brisbane had three daily newspapers and a daily state based 7.30 Report twenty years ago which contrasts with the situation now where there’s no effective state parliamentary press gallery and little coverage of local politics at all (“State of Political Blogging”). Brisbane’s situation is not unique and the gaps are there to be exploited by new players. While the high cost of market entry renders the “central square” of the public sphere inaccessible to new players (Curran 128) the ease of Web access has given the citizen journalists the chance to roam its back alleys. However even if they fill the voids left by departing news organisations, there will still be a large hole in the mediascape. No one will be doing the hardhitting investigative journalism. This gritty work requires great resources and often years of time. The final product of investigative journalism is often complicated to read, unentertaining and inconclusive (Bower in Negrine 13). Margaret Simons says that journalism is a skill that involves the ability to find things out. She says the challenge of the future will be to marry the strengths of the newsroom and the dirty work of investigative journalism with the power of the conversation of blogs (“Politics and the Internet”). One possibility is raised by the Danish project Scoop. They offer financial support to individual journalists who have good ideas for investigative journalism. Founded by the Danish Association for Investigative Journalism and funded by the Danish Foreign Ministry, Scoop supports media projects across the world with the only proviso being that a journalist has to have an agreement with an editor to publish the resulting story (ABC Media Report). But even without financial support, citizens have the ability to perform rudimentary investigative journalism. The primary tool of investigative journalism is the interview (McIlwane &amp; Bowman 260). While an interview can be arranged by anyone with access to a telephone or e-mail, it should not be underestimated how difficult a skill interviewing is. According to American journalist John Brady, the science of journalistic interviewing aims to gain two things, trust and information (Brady in White 75). In the interviews I did with politicians during the federal election, I found that getting past the “spin” of the party line to get genuine information was the toughest part of the task. There is also a considerable amount of information in the public domain which is rarely explored by reporters (Negrine 23). Knowing how to make use of this information will become a critical success factor for citizen journalists. Corporate journalists use databases such as Lexis/Nexis and Factiva to gain background information, a facility unavailable to most citizen journalists unless they are either have access through a learning institution or are prepared to pay a premium for the information. While large corporate vendors supply highly specialised information, amateurs can play a greater role in the creation and transmission of local news. According to G. Stuart Adam, journalism contains four basic elements: reporting, judging, a public voice and the here and now (13). Citizen journalism is capable of meeting all four criteria. The likelihood is that the future of communications will belong to the centralised corporations on one hand and the unsupervised amateur on the other (Bird 36). Whether the motive to continue is payment or empowerment, the challenge for citizen journalists is to advance beyond the initial success of tactical actions towards the establishment as a serious political and media alternative (Bruns 19). Nguyen et al.’s uses and gratification research project suggests there is a still a long way to go in Australia. While they found widespread diffusion of online news, the vast majority of users (78%) were still getting their news from newspaper Websites (Nguyen et al. 13). The research corroborates Mark Bahnisch’s view that “most Australians have not heard of blogs and only a tiny minority reads them (quoted in Simons, Content Makers 219). The Australian blogosphere still waits for its defining Swiftboat incident or Rathergate to announce its arrival. But Bahnisch doesn’t necessarily believe this is a good evolutionary strategy anyway. Here it is becoming more a conversation than a platform “with its own niche and its own value” (Bahnisch, “This Is Not America”). As far as my own experiments go, the citizen journalism reports I wrote gave me no financial reward but plenty of other compensations that made the experience richly rewarding. It was important to bring otherwise neglected ideas, stories and personalities into the public domain and the reports helped me make valuable connections with public-minded members of my local community. They were also useful practice to hone interview techniques and political writing skills. Finally the exercise raised my own public profile as several of my entries were picked up or hyperlinked by other citizen journalism sites and blogs. Some day, and probably soon, a model will be worked out which will make citizen journalism a worthwhile economic endeavour. In the meantime, we rely on active citizens of the blogosphere to give their evenings freely for the betterment of the public sphere. References ABC Media Report. “Scoop.” 2008. 17 Feb. 2008 &lt; http://www.abc.net.au/rn/mediareport/stories/2008/2151204.htm#transcript &gt;. Adam, G. Notes towards a Definition of Journalism: Understanding an Old Craft as an Art Form. St Petersburg, Fl.: Poynter Institute, 1993. Alia, V. “The Rashomon Principle: The Journalist as Ethnographer.” In V. Alia, B. Brennan, and B. Hoffmaster (eds.), Deadlines and Diversity: Journalism Ethics in a Changing World. Halifax: Fernwood Publishing, 1996. Bahnisch, M. “This Is Not America.” newmatilda.com 2007. 17 Feb. 2008 &lt; http://www.newmatilda.com/2007/10/04/not-america &gt;. Bahnisch, M. “The State of Political Blogging.” Larvatus Prodeo 2007. 17 Feb. 2008 &lt; http://larvatusprodeo.net/2007/09/30/the-state-of-political-blogging/ &gt;. Bartlett, A. “Leaders Debate.” The Bartlett Diaries 2007. 19 Feb. 2008 &lt; http://andrewbartlett.com/blog/?p=1767 &gt;. Batrouney, T., and J. Goldlust. Unravelling Identity: Immigrants, Identity and Citizenship in Australia. Melbourne: Common Ground, 2005. Bird, R. “News in the Global Village.” The End of the News. Toronto: Irwin Publishing, 2005. Bruns, A. “News Blogs and Citizen Journalism: New Directions for e-Journalism.” In K. Prasad (ed.), E-Journalism: New Directions in Electronic News Media. New Delhi: BR Publishing, 2008. 2 Feb. 2008 &lt; http://snurb.info/files/News%20Blogs%20and%20Citizen%20Journalism.pdf &gt;. Cowden, G. “Online News: Patterns, Participation and Personalisation.” Australian Journalism Review 29.1 (July 2007). Curran, J. “Rethinking Media and Democracy.” In J. Curran and M. Gurevitch (eds.), Mass Media and Society. 3rd ed. London: Arnold, 2000. Devine, F. “Curse of the Blog.” Quadrant 49.3 (Mar. 2005). Dutton, W. Through the Network (of Networks) – The Fifth Estate. Oxford Internet Institute, 2007. 6 April 2007 &lt; http://people.oii.ox.ac.uk/dutton/wp-content/uploads/2007/10/ 5th-estate-lecture-text.pdf &gt;. Glaser, M. “The New Voices: Hyperlocal Citizen’s Media Sites Want You (to Write!).” Online Journalism Review 2004. 16 Feb. 2008 &lt; http://ojr.org/ojr/glaser/1098833871.php &gt;. Habermas, J. The Structural Transformation of the Public Sphere: An Inquiry into a Category of Bourgeois Society. Cambridge: Polity Press, 1989 [1962]. Hills, R. “Citizen Journos Turning Inwards.” The Age 18 Nov. 2007. 17 Feb. 2008 &lt; http://www.theage.com.au/news/federal-election-2007-news/citizen-journos- turning-inwards/2007/11/17/1194767024688.html &gt;. Hirschman, A, Shifting Involvements: Private Interest and Public Action. Princeton, NJ: Princeton UP, 1982. Hunter, C. “The Internet and the Public Sphere: Revitalization or Decay?” Virginia Journal of Communication 12 (2000): 93-127. Killenberg, G., and R. Dardenne. “Instruction in News Reporting as Community Focused Journalism.” Journalism &amp; Mass Communication Educator 52.1 (Spring 1997). McIlwane, S., and L. Bowman. “Interviewing Techniques.” In S. Tanner (ed.), Journalism: Investigation and Research. Sydney: Longman, 2002. Menand, L. “The Unpolitical Animal: How Political Science Understands Voters.” The New Yorker 30 Aug. 2004. 17 Feb. 2008 &lt; http://www.newyorker.com/archive/2004/08/30/040830crat_atlarge &gt;. Meyer, P. Public Journalism and the Problem of Objectivity. 1995. 16 Feb. 2008 &lt; http://www.unc.edu/%7Epmeyer/ire95pj.htm &gt;. Milbrath, L., and M. Goel. Political Participation: How and Why Do People Get Involved in Politics? Chicago: Rand McNally M, 1975. National Forum. “Annual Report 2005.” 6 April 2008 &lt; http://www.onlineopinion.com.au/documents/reports/ annual_report_to_agm_2005.pdf &gt;. Negrine, R. The Communication of Politics. London: Sage, 1996. Nguyen, A. “Journalism in the Wake of Participatory Publishing.” Australian Journalism Review 28.1 (July 2006). Nguyen, A., E. Ferrier, M. Western, and S. McKay. “Online News in Australia: Patterns of Use and Gratification.” Australian Studies in Journalism 15 (2005). Norris, P., J. Curtice, D. Sanders, M. Scammell, and H. Setemko. On Message: Communicating the Campaign. London: Sage, 1999. Papandrea, M. “Citizen Journalism and the Reporter’s Privilege.” Minnesota Law Review 91 (2007). Pearson, M. The Journalist’s Guide to Media Law. 2nd ed. Sydney: Allen &amp; Unwin, 2004. Quinn, S., and D. Quinn-Allan. “User-Generated Content and the Changing News Cycle.” Australian Journalism Review 28.1 (July 2006). Rosen, J. “Assignment Zero: Can Crowds Create Fiction, Architecture and Photography?” Wired 2007. 6 April 2008 &lt; http://www.wired.com/techbiz/media/news/2007/07/assignment_zero_all &gt;. Ross, K., and V. Nightingale. Media Audiences: New Perspectives. Maidenhead, Berkshire: Open UP, 2003. Schaffer, J. “Citizens Media: Has It Reached a Tipping Point.” Nieman Reports 59.4 (Winter 2005). Schudson, M. Good Citizens and Bad History: Today’s Political Ideals in Historical Perspective. 1999. 17 Feb. 2008 &lt; http://www.mtsu.edu/~seig/paper_m_schudson.html &gt;. Simons, M. The Content Makers. Melbourne: Penguin, 2007. Simons, M. “Politics and the Internet.” Keynote speech at the Brisbane Writers’ Festival, 14 Sep. 2007. Tapsall, S., and C. Varley (eds.). Journalism: Theory in Practice. South Melbourne: Oxford UP, 2001. Warhurst, J. “Campaign Communications in Australia.” In F. Fletcher (ed.), Media, Elections and Democracy, Toronto: Dundurn Press, 1991. White, S. Reporting in Australia. 2nd ed. Melbourne: MacMillan, 2005. Wilson, J. “Who’s Afraid of the Big Bad Electorate.” Youdecide2007 2007. 19 Feb. 2008 &lt; http://www.youdecide2007.org/content/view/283/101/ &gt;. Young, G. “Citizen Journalism.” Presentation at the Australian Blogging Conference, 28 Sep. 2007.

Food Science and Technology : An International Journal (FSTJ) Vol.1, No.1 33 HIGH-PRESSURE PROCESSING AND ITS APPLICATIONS IN THE DAIRY INDUSTRY Ronit Mandal and Rajni Kant Agriculture and Food Engineering Department, Indian Institute of Technology Kharagpur, India ABSTRACT High-pressure processing (HPP) is a novel, non-thermal food processing technology. Processing of foods by this method offers an alternative to thermal processing as it is carried out near the ambient temperature, thus, eliminating the adverse effects of heat and keeps the sensory and nutritional attributes of the food fresh like. This paper outlines the salient principles of the high pressure processing, equipment available, microbial inactivation mechanisms, applications of high pressure in the processing of dairy products, effect of pressure treatment on the milk constituents and further research needs for adaptation of the process in the dairy industry. KEYWORDS Non-thermal processes, High pressure processing, Pascal&rsquo;s law, Isostatic principle 1. INTRODUCTION High-pressure processing (HPP) is a non-thermal method of preservation and sterilization of food products, in which a product is subjected to very high pressure, leading to the inactivation of certain microorganisms and enzymes in the food. High-pressure processing is a novel alternative to the thermal processing in the food industry. It allows food to be pasteurized at or near room temperature. High pressure (HP) treated foods have most of the &ldquo;fresh like&rdquo; attributes as it enables microorganisms to be destroyed with little or negative effects on flavour components, colours, vitamins and other nutrients, hence, does not hamper the nutritional and organoleptic properties of the product, thus offering considerable advantages as compared to thermal processing. The terms pascalization, or high hydrostatic pressure (HHP) are same as high-pressure processing [1]. Research on the application of HPP for milk preservation started several decades back [2], which showed that the pressure treatment increased shelf life of milk. Research is still going on to standardize the processing of dairy products by high pressure. Apart from preservation of the dairy products, this novel process also leads to varied physicochemical, sensory and functional changes leading to the improvement in quality of treated products. Additionally, high pressure can cause rheological changes in food that may result in beneficial sensory and structural effects. Also, the effects of HPP on the food constituents may be helpful in formulation of novel and value added dairy products. These results, in turn open new arena and offer exciting opportunities for the dairy industry. Food Science and Technology : An International Journal (FSTJ) Vol.1, No.1 34 In the past few decades, several studies on HPP applications as an alternative dairy processing have been done. This has led to vast number of literature reviews on HPP applications for milk and dairy products. Based on previous results, authors have reviewed the equipment of HPP, microbial inactivation and applications of HPP for treatment of milk and dairy products [3, 4]. The effects of pressure on milk constituents, especially on lipids and proteins and potential of HPinduced modifications of dairy products have been reported elsewhere [5, 6], with a special focus on fermented products, packaging requirements and legal concerns [7]. All these studies converge on one or the other aspects of HPP. The compilation of all the relevant topics together in this subject has been done earlier. But, since the dynamic nature of research, a need has been to review all the previous works on HPP applications in dairy industry with the latest inclusions in the field. Therefore, the purpose of this literature review is to enlighten the recent technological developments and understandings in the field of HPP. This paper highlights the principles of HPP, equipment developed for food processing, microbial inactivation mechanisms by HPP, and applications of HPP on milk and dairy products based on several published data as well as effect of processing on constituents of milk. It suggests that there is an increasing interest of HPP adoption and hence paving of way for its commercialization in dairy industry. 2. PRINCIPLES OF HPP The HPP is based on two basic principles [8]: &bull; Le Chatelier&rsquo;s principle &bull; Isostatic principle (Pascal&rsquo;s Law) The Le Chatelier&rsquo;s principle states that &lsquo;if a change in conditions is applied on a system in equilibrium, then the system will try to counteract that change and restore the equilibrium&rsquo;. The Isostatic principle states that the HPP is volume-independent; therefore, pressure is transmitted instantaneously and uniformly throughout a sample, and pressure is gradients do not exist, so that the size and geometry of the product is irrelevant. Typically, HPP of food is carried out at 300&ndash;600 Mega Pascals (MPa) at the room temperature for 2&ndash;30 min. The HPP is an alternative to thermal processing as it is operated at ambient temperatures, ensuring little or no heat induced sensory changes in its components of food. It is because of the fact that, the smaller organic molecules responsible for colours, flavours, and nutrients (e.g., vitamins) have covalent bonding dominantly or exclusively, which are hardly affected by HPP. Whereas, the large bio-molecules such as proteins, nucleic acids and polysaccharides that depend on non-covalent bonding (like hydrophobic interactions, hydrogen bonds etc.) to maintain structure and function are most affected due to this. 2.1. MECHANISM OF INACTIVATION OF MICROORGANISMS A major function of high-pressure processing of food is destruction of microorganisms. Microbial cell destruction can be attributed to the following detrimental changes that take place when pressure is applied [3]: &bull; Irreversible structural changes of the membrane proteins and other macromolecules leading to cell membranes destruction &bull; Disruption of homogeneity of the intermediate layer between the cell wall and the cytoplasmic membrane &bull; Inactivation of membrane ATPase Food Science and Technology : An International Journal (FSTJ) Vol.1, No.1 35 &bull; Disruption of nucleic acids and ribosome involved in proteins synthesis These result into permeabilization of the membranes and concomitant leakage and loss of the contents of the cells and organelles, leading to eventual death of the bacterial cell [8]. Denaturation of the critical enzymes is also responsible for cell destruction. Yeasts, moulds and most vegetative bacteria are inactivated by pressures between 300 and 600MPa. In general, In general, cells in the exponential phase of growth are less resistant to pressure than cells that are in a stationary phase of growth. Gram-negative bacteria are inactivated at a lower pressure than Gram-positive bacteria, and rod shaped bacteria are more sensitive to pressure than cocci shaped. Spores are more resistant than vegetative cells because of calcium rich dipicolinic acid which protects them from excessive ionization, surviving pressures over 100 MPa [9]. The level of microbial inactivation depends on the pressure applied, duration of treatment, temperature, environment and initial load and types of microorganisms [8]. 3. EQUIPMENT Typically, the HPP equipment has the following necessary components [3, 10]: &bull; a pressure vessel and its closure &bull; a pressure generation system &bull; a temperature control device &bull; a material handling system Most pressure vessels for HPP applications are made from a high tensile steel alloy and called &lsquo;monoblocs&rsquo; (forged from a single piece of material), able to withstand pressures of 400&ndash;600 MPa. For higher pressures, several designs and configurations like &ldquo;pre-stressed multi-layer or wire-wound vessels&rdquo; are used [11]. Vessels are sealed by a threaded steel closure, a closure having an interrupted thread, which can be removed more quickly, or by a sealed frame that is positioned over the vessel. In operation of HPP, the pressure is transmitted in two ways &bull; Direct compression: It uses a piston to compress the vessel, in which the pressure transmitting medium resides. The product is introduced in the medium. When compression takes place, the product gets treated &bull; Indirect compression After the vessel is evacuated, the pressure transmitting medium is pumped from a reservoir into the pressure vessel using a pressure intensifier until the desired pressure is reached. Pressure transmitting medium is generally any food-grade fluid. Commonly used medium are simply purified water mixed with a small percentage of soluble oil (for lubrication and anticorrosion), aqueous solution of mono-propylene glycol for high temperature, isopropyl alcohol for low temperature HPP [1]. Food Science and Technology : An International Journal (FSTJ) Vol.1, No.1 36 Slight increase in temperature (3&ndash;9&deg;C per 100 MPa, depending on the pressure transmitting medium) takes place due to adiabatic heating [12] and with a corresponding decrease during depressurization. Temperature is controlled by pumping a heating/ cooling medium through a jacket that surrounds the pressure vessel. 4. APPLICATIONS IN DAIRY INDUSTRY Research into the application of HPP for milk preservation began long back [2], when it was demonstrated that pressure treatment can extend the shelf life of milk. It was investigated such treatment as an alternative method for milk pasteurization. Several researches have been done on the applications of HPP on milk and milk products. 4.1. FLUID MILK PROCESSING HP treatment [2] was carried out at 680 MPa for 10 min at room temperature and observed 5&ndash;6 log cycle reduction in the number of microorganisms. The effect of HP on milk has been extensively studied [13]. Many researchers have studied inactivation of microorganisms Listeria monocytogenes, Staphylococcus aureus, or Listeria innocua either naturally present or inoculated in milk [14]; and this offers a promising alternative for the pasteurization of milk [15]. Milk subjected to a pressure of 350 MPa had a shelf life of 25 days at 0&deg;C, 18 days at 5 &deg;C, and 12 days at 10 &deg;C [16]. HP treatment (400 MPa for 15 min or 500 MPa for 3 min) of thermally pasteurized milk increased shelf life by 10 days [17]. Raw milk pressurized at 400 MPa for 30 min at 25&deg;C contained 7 log of these bacteria after only 15 days [18]. Further, the combination of high pressure with a bacteriocin (lacticin) was shown as a promising and natural method for increasing the efficiency and safety of HPP of milk. It resulted in a synergistic effect in controlling microbial flora of milk without significantly influencing its cheese-making properties [19]. Other antimicrobial peptides such as lactoferrin and lactoferricin (500&micro;g/ml) in combination with high pressure (155&ndash;400 MPa) also resulted in enhanced microbial inactivation [20]. The gram-negative bacteria, in this case, were found to be more sensitive to high pressure, either alone or in combination with nisin, than gram-positive bacteria [21]. It is known that fat increases the thermal-resistance of microorganisms, but in case of high pressure treatment, fat content in milk (0&ndash;5 %) had no significant effect on the destruction [22]. Although Casein and lactose present in milk were the major baro-protective agents to Escherichia coli in milk during the treatment. It was also noted that combination of high pressure with temperature for the processing of milk promoted the formation of few compounds leading to generation of &lsquo;cooked&rsquo; milk flavour and sensory acceptance of treated milk was not very high [23]. Milk treated at 400 MPa results in no significant loss of vitamins like B1 and B6 [24]. 4.2. CHEESE HPP has many applications in cheese manufacture. HPP caused casein micelle disruption, whey protein denaturation, increase in milk pH and cheese yield, and reduction in rennet coagulation time, which indicates its significant potential in the cheese-manufacturing [25, 26]. Food Science and Technology : An International Journal (FSTJ) Vol.1, No.1 37 As the heat treatment of milk, in spite of killing harmful pathogens makes the same unsuitable for cheese making leading to increased RCT (rennet coagulation time) and delayed maturing. So, HPP can be good alternative for cheese milk heat treatment. The HP also reduces RCT and improves cheese yield [27] which can be ascertained from the fact that 15% increase of yield was obtained and 30% decrease in whey proteins in whey in cheese made from HP treated milk, probably due to whey proteins and casein interaction. HP treated milk cheeses have higher moisture, salt and total free amino acids content than raw or pasteurized milk cheeses. High moisture led to pasty and weak texture defects. Such an effect was attributed to the increased water-holding capacity of the milk proteins [28]. High-pressure treatment of cheese curd rather than cheese milk had beneficial effects. Rennet coagulation time was not dependant on the pressure in the lower range (less than 150 MPa), whereas at higher pressures (200&ndash;400 MPa) it decreased [29]. HP treatment (400 MPa) of pasteurized milk resulted in decreased rennet coagulation time. At 600 MPa, the rennet coagulation time was found to decrease along with decrease in pH, initial counts of nonstarter lactic acid bacteria, protein and fat content. The treatment increased incorporation of &beta;-lactoglobulin leading to increased yield [30]. Using HPP, cheese ripening process can be greatly accelerated. A Japanese patent [31] reported possible reduction in the ripening time of Cheddar cheese to six months by HP treatment (50MPa and 25&deg;C for three days). HP induces changes in biochemical processes such as glycolysis, lipolysis, and proteolysis during ripening of cheese leading to reduction in ripening time and quality improvement. In cheese manufactured from HP treated milk (at 300 or 400 MPa for 10 minutes), &beta;-casein hydrolysis increased, as well as the level of free amino acids (FAAs). Similar results were reported for HP treated Cheddar cheese (50 MPa for 72 h); however, no textural changes in cheese were observed [32]. The ripening of cheddar cheese due to HPP accelerated the degradation of &alpha; S1 -casein and accumulation of &alpha; S1 -1-casein [33]. Levels of free fatty acids in cheese was decreased by HP treatment at 400MPa, indicating slower lipolysis [34]. There are scarce literatures on the effect of HP on the flavour development and volatiles in cheese. Lower levels of butyric acid and acetoin in Gouda cheese after HP treatment between 50 and 400 MPa were reported [35]. Further studies will be necessary to determine the full potential of HP in exploiting cheese ripening. HP-treated (500 MPa) goats&rsquo; milk had higher pH and salt content, showed faster maturation, and strong flavours generation [36]. 4.3. ICE-CREAM HPP induces fat crystallization [37], curtails the time required to achieve a desirable solid fat content and thereby reducing the ageing time of ice-cream. HP treatment has an impact on the functional properties of whey proteins. HP treatment of 300 MPa for 15 min enhanced the foaming properties of whey protein concentrate, which when added to low-fat ice cream to improved body and texture of ice-cream, showed an increased overrun and foam stability and hardness the ice cream than ice cream added with untreated whey protein [38]. A team of researchers [39] studied the applications of HP processing in ice-cream manufacture. For this purpose, ice-cream mixes were subjected to pressures of about 200&ndash;500MPa for one second to 20 minutes. HPP at 400 or 500MPa for time as low as one second showed increase in the mix viscosity, which, the researchers attributed to the formation of a proteinaceous network of micellar fragments in the mix by reduction in solubility of calcium phosphate on decompression during HPP cycle. Resultant ice-cream showed a strong increase in resistance to meltdown, greatly improved textural characteristics, like mouthfeel and creaminess. Food Science and Technology : An International Journal (FSTJ) Vol.1, No.1 38 These findings highlight that HP treatment leads to beneficial changes in proteins assists manufacturing of low-fat and stabilizer-free ice creams with improved mouthfeel. Pressure assisted freezing may be a good area of research for HP treatment of ice-cream. 4.4. YOGURT Significant structural differences between yogurt made from thermally- or HP-treated milk have been reported [29, 40]. Yogurt made from HP treated milk was less susceptible to undesirable syneresis on storage, probably due to changes in gel structure and water-binding capacity of milk proteins [41]. It was reported [42] the making of stirred yogurt from HP treated milk at pressures from 100 to 400MPa and temperatures from 25 to 90&deg;C, and concluded that HPP at higher temperatures would reduce the ultimate viscosity of yogurt. A possible application of HPP in yogurt manufacture is the treatment following fermentation, to inactivate starter cultures and yeasts and moulds and, thereby, extend the shelf life of the product by prevention of &lsquo;post-acidification&rsquo; [8]. High pressure treatment at 200 to 300 MPa for 10 min at 10 - 20&deg;C showed controlling of &lsquo;post- acidification&rsquo; of yogurt without decreasing the number of viable lactic acid bacteria (LAB) or modifying the yogurt texture. Treatment at higher pressures may destroy LAB. Potential future research of the use of high-pressure processing (HPP) in yogurt manufacture may involve combining HPP with crosslinking of milk proteins by transglutaminase [43]. Optimization of combination of HPP with heat treatment and/or addition of other ingredients, such as stabilizers or milk powders has also been suggested [8]. An extended shelf- life &lsquo;Probiotic yogurt&rsquo; has been developed using pressure treatment of 350&ndash; 650 MPa at 10&ndash;15&deg;C. HPP (550 MPa) of yogurt maintained desirable sensory characteristics longer than controls during storage for 4 weeks at refrigerated (4&deg;C) or room (20&deg;C) temperature [44, 45]. 4.5. CREAM AND BUTTER When cream was treated at pressure of 600 MPa for up to 2 min, its whipping properties improved and reduced serum loss [46] possibly due to better crystallization of milk fat. HP treatment of pasteurized cream at 450 MPa at 10 or 25&deg;C did not alter the fat globules size distribution, the pH or its flow behaviour [37]. HP processing of butter or cream may lead to rise in the temperature (8&ndash;9&deg;C/100 MPa) [47]. HPP may have a potential application in the physical ripening of dairy cream for butter making. 4.6. OTHER DAIRY PRODUCTS The use of HPP on treatment of functional dairy products has been studied [48]. They concluded that unfolding of whey proteins under pressure facilitated peptide bonds hydrolysis and reduced allergenicity. They reviewed the use of HP to preserve colostrum. Treatment of colostrum with HP has been shown better retention of biological activity than of heat treatment under certain conditions and ensuring a reasonable shelf life of product [49-51]. The Fonterra Company of New Zealand has patented processes for colostrum preservation using HP treatment [8]. Food Science and Technology : An International Journal (FSTJ) Vol.1, No.1 39 HP treatment (up to 500 MPa) reduced the turbidity of reconstituted skim milk for all combinations of pH (5.5&ndash;7.5) and temperature (5&ndash;40 &deg;C) due to micelle dissociation [52]. Attempts have been made for optimizing the levels of pressure (200&ndash;400 MPa), pressurization time (0&ndash;100 min), and coagulation temperature (30&ndash;70 &deg;C) for the preparation of chhana (Indian cottage cheese) [53]. There is a great scope for utilizing HPP for the development of probiotic dairy foods with higher viable count. HP treatment of human milk indicated that with at 400MPa for 5 minutes, helped retaining of all immunoglobulin A (IgA) in milk serum that is susceptible to thermal damage during pasteurization using heat treatment [54]. 5. EFFECT OF HPP ON MILK COMPONENTS High pressure processing of milk has shown to have a significant effect on the milk constituents. Milk constituents undergo various changes, which cause alterations on the quality and functionality of milk [8]. 5.1. WATER Water content of the food gets compressed by about 4 per cent at 100 MPa and 15 per cent at 600 MPa [55]. Depression in freezing point of water was also observed at high pressure to -4&deg;C, -8&deg;C, -22&deg;C at 50, 100 and 210 MPa, respectively [56]. Thus, this technique enables sub-zero temperature food processing without ice crystal formation. It also facilitates rapid thawing of conventional frozen food. 5.2. PROTEINS Treating skimmed milk under high pressure disrupts casein micelles, which increase with pressure increment [57]. Whey proteins do not hinder HP induced disruption of casein micelles [58]. CCP solubilization is a primary factor inducing micellar disruption on HP treatment [59]. Casein micelle size does change a little at lower pressures (15 minutes) can give significant increases in micelle size [29, 60]. HP induces changes in turbidity and lightness of skimmed milk apart from changing casein micelle. The viscosity is increased by about 20 per cent [29]. Caseins show dissociated from the micelle in the following order &beta;-casein&gt;&kappa;-casein &gt; &alpha;s1-casein &gt; &alpha;s2-casein [29]. HP treatment of milk at lower pressure (up to 100 MPa) showed no denaturation of &beta;lactoglobulin but at higher pressures (&ge;400 MPa) 90% denaturation of &beta;-lactoglobulin was observed [61]. Whereas, in whey pressures up to 300 MPa gave significant denaturation in &beta;lactoglobulin, while, further pressure increase decreased &beta;-lactoglobulin [62]. Denaturation of &alpha;lactalbumin only happens at &ge;400MPa, reaching about 70% after 30 minutes treatment at 800 MPa [63]. Milk solids concentration has relatively little effect on the denaturation of whey proteins due to HP treatment [60]. Other whey proteins like bovine serum albumin showed no denaturation in HP treated milk (at 100&ndash;400 MPa) [61]. The immunoglobulins also show stability to HPP; about 90% of immunoglobulin G (IgG) in colostrum remained in native state after HP treatment (500 MPa for five minutes) [51]. 5.3. ENZYMES Milk enzymes vary in their sensitivity to high pressure. Lipoprotein lipase, xanthine oxidase and lactoperoxidase showed resistant up to pressures up to 400 MPa [7]. Phosphohexose isomerase, &gamma;glutamyltransferase and alkaline phosphatase in milk are partially inactivated at pressures Food Science and Technology : An International Journal (FSTJ) Vol.1, No.1 40 exceeding 350, 400 and 600 MPa respectively and are completely inactivated at pressures of 550, 630 and 800 MPa respectively [64]. The effect on alkaline phosphatase is of interest in milk processing. Since complete inactivation of alkaline phosphatase occurs only at very high pressures, this specific enzyme is not an appropriate indicator of effective &lsquo;pasteurization&rsquo; by high-pressure treatment unlike thermal pasteurization. HP treatment also has influences on the proteolysis in milk. The treatment of milk at lower pressure (up to about 300 MPa) has insignificant effect on plasmin activity, but, at higher pressures (30 minutes at 600 MPa) up to about 75% inactivation is observed, [65]. Hence, proteolysis was increased during the storage for low pressure treatment, whereas after 500 MPa, the proteolysis during storage of milk was less than that observed in raw milk. The combination of HP treatment (300&ndash;600 MPa, 40&ndash;60&deg;C) and homogenization resulted in inactivation of protease activity in milk, which extended its shelf life [66]. 5.4. FAT HP treatment of milk affects the fat in milk in many ways. Treatment at 100&ndash;600MPa at 100 MPa), &alpha;-lactalbumin (at &ge;700 MPa) and &kappa;-casein (at &gt;500MPa) with the fat globule membrane take place in HP treated milk [68]. HP treatment (400 MPa for 4h at 45oC) of the high melting fraction of milk fat also significantly suppresses thermal deterioration [70]. Treatment at 100&ndash;250 MPa may promote the cold agglutination of milk fat globules, which may lead to clusters of fat globules during cold storage leading to faster creaming. However, at &gt;400 MPa, reduced cold agglutination, and hence reduced creaming occurs as a result of HP induced denaturation of immunoglobulins [8]. Critical threshold pressure between 300 and 500 MPa can trigger oxidation in lipids [71]. 5.5. MILK SUGAR Pressurization of milk (200-400 MPa for 10-60 min at 25&deg;C) lead to no changes in the lactose, suggesting no Maillard reaction and isomerization occurs due to pressure treatment [61]. 5.6. MINERAL Mineral balance of milk gets affected at high pressure and effect is on both the distribution between colloidal and soluble phase as well as on the ionization. The increase in the content of soluble calcium has been reported by following HPP. In case of previously heated milk, HPP treatment solubilizes both native and heat precipitated colloidal calcium phosphate (CCP) which leads to slight increase in pH [72]. Solubilization of CCP increases with increasing pressure up to about 400 MPa [73]. The concentration of ionic calcium in milk has been observed to slightly increase, immediately after HP treatment [74]. Food Science and Technology : An International Journal (FSTJ) Vol.1, No.1 41 6. CONCLUSIONS The HPP is a &lsquo;novel&rsquo; and non-thermal technology has the potential for use as an alternative to thermal processing. Flavour and aroma components contributing to the sensory quality, and nutritional quality remained unaffected by pressure treatment. Several researches have been done on HP treatment on milk and milk products. These have provided a detailed understanding about the complex changes that take place in milk under high pressure like the dissociation of caseins micelles from the colloidal to the soluble phase, influence turbidity of milk etc. However, it cannot be denied that the dairy industry has been comparatively slow to adopt HP processing, as compared to product meat and sea-foods, jams, juices. Principal hindrances to adoption is the high cost of equipment and low throughput of HPP equipment, meaning high value, perishable materials are treated. Recent instances of commercialization of HPP in dairy industry can include HP treated Yogurt and Cheese spread. The varied physico-chemical and sensory properties obtained using the HPP offer exciting opportunities for the dairy industry. New opportunities in HPP may include exploring HP induced changes in milk giving functional ingredients, preservation of colostrum and human milk by HPP treatment, which may be of interest to the entrepreneurs. More research is required for optimization of the process in the future to fully establish it in the dairy industry. ACKNOWLEDGEMENTS The authors would like to thank all the researchers who have previously done the scientific work in the field. This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. REFERENCES [1] Rao, P. S., Chakraborty, S., Kaushik, N., Kaur, B. P., &amp; Hulle, N. R. S. (2014) High Hydrostatic Pressure Processing of Food Materials, Introduction to Advanced Food Process Engineering, CRC Press. [2] Hite, B. H. (1899) The effect of high pressure preservation of milk. Bulletin of the West Virginia Agricultural Experimental Station, 58, pp15&ndash;35. [3] Datta, N. &amp; Deeth, H. C. 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LWT-Food Science and Technology, Vol. 29, No.7, pp606-625 [38] Lim, S. Y., Swanson, B. G., &amp; Clark, S. (2008) High hydrostatic pressure modifications of whey protein concentrate for improved functional properties. Journal of Dairy Science, Vol. 91, pp1299&ndash; 1307. [39] Huppertz, T., Smiddy, M.A., Kelly, A.L., Goff, H.D. (2011) Effect of high pressure treatment of mix on ice cream manufacture. International Dairy Journal, Vol. 21, pp718&ndash;727. [40] Harte, F., Amonte, M., Luedecke, L., Swanson, B. G., &amp; Barbosa-C&aacute;novas, G. V. (2002) Yield stress and microstructure of set yoghurt made from high hydrostatic pressure-treated full fat milk. Journal of Food Science, Vol. 67, pp2245&ndash;2250. [41] Capellas, M., &amp; Needs, E. (2003) Physical properties of yoghurt prepared from pressure-treated concentrated or fortified milks. Milchwissenschaft, Vol. 58, pp46&ndash;48. [42] Udabage, P.,Augustin, M. A.,Versteeg,C. Puvanenthiran, A.,Yoo J. A., Allen, N., McKinnon, I., Smiddy, M., &amp; Kelly A. L. (2010) Properties of low-fat stirred yoghurts made from high-pressureprocessed skim milk. Innovative Food Science and Emerging Technology, Vol. 11, pp32&ndash;38. [43] Capellas, M., Noronha, R., Mor-Muh, M., Needs, E. (2002) Effect of high pressure on the consumer liking and preference of yoghurt. High Pressure Research, Vol. 22, pp701&ndash;704. [44] Carroll, T., Ping, C., Harnett, M., &amp; Harnett, J. (2004) Pressure treating food to reduce spoilage. International Patent Wo 2004/032655. [45] Jankowska, A., Reps, A., Proszek, A., and Krasowska, M. 2005. Effect of high pressure on microflora and sensory characteristics of yoghurt. Polish Journal of Food Nutrition and Science Vol. 14/55, No 1, pp79-84. [46] Eberhard, P., Strahm, W. &amp; Eyer, H. (1999) High pressure treatment of whipped cream, Agrarforschung, Vol. 6, pp352-354. Food Science and Technology : An International Journal (FSTJ) Vol.1, No.1 44 [47] Rasanayagam, V., Balasubramaniam, V. M., Ting, E., Sizer, C. E., Bush, C., &amp; Anderson, C. (2003) Compression heating of selected fatty food materials during high-pressure processing. Journal of Food Science, Vol. 68, pp254&ndash;259. [48] Kelly, A. L., Kothari, K. I.,Voigt, D. D., &amp; Huppertz, T. (2009) Improving the technological and functional properties of milk by high-pressure processing, Dairy-Derived Ingredients: Food and Nutraceutical Uses, Woodhead Publishing Ltd. [49] Li, S. Q., Zhang, H. Q., Balasubramaniam, V. M., Lee, Y. Z., Bomser, J. A., Schwartz, S. J., &amp; Dunne, C. P. (2006) Comparison of effects of high-pressure processing and heat treatment on immunoactivity of bovine milk immunoglobulin G in enriched soymilk under equivalent microbial inactivation levels. Journal of Agricultural and Food Chemisty, Vol. 54, pp739&ndash;746. [50] Trujillo, A.J., Castro, N., Quevedo, J.M. et al. (2007) Effect of heat and high-pressure treatments on microbiological quality and immunoglobulin G stability of caprine colostrum. Journal of Dairy Science, Vol. 90: 833&ndash;839. [51] Indyk, H.E., Williams, J.W., Patel, H.A. (2008) Analysis of denaturation of bovine IgG by heat and high pressure using an optical biosensor. International Dairy Journal, Vol. 18, pp359&ndash;366. [52] Orlien, V., Knudsen, J.C., Colon, M., &amp; Skibsted, L.H. (2006) Dynamics of casein micelles in skim milk during and after high pressure treatment. Food Chemistry, Vol. 98, pp513&ndash;521. [53] Sahu, J.K. (2010) Coagulation kinetics of high pressure treated acidified milk gel for preparation chhana (an Indian soft cottage cheese). International Journal Food Properties, Vol. 13, pp1054&ndash;1065. [54] Permanyer, M., Castellote, C., Ram&iacute;rez-Santana, C., Aud&iacute;, C., P&eacute;rez-Cano, F. J., Castell, M., L&oacute;pezSabater, M. C., &amp; Franch, A. (2010) Maintenance of breast milk immunoglobulin A after highpressure processing. Journal of Dairy Science, Vol. 93, pp877&ndash;883. [55] Hinrichs, J., Rademacher, B., &amp; Kessler, H.G. (1996) Reaction kinetics of pressure induced denaturation of whey proteins. Milchwissenschaft, Vol. 51, pp504&ndash;509. [56] Kalichevsky, M.T., Knorr, D., &amp; Lillford, P.J. (1995) Potential food applications of high-pressure effects on ice-water transitions. Trends in Food Science and Technology, Vol. 6, pp253&ndash;259. [57] Huppertz, T., Fox, P. F., De Kruif, K.G., &amp; Kelly, A. L. (2006) High pressure-induced changes in bovine milk proteins: a review. Biochimica et Biophysica Acta &ndash; Proteins and Proteomics, Vol. 1764, 593&ndash;598. [58] Huppertz, T., &amp; DeKruif, C. G. (2007a) Disruption and reassociation of casein micelles during high pressure treatment: influence of whey proteins. Journal of Dairy Research, Vol. 74, pp194&ndash;197. [59] Huppertz, T., De Kruif, C. G. (2006) Disruption and reassociation of casein micelles under high pressure: influence of milk serum composition and casein micelle concentration. Journal of Agricultural and Food Chemisty, Vol. 54, pp5903&ndash;5909. [60] Anema, S.G. (2008) Effect of milk solids concentration on whey protein denaturation, particle size changes and solubilization of casein in high pressure treated skim milk. International Dairy Journal, Vol. 18, pp228&ndash;235. [61] L&oacute;pez-Fandi&ntilde;o, R., Carrascosa, A.V. &amp; Olano, A. (1996) The Effects of High Pressure on Whey Protein Denaturation and Cheese-Making Properties of Raw Milk. Journal of Dairy Science, Vol. 79, Issue 6, pp929&ndash;936. [62] Pandey, P.K., &amp; Ramaswamy, H.S. (1998) Effect of high pressure of milk on textural properties, moisture content and yield of Cheddar cheese. Paper presented at the IFT annual meeting, Atlanta. [63] Huppertz, T., Fox, P.F., Kelly, A.L. (2004) High pressure treatment of bovine milk: effects on casein micelles and whey proteins. Journal of Dairy Research, Vol. 71, pp97&ndash;106. [64] Sakharam, P., Prajapati, J.P., &amp; Jana, A.H. (2011) High Hydrostatic Pressure Treatment for Dairy Applications. National seminar &ldquo;Indian Dairy Industry - Opportunities And Challenges&rdquo;, 2014. pp176-180. URL : http://dairyknowledge.in/sites/default/files/ch17_0.pdf Accessed on 4th October 2017. Food Science and Technology : An International Journal (FSTJ) Vol.1, No.1 45 [65] Scollard, P. G., Beresford, T. P., Needs, E. C., Murphy, P. M. &amp; Kell, A. L. (2000) Plasmin activity, &beta;-lactoglobulin denaturation and proteolysis in high pressure treated milk. International Dairy Journal, Vol. 10, pp835&ndash;841. [66] Sainz, C.B., Younce, F.L., Rasco, B., &amp; Clark, S. (2009) Protease stability in bovine milk under combined thermal-high hydrostatic pressure treatment. Innovative Food Science and Emerging Technologies, Vol. 10, pp314&ndash;320. [67] Huppertz, T., Fox, P. F., &amp; Kelly, A. L. (2003) High pressure-induced changes in the creaming properties of bovine milk. Innoative Food Science and Emerging Technologies, Vol. 4, pp349&ndash;359. [68] Ye, A., Anema, S. G., &amp; Singh, H. (2004) High-pressure&ndash;induced interactions between milk fat globule membrane proteins and skim milk proteins in whole milk. Journal of Dairy Science, Vol. 87, pp4013&ndash;4022. [69] Kanno, C., Uchimura, T., Hagiwara, T., Ametani, M., &amp; Azuma, N. (1998) Effect of hydrostatic pressure on the physicochemical properties of bovine milk fat globules and the milk fat globule membrane, High Pressure Food Science, BioScience and chemistry, The Royal Society of Chemistry. [70] Abe, S., Kawashima, A., Masuda T., Suzuki, K., &amp; Suzuki, K. (1999) Evaluation of heat deterioration of hard milk fat as a function of pressurization time by chemiluminescence analysis. Food Science and Technology Research, Vol. 5, pp381&ndash;383. [71] Medina-Meza, I., Barnaba, G.C. Barbosa-C&aacute;novas, G. V. (2014) Effects of high pressure processing on lipid oxidation: A review. Innovative Food Science and Emerging Technologies, Vol. 22, pp1-10. [72] Johnston, D.E., Austin, B.A. &amp; Murphy, P.M. (1992).Effect of high hydrostatic pressure on milk. Milchwissenschaft, Vol. 47, pp760-763. [73] Huppertz, T., De Kruif, &amp; C.G. (2007b) High pressure-induced solubilisation of micellar calcium phosphate from cross-linked casein micelles. Colloids and Surfaces A, Vol. 295, pp264&ndash;268. [74] Lopez-Fandino, R., Olano, A. (1998) Effects of high pressures combined with moderate temperatures on the rennet coagulation properties of milk. International Dairy Journal, Vol. 8, pp623&ndash;627. AUTHORS Ronit Mandal is a M.Tech (specializing in Food Process Engineering) student at Agricultural and Food Engineering Department, Indian Institute of Technology, Kharagpur, India. He has completed his undergraduate studies in Dairy Technology from West Bengal University of Animal and Fishery Sciences, Kolkata, India. His research interests are food processing, novel non-thermal food processing, dairy process engineering. Dr. Rajni Kant is working as a Post-Doctoral Research Fellow in Agricultural and Food Engineering Department, Indian Institute of Technology, Kharagpur, India. He has obtained his Ph.D. from SHIATS, Allahabad, India. His research interests are dairy processing, non-destructive evaluation of food spoilage, food storage &amp; shelf life extension, food fortification &amp; bio-actives, new products and process development.

What strikes me about the habits of the people who spend so much time on the Net—well, it’s so new that we don't know what will come next—is in fact precisely how niche in character it is. You ask people what nets they are on, and they’re all so specialised! The Argentines on the Argentine Net and so forth. And it’s particularly the Argentines who are not in Argentina. (Anderson, in Gower, par. 5) The preceding quotation, taken from his 1996 interview with Eric Gower, sees Benedict Anderson reflecting on the formation of imagined, transnational communities on the Internet. Anderson is, of course, famous for his work on how nationalism, as an “imagined community,” gets constructed through the shared consumption of print media (6-7, 26-27); although its readers will never all see each other face to face, people consuming a newspaper or novel in a shared language perceive themselves as members of a collective. In this more recent interview, Anderson recognised the specific groupings of people in online communities: Argentines who find themselves outside of Argentina link up online in an imagined diaspora community. Over the course of the last decade and a half since Anderson spoke about Argentinian migrants and diaspora communities, we have witnessed an exponential growth of new forms of digital communication, including social networking sites (e.g. Facebook), Weblogs, micro-blogging (e.g. Twitter), and video-sharing sites (e.g. YouTube). Alongside these new means of communication, our current epoch of globalisation is also characterised by migration flows across, and between, all continents. In his book Modernity at Large, Arjun Appadurai recognised that “the twin forces of mass migration and electronic mediation” have altered the ways the imagination operates. Furthermore, these two pillars, human motion and digital mediation, are in constant “flux” (44). The circulation of people and digitally mediatised content proceeds across and beyond boundaries of the nation-state and provides ground for alternative community and identity formations. Appadurai’s intervention has resulted in increasing awareness of local, transnational, and global networking flows of people, ideas, and culturally hybrid artefacts. In this article, we analyse the various innovative tactics taken up by migrant youth to imagine digital diasporas. Inspired by scholars such as Appadurai, Avtar Brah and Paul Gilroy, we tease out—from a postcolonial perspective—how digital diasporas have evolved over time from a more traditional understanding as constituted either by a vertical relationship to a distant homeland or a horizontal connection to the scattered transnational community (see Safran, Cohen) to move towards a notion of “hypertextual diaspora.” With hypertextual diaspora, these central axes which constitute the understanding of diaspora are reshuffled in favour of more rhizomatic formations where affiliations, locations, and spaces are constantly destabilised and renegotiated. Needless to say, diasporas are not homogeneous and resist generalisation, but in this article we highlight common ways in which young migrant Internet users renew the practices around diaspora connections. Drawing from research on various migrant populations around the globe, we distinguish three common strategies: (1) the forging of transnational public spheres, based on maintaining virtual social relations by people scattered across the globe; (2) new forms of digital diasporic youth branding; and (3) the cultural production of innovative hypertexts in the context of more rhizomatic digital diaspora formations. Before turning to discuss these three strategies, the potential of a postcolonial framework to recognise multiple intersections of diaspora and digital mediation is elaborated. Hypertext as a Postcolonial Figuration Postcolonial scholars, Appadurai, Gilroy, and Brah among others, have been attentive to diasporic experiences, but they have paid little attention to the specificity of digitally mediated diaspora experiences. As Maria Fernández observes, postcolonial studies have been “notoriously absent from electronic media practice, theory, and criticism” (59). Our exploration of what happens when diasporic youth go online is a first step towards addressing this gap. Conceptually, this is clearly an urgent need since diasporas and the digital inform each other in the most profound and dynamic of ways: “the Internet virtually recreates all those sites which have metaphorically been eroded by living in the diaspora” (Ponzanesi, “Diasporic Narratives” 396). Writings on the Internet tend to favour either the “gold-rush” mentality, seeing the Web as a great equaliser and bringer of neoliberal progress for all, or the more pessimistic/technophobic approach, claiming that technologically determined spaces are exclusionary, white by default, masculine-oriented, and heteronormative (Everett 30, Van Doorn and Van Zoonen 261). For example, the recent study by Ito et al. shows that young people are not interested in merely performing a fiction in a parallel online world; rather, the Internet gets embedded in their everyday reality (Ito et al. 19-24). Real-life commercial incentives, power hierarchies, and hegemonies also get extended to the digital realm (Schäfer 167-74). Online interaction remains pre-structured, based on programmers’ decisions and value-laden algorithms: “people do not need a passport to travel in cyberspace but they certainly do need to play by the rules in order to function electronically” (Ponzanesi, “Diasporic Narratives” 405). We began our article with a statement by Benedict Anderson, stressing how people in the Argentinian diaspora find their space on the Internet. Online avenues increasingly allow users to traverse and add hyperlinks to their personal websites in the forms of profile pages, the publishing of preferences, and possibilities of participating in and affiliating with interest-based communities. Online journals, social networking sites, streaming audio/video pages, and online forums are all dynamic hypertexts based on Hypertext Markup Language (HTML) coding. HTML is the protocol of documents that refer to each other, constituting the backbone of the Web; every text that you find on the Internet is connected to a web of other texts through hyperlinks. These links are in essence at equal distance from each other. As well as being a technological device, hypertext is also a metaphor to think with. Figuratively speaking, hypertext can be understood as a non-hierarchical and a-centred modality. Hypertext incorporates multiplicity; different pathways are possible simultaneously, as it has “multiple entryways and exits” and it “connects any point to any other point” (Landow 58-61). Feminist theorist Donna Haraway recognised the dynamic character of hypertext: “the metaphor of hypertext insists on making connections as practice.” However, she adds, “the trope does not suggest which connections make sense for which purposes and which patches we might want to follow or avoid.” We can begin to see the value of approaching the Internet from the perspective of hypertext to make an “inquiry into which connections matter, why, and for whom” (128-30). Postcolonial scholar Jaishree K. Odin theorised how hypertextual webs might benefit subjects “living at the borders.” She describes how subaltern subjects, by weaving their own hypertextual path, can express their multivocality and negotiate cultural differences. She connects the figure of hypertext with that of the postcolonial: The hypertextual and the postcolonial are thus part of the changing topology that maps the constantly shifting, interpenetrating, and folding relations that bodies and texts experience in information culture. Both discourses are characterised by multivocality, multilinearity, openendedness, active encounter, and traversal. (599) These conceptions of cyberspace and its hypertextual foundations coalesce with understandings of “in-between”, “third”, and “diaspora media space” as set out by postcolonial theorists such as Bhabha and Brah. Bhabha elaborates on diaspora as a space where different experiences can be articulated: “These ‘in-between’ spaces provide the terrain for elaborating strategies of selfhood—singular or communal—that initiate new signs of identity, and innovative sites of collaboration, and contestation (4). (Dis-)located between the local and the global, Brah adds: “diaspora space is the point at which boundaries of inclusion and exclusion, of belonging and otherness, of ‘us’ and ‘them,’ are contested” (205). As youths who were born in the diaspora have begun to manifest themselves online, digital diasporas have evolved from transnational public spheres to differential hypertexts. First, we describe how transnational public spheres form one dimension of the mediation of diasporic experiences. Subsequently, we focus on diasporic forms of youth branding and hypertext aesthetics to show how digitally mediated practices can go beyond and transgress traditional formations of diasporas as vertically connected to a homeland and horizontally distributed in the creation of transnational public spheres. Digital Diasporas as Diasporic Public Spheres Mass migration and digital mediation have led to a situation where relationships are maintained over large geographical distances, beyond national boundaries. The Internet is used to create transnational imagined audiences formed by dispersed people, which Appadurai describes as “diasporic public spheres”. He observes that, as digital media “increasingly link producers and audiences across national boundaries, and as these audiences themselves start new conversations between those who move and those who stay, we find a growing number of diasporic public spheres” (22). Media and communication researchers have paid a lot of attention to this transnational dimension of the networking of dispersed people (see Brinkerhoff, Alonso and Oiarzabal). We focus here on three examples from three different continents. Most famously, media ethnographers Daniel Miller and Don Slater focused on the Trinidadian diaspora. They describe how “de Rumshop Lime”, a collective online chat room, is used by young people at home and abroad to “lime”, meaning to chat and hang out. Describing the users of the chat, “the webmaster [a Trini living away] proudly proclaimed them to have come from 40 different countries” (though massively dominated by North America) (88). Writing about people in the Greek diaspora, communication researcher Myria Georgiou traced how its mediation evolved from letters, word of mouth, and bulletins to satellite television, telephone, and the Internet (147). From the introduction of the Web, globally dispersed people went online to get in contact with each other. Meanwhile, feminist film scholar Anna Everett draws on the case of Naijanet, the virtual community of “Nigerians Living Abroad”. She shows how Nigerians living in the diaspora from the 1990s onwards connected in global transnational communities, forging “new black public spheres” (35). These studies point at how diasporic people have turned to the Internet to establish and maintain social relations, give and receive support, and share general concerns. Establishing transnational communicative networks allows users to imagine shared audiences of fellow diasporians. Diasporic imagination, however, goes beyond singular notions of this more traditional idea of the transnational public sphere, as it “has nowadays acquired a great figurative flexibility which mostly refers to practices of transgression and hybridisation” (Ponzanesi, “Diasporic Subjects” 208). Below we recognise another dimension of digital diasporas: the articulation of diasporic attachment for branding oneself. Mocro and Nikkei: Diasporic Attachments as a Way to Brand Oneself In this section, we consider how hybrid cultural practices are carried out over geographical distances. Across spaces on the Web, young migrants express new forms of belonging in their dealing with the oppositional motivations of continuity and change. The generational specificity of this experience can be drawn out on the basis of the distinction between “roots” and “routes” made by Paul Gilroy. In his seminal book The Black Atlantic: Modernity and Double Consciousness, Gilroy writes about black populations on both sides of the Atlantic. The double consciousness of migrant subjects is reflected by affiliating roots and routes as part of a complex cultural identification (19 and 190). As two sides of the same coin, roots refer to the stable and continuing elements of identities, while routes refer to disruption and change. Gilroy criticises those who are “more interested in the relationship of identity to roots and rootedness than in seeing identity as a process of movement and mediation which is more appropriately approached via the homonym routes” (19). He stresses the importance of not just focusing on one of either roots or routes but argues for an examination of their interplay. Forming a response to discrimination and exclusion, young migrants in online networks turn to more positive experiences such as identification with one’s heritage inspired by generational specific cultural affiliations. Here, we focus on two examples that cross two continents, showing routed online attachments to “be(com)ing Mocro”, and “be(coming) Nikkei”. Figure 1. “Leipe Mocro Flavour” music video (Ali B) The first example, being and becoming “Mocro”, refers to a local, bi-national consciousness. The term Mocro originated on the streets of the Netherlands during the late 1990s and is now commonly understood as a Dutch honorary nickname for youths with Moroccan roots living in the Netherlands and Belgium. A 2003 song, Leipe mocro flavour (“Crazy Mocro Flavour”) by Moroccan-Dutch rapper Ali B, familiarised a larger group of people with the label (see Figure 1). Ali B’s song is exemplary for a wider community of youngsters who have come to identify themselves as Mocros. One example is the Marokkanen met Brainz – Hyves (Mo), a community page within the Dutch social networking site Hyves. On this page, 2,200 youths who identify as Mocro get together to push against common stereotypes of Moroccan-Dutch boys as troublemakers and thieves and Islamic Moroccan-Dutch girls as veiled carriers of backward traditions (Leurs, forthcoming). Its description reads, “I assume that this Hyves will be the largest [Mocro community]. Because logically Moroccans have brains” (our translation): What can you find here? Discussions about politics, religion, current affairs, history, love and relationships. News about Moroccan/Arabic Parties. And whatever you want to tell others. Use your brains. Second, “Nikkei” directs our attention to Japanese migrants and their descendants. The Discover Nikkei website, set up by the Japanese American National Museum, provides a revealing description of being and becoming Nikkei: As Nikkei communities form in Japan and throughout the world, the process of community formation reveals the ongoing fluidity of Nikkei populations, the evasive nature of Nikkei identity, and the transnational dimensions of their community formations and what it means to be Nikkei. (Japanese American National Museum) This site was set up by the Japanese American National Museum for Nikkei in the global diaspora to connect and share stories. Nikkei youths of course also connect elsewhere. In her ethnographic online study, Shana Aoyama found that the social networking site Hi5 is taken up in Peru by young people of Japanese heritage as an avenue for identity exploration. She found group confirmation based on the performance of Nikkei-ness, as well as expressions of individuality. She writes, “instead of heading in one specific direction, the Internet use of Nikkei creates a starburst shape of identity construction and negotiation” (119). Mocro-ness and Nikkei-ness are common collective identification markers that are not just straightforward nationalisms. They refer back to different homelands, while simultaneously they also clearly mark one’s situation of being routed outside of this homeland. Mocro stems from postcolonial migratory flows from the Global South to the West. Nikkei-ness relates to the interesting case of the Japanese diaspora, which is little accounted for, although there are many Japanese communities present in North and South America from before the Second World War. The context of Peru is revealing, as it was the first South American country to accept Japanese migrants. It now hosts the second largest South American Japanese diaspora after Brazil (Lama), and Peru’s former president, Alberto Fujimoro, is also of Japanese origin. We can see how the importance of the nation-state gets blurred as diasporic youth, through cultural hybridisation of youth culture and ethnic ties, initiates subcultures and offers resistance to mainstream western cultural forms. Digital spaces are used to exert youthful diaspora branding. Networked branding includes expressing cultural identities that are communal and individual but also both local and global, illustrative of how “by virtue of being global the Internet can gift people back their sense of themselves as special and particular” (Miller and Slater 115). In the next section, we set out how youthful diaspora branding is part of a larger, more rhizomatic formation of multivocal hypertext aesthetics. Hypertext Aesthetics In this section, we set out how an in-between, or “liminal”, position, in postcolonial theory terms, can be a source of differential and multivocal cultural production. Appadurai, Bhabha, and Gilroy recognise that liminal positions increasingly leave their mark on the global and local flows of cultural objects, such as food, cinema, music, and fashion. Here, our focus is on how migrant youths turn to hypertextual forms of cultural production for a differential expression of digital diasporas. Hypertexts are textual fields made up of hyperlinks. Odin states that travelling through cyberspace by clicking and forging hypertext links is a form of multivocal digital diaspora aesthetics: The perpetual negotiation of difference that the border subject engages in creates a new space that demands its own aesthetic. This new aesthetic, which I term “hypertext” or “postcolonial,” represents the need to switch from the linear, univocal, closed, authoritative aesthetic involving passive encounters characterising the performance of the same to that of non-linear, multivocal, open, non-hierarchical aesthetic involving active encounters that are marked by repetition of the same with and in difference. (Cited in Landow 356-7) On their profile pages, migrant youth digitally author themselves in distinct ways by linking up to various sites. They craft their personal hypertext. These hypertexts display multivocal diaspora aesthetics which are personal and specific; they display personal intersections of affiliations that are not easily generalisable. In several Dutch-language online spaces, subjects from Dutch-Moroccan backgrounds have taken up the label Mocro as an identity marker. Across social networking sites such as Hyves and Facebook, the term gets included in nicknames and community pages. Think of nicknames such as “My own Mocro styly”, “Mocro-licious”, “Mocro-chick”. The term Mocro itself is often already multilayered, as it is often combined with age, gender, sexual preference, religion, sport, music, and generationally specific cultural affiliations. Furthermore, youths connect to a variety of groups ranging from feminist interests (“Women in Charge”), Dutch nationalism (“I Love Holland”), ethnic affiliations (“The Moroccan Kitchen”) to clothing (the brand H&amp;M), and global junk food (McDonalds). These diverse affiliations—that are advertised online simultaneously—add nuance to the typical, one-dimensional stereotype about migrant youth, integration, and Islam in the context of Europe and Netherlands (Leurs, forthcoming). On the online social networking site Hi5, Nikkei youths in Peru, just like any other teenagers, express their individuality by decorating their personal profile page with texts, audio, photos, and videos. Besides personal information such as age, gender, and school information, Aoyama found that “a starburst” of diverse affiliations is published, including those that signal Japanese-ness such as the Hello Kitty brand, anime videos, Kanji writing, kimonos, and celebrities. Also Nikkei hyperlink to elements that can be identified as “Latino” and “Chino” (Chinese) (104-10). Furthermore, users can show their multiple affiliations by joining different “groups” (after which a hyperlink to the group community appears on the profile page). Aoyama writes “these groups stretch across a large and varied scope of topics, including that of national, racial/ethnic, and cultural identities” (2). These examples illustrate how digital diasporas encompass personalised multivocal hypertexts. With the widely accepted adagio “you are what you link” (Adamic and Adar), hypertextual webs can be understood as productions that reveal how diasporic youths choose to express themselves as individuals through complex sets of non-homogeneous identifications. Migrant youth connects to ethnic origin and global networks in eclectic and creative ways. The concept of “digital diaspora” therefore encapsulates both material and virtual (dis)connections that are identifiable through common traits, strategies, and aesthetics. Yet these hypertextual connections are also highly personalised and unique, offering a testimony to the fluid negotiations and intersections between the local and the global, the rooted and the diasporic. Conclusions In this article, we have argued that migrant youths render digital diasporas more complex by including branding and hypertextual aesthetics in transnational public spheres. Digital diasporas may no longer be understood simply in terms of their vertical relations to a homeland or place of origin or as horizontally connected to a clearly marked transnational community; rather, they must also be seen as engaging in rhizomatic digital practices, which reshuffle traditional understandings of origin and belonging. Contemporary youthful digital diasporas are therefore far more complex in their engagement with digital media than most existing theory allows: connections are hybridised, and affiliations are turned into practices of diasporic branding and becoming. There is a generational specificity to multivocal diaspora aesthetics; this specificity lies in the ways migrant youths show communal recognition and express their individuality through hypertext which combines affiliation to their national/ethnic “roots” with an embrace of other youth subcultures, many of them transnational. These two axes are constantly reshuffled and renegotiated online where, thanks to the technological possibilities of HTML hypertext, a whole range of identities and identifications may be brought together at any given time. We trust that these insights will be of interest in future discussion of online networks, transnational communities, identity formation, and hypertext aesthetics where much urgent and topical work remains to be done. References Adamic, Lada A., and Eytan Adar. “You Are What You Link.” 2001 Tenth International World Wide Web Conference, Hong Kong. 26 Apr. 2010. ‹http://www10.org/program/society/yawyl/YouAreWhatYouLink.htm›. Ali B. “Leipe Mocro Flavour.” ALIB.NL / SPEC Entertainment. 2007. 4 Oct. 2010 ‹http://www3.alib.nl/popupAlibtv.php?catId=42&amp;contentId=544›. Alonso, Andoni, and Pedro J. Oiarzabal. Diasporas in the New Media Age. Reno: U of Nevada P, 2010. Anderson, Benedict. Imagined Communities: Reflections on the Origin and Spread of Nationalism. Rev. ed. London: Verso, 2006 (1983). Aoyama, Shana. Nikkei-Ness: A Cyber-Ethnographic Exploration of Identity among the Japanese Peruvians of Peru. Unpublished MA thesis. South Hadley: Mount Holyoke, 2007. 1 Feb. 2010 ‹http://hdl.handle.net/10166/736›. Appadurai, Arjun. Modernity at Large: Cultural Dimensions of Globalization. Minneapolis: U of Minnesota P, 1996. Bhabha, Homi. The Location of Culture. New York: Routledge, 1994. Brah, Avtar. Cartographies of Diaspora: Contesting Identities. London: Routledge, 1996. Brinkerhoff, Jennifer M. Digital Diasporas: Identity and Transnational Engagement. Cambridge: Cambridge UP, 2009. Cohen, Robin. Global Diasporas: An Introduction. London: U College London P, 1997. Everett, Anna. Digital Diaspora: A Race for Cyberspace. Albany: SUNY, 2009. Fernández, María. “Postcolonial Media Theory.” Art Journal 58.3 (1999): 58-73. Georgiou, Myria. Diaspora, Identity and the Media: Diasporic Transnationalism and Mediated Spatialities. Creskill: Hampton Press, 2006. Gilroy, Paul. The Black Atlantic: Modernity and Double Consciousness. London: Verso, 1993. Gower, Eric. “When the Virtual Becomes the Real: A Talk with Benedict Anderson.” NIRA Review, 1996. 19 Apr. 2010 ‹http://www.nira.or.jp/past/publ/review/96spring/intervi.html›. Haraway, Donna. Modest Witness@Second Millennium. FemaleMan Meets OncoMouse: Feminism and Technoscience. New York: Routledge, 1997. Ito, Mizuko, et al. Hanging Out, Messing Out, and Geeking Out: Kids Living and Learning with New Media. Cambridge: MIT Press, 2010. Japanese American National Museum. “Discover Nikkei: Japanese Migrants and Their Descendants.” Discover Nikkei, 2005. 4 Oct. 2010. ‹http://www.discovernikkei.org/en/›. Lama, Abraham. “Home Is Where the Heartbreak Is for Japanese-Peruvians.” Asia Times 16 Oct. 1999. 6 May 2010 ‹http://www.atimes.com/japan-econ/AJ16Dh01.html›. Landow, George P. Hypertext 3.0. Critical Theory and New Media in an Era of Globalization. Baltimore: Johns Hopkins UP, 2006. Leurs, Koen. Identity, Migration and Digital Media. Utrecht: Utrecht University. PhD Thesis, forthcoming. Miller, Daniel, and Don Slater. The Internet: An Etnographic Approach. Oxford: Berg, 2000. Mo. “Marokkanen met Brainz.” Hyves, 23 Feb. 2008. 4 Oct. 2010. ‹http://marokkaansehersens.hyves.nl/›. Odin, Jaishree K. “The Edge of Difference: Negotiations between the Hypertextual and the Postcolonial.” Modern Fiction Studies 43.3 (1997): 598-630. Ponzanesi, Sandra. “Diasporic Narratives @ Home Pages: The Future as Virtually Located.” Colonies – Missions – Cultures in the English-Speaking World. Ed. Gerhard Stilz. Tübingen: Stauffenburg, 2001. 396–406. Ponzanesi, Sandra. “Diasporic Subjects and Migration.” Thinking Differently: A Reader in European Women's Studies. Ed. Gabrielle Griffin and Rosi Braidotti. London: Zed Books, 2002. 205–20. Safran, William. “Diasporas in Modern Societies: Myths of Homeland and Return.” Diaspora 1.1 (1991): 83-99. Schäfer, Mirko T. Bastard Culture! How User Participation Transforms Cultural Production. Amsterdam: Amsterdam UP, 2011. Van Doorn, Niels, and Liesbeth van Zoonen. “Theorizing Gender and the Internet: Past, Present, and Future.” Routledge Handbook of Internet Politics. Ed. Andrew Chadwick and Philip N. Howard. London: Routledge. 261-74.

&#x0D; &#x0D; &#x0D; Synonymous with globalism, the mobile phone has become an integral part of contemporary everyday life. As a global medium, the mobile phone is a compelling phenomenon that demonstrates the importance of the local in shaping and adapting the technology. The adaptation and usage of the mobile phone can be read on two levels simultaneously – the micro, individual level and the macro, socio-cultural level. Symbolic of the pervasiveness and ubiquity of global ICTs (Information and Communication Technologies) in the everyday, the mobile phone demonstrates that the experiences of the local are divergent in the face of global convergence. The cultural significance of mobile technologies sees it often as a symbol for discussion around issues of democracy, capitalism, individualism and redefinitions of place. These debates are, like all forms of mediation, riddled with paradoxes. As Michael Arnold observes, mobile media is best encapsulated by the notion of “janus-faced” which sees an ongoing process of pushing and pulling whereby one is set free to be anywhere but is on a leash to whims of others anytime. This paradox, for Arnold, is central to all technologies; the more we try to overcome various forms of distance (geographic, temporal, cultural), the more we avoid closeness and intimacy. For Jack Qui, mobile technologies are indeed the ultimate “wireless leash”. These paradoxes see themselves played in a variety of ways. This is particularly the case in the Asia-Pacific region, which houses divergence and uneven adoption, production and consumption of mobile technologies. The region simultaneously displays distinctive characteristics and a possible future of mobile media worldwide. From the so-called ‘centres’ for mobile innovation such as Tokyo and Seoul that have gained attention in global press to Asian “tigers” such as Singapore, Hong Kong and Taiwan that demonstrate high penetration rates (Singapore has a 110% penetration rate), the region often plays out its dynamics through mobile technologies. The Philippines, for example, is known as the ‘texting capital of the world’ with 300 million text messages sent per day. Moreover, the region has taken central focus for debates around the so-called democratic potential of the mobile phone through examples such as the demise of President Joseph Estrada in the Philippines and the election of President Roh in South Korea (Pertierra, Transforming Technologies; Kim). Through the use of mobile technologies and the so-called rise of the “prosumer” (consumer as producer), we can see debates about the rhetoric and reality of democracy and capitalism in the region. In the case of nascent forms of capitalism, the rise of the mobile phone in China has often been seen as China’s embrace, and redefinition, of capitalism away from being once synonymous with westernisation. As Chua Beng Huat observes, after the 1997 financial crisis in the region notions of consumerism and modernity ceased to be equated with westernization. In the case of China, the cell phone has taken on a pivotal role in everyday life with over 220 billion messages – over half the world’s SMS – sent yearly in China. Despite the ubiquity and multi-layered nature of mobile media in the region, this area has received little attention in the growing literature on mobile communication globally. Publications often explore ‘Asia’ in the context of ‘global’ media or Asia in contrast to Europe. Examples include Katz and Aakhus’s (eds.) seminal anthology Perpetual Contact, Pertierra’s (ed.) The Social Construction and Usage of Communication Technologies: European and Asian experiences and, more recently, Castells et al., Mobile Communication and Society: A Global Perspective. When publications do focus specifically on ‘Asia’, they single out particular locations in the region, such as Ito et al.’s compelling study on Japan, Personal, Portable, Pedestrian: Mobile Phones in Japanese Life and Pertierra’s eloquent discussion of the Philippines in Transforming Technologies: Altered Selves. This issue of M/C Journal attempts to address the dynamic and evolving role of mobile technologies in the Asia-Pacific region. By deploying various approaches to different issues involving mobile media, this issue aims to connect, through a regional imaginary, some of the nuances of local experience within the Asia-Pacific. As a construct, the region of the Asia-Pacific is ever evolving with constantly shifting economic and political power distributions. The rapid economic growth of parts of the region (Japan, Singapore, Taiwan, Korea, Hong Kong, and now China, Vietnam, Malaysia, the Philippines and Indonesia) over the last two to three decades, has led to increasing linkages between these nations in creating transnational networks. The boundaries of the Asia-Pacific are indeterminate and open to contestation and social construction. Initially, the Asia-Pacific was a Euro-American invention, however, its ‘Asian’ content is now playing a greater role in self-constructions, and in influencing the economic, cultural and political entity that is the Asia-Pacific. There have been alternative terms and definitions proffered to describe or delimit the area posited as the Asia-Pacific in an attempt to acknowledge, or subsume, the hierarchies inherent within the region. For example, John Eperjesi has critiqued the ‘American Pacific’ which “names the regional imaginary through which capital looked to expand into Asia and the Pacific at the turn of the [last] century” (195). Arif Dirlik has also suggested two other terms: ‘Asian Pacific’ and ‘Euro-American Pacific.’ He suggests, “the former refers not just to the region’s location, but, more important, to its human constitution; the latter refers to another human component of the region (at least at present) and also to its invention as a regional structure.” (“Asia-Pacific Idea”, 64). Together, Rob Wilson and Arif Dirlik use the configuration ‘Asia/Pacific’ to discuss the region as a space of cultural production, social migration, and transnational innovation, whereby “the slash would signify linkage yet difference” (6). These various terms are useful only insofar as they expose the ideological bases of the definitions, and identify its centre(s). In this emphasis on geography, it is important not to obscure the temporal and spatial characteristics of human activities that constitute regions. As Arif Dirlik notes, “[an] emphasis on human activity shifts attention from physical area to the construction of geography through human interactions; it also underlines the historicity of the region’s formations” (What Is in a Rim?, 4). The three-part structure of this issue seeks to provide various perspectives on the use of mobile technologies and media – from a macro, regional level, to micro, local case studies – in the context of both historical and contemporary formations and definitions of the Asia-Pacific. In an age of mobile technologies we see that rather than erode, notions of place and locality take on increasing significance. The first four papers by Jaz Hee-jeong Choi, Gerard Raiti, Yasmin Ibrahim, and Collette Snowden &amp; Kerry Green highlight some of the key concepts and phenomena associated with mobile media in the region. Choi’s paper provides a wonderful introduction to the culture of mobile technologies in East Asia, focusing largely on South Korea, China and Japan. She problematises the rhetoric surrounding technological fetishism and techno-orientalism in definitions of ‘mobile’ and ‘digital’ East Asia and raises important questions regarding the transformation and future of East Asia’s mobile cultures. Gerard Raiti examines the behemoth of globalization from the point of view of personal intimacy. He asks us to reconsider notions of intimacy in a period marked by co-presence; particularly in light of the problematic conflation between love and technological intimacy. Yasmin Ibrahim considers the way the body is increasingly implicated through the personalisation of mobile technologies and becomes a collaborator in the creation of media events. Ibrahim argues that what she calls the ‘personal gaze’ of the consumer is contributing to the visual narratives of global and local events. What we have is a figure of the mediated mobile body that participates in the political economy of events construction. The paradoxical role of mobile technologies as both pushing and pulling us, helping and hindering us (Arnold) is taken up in Collette Snowden and Kerry Green’s paper on the role of media reporting, mobility and trauma. Extending some of Ibrahim’s comments in the specific case of the reporting of traumatic events, Snowden and Green provide a wonderful companion piece about how media reporting is being transformed by contemporary mobile practices. As an integral component of contemporary visual cultures, camera phone practices are arguably both extending and creating emerging ways of seeing and representing. In the second section, we begin our case studies exploring the socio-cultural particularities of various adaptations of mobile media within specific locations in the Asia-Pacific. Randy Jay C. Solis elaborates on Gerard Raiti’s discussion of intimacy and love by exploring how the practice of ‘texting’ has contributed to the development of romantic relationships in the Philippines in terms of its convenience and affordability. Lee Humphreys and Thomas Barker further extend this discussion by investigating the way Indonesians use the mobile phone for dating and sex. As in Solis’s article, the authors view the mobile phone as a tool of communication, identity management and social networking that mediates new forms of love, sex and romance in Indonesia, particularly through mobile dating software and mobile pornography. Li Li’s paper takes the playful obsession the Chinese and South Koreans have with lucky numbers and locates its socio-cultural roots. Through a series of semi-structured interviews, the author traces this use of lucky mobile numbers to the rise of consumerism in China and views this so-called ‘superstition’ in terms of the entry into modernity for both China and South Korea. Chih-Hui Lai’s paper explores the rise of Web 2.0. in Taiwan, which, in comparison to other locations in the region, is still relatively under-documented in terms of its usage of mobile media. Here Lai addresses this gap by exploring the burgeoning role of mobile media to access and engage with online communities through the case study of EzMoBo. In the final section we problematise Australia’s place in the Asia-Pacific and, in particular, the nation’s politically and culturally uncomfortable relationship with Asia. Described as ‘west in Asia’ by Rao, and as ‘South’ of the West by Gibson, Yue, and Hawkins, Australia’s uneasy relationship with Asia deserves its own location. We begin this section with a paper by Mariann Hardey that presents a case study of Australian university students and their relationship to, and with, the mobile phone, providing original empirical work on the country’s ‘iGeneration’. Next Linda Leung’s critique of mobile telephony in the context of immigration detention centres engages with the political dimensions of technology and difference between connection and contact. Here we reminded of the luxury of mobile technologies that are the so-called necessity of contemporary everyday life. We are also reminded of the ‘cost’ of different forms of mobility and immobility – technological, geographic, physical and socio-cultural. Leung’s discussion of displacement and mobility amongst refugees calls upon us to reconsider some of the conflations occurring around mobile telephony and new media outside the comfort of everyday urbanity. The final paper, by Peter B. White and Naomi Rosh White, addresses the urban and rural divide so pointed in Australia (with 80% of the population living in urban areas) by discussing an older, though still relevant mobile technology, the CB radio. This paper reminds us that despite the technological fetishism of urban Australia, once outside of urban contexts, we are made acutely aware of Australia as a land containing a plethora of black spots (in which mobile phones are out of range). All of the papers in this issue address, in their own way, theoretical and empirical ‘black spots’ in research and speak to the ‘future’ of mobile media in a region that, while diverse, is being increasingly brought together by technologies such as the mobile phone. Lastly, we are pleased to include a photo essay by Andrew Johnson. Entitled Zeitgeist, this series of artworks sees Johnson exploring the symbolic dimensions of the hand phone in South Korea by drawing on the metaphor of the dust mask. According the Johnson, these images refer to ‘the visibility and invisibility of communication’ that characterises the spirit of our time. The cover image is by Larissa Hjorth as part of her Snapshots: Portrait of the Mobile series conducted whilst on an Asialink residency at Ssamzie space (Seoul, South Korea) in 2005. The editors would like to offer a special note of thanks to all of our external reviewers who answered our pleas for help with willingness, enthusiasm, and especially, promptness. This issue could not have been completed without your support. References Arnold, Michael. “On the Phenomenology of Technology: The ‘Janus-Faces’ of Mobile Phones.” Information and Organization 13 (2003): 231-256. Castells, Manuel, et al., Mobile Communication and Society: A Global Perspective. Cambridge, Mass.: MIT Press, 2007. Chua, Beng Huat, ed. Consumption in Asia. London: Routledge, 2000. Dirlik, Arif. “The Asia-Pacific Idea: Reality and Representation in the Invention of a Regional Structure.” Journal of World History 3.1 (1992): 55-79. Dirlik, Arif, ed. What Is in a Rim? Critical Perspectives on the Pacific Region Idea. Boulder: Westview, 1993. Eperjesi, John. “The American Asiatic Association and the Imperialist Imaginary of the American Pacific.” Boundary 2 28.1 (Spring 2001): 195-219. Gibson, Ross. South of the West: Postcolonialism and the Narrative Construction of Australia. Bloomington and Indianapolis: Indiana UP, 1996. Ito, Mizuko, Daisuke Okabe, and Misa Matsuda, eds. Personal, Portable, Pedestrian: Mobile Phones in Japanese Life. Cambridge, Mass.: MIT Press, 2005. Katz, James E., and Mark Aakhus, eds. Perpetual Contact: Mobile Communications, Private Talk, Public Performance. Cambridge: Cambridge UP, 2002 Kim, Shin Dong. “The Shaping of New Politics in the Era of Mobile and Cyber Communication.” Mobile Democracy: Essays on Society, Self and Politics. Ed. Kristof Nyiri. Vienna: Van Passen Verlag, 2003. Pertierra, Raul, ed. The Social Construction and Usage of Communication Technologies: European and Asian Experiences. Singapore: Singapore UP, 2005. –––. Transforming Technologies: Altered Selves. Philippines: De La Salle UP, 2006. Qui, Jack. “The Wireless Leash: Mobile Messaging Service as a Means of Control.” International Journal of Communication 1 (2007): 74-91. Rao, Madanmohan, ed. News Media and New Media: The Asia-Pacific Internet Handbook. Singapore: Times Academic Press, 2004. Wilson, Robert, and Afir Dirlik, eds. Asia/Pacific as Space of Cultural Production. Durham: Duke UP, 1995. Yue, Audrey. “Asian Australian Cinema, Asian-Australian Modernity.” Diaspora: Negotiating Asian-Australia. Eds. Helen Gilbert et al. St Lucia: U of Queensland P, 2000. 190–99. Yue, Audrey, and Gay Hawkins. “Going South.” New Formations 40 (Spring 2000): 49-63. &#x0D; &#x0D; &#x0D; &#x0D; Citation reference for this article&#x0D; &#x0D; MLA Style&#x0D; Hjorth, Larissa, and Olivia Khoo. "Collect Calls." M/C Journal 10.1 (2007). echo date('d M. Y'); ?&gt; &lt;http://journal.media-culture.org.au/0703/00-editorial.php&gt;. APA Style&#x0D; Hjorth, L., and O. Khoo. (Mar. 2007) "Collect Calls," M/C Journal, 10(1). Retrieved echo date('d M. Y'); ?&gt; from &lt;http://journal.media-culture.org.au/0703/00-editorial.php&gt;. &#x0D;

The soft error rates per single-bit due to alpha particles in sub-micron technology is expectedly reducedas the feature size is shrinking. On the other hand, the complexity and density of integrated systems are accelerating which demand ecient soft error protection mechanisms, especially for on-chip communication. Using soft error protection method has to satisfy tight requirements for the area and energy consumption, therefore a low complexity and low redundancy coding method is necessary. In this work, we propose a method to enhance Parity Product Code (PPC) and provide adaptation methods for this code. First, PPC is improved as forward error correcting using transposable retransmissions. Then, to adapt with dierent error rates, an augmented algorithm for configuring PPC is introduced. The evaluation results show that the proposed mechanism has coding rates similar to Parity check’s and outperforms the original PPC.Keywords&#x0D; Error Correction Code, Fault-Tolerance, Network-on-Chip.&#x0D; References&#x0D; [1] R. Baumann, Radiation-induced soft errors in advanced semiconductor technologies, IEEETransactions on Device and materials reliability. 5-3 (2005) 305–316. https://doi.org/10.1109/tdmr.2005.853449.[2] N. Seifert, B. Gill, K. Foley, P. Relangi, Multi-cell upset probabilities of 45nm high-k + metal gateSRAM devices in terrestrial and space environments, in: IEEE International Reliability Physics Symposium 2008, IEEE, AZ, USA, 2008, pp. 181–186.[3] S. Lee, I. Kim, S. Ha, C.-s. Yu, J. Noh, S. Pae, J. Park, Radiation-induced soft error rate analyses for 14 nmFinFET SRAM devices, in: 2015 IEEE International Reliability Physics Symposium (IRPS), IEEE, CA, USA, 2015, pp. 4B–1.[4] R. Hamming, Error detecting and error correcting codes, Bell Labs Tech. 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At a time when almost every human transgression, illness, profession and other personal aspect of life has been chronicled in autobiographical writing (Rak)—in 1998 Zinsser called ours “the age of memoir” (3)—writing about fat is one of the most recent subjects to be addressed in this way. This article surveys a range of contemporary autobiographical texts that are titled with, or revolve around, that powerful and most evocative word, “fat”. Following a number of cultural studies of fat in society (Critser; Gilman, Fat Boys; Fat: A Cultural History; Stearns), this discussion views fat in socio-cultural terms, following Lupton in understanding fat as both “a cultural artefact: a bodily substance or body shape that is given meaning by complex and shifting systems of ideas, practices, emotions, material objects and interpersonal relationships” (i). Using a case study approach (Gerring; Verschuren), this examination focuses on a range of texts from autobiographical cookbooks and memoirs to novel-length graphic works in order to develop a preliminary taxonomy of these works. In this way, a small sample of work, each of which (described below) explores an aspect (or aspects) of the form is, following Merriam, useful as it allows a richer picture of an under-examined phenomenon to be constructed, and offers “a means of investigating complex social units consisting of multiple variables of potential importance in understanding the phenomenon” (Merriam 50). Although the sample size does not offer generalisable results, the case study method is especially suitable in this context, where the aim is to open up discussion of this form of writing for future research for, as Merriam states, “much can be learned from […] an encounter with the case through the researcher’s narrative description” and “what we learn in a particular case can be transferred to similar situations” (51). Pro-Fat Autobiographical WritingAlongside the many hundreds of reduced, low- and no-fat cookbooks and weight loss guides currently in print that offer recipes, meal plans, ingredient replacements and strategies to reduce fat in the diet, there are a handful that promote the consumption of fats, and these all have an autobiographical component. The publication of Jennifer McLagan’s Fat: An Appreciation of a Misunderstood Ingredient, with Recipes in 2008 by Ten Speed Press—publisher of Mollie Katzen’s groundbreaking and influential vegetarian Moosewood Cookbook in 1974 and an imprint now known for its quality cookbooks (Thelin)—unequivocably addressed that line in the sand often drawn between fat and all things healthy. The four chapter titles of this cookbook— “Butter,” subtitled “Worth It,” “Pork Fat: The King,” “Poultry Fat: Versatile and Good For You,” and, “Beef and Lamb Fats: Overlooked But Tasty”—neatly summarise McLagan’s organising argument: that animal fats not only add an unreplaceable and delicious flavour to foods but are fundamental to our health. Fat polarised readers and critics; it was positively reviewed in prominent publications (Morris; Bhide) and won influential food writing awards, including 2009 James Beard Awards for Single Subject Cookbook and Cookbook of the Year but, due to its rejection of low-fat diets and the research underpinning them, was soon also vehemently criticised, to the point where the book was often described in the media as “controversial” (see Smith). McLagan’s text, while including historical, scientific and gastronomic data and detail, is also an outspokenly personal treatise, chronicling her sensual and emotional responses to this ingredient. “I love fat,” she begins, continuing, “Whether it’s a slice of foie gras terrine, its layer of yellow fat melting at the edges […] hot bacon fat […] wilting a plate of pungent greens into submission […] or a piece of crunchy pork crackling […] I love the way it feels in my mouth, and I love its many tastes” (1). Her text is, indeed, memoir as gastronomy / gastronomy as memoir, and this cookbook, therefore, an example of the “memoir with recipes” subgenre (Brien et al.). It appears to be this aspect – her highly personal and, therein, persuasive (Weitin) plea for the value of fats – that galvanised critics and readers.Molly Chester and Sandy Schrecengost’s Back to Butter: A Traditional Foods Cookbook – Nourishing Recipes Inspired by Our Ancestors begins with its authors’ memoirs (illness, undertaking culinary school training, buying and running a farm) to lend weight to their argument to utilise fats widely in cookery. Its first chapter, “Fats and Oils,” features the familiar butter, which it describes as “the friendly fat” (22), then moves to the more reviled pork lard “Grandma’s superfood” (22) and, nowadays quite rarely described as an ingredient, beef tallow. Grit Magazine’s Lard: The Lost Art of Cooking with Your Grandmother’s Secret Ingredient utilises the rhetoric that fat, and in this case, lard, is a traditional and therefore foundational ingredient in good cookery. This text draws on its publisher’s, Grit Magazine (published since 1882 in various formats), long history of including auto/biographical “inspirational stories” (Teller) to lend persuasive power to its argument. One of the most polarising of fats in health and current media discourse is butter, as was seen recently in debate over what was seen as its excessive use in the MasterChef Australia television series (see, Heart Foundation; Phillipov). It is perhaps not surprising, then, that butter is the single fat inspiring the most autobiographical writing in this mode. Rosie Daykin’s Butter Baked Goods: Nostalgic Recipes from a Little Neighborhood Bakery is, for example, typical of a small number of cookbooks that extend the link between baking and nostalgia to argue that butter is the superlative ingredient for baking. There are also entire cookbooks dedicated to making flavoured butters (Vaserfirer) and a number that offer guides to making butter and other (fat-based) dairy products at home (Farrell-Kingsley; Hill; Linford).Gabrielle Hamilton’s Blood, Bones and Butter: The Inadvertent Education of a Reluctant Chef is typical among chef’s memoirs in using butter prominently although rare in mentioning fat in its title. In this text and other such memoirs, butter is often used as shorthand for describing a food that is rich but also wholesomely delicious. Hamilton relates childhood memories of “all butter shortcakes” (10), and her mother and sister “cutting butter into flour and sugar” for scones (15), radishes eaten with butter (21), sautéing sage in butter to dress homemade ravoli (253), and eggs fried in browned butter (245). Some of Hamilton’s most telling references to butter present it as an staple, natural food as, for instance, when she describes “sliced bread with butter and granulated sugar” (37) as one of her family’s favourite desserts, and lists butter among the everyday foodstuffs that taste superior when stored at room temperature instead of refrigerated—thereby moving butter from taboo (Gwynne describes a similar process of the normalisation of sexual “perversion” in erotic memoir).Like this text, memoirs that could be described as arguing “for” fat as a substance are largely by chefs or other food writers who extol, like McLagan and Hamilton, the value of fat as both food and flavouring, and propose that it has a key role in both ordinary/family and gourmet cookery. In this context, despite plant-based fats such as coconut oil being much lauded in nutritional and other health-related discourse, the fat written about in these texts is usually animal-based. An exception to this is olive oil, although this is never described in the book’s title as a “fat” (see, for instance, Drinkwater’s series of memoirs about life on an olive farm in France) and is, therefore, out of the scope of this discussion.Memoirs of Being FatThe majority of the other memoirs with the word “fat” in their titles are about being fat. Narratives on this topic, and their authors’ feelings about this, began to be published as a sub-set of autobiographical memoir in the 2000s. The first decade of the new millennium saw a number of such memoirs by female writers including Judith Moore’s Fat Girl (published in 2005), Jen Lancaster’s Such a Pretty Fat: One Narcissist’s Quest to Discover If Her Life Makes Her Ass Look Big, or Why Pie Is Not the Answer, and Stephanie Klein’s Moose: A Memoir (both published in 2008) and Jennifer Joyne’s Designated Fat Girl in 2010. These were followed into the new decade by texts such as Celia Rivenbark’s bestselling 2011 You Don’t Sweat Much for a Fat Girl, and all attracted significant mainstream readerships. Journalist Vicki Allan pulled no punches when she labelled these works the “fat memoir” and, although Sidonie Smith and Julia Watson’s influential categorisation of 60 genres of life writing does not include this description, they do recognise eating disorder and weight-loss narratives. Some scholarly interest followed (Linder; Halloran), with Mitchell linking this production to feminism’s promotion of the power of the micro-narrative and the recognition that the autobiographical narrative was “a way of situating the self politically” (65).aken together, these memoirs all identify “excess” weight, although the response to this differs. They can be grouped as: narratives of losing weight (see Kuffel; Alley; and many others), struggling to lose weight (most of these books), and/or deciding not to try to lose weight (the smallest number of works overall). Some of these texts display a deeply troubled relationship with food—Moore’s Fat Girl, for instance, could also be characterised as an eating disorder memoir (Brien), detailing her addiction to eating and her extremely poor body image as well as her mother’s unrelenting pressure to lose weight. Elena Levy-Navarro describes the tone of these narratives as “compelled confession” (340), mobilising both the conventional understanding of confession of the narrator “speaking directly and colloquially” to the reader of their sins, failures or foibles (Gill 7), and what she reads as an element of societal coercion in their production. Some of these texts do focus on confessing what can be read as disgusting and wretched behavior (gorging and vomiting, for instance)—Halloran’s “gustatory abject” (27)—which is a feature of the contemporary conceptualisation of confession after Rousseau (Brooks). This is certainly a prominent aspect of current memoir writing that is, simultaneously, condemned by critics (see, for example, Jordan) and popular with readers (O’Neill). Read in this way, the majority of memoirs about being fat are about being miserable until a slimming regime of some kind has been undertaken and successful. Some of these texts are, indeed, triumphal in tone. Lisa Delaney’s Secrets of a Former Fat Girl is, for instance, clear in the message of its subtitle, How to Lose Two, Four (or More!) Dress Sizes—And Find Yourself Along the Way, that she was “lost” until she became slim. Linden has argued that “female memoir writers frequently describe their fat bodies as diseased and contaminated” (219) and “powerless” (226). Many of these confessional memoirs are moving narratives of shame and self loathing where the memoirist’s sense of self, character, and identity remain somewhat confused and unresolved, whether they lose weight or not, and despite attestations to the contrary.A sub-set of these memoirs of weight loss are by male authors. While having aspects in common with those by female writers, these can be identified as a sub-set of these memoirs for two reasons. One is the tone of their narratives, which is largely humourous and often ribaldly comic. There is also a sense of the heroic in these works, with male memoirsts frequently mobilising images of battles and adversity. Texts that can be categorised in this way include Toshio Okada’s Sayonara Mr. Fatty: A Geek’s Diet Memoir, Gregg McBride and Joy Bauer’s bestselling Weightless: My Life as a Fat Man and How I Escaped, Fred Anderson’s From Chunk to Hunk: Diary of a Fat Man. As can be seen in their titles, these texts also promise to relate the stratgies, regimes, plans, and secrets that others can follow to, similarly, lose weight. Allen Zadoff’s title makes this explicit: Lessons Learned on the Journey from Fat to Thin. Many of these male memoirists are prompted by a health-related crisis, diagnosis, or realisation. Male body image—a relatively recent topic of enquiry in the eating disorder, psychology, and fashion literature (see, for instance, Bradley et al.)—is also often a surprising motif in these texts, and a theme in common with weight loss memoirs by female authors. Edward Ugel, for instance, opens his memoir, I’m with Fatty: Losing Fifty Pounds in Fifty Miserable Weeks, with “I’m haunted by mirrors … the last thing I want to do is see myself in a mirror or a photograph” (1).Ugel, as that prominent “miserable” in his subtitle suggests, provides a subtle but revealing variation on this theme of successful weight loss. Ugel (as are all these male memoirists) succeeds in the quest be sets out on but, apparently, despondent almost every moment. While the overall tone of his writing is light and humorous, he laments every missed meal, snack, and mouthful of food he foregoes, explaining that he loves eating, “Food makes me happy … I live to eat. I love to eat at restaurants. I love to cook. I love the social component of eating … I can’t be happy without being a social eater” (3). Like many of these books by male authors, Ugel’s descriptions of the food he loves are mouthwatering—and most especially when describing what he identifies as the fattening foods he loves: Reuben sandwiches dripping with juicy grease, crispy deep friend Chinese snacks, buttery Danish pastries and creamy, rich ice cream. This believable sense of regret is not, however, restricted to male authors. It is also apparent in how Jen Lancaster begins her memoir: “I’m standing in the kitchen folding a softened stick of butter, a cup of warmed sour cream, and a mound of fresh-shaved Parmesan into my world-famous mashed potatoes […] There’s a maple-glazed pot roast browning nicely in the oven and white-chocolate-chip macadamia cookies cooling on a rack farther down the counter. I’ve already sautéed the almonds and am waiting for the green beans to blanch so I can toss the whole lot with yet more butter before serving the meal” (5). In the above memoirs, both male and female writers recount similar (and expected) strategies: diets, fasts and other weight loss regimes and interventions (calorie counting, colonics, and gastric-banding and -bypass surgery for instance, recur); consulting dieting/health magazines for information and strategies; keeping a food journal; employing expert help in the form of nutritionists, dieticians, and personal trainers; and, joining health clubs/gyms, and taking up various sports.Alongside these works sit a small number of texts that can be characterised as “non-weight loss memoirs.” These can be read as part of the emerging, and burgeoning, academic field of Fat Studies, which gathers together an extensive literature critical of, and oppositional to, dominant discourses about obesity (Cooper; Rothblum and Solovay; Tomrley and Naylor), and which include works that focus on information backed up with memoir such as self-described “fat activist” (Wann, website) Marilyn Wann’s Fat! So?: Because You Don’t Have to Apologise, which—when published in 1998—followed a print ’zine and a website of the same title. Although certainly in the minority in terms of numbers, these narratives have been very popular with readers and are growing as a sub-genre, with well-known actress Camryn Manheim’s New York Times-bestselling memoir, Wake Up, I'm Fat! (published in 1999) a good example. This memoir chronicles Manheim’s journey from the overweight and teased teenager who finds it a struggle to find friends (a common trope in many weight loss memoirs) to an extremely successful actress.Like most other types of memoir, there are also niche sub-genres of the “fat memoir.” Cheryl Peck’s Fat Girls and Lawn Chairs recounts a series of stories about her life in the American Midwest as a lesbian “woman of size” (xiv) and could thus be described as a memoir on the subjects of – and is, indeed, catalogued in the Library of Congress as: “Overweight women,” “Lesbians,” and “Three Rivers (Mich[igan]) – Social life and customs”.Carol Lay’s graphic memoir, The Big Skinny: How I Changed My Fattitude, has a simple diet message – she lost weight by counting calories and exercising every day – and makes a dual claim for value of being based on both her own story and a range of data and tools including: “the latest research on obesity […] psychological tips, nutrition basics, and many useful tools like simplified calorie charts, sample recipes, and menu plans” (qtd. in Lorah). The Big Skinny could, therefore, be characterised with the weight loss memoirs above as a self-help book, but Lay herself describes choosing the graphic form in order to increase its narrative power: to “wrap much of the information in stories […] combining illustrations and story for a double dose of retention in the brain” (qtd. in Lorah). Like many of these books that can fit into multiple categories, she notes that “booksellers don’t know where to file the book – in graphic novels, memoirs, or in the diet section” (qtd. in O’Shea).Jude Milner’s Fat Free: The Amazing All-True Adventures of Supersize Woman! is another example of how a single memoir (graphic, in this case) can be a hybrid of the categories herein discussed, indicating how difficult it is to neatly categorise human experience. Recounting the author’s numerous struggles with her weight and journey to self-acceptance, Milner at first feels guilty and undertakes a series of diets and regimes, before becoming a “Fat Is Beautiful” activist and, finally, undergoing gastric bypass surgery. Here the narrative trajectory is of empowerment rather than physical transformation, as a thinner (although, importantly, not thin) Milner “exudes confidence and radiates strength” (Story). ConclusionWhile the above has identified a number of ways of attempting to classify autobiographical writing about fat/s, its ultimate aim is, after G. Thomas Couser’s work in relation to other sub-genres of memoir, an attempt to open up life writing for further discussion, rather than set in placed fixed and inflexible categories. Constructing such a preliminary taxonomy aspires to encourage more nuanced discussion of how writers, publishers, critics and readers understand “fat” conceptually as well as more practically and personally. 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The academic journal is obsolete. In a world where there are more titles than ever, this is a comment on their form – especially the print journal – rather than their quantity. Now that you can get everything online, it doesn’t really matter what journal a paper appears in; certainly it doesn’t matter what’s in the same issue. The experience of a journal is rapidly obsolescing, for both editors and readers. I’m obviously not the first person to notice this (see, for instance, "Scholarly Communication"; "Transforming Scholarly Communication"; Houghton; Policy Perspectives; Teute), but I do have a personal stake in the process. For if the journal is obsolete then it follows that the editor is obsolete, and I am the editor of the International Journal of Cultural Studies. I founded the IJCS and have been sole editor ever since. Next year will see the fiftieth issue. So far, I have been responsible for over 280 published articles – over 2.25 million words of other people’s scholarship … and counting. We won’t say anything about the words that did not get published, except that the IJCS rejection rate is currently 87 per cent. Perhaps the first point that needs to be made, then, is that obsolescence does not imply lack of success. By any standard the IJCS is a successful journal, and getting more so. It has recently been assessed as a top-rating A* journal in the Australian Research Council’s journal rankings for ERA (Excellence in Research for Australia), the newly activated research assessment exercise. (In case you’re wondering, M/C Journal is rated B.) The ARC says of the ranking exercise: ‘The lists are a result of consultations with the sector and rigorous review by leading researchers and the ARC.’ The ARC definition of an A* journal is given as: Typically an A* journal would be one of the best in its field or subfield in which to publish and would typically cover the entire field/ subfield. Virtually all papers they publish will be of very high quality. These are journals where most of the work is important (it will really shape the field) and where researchers boast about getting accepted.Acceptance rates would typically be low and the editorial board would be dominated by field leaders, including many from top institutions. (Appendix I, p. 21; and see p. 4.)Talking of boasting, I love to prate about the excellent people we’ve published in the IJCS. We have introduced new talent to the field, and we have published new work by some of its pioneers – including Richard Hoggart and Stuart Hall. We’ve also published – among many others – Sara Ahmed, Mohammad Amouzadeh, Tony Bennett, Goran Bolin, Charlotte Brunsdon, William Boddy, Nico Carpentier, Stephen Coleman, Nick Couldry, Sean Cubitt, Michael Curtin, Daniel Dayan, Ben Dibley, Stephanie Hemelryk Donald, John Frow, Elfriede Fursich, Christine Geraghty, Mark Gibson, Paul Gilroy, Faye Ginsberg, Jonathan Gray, Lawrence Grossberg, Judith Halberstam, Hanno Hardt, Gay Hawkins, Joke Hermes, Su Holmes, Desmond Hui, Fred Inglis, Henry Jenkins, Deborah Jermyn, Ariel Heryanto, Elihu Katz, Senator Rod Kemp (Australian government minister), Youna Kim, Agnes Ku, Richard E. Lee, Jeff Lewis, David Lodge (the novelist), Knut Lundby, Eric Ma, Anna McCarthy, Divya McMillin, Antonio Menendez-Alarcon, Toby Miller, Joe Moran, Chris Norris, John Quiggin, Chris Rojek, Jane Roscoe, Jeffrey Sconce, Lynn Spigel, John Storey, Su Tong, the late Sako Takeshi, Sue Turnbull, Graeme Turner, William Uricchio, José van Dijck, Georgette Wang, Jing Wang, Elizabeth Wilson, Janice Winship, Handel Wright, Wu Jing, Wu Qidi (Chinese Vice-Minister of Education), Emilie Yueh-Yu Yeh, Robert Young and Zhao Bin. As this partial list makes clear, as well as publishing the top ‘hegemons’ we also publish work pointing in new directions, including papers from neighbouring disciplines such as anthropology, area studies, economics, education, feminism, history, literary studies, philosophy, political science, and sociology. We have sought to represent neglected regions, especially Chinese cultural studies, which has grown strongly during the past decade. And for quite a few up-and-coming scholars we’ve been the proud host of their first international publication. The IJCS was first published in 1998, already well into the internet era, but it was print-only at that time. Since then, all content, from volume 1:1 onwards, has been digitised and is available online (although vol 1:2 is unaccountably missing). The publishers, Sage Publications Ltd, London, have steadily added online functionality, so that now libraries can get the journal in various packages, including offering this title among many others in online-only bundles, and individuals can purchase single articles online. Thus, in addition to institutional and individual subscriptions, which remain the core business of the journal, income is derived by the publisher from multi-site licensing, incremental consortial sales income, single- and back-issue sales (print), pay-per-view, and deep back file sales (electronic). So what’s obsolete about it? In that boasting paragraph of mine (above), about what wonderful authors we’ve published, lies one of the seeds of obsolescence. For now that it is available online, ‘users’ (no longer ‘readers’!) can search for what they want and ignore the journal as such altogether. This is presumably how most active researchers experience any journal – they are looking for articles (or less: quotations; data; references) relevant to a given topic, literature review, thesis etc. They encounter a journal online through its ‘content’ rather than its ‘form.’ The latter is irrelevant to them, and may as well not exist. The Cover Some losses are associated with this change. First is the loss of the front cover. Now you, dear reader, scrolling through this article online, might well complain, why all the fuss about covers? Internet-generation journals don’t have covers, so all of the work that goes into them to establish the brand, the identity and even the ‘affect’ of a journal is now, well, obsolete. So let me just remind you of what’s at stake. Editors, designers and publishers all take a good deal of trouble over covers, since they are the point of intersection of editorial, design and marketing priorities. Thus, the IJCS cover contains the only ‘content’ of the journal for which we pay a fee to designers and photographers (usually the publisher pays, but in one case I did). Like any other cover, ours has three main elements: title, colour and image. Thought goes into every detail. Title I won’t say anything about the journal’s title as such, except that it was the result of protracted discussions (I suggested Terra Nullius at one point, but Sage weren’t having any of that). The present concern is with how a title looks on a cover. Our title-typeface is Frutiger. Originally designed by Adrian Frutiger for Charles de Gaulle Airport in Paris, it is suitably international, being used for the corporate identity of the UK National Health Service, Telefónica O2, the Royal Navy, the London School of Economics , the Canadian Broadcasting Corporation, the Conservative Party of Canada, Banco Bradesco of Brazil, the Finnish Defence Forces and on road signs in Switzerland (Wikipedia, "Frutiger"). Frutiger is legible, informal, and reads well in small copy. Sage’s designer and I corresponded on which of the words in our cumbersome name were most important, agreeing that ‘international’ combined with ‘cultural’ is the USP (Unique Selling Point) of the journal, so they should be picked out (in bold small-caps) from the rest of the title, which the designer presented in a variety of Frutiger fonts (regular, italic, and reversed – white on black), presumably to signify the dynamism and diversity of our content. The word ‘studies’ appears on a lozenge-shaped cartouche that is also used as a design element throughout the journal, for bullet points, titles and keywords. Colour We used to change this every two years, but since volume 7 it has stabilised with the distinctive Pantone 247, ‘new fuchsia.’ This colour arose from my own environment at QUT, where it was chosen (by me) for the new Creative Industries Faculty’s academic gowns and hoods, and thence as a detailing colour for the otherwise monochrome Creative Industries Precinct buildings. There’s a lot of it around my office, including on the wall and the furniture. New Fuchsia is – we are frequently told – a somewhat ‘girly’ colour, especially when contrasted with the Business Faculty’s blue or Law’s silver; its similarity to the Girlfriend/Dolly palette does introduce a mild ‘politics of prestige’ element, since it is determinedly pop culture, feminised, and non-canonical. Image Right at the start, the IJCS set out to signal its difference from other journals. At that time, all Sage journals had calligraphic colours – but I was insistent that we needed a photograph (I have ‘form’ in this respect: in 1985 I changed the cover of the Australian Journal of Cultural Studies from a line drawing (albeit by Sydney Nolan) to a photograph; and I co-designed the photo-cover of Cultural Studies in 1987). For IJCS I knew which photo I wanted, and Sage went along with the choice. I explained it in the launch issue’s editorial (Hartley, "Editorial"). That original picture, a goanna on a cattle grid in the outback, by Australian photographer Grant Hobson, lasted ten years. Since volume 11 – in time for our second decade – the goanna has been replaced with a picture by Italian-based photographer Patrick Nicholas, called ‘Reality’ (Hartley, "Cover Narrative"). We have also used two other photos as cover images, once each. They are: Daniel Meadows’s 1974 ‘Karen &amp; Barbara’ (Hartley, "Who"); and a 1962 portrait of Richard Hoggart from the National Portrait Gallery in London (Owen &amp; Hartley 2007). The choice of picture has involved intense – sometimes very tense – negotiations with Sage. Most recently, they were adamant the Daniel Meadows picture, which I wanted to use as the long-term replacement of the goanna, was too ‘English’ and they would not accept it. We exchanged rather sharp words before compromising. There’s no need to rehearse the dispute here; the point is that both sides, publisher and editor, felt that vital interests were at stake in the choice of a cover-image. Was it too obscure; too Australian; too English; too provocative (the current cover features, albeit in the deep background, a TV screen-shot of a topless Italian game-show contestant)? Running Order Beyond the cover, the next obsolete feature of a journal is the running order of articles. Obviously what goes in the journal is contingent upon what has been submitted and what is ready at a given time, so this is a creative role within a very limited context, which is what makes it pleasurable. Out of a limited number of available papers, a choice must be made about which one goes first, what order the other papers should follow, and which ones must be held over to the next issue. The first priority is to choose the lead article: like the ‘first face’ in a fashion show (if you don’t know what I mean by that, see FTV.com. It sets the look, the tone, and the standard for the issue. I always choose articles I like for this slot. It sends a message to the field – look at this! Next comes the running order. We have about six articles per issue. It is important to maintain the IJCS’s international mix, so I check for the country of origin, or failing that (since so many articles come from Anglosphere countries like the USA, UK and Australia), the location of the analysis. Attention also has to be paid to the gender balance among authors, and to the mix of senior and emergent scholars. Sometimes a weak article needs to be ‘hammocked’ between two good ones (these are relative terms – everything published in the IJCS is of a high scholarly standard). And we need to think about disciplinary mix, so as not to let the journal stray too far towards one particular methodological domain. Running order is thus a statement about the field – the disciplinary domain – rather than about an individual paper. It is a proposition about how different voices connect together in some sort of disciplinary syntax. One might even claim that the combination of cover and running order is a last vestige of collegiate collectivism in an era of competitive academic individualism. Now all that matters is the individual paper and author; the ‘currency’ is tenure, promotion and research metrics, not relations among peers. The running order is obsolete. Special Issues An extreme version of running order is the special issue. The IJCS has regularly published these; they are devoted to field-shaping initiatives, as follows: Title Editor(s) Issue Date Radiocracy: Radio, Development and Democracy Amanda Hopkinson, Jo Tacchi 3.2 2000 Television and Cultural Studies Graeme Turner 4.4 2001 Cultural Studies and Education Karl Maton, Handel Wright 5.4 2002 Re-Imagining Communities Sara Ahmed, Anne-Marie Fortier 6.3 2003 The New Economy, Creativity and Consumption John Hartley 7.1 2004 Creative Industries and Innovation in China Michael Keane, John Hartley 9.3 2006 The Uses of Richard Hoggart Sue Owen, John Hartley 10.1 2007 A Cultural History of Celebrity Liz Barry 11.3 2008 Caribbean Media Worlds Anna Pertierra, Heather Horst 12.2 2009 Co-Creative Labour Mark Deuze, John Banks 12.5 2009 It’s obvious that special issues have a place in disciplinary innovation – they can draw attention in a timely manner to new problems, neglected regions, or innovative approaches, and thus they advance the field. They are indispensible. But because of online publication, readers are not held to the ‘project’ of a special issue and can pick and choose whatever they want. And because of the peculiarities of research assessment exercises, editing special issues doesn’t count as research output. The incentive to do them is to that extent reduced, and some universities are quite heavy-handed about letting academics ‘waste’ time on activities that don’t produce ‘metrics.’ The special issue is therefore threatened with obsolescence too. Refereeing In many top-rating journals, the human side of refereeing is becoming obsolete. Increasingly this labour-intensive chore is automated and the labour is technologically outsourced from editors and publishers to authors and referees. You have to log on to some website and follow prompts in order to contribute both papers and the assessment of papers; interactions with editors are minimal. At the IJCS the process is still handled by humans – namely, journal administrator Tina Horton and me. We spend a lot of time checking how papers are faring, from trying to find the right referees through to getting the comments and then the author’s revisions completed in time for a paper to be scheduled into an issue. The volume of email correspondence is considerable. We get to know authors and referees. So we maintain a sense of an interactive and conversational community, albeit by correspondence rather than face to face. Doubtless, sooner or later, there will be a depersonalised Text Management System. But in the meantime we cling to the romantic notion that we are involved in refereeing for the sake of the field, for raising the standard of scholarship, for building a globally dispersed virtual college of cultural studies, and for giving everyone – from unfavoured countries and neglected regions to famous professors in old-money universities – the same chance to get their research published. In fact, these are largely delusional ideals, for as everyone knows, refereeing is part of the political economy of publicly-funded research. It’s about academic credentials, tenure and promotion for the individual, and about measurable research metrics for the academic organisation or funding agency (Hartley, "Death"). The IJCS has no choice but to participate: we do what is required to qualify as a ‘double-blind refereed journal’ because that is the only way to maintain repute, and thence the flow of submissions, not to mention subscriptions, without which there would be no journal. As with journals themselves, which proliferate even as the print form becomes obsolete, so refereeing is burgeoning as a practice. It’s almost an industry, even though the currency is not money but time: part gift-economy; part attention-economy; partly the payment of dues to the suzerain funding agencies. But refereeing is becoming obsolete in the sense of gathering an ‘imagined community’ of people one might expect to know personally around a particular enterprise. The process of dispersal and anonymisation of the field is exacerbated by blind refereeing, which we do because we must. This is suited to a scientific domain of objective knowledge, but everyone knows it’s not quite like that in the ‘new humanities’. The agency and identity of the researcher is often a salient fact in the research. The embedded positionality of the author, their reflexiveness about their own context and room-for-manoeuvre, and the radical contextuality of knowledge itself – these are all more or less axiomatic in cultural studies, but they’re not easily served by ‘double-blind’ refereeing. When refereeing is depersonalised to the extent that is now rife (especially in journals owned by international commercial publishers), it is hard to maintain a sense of contextualised productivity in the knowledge domain, much less a ‘common cause’ to which both author and referee wish to contribute. Even though refereeing can still be seen as altruistic, it is in the service of something much more general (‘scholarship’) and much more particular (‘my career’) than the kind of reviewing that wants to share and improve a particular intellectual enterprise. It is this mid-range altruism – something that might once have been identified as a politics of knowledge – that’s becoming obsolete, along with the printed journals that were the banner and rallying point for the cause. If I were to start a new journal (such as cultural-science.org), I would prefer ‘open refereeing’: uploading papers on an open site, subjecting them to peer-review and criticism, and archiving revised versions once they have received enough votes and comments. In other words I’d like to see refereeing shifted from the ‘supply’ or production side of a journal to the ‘demand’ or readership side. But of course, ‘demand’ for ‘blind’ refereeing doesn’t come from readers; it comes from the funding agencies. The Reading Experience Finally, the experience of reading a journal is obsolete. Two aspects of this seem worthy of note. First, reading is ‘out of time’ – it no longer needs to conform to the rhythms of scholarly publication, which are in any case speeding up. Scholarship is no longer seasonal, as it has been since the Middle Ages (with university terms organised around agricultural and ecclesiastical rhythms). Once you have a paper’s DOI number, you can read it any time, 24/7. It is no longer necessary even to wait for publication. With some journals in our field (e.g. Journalism Studies), assuming your Library subscribes, you can access papers as soon as they’re uploaded on the journal’s website, before the published edition is printed. Soon this will be the norm, just as it is for the top science journals, where timely publication, and thereby the ability to claim first discovery, is the basis of intellectual property rights. The IJCS doesn’t (yet) offer this service, but its frequency is speeding up. It was launched in 1998 with three issues a year. It went quarterly in 2001 and remained a quarterly for eight years. It has recently increased to six issues a year. That too causes changes in the reading experience. The excited ripping open of the package is less of a thrill the more often it arrives. Indeed, how many subscribers will admit that sometimes they don’t even open the envelope? Second, reading is ‘out of place’ – you never have to see the journal in which a paper appears, so you can avoid contact with anything that you haven’t already decided to read. This is more significant than might first appear, because it is affecting journalism in general, not just academic journals. As we move from the broadcast to the broadband era, communicative usage is shifting too, from ‘mass’ communication to customisation. This is a mixed blessing. One of the pleasures of old-style newspapers and the TV news was that you’d come across stories you did not expect to find. Indeed, an important attribute of the industrial form of journalism is its success in getting whole populations to read or watch stories about things they aren’t interested in, or things like wars and crises that they’d rather not know about at all. That historic textual achievement is in jeopardy in the broadband era, because ‘the public’ no longer needs to gather around any particular masthead or bulletin to get their news. With Web 2.0 affordances, you can exercise much more choice over what you attend to. This is great from the point of view of maximising individual choice, but sub-optimal in relation to what I’ve called ‘population-gathering’, especially the gathering of communities of interest around ‘tales of the unexpected’ – novelty or anomalies. Obsolete: Collegiality, Trust and Innovation? The individuation of reading choices may stimulate prejudice, because prejudice (literally, ‘pre-judging’) is built in when you decide only to access news feeds about familiar topics, stories or people in which you’re already interested. That sort of thing may encourage narrow-mindedness. It is certainly an impediment to chance discovery, unplanned juxtaposition, unstructured curiosity and thence, perhaps, to innovation itself. This is a worry for citizenship in general, but it is also an issue for academic ‘knowledge professionals,’ in our ever-narrower disciplinary silos. An in-close specialist focus on one’s own area of expertise need no longer be troubled by the concerns of the person in the next office, never mind the next department. Now, we don’t even have to meet on the page. One of the advantages of whole journals, then, is that each issue encourages ‘macro’ as well as ‘micro’ perspectives, and opens reading up to surprises. This willingness to ‘take things on trust’ describes a ‘we’ community – a community of trust. Trust too is obsolete in these days of performance evaluation. We’re assessed by an anonymous system that’s managed by people we’ll never meet. If the ‘population-gathering’ aspects of print journals are indeed obsolete, this may reduce collegiate trust and fellow-feeling, increase individualist competitiveness, and inhibit innovation. In the face of that prospect, I’m going to keep on thinking about covers, running orders, referees and reading until the role of editor is obsolete too. ReferencesHartley, John. "'Cover Narrative': From Nightmare to Reality." International Journal of Cultural Studies 11.2 (2005): 131-137. ———. "Death of the Book?" Symposium of the National Scholarly Communication Forum &amp; Australian Academy of the Humanities, Sydney Maritime Museum, 2005. 26 Apr. 2009 ‹http://www.humanities.org.au/Resources/Downloads/NSCF/RoundTables1-17/PDF/Hartley.pdf›. ———. "Editorial: With Goanna." International Journal of Cultural Studies 1.1 (1998): 5-10. ———. "'Who Are You Going to Believe – Me or Your Own Eyes?' New Decade; New Directions." International Journal of Cultural Studies 11.1 (2008): 5-14. Houghton, John. "Economics of Scholarly Communication: A Discussion Paper." Center for Strategic Economic Studies, Victoria University, 2000. 26 Apr. 2009 ‹http://www.caul.edu.au/cisc/EconomicsScholarlyCommunication.pdf›. Owen, Sue, and John Hartley, eds. The Uses of Richard Hoggart. International Journal of Cultural Studies (special issue), 10.1 (2007). Policy Perspectives: To Publish and Perish. (Special issue cosponsored by the Association of Research Libraries, Association of American Universities and the Pew Higher Education Roundtable) 7.4 (1998). 26 Apr. 2009 ‹http://www.arl.org/scomm/pew/pewrept.html›. "Scholarly Communication: Crisis and Revolution." University of California Berkeley Library. N.d. 26 Apr. 2009 ‹http://www.lib.berkeley.edu/Collections/crisis.html›. Teute, F. J. "To Publish or Perish: Who Are the Dinosaurs in Scholarly Publishing?" Journal of Scholarly Publishing 32.2 (2001). 26 Apr. 2009 ‹http://www.utpjournals.com/product/jsp/322/perish5.html›."Transforming Scholarly Communication." University of Houston Library. 2005. 26 Apr. 2009 ‹http://info.lib.uh.edu/scomm/transforming.htm›.

<strong>Dolomatov S.I., Zukow W. </strong><strong>Эпигенетика почек</strong><strong> = Kidney</strong><strong>s</strong><strong> epigenetics</strong><strong>. </strong><strong>RSW. Radom,</strong><strong> 144 </strong><strong>p. ISBN </strong><strong>9780359774524</strong><strong>.</strong><strong> DOI </strong><strong>http://dx.doi.org/10.5281/zenodo.3270699</strong><strong> PBN Poland </strong><strong>https://pbn.nauka.gov.pl/sedno-webapp/works/917606</strong> &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; <strong>Radomska Szkoła Wyższa w Radomiu, Radom, Poland</strong> &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; <strong>С.И. Доломатов, В.А. Жуков </strong> <strong>S.I. </strong><strong>Dolomatov, </strong><strong>W. </strong><strong>Zukow</strong> &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; <strong>Эпигенетика почек</strong> <strong>Kidneys epigenetics</strong> &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; <strong>Radom, 2019</strong> <strong>Dolomatov S.I., Zukow W. </strong><strong>Эпигенетика почек</strong><strong> = Kidneys epigenetics</strong><strong>. </strong><strong>RSW. Radom,</strong><strong> 144 </strong><strong>p. ISBN </strong><strong>9780359774524</strong><strong>.</strong><strong> DOI </strong><strong>http://dx.doi.org/10.5281/zenodo.3270699</strong><strong> PBN Poland </strong><strong>https://pbn.nauka.gov.pl/sedno-webapp/works/917606</strong> &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; <strong>Radomska Szkoła Wyższa w Radomiu, Radom, Poland</strong> &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; <strong>С.И. Доломатов, В.А. Жуков </strong> <strong>S.I. </strong><strong>Dolomatov, </strong><strong>W. </strong><strong>Zukow</strong> &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; <strong>Эпигенетика почек</strong> <strong>Kidneys epigenetics</strong> &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; <strong>Radom, 2019</strong> <strong>Dolomatov S.I., Zukow W. </strong><strong>Эпигенетика почек</strong><strong> = Kidneys epigenetics</strong><strong>. </strong><strong>RSW. Radom,</strong><strong> 144 </strong><strong>p. ISBN </strong><strong>9780359774524</strong><strong>.</strong><strong> DOI </strong><strong>http://dx.doi.org/10.5281/zenodo.3270699</strong><strong> PBN Poland </strong><strong>https://pbn.nauka.gov.pl/sedno-webapp/works/917606</strong> &nbsp; <strong>Reviewers:</strong> <strong>dr hab. </strong><strong>R</strong><strong>. </strong><strong>Muszkieta, prof. nadzw.</strong><strong> (</strong><strong>Poland</strong><strong>)</strong> <strong>dr hab. M</strong><strong>. </strong><strong>Napierała, prof. nadzw</strong><strong> (</strong><strong>Poland</strong><strong>)</strong> &nbsp; <strong>АННОТАЦИЯ</strong> В книге представлены сведения о роли эпигенетических механизмов в системе контроля функции почек в норме и при патологии. Результаты анализа роли эпигенетического контроля экспрессии генов транспортных и регуляторных белков почки в норме указывают, во-первых, на высокую пластичность процессов изменения экспрессии генов. Во-вторых, иллюстрируют их способность адекватно реагировать на изменения параметров гомеостатических функций почек, что, в свою очередь, позволяет рассматривать данные процессы в качестве еще одного звена управления деятельностью органа наряду с нейро-эндокринными и внутриорганными уровнями гуморального контроля водно-солевого баланса организма. Приведены факты, подчеркивающие вовлеченность гуморальных факторов системного действия и внутрипочечных систем гуморального контроля в процессы эпигенетической перестройки экспрессии генов ренальной паренхимы в норме и при патологии. Также анализируется роль факторов среды в регуляции экспрессии генов. &nbsp; <strong>ANNOTATION</strong> The book provides information about the role of epigenetic mechanisms in the system of monitoring renal function in normal and pathological conditions. The results of the analysis of the role of epigenetic control of gene expression of kidney transport and regulatory proteins normally indicate, firstly, the high plasticity of gene expression change processes. Secondly, they illustrate their ability to adequately respond to changes in the parameters of homeostatic functions of the kidneys, which, in turn, makes it possible to consider these processes as another element in the management of organ activity along with neuro-endocrine and intraorgan levels of the humoral control of the body&rsquo;s water-salt balance. The facts that emphasize the involvement of humoral factors of systemic action and intrarenal systems of humoral control in the processes of epigenetic rearrangement of the expression of renal parenchyma genes in normal and pathological conditions are presented. The role of environmental factors in the regulation of gene expression is also analyzed. &nbsp; <strong>Ключевые слова: почки, эпигенетика.</strong> <strong>Key words: kidneys, epigenetics.</strong> &nbsp; &copy; The Author(s) 2019. This monograph is published with Open Access. Open Access This monograph is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. &nbsp; &nbsp; Attribution &mdash; You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Noncommercial &mdash; You may not use this work for commercial purposes. Share Alike &mdash; If you alter, transform, or build upon this work, you may distribute the resulting work only under the same or similar license to this one. &nbsp; Zawartość jest objęta licencją Creative Commons Uznanie autorstwa-Użycie niekomercyjne-Na tych samych warunkach 4.0 &nbsp; <strong>ISBN 9780359774524</strong> &nbsp; <strong>DOI </strong><strong>http://dx.doi.org/10.5281/zenodo.</strong><strong>3270699</strong> &nbsp; <strong>PBN Poland </strong><strong>https://pbn.nauka.gov.pl/sedno-webapp/works/917606</strong> &nbsp; Radomska Szkoła Wyższa w Radomiu, Polska ul. 1905 roku 26/28 26-600 Radom Tel: 048 383 66 05 mail: med@rsw.edu.pl &nbsp; <strong>144</strong><strong> p. Number of characters: 2</strong><strong>50</strong><strong> 000 (with abstracts). Number of images:</strong><strong> 4 </strong><strong>x 1000 characters (lump sum) =</strong><strong> 4 </strong><strong>000 characters.</strong> <strong>Total: Number of characters: 2</strong><strong>54</strong><strong> 000 (with abstracts, summaries and graphics) = 6,</strong><strong>35</strong><strong> sheet publications.</strong> <strong>СОДЕРЖАНИЕ</strong> &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; <strong>ВВЕДЕНИЕ</strong> <strong>7</strong> <strong>ОБЩИЕ СВЕДЕНИЯ ОБ ЭПИГЕНЕТИЧЕСКИХ МЕХАНИЗМАХ</strong> <strong>8</strong> <strong>INTRODUCTION</strong> <strong>11</strong> <strong>GENERAL INFORMATION ON EPIGENETIC MECHANISMS</strong> <strong>13</strong> <strong>СПИСОК ЛИТЕРАТУРЫ К ГЛАВЕ &laquo;ВВЕДЕНИЕ И ОБЩИЕ СВЕДЕНИЯ ОБ ЭПИГЕНЕТИЧЕСКИХ МЕХАНИЗМАХ&raquo;</strong> <strong>16</strong> &nbsp; &nbsp; <strong>ГЛАВА 1. </strong><strong>ЭПИГЕНЕТИЧЕСКИЕ</strong><strong> </strong><strong>МЕХАНИЗМЫ В СИСТЕМЕ КОНТРОЛЯ</strong><strong> </strong><strong>ФУНКЦИИ</strong><strong> </strong><strong>ПОЧЕК</strong><strong> </strong><strong>В</strong><strong> </strong><strong>НОРМЕ</strong> <strong>2</strong><strong>1</strong> <strong>1.1. ПЛАСТИЧНОСТЬ СИСТЕМ ЭПИГЕНЕТИЧЕСКОЙ МОДУЛЯЦИИ ЭКСПРЕССИИ ГЕНОВ РЕНАЛЬНОЙ ПАРЕНХИМЫ</strong> <strong>23</strong> <strong>1.2. ЭНДОКРИННЫЕ ФАКТОРЫ РЕГУЛЯЦИИ ВОДНО-СОЛЕВОГО БАЛАНСА ОРГАНИЗМА В СИСТЕМЕ ЭПИГЕНЕТИЧЕСКИХ МЕХАНИЗМОВ КОНТРОЛЯ ГОМЕОСТАЗА</strong> <strong>27</strong> <strong>1.2.1. АРГИНИН-ВАЗОПРЕССИН (АВП)</strong> <strong>27</strong> <strong>1.2.2. НАТРИЙУРЕТИЧЕСКИЕ ПЕПТИДЫ</strong> <strong>30</strong> <strong>СПИСОК ЛИТЕРАТУРЫ К ГЛАВЕ &laquo;ЭПИГЕНЕТИЧЕСКИЕ</strong><strong> </strong><strong>МЕХАНИЗМЫ </strong><strong>В СИСТЕМЕ КОНТРОЛЯ ФУНКЦИИ ПОЧЕК В НОРМЕ&raquo;</strong> <strong>33</strong> <strong>ГЛАВА 2. НЕКОТОРЫЕ ФАКТОРЫ АКТИВАЦИИ </strong><strong>ЭПИГЕНЕТИЧЕСКИХ МЕХАНИЗМОВ</strong> <strong>47</strong> <strong>2.1. ИЗМЕНЕНИЕ ТЕМПЕРАТУРНОГО РЕЖИМА, КАК ФАКТОР ИНДУКЦИИ ЭПИГЕНЕТИЧЕСКИХ МЕХАНИЗМОВ</strong> <strong>49</strong> <strong>2.2. ГИПОКСИЯ</strong> <strong>50</strong> <strong>2.3. ГИПЕРГЛИКЕМИЯ</strong> <strong>52</strong> <strong>2.4. ТЯЖЕЛЫЕ МЕТАЛЛЫ</strong> <strong>54</strong> <strong>2.5. ЭНДОКРИНОПАТИИ</strong> <strong>57</strong> <strong>2.6. </strong><strong>ЭПИГЕНЕТИЧЕСКИЕ ПРОЦЕССЫ, ИНДУЦИРОВАННЫЕ ПАТОГЕННЫМИ МИКРООРГАНИЗМАМИ</strong> <strong>59</strong> <strong>СПИСОК ЛИТЕРАТУРЫ К ГЛАВЕ &laquo;НЕКОТОРЫЕ ФАКТОРЫ АКТИВАЦИИ ЭПИГЕНЕТИЧЕСКИХ МЕХАНИЗМОВ&raquo;</strong> <strong>61</strong> &nbsp; &nbsp; <strong>ГЛАВА 3. ФАКТОРЫ ВНУТРИОРГАННОЙ ГУМОРАЛЬНОЙ РЕГУЛЯЦИИ ДЕЯТЕЛЬНОСТИ ПОЧЕК. ИХ МЕСТО И РОЛЬ В ЭПИГЕНЕТИЧЕСКИХ МЕХАНИЗМАХ НАРУШЕНИЯ ФУНКЦИОНАЛЬНОГО СОСТОЯНИЯ РЕНАЛЬНОЙ ПАРЕНХИМЫ</strong> <strong>69</strong> <strong>3.1. РЕНИН-АНГИОТЕНЗИНОВАЯ СИСТЕМА (РАС)</strong> <strong>70</strong> <strong>3.2. МИНЕРАЛОКОРТИКОИДЫ.</strong> <strong>76</strong> <strong>3.3. ТРАНСФОРМИРУЮЩИЙ ФАКТОР РОСТА-бета1 </strong> <strong>79</strong> <strong>3.4. МОЛЕКУЛА ОКСИДА АЗОТА (</strong><strong>NO</strong><strong>)</strong> <strong>83</strong> <strong>3.5. ПАТОФИЗИОЛОГИЧЕСКОЕ ЗНАЧЕНИЕ ЭПИГЕНЕТИЧЕСКОЙ ТРАНСФОРМАЦИИ СИСТЕМЫ ВНУТРИОРГАННОЙ ГУМОРАЛЬНОЙ РЕГУЛЯЦИИ ДЕЯТЕЛЬНОСТИ ПОЧЕК</strong> <strong>87</strong> <strong>СПИСОК ЛИТЕРАТУРЫ К ГЛАВЕ &laquo;ФАКТОРЫ ВНУТРИОРГАННОЙ ГУМОРАЛЬНОЙ РЕГУЛЯЦИИ ДЕЯТЕЛЬНОСТИ ПОЧЕК. ИХ МЕСТО И РОЛЬ В ЭПИГЕНЕТИЧЕСКИХ МЕХАНИЗМАХ НАРУШЕНИЯ ФУНКЦИОНАЛЬНОГО СОСТОЯНИЯ РЕНАЛЬНОЙ ПАРЕНХИМЫ&raquo;</strong> <strong>89</strong> &nbsp; &nbsp; <strong>ГЛАВА 4. БЕЛКИ РЕНИН-АНГИОТЕНЗИНОВОЙ СИСТЕМЫ ПРИ ОНКОЛОГИЧЕСКИХ ЗАБОЛЕВАНИЯХ </strong> <strong>105</strong> <strong>4.1. ДИАГНОСТИЧЕСКАЯ ЦЕННОСТЬ АНАЛИЗА ЭКСПРЕССИИ БЕЛКОВ-КОМПОНЕНТОВ РАС В ОНКОЛОГИИ</strong> <strong>107</strong> <strong>4.1.1. Рецепторы к А-</strong><strong>II</strong> <strong>107</strong> <strong>4.1.2. Ангиотензин-</strong><strong>I</strong><strong>-превращающий фермент (АСЕ-1)</strong> <strong>108</strong> <strong>4.1.3. Ангиотензин-</strong><strong>I</strong><strong>-превращающий фермент-2 (АСЕ-2) и ось ACE2/Ang-(1&ndash;7)/M</strong><strong>AS</strong><strong>1</strong> <strong>110</strong> <strong>4.1.4. Ангиотензиноген</strong> <strong>111</strong> <strong>4.1.5. (Про)Ренин</strong> <strong>112</strong> <strong>4.2. ЭПИГЕНЕТИЧЕСКИЕ МЕХАНИЗМЫ, КАК ВОЗМОЖНЫЕ РЕГУЛЯТОРЫ ЭКСПРЕССИИ ПРОТЕИНОВ-КОМПОНЕНТОВ РАС ПРИ ОНКОЛОГИЧЕСКИХ ЗАБОЛЕВАНИЯХ</strong> <strong>114</strong> <strong>4.3. ОНКОЛОГИЧЕСКИЕ АСПЕКТЫ ЭКСПРЕССИИ КОМПОНЕНТОВ РАС И ЛОКАЛЬНАЯ РЕНИН-АНГИОТЕНЗИНОВАЯ СИСТЕМА</strong> <strong>116</strong> <strong>СПИСОК ЛИТЕРАТУРЫ К ГЛАВЕ &laquo;БЕЛКИ РЕНИН-АНГИОТЕНЗИНОВОЙ СИСТЕМЫ ПРИ ОНКОЛОГИЧЕСКИХ ЗАБОЛЕВАНИЯХ&raquo;</strong> <strong>120</strong> &nbsp; &nbsp; <strong>ВВЕДЕНИЕ</strong> &nbsp; Заболевание почек является одной из наиболее актуальных глобальных проблем современной медицины. Данные медицинской статистики показывают неуклонный рост числа нефрологических пациентов, нуждающихся в диализе и трансплантации органа (Reddy M.A, Natarajan R., 2015; Uwaezuoke S.N. et al., 2016; Zununi Vahed S. et al., 2016). Дополняет драматичность картины тот факт, что данная тенденция демонстрирует актуальность и в отношении детей, включая новорожденных (Woroniecki R. et al., 2011; Uwaezuoke S.N. et al., 2016; Lee-Son K., Jetton J.G., 2016). Проблема носит серьезный характер, но даже привлечение к ее преодолению подходов генетики, основанных на законах моногенного наследования Г. Менделя, не в полной мере решает поставленную задачу. Выводы специалистов по медицинской генетике, изучающих наследование патологических признаков, способствующих повышению рисков возникновения почечной недостаточности, сопровождаются констатацией практической значимости выяснения эпигенетических механизмов поражения ткани почек (K&ouml;ttgen A. et al., 2010; Lee-Son K., Jetton J.G., 2016; Ma R.C.W., 2016). Действительно, обзоры результатов экспериментальных и клинических исследований, указывают на необходимость более глубокого развития научного направления, связанного с эпигенетическими механизмами патогенеза почечной недостаточности (Thomas M.C., 2016; Witasp A. et al., 2017). Вместе с тем, особенность деятельности почки заключается в том, что различные сегменты ее структурно-функциональной единицы &ndash; нефрона, обладают существенными отличиями между собой, связанными с их транспортными возможностями, набором гуморальных факторов регуляции их активности и физико-химическими параметрами среды микроокружения. Действительно, гомеостатические функции разных отделов нефрона, координируются достаточно сложной системой гуморальных факторов, определяющих физиологические и патофизиологические механизмы реакции почек на изменения параметров водных бассейнов организма и внешних неблагоприятных воздействий. К числу таких гуморальных факторов следует отнести ренин-ангиотензин-альдостероновую систему (РААС) (Lee-Son K., Jetton J.G, 2016), оксид азота (Shirodkar A.V., Marsden P.A., 2011), трансформирующий фактор роста-бета (Shi M. et al., 2011) и т.д. Перечисленные гуморальные факторы контроля органогенеза и гомеостатических функций почек требуют более глубокого изучения, поскольку могут также являться медиаторами структурно-функциональных нарушений ренальной паренхимы, связанных, в том числе и с эпигенетическими преобразованиями процессов транскрипции и трансляции в условиях острой и хронической почечной недостаточности. С этих позиций, актуальность эпигенетического подхода обусловлена тем, что, во-первых, сведения об изменениях экспрессии генов, осуществляющих контроль над биосинтезом и экспрессией протеинов ренальной паренхимы, а также системных и внутрипочечных гуморальных факторов регуляции гомеостатической функции почек могут быть использованы в совершенствовании методов современной лабораторной диагностики заболеваний почек (Kobori H. et al., 2008). Во-вторых, выяснение эпигенетических механизмов патогенеза почечной недостаточности открывает перспективу в разработке принципиально новых фармакологических препаратов, в том числе, контролирующих синтез ренальной паренхимой различных физиологически активных молекул (Marumo T. et al., 2008; Reddy M.A, Natarajan R., 2015). В-третьих, выяснение эпигенетических механизмов прогрессирования почечной недостаточности позволяет по-новому оценить спектр применения и нефропротекторных свойств уже известных и широко применяемых препаратов, действие которых основано на коррекции активности гуморальных систем контроля гомеостатических функций органа (Reddy M.A. et al., 2014; Hayashi K. et al., 2015). Таким образом, мы видим свою задачу в том, чтобы попытаться интегрировать современные достижения молекулярной биологии и биохимии в уже существующую систему научных представлений о физиологических механизмах регуляции деятельности почек и патофизиологических процессах патогенеза становления и прогрессирования почечной недостаточности. Следовательно, цель нашей работы сводится к выяснению нескольких вопросов. Во-первых, какова роль эпигенетической трансформации хроматина и микро РНК в физиологических механизмах регуляции деятельности почек? Во-вторых, какова роль эпигенетических процессов нарушения баланса системного и внутрипочечного метаболизма гуморальных факторов регуляции функции почек в становлении и прогрессировании почечной недостаточности? <strong>ОБЩИЕ СВЕДЕНИЯ ОБ ЭПИГЕНЕТИЧЕСКИХ МЕХАНИЗМАХ</strong> &nbsp; Эпигенетика &mdash; современное научное направление, изучающее механизмы регуляции экспрессии генов. Эпигенетические механизмы не оказывают влияния на первичную структуру нуклеиновых кислот (Beckerman P. et al., 2014) и реализуются процессами метилирования и деметилирования ДНК (van der Wijst M.G.P. et al., 2015), РНК (Saletore Y. et al., 2013) и посттрансляционным процессингом гистоновых белков (Voon H.P.J., Wong L.H., 2016; Jamal A. et al., 2012). По нашему мнению, важно отметить, что процессы синтеза и постранскрипционного процессинга микро РНК находятся под жестким контролем энзимов (Treiber T. et al., 2019; Michlewski G., C&aacute;ceres J.F., 201(). Помимо этого, микро РНК могут выполнять важную функцию в регуляции биосинтеза белка на уровне транскрипции или трансляции (Petrillo F. et al., 2017; Thomas M.J. et al., 2018). По данным авторов цитируемых обзоров, изучение метаболизма микро РНК в различных биологических средах организма способствует разработке принципиально новых методов диагностики и лечения заболеваний почек. Помимо этого, эпигенетические механизмы принимают участие в адаптивных реакциях организма и популяции в ответ на изменения факторов среды через модуляцию экспрессии генов (Zama A.M., Uzumcu M., 2010). Одним из наиболее изученных эпигенетических механизмов регуляции экспрессии генов является процесс метилирования ДНК. Данный процесс заключается в присоединении метиловых групп (CH₃) к одному из четырех видов нуклеотидов ДНК путем образования между ними ковалентной связи, однако, порядок последовательности нуклеотидов в цепи ДНК при этом не меняется (Lister R. et al., 2009;Woroniecki R. et al., 2011). За присоединение метиловых групп к нуклеотидам отвечает один из четырех изоферментов ядерных ДНК-метилтрансфераз (DNMT) &ndash; а именно DNMT1, DNMT2, DNMT3a или DNMT3b (Reddy M.A., Natarajan R., 2015; Efimova O.A. et al., 2012). DNMT1 распознает полуметилированную ДНК (каждую из её цепей) во время репликации (Bechtel W. et al., 2010). DNMT3a и DNMT3b обеспечивают метилирование ДНК de novo, т. е. повторно, в новых сайтах. Функция DNMT2 до сих пор является предметом дискуссии (Jamal A. et al., 2012). ДНК-метилтрансферазы обладают способностью встраивать особые &laquo;метки&raquo; в нуклеиновые кислоты, что приводит к изменению экспрессии данных генов (van der Wijst M.G.P. et al., 2015). Высказывается предположение о том, что эпигенетические &laquo;метки&raquo; в цепи ДНК блокируют процесс транскрипции более двух третей объема ДНК (Ponnaluri V.K.C. et al., 2016). Таким образом, клетки организма применяют ковалентную модификацию ДНК с целью регуляции экспрессии генов по так называемому принципу &ldquo;on/off&rdquo; (Quarta C. et al., 2016). Метилирование ДНК в большинстве случаев происходит на цитозиновом азотистом основании (С), располагающимся в паре с гуанином (G), на так называемых CpG-участках, или CpG-кластерах (Dwivedi R.S. et al., 2011). В человеческом организме около 70-80% CpG-динуклеотидов находятся в метилированном состоянии (Ziller M.J. et al., 2013). Участки цепи ДНК, где плотность CpG-кластеров особенно высока, называют CpG-островками (Zhang D. et al., 2009). Однако процесс метилирования ДНК осуществляется на участках с пониженной плотностью CpG-динуклеотидов (Ziller M.J. et al., 2013). Наряду с процессом метилирования ДНК большое значение в эпигенетической регуляции экспрессии генов имеет процесс деметилирования (van der Wijst M.G.P. et al., 2015; Yefimova O.A. et al., 2012). Деметилирование ДНК &ndash; процесс высвобождения ДНК от метильных групп, который осуществляется при помощи специальных ферментов демиталаз (Auclair G., Weber M., 2012). Кроме того, к эпигенетическим механизмам относят также модификации белков-гистонов в результате процессов ацетилирования, деацетилирования, фосфорилирования, убиквитинирования и др. (Ganai S.A. et al., 2016; Araki Y., Mimura T., 2017;). Процессы ацетилирования и деацетилирования возможны благодаря ряду специфических ферментов &mdash; гистонацетилазам (ацетилтрансферазам, HAT) и гистондеацетилазам (HDAC) (Gong F. et al., 2016). Фосфорилирование же происходит за счет ферментов киназ (фосфотрансфераз) (Araki Y., Mimura T., 2017; Nathan D. et al., 2012). Ковалентная модификация гистонов, как правило, ослабляет связь гистонового кора нуклеосомы и ДНК, что способствует доступности молекулы ДНК для процесса транскрипции (Rossetto D. et al., 2012). Однако, это правило не носит универсальный характер. Например, ацетилирование лизина в положении 27 гистона H3 (H3K27ac) ведёт к усилению экспрессии генов. Сочетание ацетилированного лизина 14 (H3K14ac) и фосфорилированного серина 10 гистона H3 (H3S10ph) так же говорит о повышенной экспрессии генов (Chen K.W., Chen L., 2017). Наряду с этим другие модификации, а именно ацетилирование лизина H2AK5, H2BK20, H3K14, H4K5 и др. и фосфорилирование треонина H3T3 и серина H3S28 и H4S1 аналогично приводят к активированию гена (Araki Y., Mimura T., 2017; Wang Z. et al., 2008; Rossetto D. et al., 2012). Деацетилирование гистонов, напротив, сопровождается инактивацией гена, поскольку влечет за собой конденсацию ДНК и невозможность протекания транскрипции (Ganai S.A. et al., 2016). Посттранскрипционная экспрессия генов регулируется таким эпигенетическим механизмом как интерференция РНК. Интерференция РНК &mdash; это процесс подавления процесса биосинтеза белка (транскрипция, трансляция) при помощи микро РНК (Nabzdyk C.S. et al., 2017). При этом микро РНК может оказывать непосредственное влияние на состояние трансляции (Dwivedi R.S. et al., 2011; Cui J. et al., 2017). Собственно процесс подавления экспрессии генов именуется сайленсингом (Cui J. et al., 2017). МикроРНК &mdash; это класс коротких одноцепочечных некодирующих РНК длиной в 21-24 нуклеотида (Dwivedi R.S. et al., 2011; Zhang D. et al., 2009). За производство микро РНК ответственны эндогенные некодирующие участки ДНК. Помимо трансляции микро РНК обладает способностью подавлять экспрессию генов на стадии транскрипции (Dwivedi R.S. et al., 2011). Все регуляторные эффекты микро РНК встроены в определенную систему эпигенетического контроля функций данной популяции клеток (Dwivedi R.S. et al., 2011). Анализируя современные данные литературы, можно предположить, что эпигенетические механизмы являются важным элементом адаптации на популяционном и организменном уровне, осуществляющим координацию экспрессии генов адекватно изменениям факторов внешней среды. В нашем сознании понятие &laquo;эпигенетические механизмы&raquo; чаще ассоциируются с феноменом фетального программирования. Между тем, их деятельность активно протекает и в постнатальный период онтогенеза. Сложно сказать в чем именно может иметь место несовершенство этой формы адаптивного ответа, однако, ее реализация зачастую сопряжена с активацией патогенетических механизмов различных систем органов. <strong>INTRODUCTION</strong> &nbsp; Kidney disease is one of the most pressing global problems of modern medicine. Medical statistics show a steady increase in the number of nephrological patients in need of dialysis and organ transplantation (Reddy M.A, Natarajan R., 2015; Uwaezuoke S.N. et al., 2016; Zununi Vahed S. et al., 2016). The drama of the picture complements the fact that this trend also shows relevance for children, including newborns (Woroniecki R. et al., 2011; Uwaezuoke S.N. et al., 2016; Lee-Son K., Jetton J.G., 2016). The problem is serious, but even the attraction of genetics approaches based on the monogenic inheritance laws of G. Mendel to its overcoming does not fully solve the problem posed. The findings of specialists in medical genetics who study the inheritance of pathological signs that increase the risk of renal failure are accompanied by a statement of the practical importance of finding out the epigenetic mechanisms of kidney tissue damage (K&ouml;ttgen A. et al., 2010; Lee-Son K., Jetton JG, 2016; Ma RCW, 2016). Indeed, reviews of the results of experimental and clinical studies indicate the need for a deeper development of the scientific direction related to the epigenetic mechanisms of the pathogenesis of renal failure (Thomas M.C., 2016; Witasp A. et al., 2017). At the same time, the peculiarity of the kidney activity is that the various segments of its structural and functional unit, the nephron, have significant differences among themselves related to their transport capabilities, a set of humoral factors regulating their activity and the physicochemical parameters of the microenvironment. Indeed, the homeostatic functions of different parts of the nephron are coordinated by a rather complex system of humoral factors that determine the physiological and pathophysiological mechanisms of the reaction of the kidneys to changes in the parameters of the body&rsquo;s water basins and external adverse effects. These humoral factors include the renin-angiotensin-aldosterone system (RAAS) (Lee-Son K., Jetton JG, 2016), nitric oxide (Shirodkar AV, Marsden PA, 2011), transforming growth factor-beta (Shi M. et al., 2011), etc. The listed humoral factors controlling organogenesis and homeostatic functions of the kidneys require further study, since they can also mediate structural and functional disorders of the renal parenchyma, including epigenetic transformations of transcription and translation in conditions of acute and chronic renal failure. From this point of view, the relevance of the epigenetic approach is due to the fact that, firstly, information on changes in the expression of genes that control biosynthesis and expression of renal parenchyma proteins, as well as systemic and intrarenal humoral factors regulating the homeostatic function of the kidneys can be used to improve modern methods. laboratory diagnosis of kidney disease (Kobori H. et al., 2008). Secondly, the clarification of the epigenetic mechanisms of renal failure pathogenesis opens up the prospect of developing fundamentally new pharmacological drugs, including those controlling the synthesis of renal parenchyma of various physiologically active molecules (Marumo T. et al., 2008; Reddy MA, Natarajan R., 2015) . Third, clarifying the epigenetic mechanisms of progression of renal failure allows us to re-evaluate the range of applications and the nephroprotective properties of already known and widely used drugs, which are based on correcting the activity of the humoral control systems of homeostatic organ functions (Reddy MA et al., 2014; Hayashi K . et al., 2015). Thus, we see our task in trying to integrate modern advances in molecular biology and biochemistry into the already existing system of scientific ideas about the physiological mechanisms of regulating kidney activity and the pathophysiological processes of the pathogenesis of renal failure and progression. Consequently, the goal of our work comes down to clarifying a few questions. First, what is the role of epigenetic transformation of chromatin and micro RNA in the physiological mechanisms of regulation of kidney activity? Secondly, what is the role of the epigenetic processes of imbalance of the systemic and intrarenal metabolism of humoral factors regulating the function of the kidneys in the formation and progression of renal failure? <strong>GENERAL INFORMATION ON EPIGENETIC MECHANISMS</strong> &nbsp; Epigenetics is a modern scientific direction that studies the mechanisms of regulation of gene expression. Epigenetic mechanisms do not affect the primary structure of nucleic acids (Beckerman P. et al., 2014) and are implemented by DNA methylation and demethylation processes (van der Wijst MGP et al., 2015), RNA (Saletore Y. et al., 2013) and post-translational processing of histone proteins (Voon HPJ, Wong LH, 2016; Jamal A. et al., 2012). In our opinion, it is important to note that the processes of synthesis and post-transcriptional processing of micro RNA are tightly controlled by enzymes (Treiber T. et al., 2019; Michlewski G., C&aacute;ceres JF, 201 (). In addition, micro RNA can perform an important function in the regulation of protein biosynthesis at the level of transcription or translation (Petrillo F. et al., 2017; Thomas MJ et al., 2018). According to the authors of the cited reviews, the study of the metabolism of micro RNA in various biological media of the body contributes to the development of fundamentally new diagnostic methods and kidney disease treatment. of this, epigenetic mechanisms are involved in adaptive responses of the organism and population in response to changes in environmental factors through modulation of gene expression (Zama A.M., Uzumcu M., 2010). One of the most studied epigenetic mechanisms of regulation of gene expression is the process of DNA methylation. This process involves the addition of methyl groups (CH3) to one of four types of DNA nucleotides by forming a covalent bond between them, however, the order of the sequence of nucleotides in the DNA chain does not change (Lister R. et al., 2009; Woroniecki R. et al., 2011). One of the four nuclear DNA methyltransferase isoenzymes (DNMT), namely DNMT1, DNMT2, DNMT3a or DNMT3b, is responsible for the addition of methyl groups to nucleotides (Reddy M.A., Natarajan R., 2015; Efimova O.A. et al., 2012). DNMT1 recognizes semi-methylated DNA (each of its chains) during replication (Bechtel W. et al., 2010). DNMT3a and DNMT3b provide de novo methylation of DNA, i.e., repeatedly, in new sites. The DNMT2 function is still under discussion (Jamal A. et al., 2012). DNA methyltransferases have the ability to embed specific &ldquo;tags&rdquo; in nucleic acids, which leads to changes in the expression of these genes (van der Wijst M.G.P. et al., 2015). It is suggested that epigenetic &ldquo;tags&rdquo; in the DNA chain block the transcription process for more than two thirds of the DNA volume (Ponnaluri V.K.C. et al., 2016). Thus, the cells of an organism use covalent modification of DNA in order to regulate gene expression according to the so-called &ldquo;on / off&rdquo; principle (Quarta C. et al., 2016). DNA methylation in most cases occurs on the cytosine nitrogen base (C), which is paired with guanine (G), on the so-called CpG sites, or CpG clusters (Dwivedi R.S. et al., 2011). In the human body, about 70-80% of CpG dinucleotides are in the methylated state (Ziller M.J. et al., 2013). The portions of the DNA chain where the density of CpG clusters is particularly high are called CpG islands (Zhang D. et al., 2009). However, the process of DNA methylation is carried out in areas with low density of CpG dinucleotides (Ziller M.J. et al., 2013). Along with the process of DNA methylation, the process of demethylation is of great importance in the epigenetic regulation of gene expression (van der Wijst M.G.P. et al., 2015; Yefimova O.A. et al., 2012). DNA demethylation is the process of DNA release from methyl groups, which is carried out with the help of special enzymes of demetallas (Auclair G., Weber M., 2012). In addition, modifications of histone proteins as a result of acetylation, deacetylation, phosphorylation, ubiquitination, etc. are also considered epigenetic mechanisms (Ganai S.A. et al., 2016; Araki Y., Mimura T., 2017;). Acetylation and deacetylation processes are possible due to a number of specific enzymes - histone acetylases (acetyltransferases, HAT) and histone deacetylases (HDAC) (Gong F. et al., 2016). Phosphorylation occurs at the expense of enzymes of kinases (phosphotransferases) (Araki Y., Mimura T., 2017; Nathan D. et al., 2012). Covalent modification of histones, as a rule, weakens the connection between the histone core of the nucleosome and DNA, which contributes to the availability of the DNA molecule for the transcription process (Rossetto D. et al., 2012). However, this rule is not universal. For example, acetylation of lysine at position 27 of histone H3 (H3K27ac) leads to increased gene expression. The combination of acetylated lysine 14 (H3K14ac) and phosphorylated serine 10 histone H3 (H3S10ph) also indicates increased gene expression (Chen K.W., Chen L., 2017). Along with this, other modifications, namely acetylation of lysine H2AK5, H2BK20, H3K14, H4K5, etc., and threonine H3T3 and serine H3S28 and H4S1 phosphorylation similarly lead to gene activation (Araki Y., Mimura T., 2017; Wang Z. et al ., 2008; Rossetto D. et al., 2012). On the contrary, histone deacetylation is accompanied by gene inactivation, as it entails DNA condensation and the impossibility of transcription (Ganai S.A. et al., 2016). Post-transcriptional gene expression is regulated by such an epigenetic mechanism as RNA interference. RNA interference is the process of suppressing the process of protein biosynthesis (transcription, translation) using micro RNA (Nabzdyk C.S. et al., 2017). At the same time, micro RNA can have a direct impact on the state of translation (Dwivedi R.S. et al., 2011; Cui J. et al., 2017). The actual process of suppressing gene expression is called silencing (Cui J. et al., 2017). MicroRNA is a class of short single-stranded non-coding RNAs of 21-24 nucleotides in length (Dwivedi R.S. et al., 2011; Zhang D. et al., 2009). Endogenous noncoding regions of DNA are responsible for the production of micro RNA. In addition to translation, micro RNA has the ability to suppress gene expression at the transcription stage (Dwivedi R.S. et al., 2011). All regulatory effects of micro RNA are embedded in a specific system of epigenetic control of the functions of a given cell population (Dwivedi R.S. et al., 2011). Analyzing modern literature data, it can be assumed that epigenetic mechanisms are an important element of adaptation at the population and organism level, coordinating the expression of genes adequately to changes in environmental factors. In our consciousness, the concept of &ldquo;epigenetic mechanisms&rdquo; is more often associated with the phenomenon of fetal programming. Meanwhile, their activity is actively proceeding in the postnatal period of ontogenesis. It is difficult to say exactly what the imperfection of this form of adaptive response can take place, however, its implementation often involves activation of the pathogenetic mechanisms of various organ systems. <strong>СПИСОК ЛИТЕРАТУРЫ К ГЛАВЕ &laquo;ВВЕДЕНИЕ&raquo;</strong> &nbsp; 1.Reddy M.A, Natarajan R. Recent Developments in Epigenetics of Acute and Chronic Kidney Diseases Kidney Int. 2015 88(2): 250&ndash;261 doi:10.1038/ki.2015.148 &nbsp; 2.Uwaezuoke S.N., Okafor H.U., Muoneke V.N. et al. Chronic kidney disease in children and the role of epigenetics: Future therapeutic trajectories. Biomed Rep. 2016; 5(6): 660&ndash;664 doi: 10.3892/br.2016.781 &nbsp; 3.Zununi Vahed S., Samadi N., Mostafidi E. et al. Genetics and Epigenetics of Chronic Allograft Dysfunction in Kidney Transplants. Iran J Kidney Dis. 2016;10(1):1-9 &nbsp; 4.Lee-Son K., Jetton J.G. AKI and Genetics: Evolving Concepts in the Genetics of Acute Kidney Injury: Implications for Pediatric AKI. J Pediatr Genet. 2016; 5(1): 61&ndash;68 doi:10.1055/s-0035-1557112 &nbsp; 5.Woroniecki R., Gaikwad A., Susztak K. Fetal environment, epigenetics, and pediatric renal disease. Pediatr Nephrol. 2011; 26(5): 705&ndash;711 doi: 10.1007/s00467-010-1714-8 &nbsp; 6.K&ouml;ttgen A., Pattaro C., B&ouml;ger C.A. et al. Multiple New Loci Associated with Kidney Function and Chronic Kidney Disease: The CKDGen consortium. Nat Genet. 2010; 42(5): 376&ndash;384 doi: 10.1038/ng.568 &nbsp; 7.Ma R.C.W. Genetics of cardiovascular and renal complications in diabetes. J Diabetes Investig. 2016; 7(2): 139&ndash;154 doi: 10.1111/jdi.12391 &nbsp; 8.Thomas M.C. Epigenetic Mechanisms in Diabetic Kidney Disease. Curr Diab Rep. 2016;16:31 doi 10.1007/s11892-016-0723-9 &nbsp; 9.Witasp A., Van Craenenbroeck A.H., Shiels P.G. et al. Current epigenetic aspects the clinical kidney researcher should embrace. Clinical Science. 2017; 131:1649&ndash;1667 doi:10.1042/CS20160596 &nbsp; 10.Shirodkar A.V., Marsden P.A. Epigenetics in cardiovascular disease. Curr Opin Cardiol. 2011; 26(3): 209&ndash;215 doi:10.1097/HCO.0b013e328345986e &nbsp; 11.Shi M., Zhu J., Wang R. et al. Latent TGF-&beta; structure and activation. Nature. 2011; 474(7351): 343&ndash;349 doi: 10.1038/nature10152 &nbsp; 12.Kobori H., Katsurada A., Miyata K. et al. Determination of plasma and urinary angiotensinogen levels in rodents by newly developed ELISA. Am J Physiol Renal Physiol. 2008; 294(5): F1257&ndash;F1263 doi: 10.1152/ajprenal.00588.2007 &nbsp; 13.Marumo T., Hishikawa K., Yoshikawa M., Fujita T. Epigenetic Regulation of BMP7 in the Regenerative Response to Ischemia. J Am Soc Nephrol. 2008; 19(7): 1311&ndash;1320 doi: 10.1681/ASN.2007091040 &nbsp; 14.Hayashi K., Sasamura H., Nakamura M. et al. Renin-angiotensin blockade resets podocyte epigenome through Kruppel-like Factor 4 and attenuates proteinuria. Kidney Int. 2015;88(4):745-753 doi: 10.1038/ki.2015.178 &nbsp; 15.Reddy M.A., Sumanth P., Lanting L. et al. Losartan reverses permissive epigenetic changes in renal glomeruli of diabetic db/db mice. Kidney Int. 2014; 85(2): 362&ndash;373 doi: 10.1038/ki.2013.387 &nbsp; 16.Kobori H., Kamiyama M., Harrison-Bernard L.M., Navar L.G. Cardinal Role of the Intrarenal Renin-Angiotensin System in the Pathogenesis of Diabetic Nephropathy. J Investig Med. 2013; 61(2): 256&ndash;264 doi:10.231/JIM.0b013e31827c28bb &nbsp; 17.Beckerman P., Ko Y.-A., Susztak K. Epigenetics: a new way to look at kidney diseases. Nephrol Dial Transplant. 2014; 29(10): 1821&ndash;1827 doi: 10.1093/ndt/gfu026 &nbsp; 18.van der Wijst M.G.P., Venkiteswaran M., Chen H. et al. Local chromatin microenvironment determines DNMT activity: from DNA DNMT activity: from DNA methyltransferase to DNA demethylase or DNA dehydroxymethylase. Epigenetics. 2015; 10(8): 671&ndash;676 doi:10.1080/15592294.2015.1062204 &nbsp; 19.Saletore Y.,Chen-Kiang S., Mason C.E. Novel RNA regulatory mechanisms revealed in the epitranscriptome. RNA Biol. 2013; 10(3): 342&ndash;346 doi: 10.4161/rna.23812 &nbsp; 20.Voon H.P.J., Wong L.H. New players in heterochromatin silencing: histone variant H3.3 and the ATRX/DAXX chaperone. Nucleic Acids Res. 2016; 44(4): 1496&ndash;1501 doi: 10.1093/nar/gkw012 &nbsp; 21.Jamal A., Man H.S.J., Marsden P.A. Gene Regulation in the Vascular Endothelium: Why Epigenetics Is Important for the Kidney. Semin Nephrol. 2012; 32(2): 176&ndash;184 doi: 10.1016/j.semnephrol.2012.02.009 &nbsp; 22.Treiber T., Treiber N., Meister G. Publisher Correction: Regulation of microRNA biogenesis and its crosstalk with other cellular pathways. Nat Rev Mol Cell Biol. 2019;20(5):321 doi: 10.1038/s41580-019-0106-6 23.Michlewski G., C&aacute;ceres J.F. Post-transcriptional control of miRNA biogenesis. RNA. 2019;25(1):1-16 doi: 10.1261/rna.068692.118 &nbsp; 24.Petrillo F., Iervolino A., Zacchia M., Simeoni A., Masella C., Capolongo G., Perna A., Capasso G., Trepiccione F. MicroRNAs in Renal Diseases: A Potential Novel Therapeutic Target. Kidney Dis (Basel). 2017;3(3):111-119 doi: 10.1159/000481730 25.Thomas M.J., Fraser D.J., Bowen T. Biogenesis, Stabilization, and Transport of microRNAs in Kidney Health and Disease. Noncoding RNA. 2018;4(4). pii: E30 doi: 10.3390/ncrna4040030 &nbsp; 26.Zama A.M., Uzumcu M. Epigenetic effects of endocrine-disrupting chemicals on female reproduction: An ovarian perspective. Front Neuroendocrinol. 2010; 31(4): 420&ndash;439 doi:10.1016/j.yfrne.2010.06.003 &nbsp; 27.Lister R., Pelizzola M., Dowen R.H. et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature. 2009; 462: 315-322 doi: 10.1038/nature08514 &nbsp; 28.Efimova O.A., Pendina A.A., Tikhonov A.V. et al. DNA methylation - a major mechanism of human genome reprogramming and regulation. Medical Genetics. 2012; 4(118): 10-18 &nbsp; 29.Bechtel W., McGoohan S., Zeisberg E.M. et al. Methylation determines fibroblast activation and fibrogenesis in the kidney. Nat Med. 2010; 16(5): 544&ndash;550 doi: 10.1038/nm.2135 &nbsp; 30.Ponnaluri V.K.C., Ehrlich K.C., Zhang G., Lacey M., Johnston D., Pradhan S., Ehrlich M. Association of 5-hydroxymethylation and 5-methylation of DNA cytosine with tissue-specific gene expression. Epigenetics. 2016; 12(2): 123-138 doi: 10.1080/15592294.2016.1265713 &nbsp; 31.Quarta C., Shneider R., Tschӧp M.H. Epigenetic ON/OFF Switches for Obesity. Cell. 2016; 164(3): 341-342 doi: 10.1016/j.cell.2016.01.006 &nbsp; 32.Dwivedi R.S., Herman J.G., McCaffrey T. et al. Beyond genetics: epigenetic code in chronic kidney disease. Kidney Int. 2011; 79(1): 23-32 doi: 10.1038/ki.2010.335 &nbsp; 33.Ziller M.J., Gu H., M&uuml;ller F., Donaghey J. et al. Charting a dynamic DNA methylation landscape of the human genome. Nature. 2013; 500(7463): 477-481. doi: 10.1038/nature12433 <strong>ГЛАВА 1. </strong><strong>ЭПИГЕНЕТИЧЕСКИЕ</strong><strong> </strong><strong>МЕХАНИЗМЫ </strong> <strong>В СИСТЕМЕ КОНТРОЛЯ</strong><strong> </strong><strong>ФУНКЦИИ</strong><strong> </strong><strong>ПОЧЕК</strong><strong> </strong><strong>В</strong><strong> </strong><strong>НОРМЕ</strong><strong> </strong> &nbsp; &nbsp; Эпигенетические системы управления экспрессии генов выполняют принципиально важную функцию на разных этапах онтогенеза. Например, у плода они координируют нормальное течение нефрогенеза. В зрелом возрасте эпигенетические механизмы тесно вовлечены в систему контроля гомеомтатических функций органа. Процессы снижения функции почек у пожилых людей также тесно связаны с эпигенетическими механизмами. Оценивая роль эпигенетических механизмов в процессах органогенеза почки, необходимо отметить роль метилирования гистонов в цитодифференцировке эмбриональных клеток (Adli M. et al., 2015). Наряду с этим, подчеркивается роль баланса активности гистоновых ацетилтрансфераз и деацетилаз в регуляции экспрессии генов на ранних стадиях органогенеза почки (Hilliard S.A, El-Dahr S.S., 2016). Высказывается мнение о том, что некоторые деацетилазы гистоновых белков (HDAC1 and HDAC2) могут быть критически важны для процессов развития канальцевого и сосудистого компонентов нефрона на ранних стадиях онтогенеза почки (Liu H. et al., 2018). Наряду с этим, в литературе имеются даные о том, что синтез некодидурющих РНК и реакции ацетилирования гистонов выполняют важную роль в формировании юкста-гломерулярного аппарата (ЮГА) в процессе нефрогенеза (Martini A.G., Danser A.H.J., 2017). С другой стороны, внутриорганная продукция компонентов ренин-ангиотензиновой системы (РАС) на ранних этапах онтогенеза также критически важна для координации гисто- и органогенеза. Установлено, что избыточное потребление хлорида натрия во время беременности может нарушать эти процессы через изменения активности внутриорганной экспрессии компонентов РАС и продукции оксида азота в тканях плода (Stocher D.P. et al., 2018). Анализ роли метилтрансфераз и деметилаз, а также гистон-ацетилтрансфераз и гистон-деацетилаз в процессах нефрогенеза позволил выявить определенные закономерности динамики активности данных групп ферментов по мере формирования нефрона (Hilliard S.A., El-Dah S.S., 2016). Авторы цитируемого обзора сопоставляют процессы нефрогенеза с топологией и динамикой во времени активности систем ковалентной модификации хроматина: остатков лизина в составе гистонов (H3K), остатков аргинина в составе гистонов (H3R) и молекулы ДНК. Наряду с процессами ковалентной модификации хроматина, механизмы транскрипции и метаболизма некодирующих РНК также могут иметь принципиально важное значение для нормального течения морфогенеза почки млекопитающих (Ho J., Kreidberg J.A., 2012). В настоящее время роль микро РНК в процессах органогенеза почки изучено достаточно подробно. В литературе имеются сведения о том, что некоторые семейства микро РНК критически важны для морфогенеза сосудисто-клубочкового и канальцевого отделов почки (Trionfini P., Benigni A., 2017). Возможно, эпигенетические механизмы органогенеза почки находятся под контролем гормонов системного действия материнского организма. В частности, показано, что такой способностью может обладать мелатонин (Tain Y.-L. et al., 2017). По мнению авторов, мелатонин обладает способностью контролировать не только формирование архитектуры нефрона, но и регулировать уровень активности внутрипочечной системы NO-синтаз и ренин-ангиотензиновой системы плода через интенсивность метилирования ДНК и ацетилирования белков гистонов. Кроме того, показано, что инсулин также обладает выраженным влиянием на состояние эпигенетических механизмов в тканях почки человека (Lay A.C., Coward R.J.M., 2018). В литературе приводятся данные о том, что гипометилирование хроматина на уровне нейро-эндокринного звена контроля деятельности почки &mdash; одна из причин увядания гомеостатической функции органа в преклонном возрасте (Murgatroyd C. et al., 2010). Дальнейшие исследования позволили установить важное значение роли метилирования хроматина в возрастных изменениях системы контроля водно-солевого баланса у млекопитающих (Greenwood M.P. et al., 2018). В литературе уделяется внимание роли микро РНК и ковалентной модификации хроматина в процессах возрастных нарушений функции почек человека (Shiels P.G. et al., 2017). На основе анализа роли деацетилаз гистонов SIRT1 и SIRT3 в регуляции обменных процессов почки, делается вывод о том, что данная группа ферментов обладает выраженным нефропротекторным свойством, обеспечивая сдерживание процессов старения тканей органа (Morigi M. et al., 2018). <strong>1.1. ПЛАСТИЧНОСТЬ СИСТЕМ ЭПИГЕНЕТИЧЕСКОЙ МОДУЛЯЦИИ ЭКСПРЕССИИ ГЕНОВ РЕНАЛЬНОЙ ПАРЕНХИМЫ </strong> &nbsp; Как уже отмечалось выше, в зрелом возрасте эпигенетические механизмы сохраняют за собой важное место в регуляции функции почек, в частности, адаптивных реакций ренальной паренхимы. Необходимо подчеркнуть, что эпигенетические механизмы контроля биосинтеза белка сохраняют высокий уровень пластичности в зрелом возрасте. Иллюстрируя высокие показатели пластичности обсуждаемых процессов, можно упомянуть роль метилирования ДНК в формировании суточного ритма поведенческой активности млекопитающих (Azzi A. et al., 2014). Следовательно, есть основания полагать, что молекулярные механизмы регуляции экспрессии генов могут непосредственно координировать адаптивные реакции ренальной паренхимы. Возможно, эпигенетические механизмы, наряду с нейро-гуморальными системами контроля водно-солевого обмена, принимают участие в регуляции гомеостатических функций почек. В ряде публикаций указывается, что стимулом для молекулярных механизмов управления экспрессии генов, как правило, является динамика параметров констант водно-солевого баланса организма. Результаты более ранних исследований показали, что метилтрансфераза гистонов Dot1a непосредственно определяет альдостерон-зависимую транскрипцию гена EnaC-альфа в дистальных отделах нефрона (Zhang D. et al., 2009). Согласно данным литературы, состояние посттранскрипционного процессинга предшественника микроРНК в проксимальных нефроцитах может выполнять ключевую роль в адаптации канальцевого эпителия к ишемии, возможно, участвуя в патогенезе реперфузионного поражения S3-сегмента (Wei Q. et al., 2010). Подчеркивается, что интенсивность репаративных реакций ренальной паренхимы может контролироваться некодирующими РНК и состоянием метилирования Н2А и Н3 гистонов (Chou Y.-H. et al., 2017). В литературе имеются сведения о том, что у некоторых видов млекопитающих в адаптивных реакциях почки на острые изменения системных параметров водно-солевого обмена могут принимать участие механизмы регуляции экспрессии генов (MacManes M.D., 2017). Наряду с этим, в проксимальном сегменте нефрона объектом регуляторного влияния эпигенетических механизмов являются гены субъединиц натрий\калиевой АТФазы базолатеральной мембраны эпителия (Taub M., 2018). По мнению автора цитируемого обзора, сигналом для активации/инктивации транскрипции указанных генов может служить концентрация натрия в люминальной жидкости, а непосредственная реализация поступающих сигналов определяется интенсивностью ацетилирования гистонов. Наряду с этим, изменения внутриклеточной концентрации натрия в эпителии проксимального сегмента нефрона и тонкой восходящей петли Генле также может оказывать прямое влияние на состояние транскрипции генов транспортных белков, экспрессируемых данной популяцией нефроцитов (Gildea J.J. et al., 2018). Приводятся данные о том, что содержание натрия в рационе питания оказывает влияние на экспрессию генов белков-транспортеров натрия (ENaC и Na-Cl-котранспортер) в дистальном отделе нефрона (Ivy J.R. et al., 2018). С другой стороны, показано, что гипонатриевая диета стимулирует гипометилирование гена альдостерон-синтазы через активацию РАС (Takeda Y. et al., 2018). Привлекает внимание тот факт, что ядерные деацетилазы ренальной паренхимы (SIRT1,3,6,7) обладают способностью регулировать экспрессию ряда белков в тканях почки, имеющих фундаментальное значение для гомеостатических функций органа (Morigi M. et al., 2018). В частности, авторы обзора сообщают, что SIRT1 непосредственно регулирует экспрессию альфа-субъединицы эпителиального натриевого канала, эндотелиальной NO-синтазы и рецептора к ангиотензину-2 (AT1R) в подоцитах и гладкомышечных волокнах кровеносных сосудов почки. По данным авторов SIRT3 участвует в регуляции обменных процессов в митохондриях, обладает противовоспалительным и противосклерозирующим действием. Белок SIRT6 также необходим для сдерживания просклерозирующих факторов. Следует отметить, что, наряду с ковалентной модификацией хроматина, важная роль в эпигенетическом контроле ренального транспорта веществ отводится некодирующим РНК (Hua J.X. et al., 2012). Показана важная роль микро РНК в регуляции транспорта натрия в эпителии нефрона (Mladinov D. et al., 2013). Наряду с ионорегулирующей функцией почек, установлено, что некодирующие РНК могут принимать участие в управлении осморегулирующей функцией почек млекопитающих (Huang W. et al., 2011; Luo Y. et al., 2014). Авторы цитируемых публикаций указывают на роль микро РНК в регуляции экспрессии транспортных белков медуллярных сегментов нефрона в ответ на острый гиперосмотический стимул. Следует отметить, что в норме экспрессия некоторых типов микро РНК в корковом и мозговом слое почки имеет четкие отличия (Chandrasekaran K. et al., 2012; Ichii O., Horino T., 2018). Приводится информация о непосредственном влиянии гиперосмотического стимула на экспрессию строго определенных типов микро РНК во внутренней медулле почки (Chandrasekaran K. et al., 2012). Вместе с тем, авторы обращают внимание на тот факт, что состояние метаболизма микро РНК в ренальной паренхиме может регулироваться гуморальными факторами нейро-эндокринного звена контроля гомеостатических функций почек. При этом микро РНК осуществляют контроль транспорта ионов не только в почке, но и системные параметры ионного гомеостаза (Hua J.X. et al., 2012). В ряде публикаций подчеркивается тезис о том, что микро РНК могут осуществлять постоянную тонкую регуляцию обменных процессов в ренальной паренхиме. Например, имеются сообщения о роли микро РНК в регуляции обменных процессов в подоцитах, в зависимости от возможных изменений величины гидростатического давления в клубочке и химического состава ультрафильтрата (Trionfini P., Benigni A., 2017). Одним из наиболее перспективных направлений исследований роли микро РНК в регуляции деятельности почки является анализ взаимосвязи внутриорганного метаболизма микро РНК и их содержания в биологических средах организма (Thomas M.J. et al., 2018). С точки зрения практической медицины, ценность таких исследований обусловлена необходимостью внедрения новых методов диагностики и терапии заболеваний почек (Trionfini P., Benigni A., 2017; Thomas M.J. et al., 2018). Вместе с тем, значительное внимание уделяется роли гуморальных систем контроля гомеостатических функций почек в регуляции эспрессии генов в ренальной паренхиме (Hirohama D. et al., 2018; Lu C.C. et al., 2018). Приводятся данные о молекулярных механизмах регуляции локальной экспрессии белков-компонентов РАС (Martini A.G. et al., 2017; Lu C.C. et al., 2018). Ранее была показана роль микро РНК в регуляции экспрессии генов ренина (Sequeira-Lopez M.L.S. et al., 2010). В настоящее время установлена роль метилирования ДНК, ацетилирования и метилирования гистонов канальцевого эпителия в управлении экспрессией гена ангиотензиногена (Marumo T. et al., 2015). Показано участие метилирования ДНК, ковалентной модификации гистонов и метаболизма микро РНК в экспрессии генов ренина в почке (Martini A.G., Danser A.H.J., 2017). С другой стороны, выявлено значение ренин-ангиотензин-альдостероновой системы в регуляции экспрессии генов транспортных белков канальцевого отдела нефрона в ответ на изменение физиологических констант водно-солевого баланса (Hirohama D. et al., 2018). Установлено, что ковалентная модификация гистонов (метилирование и ацетилирование) может принимать участие в контроле экспрессии гена атриального натрийуретического пептида (Hohl M. et al., 2013). Сообщается, что эпигенетический контроль экспрессии гена атриального натрийуретического пептида способствует адаптивным изменениям продукции гормона (Sergeeva I.A. et al., 2016). При этом, атриальный натрийуретический пептид также рассматривается в качестве индуктора эпигенетических механизмов, реализуемых через специфические микро РНК (Li Y. et al., 2016). Не меньший интерес привлекают сведения о влиянии острого осмотического стимула на эпигенетические системы контроля синтеза аргинин-вазопрессина - АВП (Hayashi M. et al., 2006; Greenwood M.P. et al., 2016). Необходимо отметить, что половые стероидные гормоны также могут оказывать влияние на экспрессию гена АВП при участии эпигенетических механизмов (Augera C.J. et al., 2011). Поскольку канальцевые эффекты АВП реализуются при участии специфических поробразующих белков &mdash; аквапоринов, в частности, при участии аквапорина-2 (AQP2), привлекают интерес сведения о значении эпигенетического контроля данного белка (Park E.-J., Kwon T.H.; 2015; Jung H.J., Kwon T.-H.; 2016). <strong>1.2. ЭНДОКРИННЫЕ ФАКТОРЫ РЕГУЛЯЦИИ ВОДНО-СОЛЕВОГО БАЛАНСА ОРГАНИЗМА В СИСТЕМЕ ЭПИГЕНЕТИЧЕСКИХ МЕХАНИЗМОВ КОНТРОЛЯ ГОМЕОСТАЗА </strong> &nbsp; Предполагая определенную роль эпигенетических механизмов в регуляции гомеостатических функций почек и адаптивных изменениях органа, по нашему мнению, необходимо проанализировать, во-первых, информацию о роли эпигенетических механизмов в модуляции экспрессии генов белковых гормонов-регуляторов водно-солевого обмена. Во-вторых, свойства гуморальных факторов системного действия, как возможных индукторов эпигенетической трансформации ренальной паренхимы. Общеизвестна роль аргинин-вазопрессина, как системного регулятора осмотического гомеостаза, определяющего острую и точную реакцию организма на изменение пищевого и внутривенного поступления жидкости осмотически активных веществ (Bourque C.W., 2008; Thornton S.N.; 2010; Greenwood M.P. et al., 2015; Park E.-J., Kwon T.-H., 2015). Физиологическая роль ренин-ангиотензиновой системы определяется, как контролем реабсорбции весьма значительного объема ультрафильтрата, растворенных в нем натрия и калия, а также других жизненно важных компонентов ультрафильтрата (Zhuo J.L., Li X.C., 2001; Kurtz A., 2012; Gomez R.A., Sequeira-Lopez M.L.S., 2018). Таким образом, Ангиотензин-II принимает участие в регуляции показателей ионного, осмотического, волемического, кислотно-основного гомеостаза, а также регулирует тонус кровеносных сосудов. Атриальный (мозговой) натрий уретический пептид &mdash; важнейший гуморальный регулятор волемического гомеостаза, определяющий выведение натрия и жидкости на уровне дистального отдела нефрона (Kuwahara К., Nakao К., 2010; Nakagawa Y. et al., 2019). &nbsp; 1.2.1. АРГИНИН-ВАЗОПРЕССИН (АВП) &nbsp; Результаты более ранних исследований позволили установить, что изменения физиологических констант осмотического и волемического гомеостаза оказывают влияние на уровни транскрипции гена аргинин вазопрессина (АВП) (Kondo N. et al., 2004). Кроме того, авторами цитируемой публикации была выявлена корреляция между концентрацией катионов натрия во внеклеточной жидкости и уровнем экспрессии гена аргинин вазопрессина. Было продемонстрировано также резкое усиление транскрипции гена АВП под влиянием осмотического стимула (Hindmarch C.C.T., Murphy D., 2010). Наряду с этим, было показано, что гиперосмотический стимул усиливает транскрипцию ряда генов, белки которых аккумулируются в задней доли гипофиза (Hindmarch C. et al., 2006). Выявлено, что активация транскрипции гена аргинин вазопрессина, под влиянием осмотического воздействия, демонстрирует более выраженную чувствительность к стимулу, в сравнении с другими нейропептидами задней доли гипофиза (Yue C. et al., 2008). Сложность вопроса в том, что к осмотическому стимулу чувствительны также гены гипоталамо-гипофизарной оси, принимающие участие в регуляции репродуктивной сферы (Qiu J. et al., 2007). В то же время, было установлено, что осмотические нагрузки оказывают специфическое влияние на экспрессию вполне определенной группы генов в супраоптическом ядре крысы (Johnson K.R. et al., 2015). При этом, необходимо отметить, что, вероятно, ген аргинин вазопрессина содержит нуклеотидную последовательность в области промотора, обладающей чувствительностью к изменениям показателей осмотического гомеостаза (Ponzio T.A. et al., 2012). Авторами установлено отличие в первичной последовательности нуклеотидов данного участка генов аргинин вазопрессина и окситоцина. Далее, сопоставляя классическую схему физиологического контроля осмотического гомеостаза и факты, подтверждающие участие эпигенетических механизмов, опираясь на выше изложенные результаты исследований, мы констатируем, что показатель экспрессии гена аргинин вазопрессина обладает чувствительностью к сдвигам осмоляльности внеклеточной жидкости организма. Вероятный механизм влияния физико-химических условий внеклеточной жидкости (концентрации хлорида натрия во внеклеточной жидкости) на состояние транскрипции гена аргинин вазопрессина, в основном, подтвердили ранее выполненные наблюдения (Kondo N. et al., 2004; Hindmarch C.C., Murphy D., 2010). При этом отмечается, что АВП, помимо регуляции осмотического гомеостаза, может отвечать за поведенческие реакции, поэтому, с точки зрения авторов, нарушения осмотического гомеостаза могут негативно отражаться на адаптивных поведенческих реакциях (Mitchell N.C. et al., 2018). Показано, что экспрессия гена аргинин вазопрессина демонстрирует высокий уровень пластичности, и что интенсивность метилирования ДНК в области помотора гена гормона может существенно изменяться в зависимости от состояния показателей осмотического гомеостаза организма (Greenwood M.P. et al., 2016). Сообщается о видоспецифических молекулярных механизмах, вовлеченных в индукцию транскрипции аргинин вазопрессина, на фоне дегидратации организма (Stewart L. et al., 2011). Высокий уровень пластичности эпигенетических систем контроля биосинтеза аргинин вазопрессина подтверждается тем фактом, что усиление транскрипции гена гормона регистрируется в условиях острого гиперосмотического стимула раствором хлорида натрия (Kawasaki M. et al., 2009). В настоящее время имеются данные и о том, какие энзиматические системы, отвечающие за ковалентную трансформацию хроматина принимают участие в изменении транскрипции гена аргинин вазопрессина (Archer T., 2015). Дальнейшие исследования, проведенные научными сотрудниками групппы Murphy D. показали, что к чувствительностью к осмотическому стимулу обладают целый ряд генов (Caprin2), белки которых могут быть важны в формировании адаптивного ответа супраоптических ядер гипоталамуса на изменения осмотического гомеостаза организма (Loh S.-Y. et al., 2017). При том, что показана роль гена Caprin2 в механизмах стабилизации матричной РНК аргинин вазопрессина (Konopacka A. et al., 2015). Высказанный тезис можно дополнить сведениями о том, что микро РНК также принимают участие в эпигенетической модуляции активности нейро-эндокринного контроля осмотического гомеостаза (Luo Y. et al., 2014). В этом блоке анализа данных литературы необходимо выделить тот факт, что аргинин вазопрессин может непосредственно контролировать экспрессию транспортного белка Na+,K+,2Cl- котранспортера в восходящей петле Генле нефрона (Konopacka A. et al., 2015). Однако, непосредственно усиление экспрессии гена Na+,K+,2Cl-котранспортера по влиянием аргинин вазопрессина, рассматривается в качестве долговременной АВП- зависимой стимуляции белка (Knepper M.A. et al., 2015). Вместе с тем, авторы обзора подчеркивают, что аргинин вазопрессин может контролировать в дистальных сегментах нефрона экспрессию таких транспортных белков, как: натрий-хлор котранспортирующий протеин, переносчик мочевины, некоторые субъединицы эпителиального натриевого канала порообразующих белков аквапоринов. Подчеркивается актуальность данных механизмов в изучении патогенеза заболеваний почек и сердечно-сосудистой системы (Qian Q., 2018). Также анализируется АВП-зависимые системы внутриклеточной передачи сигнала (через протеин киназы) в эпителии собирательных трубочек канальцевого отдела нефрона, как звено индукции эпигенетического контроля экспрессии генов транспортных белков (Sanghi A. et al., 2014). С другой стороны, анализируется взаимосвязь различных изоформ аденилатциклаз и протеинкиназ в системе регуляции генов транспортных белков в эпителии собирательных трубочек (Roos K.P. et al., 2013). Завершая рассмотрение роли эпигенетических механизмов в поддержании осмотического гомеостаза, необходимо отметить участие АВП в долговременной стимуляции биосинтеза и экспрессии аквапорина-2 в эпителии собирательных трубочек канальцевого отдела нефрона (Wilson J.L.L. et al., 2013). Были установлены механизмы активации транскрипции гена аквапорина-2, включающие в себя механизмы внутриклеточной передачи сигнала, а также идентифицированы участки ДНК предполагаемого связывания регулятора транскрипции (Yua M.-J. et al., 2009). Проведен анализ метаболизма белка аквапорина-2 в эпителии собирательных трубочек канальцевого отдела нефрона и роль АВП в управлении транскрипции гена AQP2 (Jung H.J., Kwon T.H., 2016). Наряду с этим, авторы цитируемого обзор указывают на роль микро РНК (miR-32 и miR-137) в процессах внутриклеточного метаболизма протеина аквапорина-2. Оценивая роль эпигенетического контроля физиологических функций собирательных трубочек (Xiao Z. et al., 2016), авторы приходят к выводу, что баланс активности ядерных метилтрансфераз (Dot1lAC и Dot1lf/f) в эпителии данного сегмента нефрона, может оказывать существенное влияние на экспрессию белка аквапорин-2. Поскольку изменения в продукции и биологических эффектах аргинин вазопрессина имеют отношение не только к регуляции водно-солевого гомеостаза, но и к поведенческим реакциям человека, изучение эпигенетических процессов контроля, например, рецепторов АВП также является объектом междисциплинарных исследований (Bodden C. et al., 2017). &nbsp; 1.2.2. НАТРИЙУРЕТИЧЕСКИЕ ПЕПТИДЫ &nbsp; Интерес к эпигенетическим системам контроля АНП также в некоторой степени обусловлен нейротропными эффектами гормона (Frieling H. et al., 2008). Были достаточно подробно изучены физиологически активные вещества, способные индуцировать транскрипцию генов атриального (АНП), мозгового (БНП) и С-типа натрийуретических пептидов, структура генов и самих натрийуретических гормонов (Gardner D.G. et al., 2007; Kuwahara К., Nakao К., 2010; Ichiki T., Burnett J.C., 2017; Nakagawa Y. et al., 2019). Вместе с тем, имеются данные о том, что АНП может синтезироваться эпителием канальцевого отдела нефрона (Dong L. et al., 2016; Pandey K.N., 2018). При этом, в некоторых обзорных публикациях высказывается тезис о важной практической значимости исследований эпигенетического контроля экспрессии генов натрийуретических пептидов (DiSalvo T.G., 2015; Man J. et al., 2018). Поскольку представляет интерес несколько аспектов данной проблемы: эффективность использования параметров синтеза и секреции натрийуретических пептидов в качестве диагностических маркеров ряда актуальных нозологий, исследования собственно механизмов контроля экспрессии генов этих гормонов и вовлеченность в этот процесс некоторых гормонов и цитокинов, участвующих в патогенезе заболеваний сердечно-сосудистой системы и почек: ангиотензин-2, трансформирующий фактор роста-бета1, гормоны щитовидной железы (Sergeeva I.A., Christoffels V.M., 2013). Вместе с тем, данные литературы подчеркивают значение уровня экспрессии рецепторов натрийуретических пептидов в сердечно-сосудистой системе и ренальной паренхиме для понимания физиологических и патофизиологических эффектов гормонов (Pandey K.N., 2011; Kumar P. et al., 2014). Результаты экспериментальных исследований показали, что гипертрофия кардиомиоцитов токсического генеза сопровождается снижением продукции miR-133a на фоне усиления метилирования ДНК метилтрансферазами ДНК DNMT1 и DNMT3b, а также дозозависимым увеличением уровня м-РНК АНП и БНП (Huang L. et al., 2016). Снижение уровня miR-133a в миокарде было обнаружено у лабораторных крыс, подвергавшихся продолжительной инфузии ангиотензина-2 (Li Y. et al., 2016). Вместе с тем, авторы сообщают, что предварительное введение животным рекомбинантного АНП благоприятно сказывалось на динамике miR-133a. Показано, что в условиях неишемической кардиомиопатии наблюдается снижение экспрессии генов АНП и БНП в кардиомиоцитах на фоне усиления метилирования остатка лизина H3 гистона нуклеосомы (Ito E. et al., 2017). Наряду с этим, авторами публикации выявлена взаимосвязь биосинтеза АНП в кардиомиоцитах и продукции (miR-133a) микро РНК. Результаты дальнейших наблюдений показали, что микро РНК-30 может принимать участие в регуляции синтеза БНП (Nakagawa Y. et al., 2019). Важная роль в регуляторном эффекте гормона отводится его рецепторам. В этом смысле, привлекают внимание сведения о том, что уровень экспрессии рецептора популяции А к натрийуретическому пептиду, наиболее физиологически активных рецепторов к АНП (БНП), негативно коррелирует с параметрами энзиматической активности изоформы ДНК-метилтрансферазы DNMT3B (Shen K. et al., 2017). Наряду с этим, высказывается мнение о ключевой роли деацетилаз гистоновых белков (HDAC4) в регуляции экспрессии генов АНП И БНП в норме и при патологии (Hohl M. et al., 2013). В результате экспериментальных исследований установлено, что нарушения функционального состояния миокарда сопровождаются изменениями продукции АНП и БНП на фоне деметилирования H3K9 в промоторных областях генов данных белков и умеренного повышения ацетилирования Н3 гистона (H3K27ac) (Sergeeva I.A. et al., 2016). Тогда показатели ацетилирования H3K9 существенно не были изменены. Ранее полученные сведения также подчеркивают роль ацетилирования белков гистонов в регуляции экспрессии рецептора популяции А к натрийуретическому пептиду в ренальной паренхиеме, (Kumar P., Pandey K.N., 2009). По мнению авторы цитируемой публикации, ацетилтрансфераза белков гистонов (Р300), при участии специализированной микро РНК, может регулировать экспрессию гена гуанилил циклазы-А/рецептора-А натрийуретического пептида. В дальнейших исследованиях авторами было показано, что эпигенетический механизм регуляции экспрессии гуанилил циклазы-А/рецептора-А натрийуретического пептида (<em>Npr1</em>) на основе баланса ацетилирования гистонов (ацетилтрансфераза Р300 и деацетилазы гистонов HDAC1/2), может выполнять важную роль в поддержании физиологических констант волемического гомеостаза организма (Kumar P. et al., 2014). Вместе с тем, авторами высказывается мысль о том, что величина осмотического давления во неклеточной жидкости, уровень продукции ангиотензина-II и витамин Д могут оказывать влияние на показатели экспрессии гена <em>Npr1</em>. Подчеркивается, что амплификация генов (Nppa и Nppb), а также рецепторов натрийуретических пептидов, препятствует повышению кровяного давления, способствует усилению почечного кровотока, увеличению скорости клубочковой фильтрации, ограничивает процессы воспаления и фиброза в ренальной паренхме (Pandey K.N., 2018). Авторы цитируемого обзора также указывают, что натрийуретические пептиды обладают способностью сдерживать активность ренин-ангиотензин-альдостероновой системы. Обращает на себя внимание тот факт, что значительное количество публикаций по данной тематике отмечают антагонизм физиологических эффектов натрийуретических пептидов и трансформирующего фактора роста бета1 (ТФР бета1). Причина такого внимания, по нашему мнению, объясняется фундаментальной ролью ТФР бета1 в ряде патогенетических механизмов нарушения функций органов сердечно-сосудистой системы и почек (Chen L. et al., 2018). В этом смысле, уместно привести данные о том, что ТФР бета1 может оказывать влияние на систему натрийуретических пептидов, подавляя транскрипцию гена <em>Npr1 -</em> основного рецептора гормонов<em> </em>в мышечном слое стенки аорты лабораторных мышей (Sen A. et al., 2016). <strong>СПИСОК ЛИТЕРАТУРЫ К ГЛАВЕ</strong> <strong>&laquo;ЭПИГЕНЕТИЧЕСКИЕ</strong><strong> </strong><strong>МЕХАНИЗМЫ </strong><strong>В СИСТЕМЕ КОНТРОЛЯ ФУНКЦИИ ПОЧЕК В НОРМЕ&raquo; </strong> &nbsp; 1.Adli M., Parlak M., Li Y., El-Dahr S. Epigenetic States of Nephron Progenitors and Epithelial Differentiation. J Cell Biochem. 2015;116(6): 893&ndash;902 doi:10.1002/jcb.25048 &nbsp; 2.Hilliard S.A, El-Dahr S.S. Epigenetics mechanisms in renal development. Pediatr Nephrol. 2016;31(7):1055&ndash;1060 doi:10.1007/s00467-015-3228-x &nbsp; 3.Liu H., Chen S., Yao X., Li Y., Chen C.-H., Liu J., Saifudeen Z., El-Dahr S.S. 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Journal of biological chemistry. 2014; 289(10):6991-7002 doi: 10.1074/jbc.M113.511444 &nbsp; 96.Chen L., Yang T., Lu D.W., Zhao H., Feng Y.L., Chen H., Chen D.Q., Vaziri N.D., Zhao Y.Y. Central role of dysregulation of TGF-&beta;/Smad in CKD progression and potential targets of its treatment. Biomed Pharmacother. 2018;101:670-681 doi: 10.1016/j.biopha.2018.02.090 &nbsp; 97.Sen A., Kumar P., Garg R., Lindsey S.H., Katakam P.V.G., Bloodworth M., Pandey K.N. Transforming growth factor b1 antagonizes the transcription, expression and vascular signaling of guanylyl cyclase/natriuretic peptide receptor A &ndash; role of dEF1. FEBS Journal. 2016; 283(9):1767&ndash;1781 doi:10.1111/febs.13701 PMID:26934489 &nbsp; <strong>ГЛАВА 2. НЕКОТОРЫЕ ФАКТОРЫ АКТИВАЦИИ </strong> <strong>ЭПИГЕНЕТИЧЕСКИХ МЕХАНИЗМОВ</strong> &nbsp; Приступая к обсуждению данной темы, считаем необходимым подчеркнуть, что, во-первых, в отличие от предыдущих разделов, при рассмотрении большинства факторов, включая факторы внешней среды, способных оказывать влияние на экспрессию генов, речь идет о надорганизменном уровне, чаще всего, о популяции. Во-вторых, косвенно затронуты вопросы участия эпигенетических механизмов в процессах наследования приобретенных признаков и их роли в эволюционных преобразований. Возможно, сочетание новых признаков, обусловленных не только мутациями, но и эпигенетическими механизмами внутри популяции, может оказать влияние на процессы микроэволюции. Необходимо отметить, что в современной литературе уделяется внимание поставленным вопросам. В частности, указывается, что предполагаемые глобальные изменения климатических условий учитывают актуальность эпигенетических преобразований для динамики адаптивных изменений популяций человека (Hu J., Barrett R.D.H., 2017). Поэтому, экспериментальные данные, полученные в исследованиях на животных позволяют, с одной стороны, расширить наши представления о роли эпигенетических система контроля адаптивных реакций на изменения факторов среды. С другой стороны, предположить возможность закрепления этих адаптивных преобразований экспрессии генов в ряду поколений. При этом, авторы цитируемого обзора, во-первых, подчеркивают важное значение эпигенетических механизмов для экологической пластичности различных видов животных. Во-вторых, приводят конкретные примеры передачи в последующие поколения эпигенетических изменений хроматина у некоторых видов млекопитающих. Наряду с этим, привлекают внимание сведения об устойчивых сочетаниях генов, выполняющих ведущую роль в формировании экологической пластичности животных к изменениям, например, температурного режима окружающей среды (Wollenberg Valero K.C. et al., 2014). Заслуживает внимания и тот факт, что комбинация данных генов в ряду позвоночных животных обладает достаточно высокой эволюционной консервативностью. Поэтому, необходимо отметить, что в современной литературе высказываются мнения о том, что эпигенетические преобразования, сформированные в генотипе родительских особей, могут выполнять принципиально важную функцию в эволюционном процессе, поскольку могут передаваться потомству и играть существенную роль в адаптивных реакциях потомства (Wang Y. et al., 2017). Авторы приводят ряд аргументов, подтверждающих возможность наследования эпигенетических трансформаций хроматина и у человека. Аналогичная точка зрения, относительно возможности наследования в поколениях эпигенетической модуляции экспрессии генов, обусловленной, в первую очередь, ковалентной модификацией хроматина, высказывается и в последующих публикациях (Norouzitallab P. et al., 2019). Вместе с тем, приведенные сведения о наследовании эпигенетических модификаций генома, во-первых, не являются общепризнанными. Во-вторых, возможный тип наследования эпигенетических трансформаций также мало изучен. Тем не менее, мы посчитали необходимым включить в обзор краткое упоминание об этих аспектах эпигенетики, поскольку возможность их реализации существует. Следовательно, рассматривая роль эпигенетических механизмов в адаптивных реакциях почки на факторы среды (не только внешних) в масштабе популяций, уместно принять к сведению возможность наследования эпигенетических перестроек в системе регуляции экспрессии генов, равно, как и их возможное участие в эволюционных процессах. Также, на наш взгляд, необходимо учитывать интересы практической медицины, особенно, если речь идет об участии эпигенетических процессов в патофизиологических механизмах заболеваний почек. В данном разделе, в качестве тем для обсуждения нами выбраны факторы внешней среды, в том числе и антропогенной природы. Наряду с этим, некоторые факторы, связанные с устойчивыми нарушениями физиологических констант организма также достаточно широко распространены в популяциях человека и заслуживают рассмотрения. <strong>2.1. ИЗМЕНЕНИЕ ТЕМПЕРАТУРНОГО РЕЖИМА, КАК ФАКТОР ИНДУКЦИИ ЭПИГЕНЕТИЧЕСКИХ МЕХАНИЗМОВ</strong> &nbsp; Даже принимая к сведению тот факт, что представители биологического вида Homo sapiens sapiens проживают в искусственно созданной среде и используют различные способы формирования микроклимата своих жилищ, климатические факторы среды, в частности, температура среды, до настоящего времени оказывает исключительно важное влияние на человека (Cheshire W.P. Jr., 2016; Beker B.M. et al., 2018). Обсуждение вероятности стремительных изменений климатических условий на планете Земля не входит в круг наших задач. Вместе стем, мы исходим из того, что уже существующее разнообразие климатических условий различных географических широт, а также межсезонные флуктуации климатических условий можно рассматривать, как важный стимул в изучении роли эпигенетических процессов в адаптации организма к изменению температурного режима среды (Franks S.J., Hoffmann A.A., 2012; Wollenberg Valero K.C. et al., 2014). В данном случае, температурный режим рассматривается в качестве одного из основных факторов среды, способных вызывать устойчивые эпигенетические изменения структуры ДНК человека, которые, направлены на повышение адаптивных возможностей популяции и, вероятно, могут передаваться по наследству (Giuliani C. et al., 2015). Вместе с тем, авторы иллюстрируют адаптивный характер эпигенетических изменений, адекватных геофизическим условиям проживания данных популяций человека. Почки наземных позвоночных животных (амниот) выполняют жизненно важную функцию поддержания постоянства внутренней среды организма. При этом, с одной стороны, физиологические и патофизиологические аспекты адаптации почек человека к изменению температурного режима среды постоянно находятся в центре внимания современной науки (Johnson R.J. et al., 2016; de Lorenzo A., Lia&ntilde;o F., 2017). С другой стороны, участие эпигенетических механизмов в этих процессах требует более глубокого изучения. Тем не менее, установлено, что, в частности, тепловой стресс оказывает мощное влияние на перестройку метаболизма микро РНК в почках (Permenter M.G. et al., 2019). Вместе с тем, авторы цитируемой публикации отмечают органоспецифический характер изменений метаболизма микро РНК под влиянием повышения температуры. Ранее было показано, что белки семейства аквапорины также могут изменять свою экспрессию в почках и слюнных железах под влиянием температурного фактора (как повышение, так и понижение температуры) у позвоночных животных (Wollenberg Valero K.C. et al., 2014). <strong>2.2. </strong><strong>ГИПОКСИЯ.</strong> &nbsp; Наряду с температурным фактором, одним из важнейших факторов среды, способным оказать влияние на состояние эпигенетических механизмов человека, является гипоксия (Giuliani C. et al., 2015). Гипоксическая гипоксия может оказывать влияние на структурные показатели и функциональное состояние ренальной паренхимы через систему HIFs-протеинов, контролируя экспрессию генов, белки которых критически важны для регуляции деятельности почек (Poonit N.D. et al., 2018). С другой стороны, гипоксия ренальной паренхимы различного генеза рассматривается в качестве одного из базовых индукторов эпигенетических механизмов трансформации гуморальных систем контроля гомеостатических функций почек человека (Clarke N.E., Turner A.J., 2012; Macconi D. et al., 2014). Известно, что, стимулируемый гипоксией HIF-1альфа, является одним из ведущих активаторов эпигенетических механизмов (Perez-Perri J.I. et al., 2011). Являясь важным звном в системе адаптации почки к гипоксии, HIF-1альфа может быть непосредственно вовлечен в патогенетические механизмы хронизации и прогрессирования почечной недостаточности (Shoji K. et al., 2014). Установлено, что HIFs-зависимое угнетение метилирования гистонов (H3K9me3 и H3K27me3) может сопутствовать прогрессированию почечной недостаточности (Nangaku M. et al., 2017). Сообщается, что эпигенетические механизмы активации ренин-ангиотензиновой системы могут выполнять ключевую роль в хронизации и прогрессирования почечной недостаточности (Chou Y.H. et al., 2017). Вместе с тем, показано, что HIF-1альфа на уровне транскрипции изменяет баланс экспрессии компонентов РАС в направлении стимуляции биосинтеза компонентов оси Ангиотензин-I-превращающий фермент (АСЕ)/Ангиотензин-2/АТ1-рецепторы против угнетения контура отрицательной обратной связи РАС АСЕ-2/Ангиотензин-1-7/MASS1-рецепторов (Clarke N.E., Turner A.J., 2012; Macconi D. et al., 2014). Помимо того, что HIF-1альфа усиливает экспрессию АТ1-рецепторов и АСЕ, в условиях гипоксии в почке наблюдается резкая активация АСЕ-независимого пути образования Ангиотензин-I в присутствии индуцированного гипоксией лактат-химаза-зависимого механизма (Xie G. et al., 2017). В совокупности, индуцированное гипоксией смещение баланса в пользу оси Ангиотензин-I-превращающего фермента (АСЕ)/Ангиотензин-2/АТ1-рецептор против угнетения контура отрицательной обратной связи РАС АСЕ-2/Ангиотензин-1-7/MASS1 способствует активации воспаления, нарушению клеточного цикла клеток ренальной паренхимы, состоянию энергетического обмена нефроцитов, а также активации эпителиально-мезенхимальной трансформации (Macconi D. et al., 2014; Chou Y.H. et al., 2017). Эпигенетические механизмы, стимулированные гипоксией, выполняют важную роль в хронизации и прогрессирования почечной недостаточности, индуцируя нарушение функции подоцитов (Lin C.-L. et al., 2014) и мезангиума (Lu Z. et al., 2017). По мнению ряда исследователей, ключевым звеном в этом процессе является поражение проксимального отдела нефрона (Matsusaka T. et al., 2012; Kobori H. et al., 2013). Наряду с этим приводятся аргументы о том, что стимулируемые семейством HIF-протеинов эпигенетический процессы являются перспективным объектом фармакологических методов сдерживания прогрессирующей почечной недостаточности (Shoji K. et al., 2014). <strong>2.3. ГИПЕРГЛИКЕМИЯ</strong> &nbsp; Гипергликемия, в подавляющем большинстве случаев, рассматривается в качестве симптома, сопутствующего течению сахарного диабета. Тем не менее, устойчиво повышенный уровень глюкозы во внеклеточной жидкости выступает в качестве самостоятельного патогенетического фактора ренальных дисфункций (Dounousi E. et al., 2015), способного инициировать дальнейшее прогрессирование почечной недостаточности при участии ковалентной трансформации хроматина (Reddy M.A, Natarajan R., 2015; Lu Z. et al., 2017). Обсуждая роль гипергликемии в эпигенетических механизмах перестройки функции почки, необходимо отметить, что данному симптому сахарного диабета 2-го типа сопутствует также изменения секреции инсулина, нарушения обменных процессов, усиление продукции активных форм кислорода, нарушение параметров системной и внутриорганной гемодинамики, повышение уровня HIF-1 (Reddy M.A, Natarajan R., 2015). По мнению цитируемых авторов, HIF-1 обладает способностью стимулировать эпигенетические механизмы активации экспрессии ферментов деметилаз гистонов. Высказывается мнение о том, что гипергликемия в значительной степени ответственна за ряд характерных изменений систем передачи внутриклеточного сигнала в целом ряде различных популяций клеток почки, включая клетки канальцевого эптелия, фибробласты, эндотелиоциты, клетки мезангиума и подоциты (Reddy M.A, Natarajan R., 2015). В обзоре указывается, что стимуляция фиброза тканей почки может усиливаться TGF-&beta;, индуцирующего повышение таких эпигенетических меток, как miR-29, H3K9/14Ac,<strong> </strong>H3K9Ac, H3K4me1 и H3K4me3, на фоне снижения H3K9me3. Указанные изменения могут сопровождаться усилением экспрессии гена <em>Agt</em> (ангиотензиногена) в проксимальных нефроцитах, вызванной ингибированием DNMT и повышением активности HDAC. С другой стороны, следует учитывая роль сопутствующих сахарному диабету изменений гемодинамических параметров на эпигенетические процессы. Известно, что устойчивое повышение кровяного давления может способствовать повышению экспрессии гена Асе (ангиотензин-превращающего фермента) в том числе и в почках через повышение уровня меток H3KAc и H3K4me, на фоне снижения экспрессии метки H3K9me2 (Liang M. et al., 2013; Reddy M.A, Natarajan R., 2015). Эпигенетические механизмы патогенеза и прогрессирования гипертонической болезни рассмотрены в ряде обзорных публикаций (Friso S. et al., 2013; Wise I.A., Charchar F.J., 2016). Авторами цитируемых работ указан ряд генов, экспрессия которых тесно связана с течением гипертонии, включая гены ренина, АСЕ, рецепторов ангиотензина-2 и эндотелиальной NO-синтазы. Эпигенетическая перестройка экспрессии генов системы NO-синтаз может индуцироваться гипоксией (Fish J.E. et al., 2010) и гипергликемией (Advani A. et al., 2011; Schmidt Dellamea B. et al., 2014). Показано, что ингибитор ферментов деацетилаз белков гистонов vorinostat способствует снижению албуминурии, отложению коллагена IV клетками мезангиума, а также оксидативный стресс в экспериментальной модели сахарного диабета 1 типа (Advani A. et al., 2011). <strong>2.4. ТЯЖЕЛЫЕ МЕТАЛЛЫ</strong> &nbsp; Широко известно нефротоксическое действие тяжелых металлов. Наряду с этим в литературе имеются отдельные сведения об их эпигенетических эффектах (Ruiz-Hernandez A. et al., 2015). В частности, авторами показано усиление метилирование ДНК в зависимости от продолжительности экспозиции к кадмию, a также общая тенденция к гипометилированию ДНК на фоне повышения свинца в крови. Относительно ртути, экспериментальные исследования свидетельствуют о том, что ртуть может изменить характер метилирования ДНК. В эмбриональных стволовых клетках крысы метилртуть уменьшала пролиферацию нервных клеток, в связи с гипометилированием ДНК. Также авторы цитируемой публикации сообщают, что механизмы индукции тяжелыми металлами эпигенетической перестройки ДНК остаются крайне мало изучены. Поскольку высоко токсичные тяжелые металлы (ртуть, кадмий и свинец) в организм человека поступают, как правило, в следовых количествах не вызывая острого токсического эффекта, представляет интерес анализ их влияния на изменение обменных процессов в организме, эндокринных функций поджелудочной железы, в патогенезе резистентности тканей к инсулину и избыточной массы тела (Kuo C.-C. et al., 2013). Действительно, эпигенетические эффекты тяжелых металлов могут быть индуцированы достаточно низкими уровнями поступления ксенобиотиков, как правило, не превышающие санитарные нормы. Наиболее ранние публикации, посвященные данной тематике, содержат информацию о том, что, например, тяжелый металл Со2+ может стимулировать процессы транскрипции некоторых белков независимо от внутриклеточной эндогенной продукции активных форм кислорода (Salnikow K. et al., 2000). В дальнейшем были непосредственно указаны индуцированные Со2+, HIF‐1&alpha;-зависимые эпигенетические механизмы, связанные с ферментными системами метилирования ДНК и ацетилирования гистонов (Maxwell P., Salnikow K., 2004). В современной литературе роль эпигенетических механизмов в реализации токсических и канцерогенных эффектов тяжелых металлов широко признана (Salnikow K., Zhitkovich A., 2008; Chervona Y, Costa M., 2012; Brocato J.,<strong> </strong>Costa M., 2013). Также широко признана важность роли HIF‐1&alpha;-зависимых эпигенетических механизмов, индуцируемых тяжелыми металлами (Salnikow K. et al., 2008; Nagasawa H., 2011; Brocato J.,<strong> </strong>Costa M., 2013; Eskandani M. et al., 2017). Было также показано, что стимуляция кобальтом отложений белков внеклеточного матрикса, а также индукция регуляторных пептидов VEGF и эритропоэтина связаны с HIF‐1&alpha; (Tanaka T. et al., 2005). По нашему мнению, научная новизна предлагаемого подхода, состоит в том, что впервые было предложено теоретически обоснованное эпигенетическими механизмами контроля экспрессии генов объяснение патогенеза смертельно опасных онкологических заболеваний, индуцированных тяжелыми металлами. При этом, патогенез этих заболеваний не рассматривался, как результат прямого повреждения ДНК. Был разработан подход, основанный на малигнизирующих эффектах тяжелых металлов, обусловленных специфической ковалентной модификацией хроматина, изменяющей экспрессию генов (Salnikow K., Zhitkovich A., 2008; Salnikow K. et al., 2008). Продуктивность такого подхода была подтверждена последующими результатами исследований (Chervona Y, Costa M., 2012; Brocato J.,<strong> </strong>Costa M., 2013). Результаты исследований in vitro на культуре малигнизированных клеток показали, что присутствие в среде тяжелых металлов оказывает существенное влияние на уровни HIF-1&alpha; в клетках, а также на состояние экспрессии генов, идентифицированных, как гены факторов транскрипции, маркеров дифференциации клеток, цитокинов и факторов роста, протеинкиназ, супрессоров опухолей и онкогенов (Bae S. et al., 2012). По данным авторов, им удалось выделить группу генов, чувствительных, в частности, к ионам Со2+. Установлено также, что тяжелые металлы, через процессы ацетилирования белков-гистонов, регулирует экспрессию гена внеклеточной супероксид дисмутазы<strong> </strong> (Hattori S. et al., 2016). С другой стороны, показано, что применение в эксперименте ингибитора деацетилаз гистоновых белков (valproic acid), способствует ослаблению патофизиологических эффектов HIF-1&alpha; (Luo H.-M. et al., 2013; Kim Y.J. et al., 2017). Наряду с этим, показано, что HIF-1&alpha; может регулировать не только ковалентную модификацию хроматина, но и биосинтез малых некодирующих РНК, способных определять биосинтез белка на уровне транскрипции или трансляции (Kwak J. et al., 2018). Действительно, в литературе имеются данные о том, что HIF могут оказывать влияние на системы метаболизма некодирующих малых РНК (Ho J.J. et al., 2012; Ibrahim A.A. et al., 2017). При этом, в исследованиях in vitro установлена связь между присутствием в среде дихлорида кобальта, HIFs протеинами и показателями экспрессии клетками микро РНК (Silakit R. et al., 2018). Приводятся данные о том, что HIF-зависимые механизмы, через систему микро РНК принимают участие в регуляции экспрессии провоспалительных цитокинов (Kwak J. et al., 2018). Механизмы индукции Со2+ процессов воспаления занимают важное место в патогенезе кобальтовой интоксикации, однако роль эпигенетических механизмов, определяющих синтез (в том числе и микро РНК) провоспалительных факторов белковой природы изучены пока не достаточно (Kumanto M. et al., 2017). По нашему мнению, в литературе проведен достаточно детальный анализ роли тяжелых металлов в индукции базовых механизмов эпигенетической трансформации систем контроля экспрессии генов. В тоже время, нельзя исключить определенных органоспецифических особенностей их реализации. Например, в почках. При том, что ренальная паренхима является одной из основных мишеней для данной группы ксенобиотиков. <strong>2.5. ЭНДОКРИНОПАТИИ</strong> &nbsp; Течение эндокринопатий связано с тем, что на состояние эпигенетических механизмов может одновременно оказывать существенное влияние несколько факторов. Пример такого комбинированного влияния мы уже рассматривали, анализируя эпигенетические эффекты гипергликемии. Вместе с тем, фактор неадекватной секреции инсулина и изменение чувствительности тканей к гормону не является второстепенным и может участвовать в эпигенетических механизмах регуляции деятельности почки (Shiels P.G. et al., 2017). Авторы цитируемого обзора рассматривают роль инсулина в эпигенетической системе контроля деятельности почек в процессе возрастных изменений функции органа. В этом смысле, представляют интерес сведения о базовых эпигенетических механизмах, способных детерминировать резистентность тканей к регуляторному воздействию инсулина (Seok S. et al., 2018). Сложность точной оценки степени влияния различных факторов (гипергликемия, изменение чувствительности тканей к инсулину, оксидативный стресс и т.д) течения сахарного диабета второго типа на перестройку экспрессии генов &mdash; вполне объективная проблема. Вместе с тем, в литературе имеются данные о том, что собственно резистентность к инсулину может, через регуляцию метилирования гистонов, принимать участие в патофизиологических механизмах нарушении целостности слоя подоцитов, провоцируя усиление альбуминурии и прогрессирование нефропатии (Lizotte F. et al., 2016). Действительно, ранее экспериментально было подтверждено участие инсулина в регуляции экспрессии генов мыши и человека через систему метилирования ДНК (Kuroda A. et al., 2009). Известно также, что альдостерон через систему метилирования гистонов может непосредственно регулировать экспрессию гена альфа-субъединицы эпителиального натриевого канала дистального отдела нефрона &alpha;<em>ENaC</em> (Kone B.C., 2013), а также эндотелина-1 (Welch A.K. et al., 2016). Высказывается мнение, что понимание этих эпигенетических механизмов альдостерона представляет интерес, как в лечении гипертонической болезни, так и в борьбе с избыточным весом (Kawarazaki W., Fujita T., 2016). В качестве потенциального индуктора эпигенетической трансформации гуморальных систем контроля гомеостатических функций почек можно упомянуть гормоны щитовидной железы. В литературе имеются указания на регуляторные эффеты гормонов щитовидной железы, рассматриваемых, как природные ингибиторы ацетилазы белков-гистонов (Re A. et al., 2016). Также установлено, что эпигенетические эффекты тироксина стимуляции деацетилазы гистонов-5 (HDAC5) могут реализовываться через путь передачи сигнала, сопряженный с интегрином &alpha;v&beta;3/PKD/HDAC5 (Liu X. et al., 2014). В литературе представлены данные и о том, что в условиях гипофункции щитовидной железы также наблюдается закономерное изменение экспрессии некоторых генов через механизм импринтинга (Hu Z. et al., 2014; Leow M.K., 2016). Следовательно, как гипо- так и гипертиреоз могут рассматриваться в качестве потенциальных индукторов эпигенетической перестройки гуморальных систем контроля деятельности почки. Поскольку широко известен тот факт, что нарушение тиреоидного статуса организма усиливает риск заболевания почек через активацию РАС (Kobori H. et al., 1999). <strong>2.6. </strong><strong>ЭПИГЕНЕТИЧЕСКИЕ ПРОЦЕССЫ, ИНДУЦИРОВАННЫЕ ПАТОГЕННЫМИ МИКРООРГАНИЗМАМИ</strong> &nbsp; Воспалительные реакции тканей почки человека в ответ на инфекционные и неинфекционные заболевания, анализируются с учетом их популяционных особенностей с позиций современных взглядов на филогенез выделительной системы и принципы антропогенеза (Chevalier R.L., 2017). В литературе подчеркивается роль эпигенетических механизмов в эволюционных аспектах формирования адаптивных реакций иммунной системы и тканей почки. При этом, особое внимание уделяется механизмам иммунопатологии почки. В связи с этим в ряде публикаций высказывается мнение о том, что у человека эпигенетическая перестройка (метилирование и ацетилирование гистонов) клеток моноцитарного ряда, направленная на регуляцию выработки провоспалительных факторов, может сохранятся и передаваться дочерним клеткам, определяя особенности течения заболевания (Venet F., Monneret G., 2018). По мнению некоторых исследователей, эпигенетические изменения в иммунной системе, вызванные хроническим воспалением и повышенным окислительным стрессом, могут рассматриваться в качестве базового патогенетического механизма патологии почек и могут приводить к необратимым нарушениям ренальной паренхимы (Syed-Ahmed M., Narayanan M., 2019). Помимо этого, на основе результатов популяционных исследований была проанализирована возможная роль микрофлоры организма человека в эпигенетической перестройке иммунных реакций, связанных с риском заболеваний почек (Uy N. et al., 2015). Дальнейшие исследования показали, что состояние микрофлоры организма может оказывать влияние на риск заболевания почек через эпигенетические механизмы перестройки внутрипочечной РАС (Marques F.Z. et al., 2017). По мнению некоторых авторов, нарушения функции почек могут быть тесно связаны с нарушениями микрофлоры кишечника, поскольку данный показатель оказывает влияние на состояние иммунитета кишечника таким образом, что он больше не может поддерживать физиологический контроль микробиоты (Syed-Ahmed M., Narayanan M., 2019). Авторы цитируемого обзора рассматривают эпигенетическую активацию провоспалительных реакций, возможно, за пределами почечной паренхимы, как мощный индуктор патологических изменений органа. Аналогичную точку зрения высказывают и другие авторы, обращая внимание на тот факт, что эпигенетические механизмы могут выполнять определенную роль в патогенезе прогрессирующей почечной недостаточности на фоне нарушений микрофлоры кишечника (Lu C.C. et al., 2018). Наряду с эим, привлекают внимание сведения о том, что метаболиты микрофлоры кишечника могут оказывать влияние на состояние внутрипочечной ренин-ангиотензиновой системы. Предполагая наличие патогенетических механизмов активации внутрипочечных систем гуморального контроля гомеостатических функций почек &mdash; ренин-ангиотензиновой системы. Показана актуальность эпигенетической индукции РАС и при вирусной инвазии (Chandel N. et al., 2013). <strong>СПИСОК ЛИТЕРАТУРЫ К ГЛАВЕ</strong> &laquo;<strong>НЕКОТОРЫЕ ФАКТОРЫ АКТИВАЦИИ ЭПИГЕНЕТИЧЕСКИХ МЕХАНИЗМОВ&raquo;</strong> &nbsp; 1.Hu J., Barrett R.D.H. Epigenetics in natural animal populations. J Evol Biol. 2017;30(9):1612-1632 doi: 10.1111/jeb.13130 2.Wollenberg Valero K.C., Pathak R., Prajapati I., Bankston S., Thompson A., Usher J., Isokpehi R.D. A candidate multimodal functional genetic network for thermal adaptation. PeerJ. 2014;2:e578 doi: 10.7717/peerj.578 &nbsp; 3.Wang Y., Liu H., Sun Z. 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ИХ МЕСТО И РОЛЬ В ЭПИГЕНЕТИЧЕСКИХ МЕХАНИЗМАХ НАРУШЕНИЯ ФУНКЦИОНАЛЬНОГО СОСТОЯНИЯ РЕНАЛЬНОЙ ПАРЕНХИМЫ</strong> &nbsp; Обсуждая физиологические и патофизиологические аспекты эпигенетического контроля экспрессии генов клеток ренальной паренхимы, необходимо учитывать, что почки обладают автономной системой продукции тканевых гормонов, с одной стороны, принимающих участие в системе ауторегуляции гомеостатических функций и внутриорганного кровотока. С другой стороны, способных индуцировать эпигенетическую трансформацию экспрессии регуляторных и транспортных белков в интересах системного контроля гомеостаза. Вместе с тем, данным эпигенетическим системам контроля экспрессии генов отводится важная патофизиологическая роль в индукции патогенеза почечной недостаточности. Следовательно, еще одним объектом исследований молекулярной биологии и генетики в изучении патогенеза заболеваний почек являются принципиально новые фармакологические методы сдерживания прогрессирования почечной недостаточности. <strong>3.1. РЕНИН-АНГИОТЕНЗИНОВАЯ СИСТЕМА (РАС)</strong> &nbsp; Для того чтобы более полно охарактеризовать результаты эпигенетической перестройки локальной внутрипочечной РАС для функции почек, мы позволим себе кратко упомянуть широко известную схему функционирования внутрипочечной РАС. Согласно существующим представлениям, утвердившимся в мировой литературе, в почке основным местом синтеза ренина являются специализированные клетки ЮГА. Субстрат ренина &ndash; ангиотензиноген, синтезируется в печени. Основные регуляторные эффекты ангиотензина-II, образующегося в результате поступательной конверсии ангиотензиногена в ангиотензин-I при участии ренина, а затем и в ангиотензин-II при участии ангиотензин-превращающего фермента-1 (АПФ-1), сосредоточены на уровне проксимального сегмента нефрона и кровеносных сосудов почки, главным образом, через АТ1-популяцию рецепторов. Благодаря этим эффектам ангиотензин-II осуществляет контроль кровяного давления, волемического гомеостаза, параметров ионного и кслотно-основного гомеостаза организма, а также принимает участие в ауторегуляции почечного кровотока. Некоторые авторы не исключают, что в норме ангиотензиноген может в небольших количествах синтезироваться нефроцитами проксимального отдела нефрона (Kobori H. et al., 2013). Вместе с тем, результаты экспериментальных исследований указывают, что главным источником ангиотензиногена в норме является печень (Matsusaka T. et al., 2012). Помимо АПФ-1, в почке достаточно высокие уровни активности АПФ-2, отвечающего за образование ангиотензина-1-7, отвечающего за механизмы отрицательной обратной связи к ангиотензину-II, хотя, строго говоря, ангиотензин-1-7 антагонистом октапептида не является. В указанной схеме преобразования ангиотензиногена в ангиотензин-II регуляторным ферментом является ренин. При этом, более ранние источники литературы указывали роль процесса ацитилирования гистонов в контроле прогрессирования заболеваний почек, сердца, легких (Bush E.W., McKinsey T.A., 2010). В обсуждении роли преобразований экспрессии компонентов ренин-ангиотензиновой системы почки в процессах патогенеза почечной недостаточности, высказывается мысль о том, что индукцию экспрессии ренина/проренина в канальцевом эпителии следует рассматривать в качестве одного из ключевых событий (Prieto M.C. et al., 2013). Рассматривается роль усиления экспрессии ренина в канальцевом отделе нефрона в патогенезе фиброза почки и гипертонической болезни (Prieto M.C. et al., 2013; Gonzalez A.A., Prieto M.C., 2015). В литературе анализируются возможные молекулярные механизмы индукции экспрессии гена ренина в почках, включая механизмы экспансии ренин-секретирующих клеток за пределы юкста-гломерулярного аппарата (Sequeira Lopez M.L., Gomez R.A., 2010; Kurtz A., 2012). В настоящее время экспансия экспрессии гена ренина (за счет рекрутирования новых, ранее не синтезировавших ренин клеток) рассматривается, как результат, в основном, деятельности эпигенетических механизмов (Gomez R.A., 2017). С другой стороны, эпигенетические системы контроля экспрессии гена ренина сохраняют свою актуальность не только для тканей почки, но и для процессов кроветворения, иммунокомпетентных клеток и т. д. (Gomez R.A., Sequeira-Lopez M.L.S., 2018). Необходимо отметить, что, по мнению ряда авторов, ангиотензин-II следует рассматривать в качестве одного из основных факторов, способствующих прогрессированию почечной недостаточности, через нарушение внутриорганной гемодинамики, стимуляцию фиброза органа, активацию провоспалительных факторов, ограничение клеточного цикла канальцевого эпителия и нарушение обменных процессов в нефроцитах (Kobori H. et al., 2013). Указывается, что по мере прогрессирования почечной недостаточности концентрации ангиотензина-II в тканях почки могут существенно повышаться, на фоне незначительных изменений уровня октапептида в системном кровотоке (Matsusaka T. et al., 2012; Kobori H. et al., 2013). Привлекает внимание тот факт, что существенному увеличению внутриренальной продукции ангиотензина-II, на фоне прогрессирования почечной недостаточности, сопутствует отчетливый прирост биосинтеза белков-компонентов РАС: ангиотензиногена, проренина, АПФ-1 и АТ1-рецепторов ангиотензина-II (основной популяции рецепторов, отвечающих за большинство физиологических и патофизиологических эффектов ангиотензина-II), не только в проксимальных нефроцитах, но и в атипичных очагах активности РАС - эпителии дистальных отделов нефрона (Kobori H. et al., 2013). Авторы цитируемой публикации детально не обсуждают возможную роль эпигенетических механизмов в перестройке внутрипочечной РАС по мере нарастания патологических изменений ренальной паренхимы. Тем не менее, сама логика излагаемых фактов подводит к этому вопросу. Постараемся выяснить, насколько обосновано такое предположение. Действительно, дальнейшие исследования показали, что экспрессия компонентов РАС может регулироваться эпигенетическими механизмами на разных этапах онтогенеза (Tain Y.-L. et al., 2017; Tain Y.L., Hsu C.N., 2017, Witasp A. et al., 2017). При этом, эпигенетическая модуляция экспрессии компонентов РАС рассматривается, в качестве одного из ведущих патогенетических механизмов целого ряда опасных заболеваний (Tain Y.-L. et al., 2017). В частности, показано, что эпигенетические изменения критически важны для понимания перехода острой почечной недостаточности в хроническую форму (Rodr&iacute;guez-Romo R. et al., 2015). В литературе мы встречаем данные о том, что в условиях экспериментальной модели фетального программирования подтверждено участие эпигенетических факторов в регуляции уровней экспрессии АТ1 рецепторов ангиотензина-II (Bogdarina I. et al., 2007; Wu L. et al., 2016). Важным является тот факт, что эпигенетические механизмы, усиливая синтез компонентов РАС, создают условия для активации внутриклеточных (аутокринных) эффектов ангиотензина-II, что, по мнению некоторых авторов, является базовым патогенетическим механизмом РАС-зависимых повреждений тканей почек и сердца (De Mello W.C., 2015). В качестве иллюстрации к высказанному мнению можно привести данные о том, что ацетилирование гистонов 3 (H3Ac), а также их триметилирование (H3K4me3) и диметилирование (H3K9me2) может способствовать высвобождению промотора гена АПФ-1 в почечной паренхиме, обеспечивая биосинтез фермента (Liang M. et al., 2013). С одной стороны, в соответствии с классическим представлением о деятельности РАС, АПФ-1 в нашем организме присутствует в избытке и не является лимитирующим фактором в процессах образования ангиотензина-II. Но если оценивать упомянутый факт с позиций формирования полноценно функционирующей внутриклеточной РАС, то он приобретает совершенно иное значение (Abadir P.M. et al., 2012; Ellis B. et al., 2012). Действительно, по данным литературы, повышение экспрессии в тканях почки гена АПФ-1 является маркером неблагоприятного течения диабетической нефропатии (Thomas M.C., 2016). В дополнение к сказанному, можно привести сообщение группы исследователей, выявивших в условиях диабетической нефропатии усиление внутриклеточной продукции ангиотензиногена в проксимальных нефроцитах, обусловленное ацетилированием (H3K9) и триметилированием (H3K4me3) белка гистона-3 (Marumo T. et al., 2015). По мнению авторов, выявленный эффект может в равной степени свидетельствовать, как о повышении функциональной нагрузки на данный сегмент нефрона, так и о включении патофизиологических механизмов, индуцирующих повреждение данной популяции клеток канальцевого эпителия. Мнение о том, что повышение экспрессии ангиотензиногена в проксимальных нефроцитах может рассматриваться в качестве маркера прогрессирования почечной недостаточности, высказывают и другие авторы (O&#39;Leary R. et al., 2016; Bourgeois C.T. et al., 2017). Патофизиологические и эпигенетические механизмы этого феномена требуют более глубокого исследования. Однако, установлено, что на процессы эпигенетического контроля синтеза ангиотензиногена проксимальными нефроцитами могут оказывать влияние такие факторы, как интерферон-гамма (Satou R. et al., 2013), IL-6 (O&#39;Leary R. et al., 2016) и половые стероидные гормоны (Bourgeois C.T. et al., 2017). Наряду с этим, ангиотензин-II также обладает способностью модулировать состояние экспрессии белков в тканях почки, стимулируя повышение экспрессии АТ1 рецепторов и трансформирующего фактора роста-бета1, на фоне угнетения АПФ-2 (Macconi D. et al., 2014). Эпигенетические механизмы, инициируемые на стадии острой почечной недостаточности, могут рассматриваться в качестве фактора, создающего предпосылки прогрессирования почечной недостаточности, формируя неблагоприятный прогноз течения заболевания (Beckerman P. et al., 2014; Tang J., Zhuang S., 2015; Lee-Son K., Jetton J.G., 2016). В контексте обсуждаемой темы уместно напомнить, что фармакологические ингибиторы РАС (ингибиторы АПФ-1, антагонисты АТ1 рецепторов и ингибиторы ренина) довольно широко и успешно применяются, в том числе, при решении проблемы сдерживания прогрессирующей почечной недостаточности. Применение данной группы препаратов способствует ослаблению протеинурии, предотвращает поражение канальцевого эпителия, содействует ограничению воспаления и фиброза почки (Macconi D. et al., 2014). Поэтому, вполне логичным является вопрос о возможном участии блокаторов РАС в нормализации изменений, индуцированных эпигенетической перестройкой хроматина. Установлено, что в условиях острой почечной недостаточности токсического генеза, ренопротекторные свойства антагониста АТ1 рецепторов (лозартана) обусловлены сдерживанием, в том числе, эпигенетических механизмов, индуцирующих десквамацию подоцитов и усиление протеинурии (Hayashi K. et al., 2015). В частности, авторами выявлено, что лозартан влияет на состояние метилирования промотора гена белка нефрина. По некоторым данным, в условиях экспериментальной модели диабетической нефропатии, лозартан может оказывать умеренный благоприятный эффект на состояние эпигенетических механизмов в тканях почки крыс (Reddy M.A. et al., 2013). В дальнейшем, в условиях ранее примененной экспериментальной модели, авторы показали, что лозартан эффективно блокирует эпигенетические механизмы (через регуляцию процессов ацетилирования H3K9/14Ac) экспрессии генов, ответственных за стимуляцию синтеза ингибитора активатора плазминогена-1 (PAI-1) и моноцитарного хемоаттрактанта протеина-1 (MCP-1), являющихся важными медиаторами повреждения тканей почек (Reddy M.A. et al., 2014). На основании полученных данных авторы цитируемой публикации делают вывод о том, что наиболее эффективная фармакологическая терапия почечной недостаточности должна базироваться на комбинированном применении ингибиторов РАС и специфических модуляторов эпигенетических механизмов. Аналогичную точку зрения высказываются и другие авторы, предполагая, что к наиболее благоприятным терапевтическим результатам может привести сочетанное назначение нефрологическим пациентам ингибитора АПФ-1 и ингибитора деацетилазы гистонов (HDACI) (Zhong Y. et al., 2013). Признавая эффективность лозартана в ограничении метилирования гистонов Harshman L.A. и Zepeda-Orozco D. (2016) видят перспективность клинического использования в нефрологической практике препаратов, относящихся к группе ингибиторов HDACI. Наряду с этим, высказывается мнение о роли микро-РНК в эпигенетических механизмах активации локальной РАС почек при хронической почечной недостаточности (Witasp A. et al., 2017). В литературе высказывается мнение о том, что изучение эпигенетических механизмов функционирования внутриклеточной РАС является фундаментальным направлением современной медицинской науки, призванное решать наиболее актуальные практические задачи в области нефрологии и заболеваний сердечно-сосудистой системы (De Mello W.C., 2017). Таким образом, проведенный анализ данных литературы показал, что эпигенетические аспекты перестройки внутрипочечной (внутриорганной) РАС принципиально важны для понимания патофизиологических механизмов нарушения деятельности почек, сопряженных с усилением внутриклеточной продукции ангиотензина-II. Во-первых, эпигенетическая модификация хроматинового комплекса приводит к появлению новых атипичных очагов интенсивной продукции ангиотензина-II в канальцевом эпителии проксимального и дистального отдела нефрона. Во-вторых, самодостаточная (содержащая все основные компоненты) внутриклеточная РАС канальцевого эпителия переключается на аутокринный и паракринный механизмы, с одной стороны, ослабляет свою роль в физиологической регуляции гомеостатических функций почек. С другой стороны, активация внутриклеточной РАС все более нацелена на патофизиологические механизмы усиления повреждения ткани через нарушения энергетического обмена клетки (De Mello W.C., 2017). Кроме того, активируемые эпигенетическими механизмами гены белков-компонентов РАС, через повышение продукции ангиотензина-II, запускают новый виток каскадного усиления ковалентной модификации хроматина, где в качестве индуктора эпигенетических преобразований, напрямую или опосредовано выступает сам ангиотензин-II. Об этом убедительно свидетельствует эффективность применения блокаторов РАС в отношении эпигенетической трансформации хроматина клеток почки. В-третьих, в доступной нам литературе имеются единичные косвенные данные, позволяющие судить о том, насколько эффективно проникают внутрь клеток (в том числе в эпителий канальца) фармакологические ингибиторы РАС (Foster D.R. et al., 2009). При том, что существует очевидная потенциальная возможность с помощью ингибиторов РАС оказывать влияние на внутриклеточные эффекты ангиотензина-II, нацеленные на регуляцию экспрессии генов (da Silva Novaes A. et al., 2018). При этом мы можем только предполагать характер возможного терапевтического действия селективных ингибиторов на внутриклеточную РАС. В-четвертых, данное направление исследований способствует разработке принципиально новых фармакологических препаратов, способствующих более эффективному решению практических задач не только в нефрологии, но и в борьбе с заболеваниями сердечно-сосудистой системы и в области онкологии. <strong>3.2. МИНЕРАЛОКОРТИКОИДЫ.</strong> &nbsp; Анализ фармакологических способов контроля метаболизма минералокортикоидов вовлечен в довольно широкий круг задач, далеко выходящий за пределы изучения патогенеза почечной недостаточности (Zhang D. et al., 2009; Welch A.K. et al., 2016; Bavishi C. et al., 2016; Kawarazaki W., Fujita T., 2016; Azzam Z.S. et al., 2017). Однако, роль альдостерона в патогенезе заболеваний почек, по-прежнему занимает одно из центральных мест (Currie G. et al., 2016). Строго говоря, альдостерон синтезируется вне почки. Тем не менее, мы посчитали возможным рассмотрение эпигенетических эффектов, связанных с его метаболизмом в контексте анализируемого вопроса, поскольку его физиологические, патофизиологические и фармакологические аспекты тесно связаны с функционированием локальной РАС коры надпочечников и внутрипочечной РАС (Feraille E., Dizin E., 2016; Kawarazaki W., Fujita T., 2016; Nehme A., Zibara K., 2017). Возможно, такое объединение может иметь и более обоснованный аргументы, однако, данный вопрос требует дополнительного изучения (De Mello W.C., 2017). Тем не менее, уже известные факты, широко применяемые в практической медицине (Bavishi C. et al., 2016; Currie G. et al., 2016), дают нам право дополнить выше изложенные аргументы сведениями о роли эпигенетических механизмов в патофизиологии альдостерона и РААС. Позволим себе еще одно краткое замечание. В процессе филогенеза появление у амниот минералокортикоидов произошло относительно недавно &ndash; в связи с выходом позвоночных животных на сушу. В то время, как у низших позвоночных (анамний) функцию минералокортикоидов выполнял кортизол (Dolomatov S.I. et al., 2012). Вероятно, поэтому мы наблюдаем интерференцию эффектов альдостерона и глюкокортикоидов на процессы реабсорбции натрия в дистальном отделе нефрона человека (Feraille E., Dizin E., 2016; Nehme A., Zibara K., 2017). В данном случае упоминание о минералокортикоидной функции глюкокортикоидов следует рассматривать, как попытку более полно оценить обсуждаемые процессы. Возможно, рассмотрение регуляторных эффектов альдостерона, необходимо начать с того, что наиболее важными стимулами интенсивности его секреции в коре надпочечников являются: повышение содержания ионов калия во внеклеточной (внутрисосудистой) жидкости и ангиотензина-II, образующийся в локальной (внутриорганной) РАС надпочечников и почек (Feraille E., Dizin E., 2016; Kawarazaki W., Fujita T., 2016; Nehme A., Zibara K., 2017). Поскольку стимулирующее действие ангиотензина-II на уровень секреции альдостерона реализуется через АТ1-популяцию рецепторов, уместно напомнить, что ранее была установлена роль эпигенетических механизмов в управлении экспрессией АТ1 рецепторов, в том числе и в корковом веществе надпочечников (Bogdarina I. et al., 2007; Liang M. et al., 2013). Кроме того, показано, что механизмы фетального программирования, обусловленные даже непродолжительным повышением кортизола в крови матери могут усиливать экспрессию их рецепторов у плода (Liang M. et al., 2013; Tain Y.L., Hsu C.N., 2017). По мнению авторов цитируемых публикаций, такой механизм может способствовать неадекватной стимуляции реабсорбции натрия в зрелом возрасте, приводя к системным нарушениям параметров гемодинамики. Кроме того, авторы отмечают, что активация реабсорбции натрия в дистальном отделе нефрона может осуществляться и за счет триметилирования of H3K36, сопровождающегося подавлением экспрессии гена 11&beta;-гидроксистероид дегидрогеназы-2, отвечающей за метаболический клиренс глюкокортикоидов. Необходимо подчеркнуть, что патофизиологические механизмы альдостерона в почках непосредственно сопряжены со стимуляцией фиброгенеза в тканях органа, повреждением подоцитов и нарастанием протеинурии (Kawarazaki W., Fujita T., 2016). В современной литературе мы наблюдаем повышение интереса к эпигенетическим механизмам перестройки работы почки, связанных с изменением экспрессии транспортных систем натрия, калия и хлора в различных сегментах нефрона (Tain Y.L., Hsu C.N., 2017). Одно из центральных мест этого направления исследований прочно занимает эпителиальный натриевый канал (ENaC) дистального отдела нефрона (Duarte J.D. et al., 2012; Kone B.C., 2013; Yu Z. et al., 2013). В цитируемых источниках сообщается, что альдостерон стимулирует транскрипцию гена белка альфа-субъединицы EnaC (&alpha;ENaC) через активацию фермента глюкокортикоид-индуцируемую киназу-1, подавляющую активность Dot1a (метилтрансферазу белков-гистонов H3K79), транскрипционного фактора Af9 и гистоновой деацетилазы Sirt1, изменяя активность комплекса Dot1/Af9. Кроме того, в литературе имеются данные о том, что индуцированная альдостероном модификация хроматина может способствовать усилению экспрессии гена эндотелина-1 в соединительных трубочках внутренней медуллы (Welch A.K. et al., 2016). Поскольку рецепторам минералокортикоидов отводится важная роль в реализации эпигенетических эффектов альдостерона, могут представлять интерес данные о том, какова роль данной популяции рецепторов в регуляции экспрессии генов, чувствительных к влиянию альдостерона (Ueda K. et al., 2014). Привлекают внимание сообщения о том, что эпигенетические изменения в системе РААС могут принципиально нарушать механизмы стимуляции секреции альдостерона в корковом веществе надпочечников, ослабляя регуляторную роль внутриорганной РАС почек и надпочечников, выводя на первые позиции совершенно иные факторы (например, лептин), непосредственно не связанные с функциональным состоянием почек и не привязанные к параметрам водно-солевого обмена (Kawarazaki W., Fujita T., 2016). Таким образом, проведенный анализ данных литературы показал, что эпигенетические механизмы перестройки метаболизма альдостерона являются важным фактором в патогенезе ренальных дисфункций и патологических нарушений системной гемодинамики. Установлено, что эпигенетические механизмы затрагивают: систему регуляции метаболизма неполовых стероидов; контролирующих экспрессию транспортных белков дистального отдела нефрона; секрецию физиологически активных пептидов в канальцевом отделе нефрона. Кроме того, есть основания предполагать, что процессы регулирования секреции альдостерона также могут подвергаться эпигенетическим изменениям, приводя к неадекватной стимуляции продукции гормона. Возможно, совокупность выявленных закономерностей позволяет некоторым авторам утверждать, что вызванная эпигенетической перестройкой хроматина неограниченная активация РААС и взаимное усиление патофизиологических эффектов ангиотензина-II и альдостерона является одним из базовых патогенетических механизмов хронических заболеваний почек и органов сердечно-сосудистой системы (De Mello W.C., 2017). <strong>3.3. ТРАНСФОРМИРУЮЩИЙ ФАКТОР РОСТА-бета1 </strong> &nbsp; Согласно данным литературы, трансформирующий фактор роста-бета1 (ТФР-бета1) принадлежит к суперсемейству цитокинов, в состав которых, помимо ТФР-бета, входит большое количество белков, например, ВМР, в норме имеющих важное значение для цитодифференцировки тканей и процессов заживления ран (Shi M. et al., 2011). В стимуляции внутриренального синтеза ТФР-бета1важную роль играет ангиотензин-II через АТ1 популяцию рецепторов (Reddy M.A. et al., 2014). Между тем, авторы цитируемого источника отмечают, что антагонисты АТ1 рецепторов и блокаторы АПФ-1 оказывают умеренное благоприятное воздействие на процессы фиброза органа при хронической почечной недостаточности, поскольку существуют РАС-независимые пути индукции ТФР-бета1. Известно, что ТФР-бета1 и ТФР-бета3 является ключевым фактором стимуляции фиброгенеза ткани почки в условиях хронической почечной недостаточности (Wing M.R. et al., 2013). Обнаружено, что патологические нарушения почек в условиях экспериментальных моделей острой почечной недостаточности сопровождаются достаточно быстрым приростом продукции ТФР-бета1 в тканях почки, в том числе, благодаря активации эпигенетических механизмов (Zager R.A. et al., 2011), нарушая нормальное течение репаративных процессов в почке (Bonventre J.V., Yang L., 2011). В экспериментальных условиях острой почечной недостаточности in vivo и в моделировании острого токсического воздействия на культивируемые проксимальные нефроциты было установлено, что стимуляция метилирования Н3 (H3K4mе3) предшествует резкому повышению уровня мРНК ТФР-бета1 в ткани (Zager R.A., Johnson A.C.M., 2010). Результаты экспериментальных исследований подтверждают, что эпигенетическая активация гена ТФР-бета1 происходит в условиях острой почечной недостаточности, способствуя хронизации заболеваний почек (Sun G. et al., 2014). Поскольку ТФР-бета1 может участвовать в метастазировании злокачественных опухолей, является одним из основных индукторов фиброза почек, печени, легких, кожи, проблеме клинического применения анти-ТФР-бета терапии, основанной, в том числе, на эпигенетических механизмах, уделяется значительное внимание, как наиболее перспективному направлению в лечении целого ряда опасных заболеваний (Zeisberg M., Zeisberg E.M., 2015). В частности, анализируется эффективность различных способов подавления патогенетических ТФР-бета1-заисимых механизмов через селективное ингибирование популяции II-типа рецепторов цитокина (Doi S. et al., 2011), применение антисывороток ТФР-бета1 протеина (Zeisberg M., Zeisberg E.M., 2015), использование селективных блокаторов активности деацетилаз гистоновых белков (HDAC) (Guo W. et al., 2009). Хотя, по мнению некоторых авторов, в качестве основной мишени специфических блокаторов деацетилаз гистонов следует рассматривать фермент HDAC класса I, которая, возможно, критически важна для стимуляции ТФР-бета1-зависимого фиброза почек (Liu N. et al., 2013). Также, некоторыми авторами высказывается мнение о целесообразности фармакологической коррекции баланса активности феерментов ацетилтрансфераз гистонов (HATs) и ферментов деацетилаз гистонов (HDACs) (Yuan H. et al., 2013). Необходимо отметить, что в литературе представлены обзоры, содержащие достаточно глубокий и всесторонний анализ возможных системных терапевтических эффектов ингибиторов энзиматической активности HDACs, нацеленных на предотвращение фиброза внутренних органов, включая почки, а также других модуляторов эпигенетических изменений в ренальной паренхиме (Van Beneden K. et al., 2013; Tang J., Zhuang S., 2015). Приводятся аргументы в пользу терапевтической эффективности ингибиторов метилирования в развитии ТФР-бета1-зависимого фиброгенеза почки (Bechtel W. et al., 2010). При этом, в качестве наиболее актуальной мишени перспективных препаратов предлагается фермент метилтрансфераза 7/9 (SET7/9), осуществляющая монометилирование остатка лизина 4 белка-гистона H3 (H3K4me1) (Sasaki K. et al., 2016). На том основании, что некоторые виды микроРНК (в частности, miR-29b) обладают способностью подавлять некоторые просклерозирующие эффекты ТФР-бета1, предполагается, данное направление также может быть в перспективе применено для сдерживания прогрессирующей почечной недостаточности (Wing M.R. et al., 2013). Установлено, что некоторые микроРНК (микро РНК-21 и микро ТНК-192), могут рассматриваться в качестве индукторов ТФР-бета1-зависимого тубулоинтерстициального фиброза и гломерулосклероза (Liu R. et al., 2015). Стимулированное ТФР-бета1 повышение транскрипции микро РНК<em>-192</em> подтверждено в опытах in vitro в культуре клеток (человека и мыши) мезангиума, подоцитов, эндотелиоцитов и канальцевого эпителия (Kato M. et al., 2013). Авторам также удалось установить, что стимуляция ТФР-бета1 транскрипции микро РНК<em>-192</em> зависит от нескольких участков ацетилирования гистона Н3 (H3K9, H3K14 и H3K27). Кроме того, авторами данной публикации высказывается мысль о том, что микро РНК-192 принадлежит особая роль в каскадном усилении просклерозирующих эффектов ТФР-бета1 через активацию транскрипции микро РНК-200b и микро РНК-200c, повышающих экспрессию генов коллагена-1альфа2 (<em>Col1a2</em><em>), коллагена-4альфа1(</em><em>Col4a1</em>) и самого ТФР-бета1 (<em>TGF-</em>&beta;<em>1</em>). С другой стороны, известно, что ТФР-бета1 через Smad3-протеин, стимулирует образование микро РНК-21, активирующей, в свою очередь, экспрессию генов collagen I и fibronectin, а также способствующей повышению уровня &alpha;-SMA в почке (Wing M.R. et al., 2013). Показано, что ТФР-бета1 через активацию фермента метилирования гистонов H3K4-метилтрансферазы SET7/9, повышает экспрессию генов, запускающих, процессы фиброгенеза в почке. Напротив, подавление SET7/9 ингибирует экспрессию индуцируемых ТФР-бета1 генов фиброза (Reddy M.A, Natarajan R., 2015; Dressler G.R., Patel S.R., 2015; Hilliard S.A., El-Dahr S.S., 2016). Возможно, медиаторами эффекта ТФР-бета1 в отношении активности SET7/9 являются продукты реакции, катализируемой ферментом 12/15-липоксигеназы (Yuan H. et al., 2016). Наряду с этим, сообщается о том, что ТФР-бета1-зависимая активация фиброгенеза осуществляется через систему внутриклеточной передачи сигнала Smad-протеинами (Reddy M.A, Natarajan R., 2015). Авторы указывают, что, например, Smad2-протеин причастен к стимуляции ацетилирования молекулы гистона Н3 (H3K9/14Ac). Наряду с ранее названными эпигенетическими изменениями, отмечается, что метилирование гистона Н3 (H3K9me2 и H3K9me3) является важным механизмом в регуляции экспрессии генов коллагена-1альфа1 (Col1&alpha;1) и ингибитора активатора плазминогена (PAI-1) (Reddy M.A. et al., 2013; Sun G. et al., 2014). Одним из базовых патогенетических механизмов тубулоинтерстициальных повреждений канальцевого отдела нефрона является эпителиально-мезенхимальная трансформация, маркером интенсивности которого служит экспрессия &alpha;-актина (&alpha;SMA). В связи с этим представляет интерес сообщение о том, что в условиях экспериментальной модели односторонней обструкции мочеточника у мышей TGF-&beta;1 не оказывал существенного влияния на состояние H3K9Ac в проксимальных нефроцитах и миофибробластах. Наряду с этим, цитокин приводил к перераспределению метки H3K9Me3 в хроматине ядра фибробластов, что коррелировало с увеличением экспрессии &alpha;-SMA (Hewitson T.D. et al., 2017). Таким образом, обзор литературы показал, что эпигенетические эффекты TGF-&beta;1 оказывают весьма значительное влияние на процессы фиброгенеза в тканях почек, затрагивая, фактически, все известные механизмы импринтинга: метилирование и ацетилирование гистоновых белков, а также перестройку экспрессии некоторых специфических микро РНК. Следует отметить, что эпигенетические механизмы, инициируемые TGF-&beta;1 в ренальной паренхиме, не только непосредственно участвуют в реализации просклерозирующего эффекта цитокина, но и способствуют резкому усилению TGF-&beta;1-зависимых патогенетических механизмов ремоделирования ренальной паренхимы. При этом, ингибирование TGF-&beta;1-зависимой модификации хроматина способствует сдерживанию патологических изменений деятельности почек. Что, с одной стороны, доказывает важную патогенетическую роль TGF-&beta;1 в хронизации и прогрессировании почечной недостаточности. С другой стороны, это открывает новые перспективы использования селективных модуляторов эпигенетических процессов в практической медицине, что подтверждается сведениями о готовности их применения в доклинических испытаниях (Van Beneden K. et al., 2013). <strong>3.4. МОЛЕКУЛА ОКСИДА АЗОТА (</strong><strong>NO</strong><strong>)</strong> &nbsp; По данным литературы, эпигенетические механизмы выполняют очень важную функцию в регуляции аргинин-зависимого пути синтеза NO в системе изоформ NO-синтаз: эндотелиальной (nNOS - NOS-1), индуцибельной (iNOS - NOS-2), и нейрональной (eNOS - NOS-3). Некоторые авторы выделяют еще одну изоформу &ndash; митохондриальную mtNOS. Имеются данные о том, что гипоксия, один из наиболее мощных активаторов эпигенетической модификации хроматина, способствует изменению экспрессии генов различных изоформ фермента NOS (Shirodkar A.V., Marsden P.A., 2011). Согласно данным цитируемого обзора, ишемия может сопровождаться репрессией гена eNOS в эндотелиоцитах, на фоне активации транскрипции всех трех изоформ NOS в неоинтиме, включая транскрипцию гена eNOS в мышечных волокнах стенки кровеносных сосудов. Авторы отмечают, что добавление к культивируемым клеткам гладкой мускулатуры сосудистой стенки ингибитора метилтрансферазы ДНК (5-azacytidine), а также как ингибитора HDAC (Trichostatin A), приводило к стимуляции транскрипцию гена eNOS в этих клетках, также, способствуя увеличению мРНК eNOS. В исследованиях in vitro на культуре проангиогенных клеток (early EPCs) и мезангиобластов было установлено, что добавление в среду только 3-deazaneplanocin A (DZNep), ингибитора триметилирования H3K27, не оказывало существенного влияния на экспрессию гена eNOS, тогда, как сочетанное воздействие на клетки DZNep и ингибитора гистоновой дезацетилазы Trichostatin A (TSA) увеличивает экспрессию eNOS (Ohtani K. et al., 2011). Результаты клинических наблюдений, подтверждая роль метилирования и ацетилирования гистонов в регуляции экспресси гена eNOS, также акцентируют внимание на процессах метилирования ДНК (Kheirandish-Gozal L. et al., 2013). Возможно, анализ процесса метилирования промотора гена eNOS представляет интерес в составлении прогноза рисков патологических нарушений некоторых показателей минерального обмена человека (Harvey N.C. et al., 2012). Эпигенетические механизмы контроля экспрессия eNOS в эндотелии кровеносных сосудов почки критически важны в процессе органогенеза, а также адаптации почки к гипоксии и изменениям параметров внутрипочечной гемодинамики (Jamal A. et al., 2012). По данным источника, эндотелиоциты могут не проявлять чувствительность к действию цитокинов, стимулирующих экспрессию iNOS, в том случае, если промотор этого гена обильно метилирован, В норме в тканях почки преимущественно представлены nNOS (NOS-1), в основном в области macula densa, а также eNOS (NOS-3) в эндотелиоцитах и в канальцевом эпителии. Известно, что NO участвует в регуляции ренальной гемодинамики, канальцевого транспорта натрия, регуляции величины скорости клубочковой фильтрации. Является важным фактором контроля тубуло-гломерулярной обратной связи, регулятором агрегатного состояния крови и процессов воспаления. Однако, динамика изменения внутрипочечной продукции NO не всегда совпадает с уровнем экспрессии генов NO-синтаз. Так, как интенсивность внутрипочечного синтеза NO, по мере прогрессирования почечной недостаточности, может снижаться в результате поражения сосудистого русла, фиброза коркового слоя почек, изменения метаболизма субстрата (L-Arginine), повышения концентрации эндогенного блокатора NO-синтаз (асимметричного диметиларгинина - ADMA) и доступности кофакторов NOS-синтазных энзиматических комплексов. Установлено, что прогрессирование почечной недостаточности сопровождается снижением внутрипочечной продукции NO, что коррелирует с интенсивностью фиброгенеза в почке (Schmidt Dellamea B. et al., 2014). Вместе с тем, авторы отмечают роль некоторых биологически активных веществ (инсулина, фактора некроза опухоли-альфа, ангиотензина-II) в регуляции экспрессии генов NO-синтаз. Возможно, стимулируемая инсулином избыточная экспрессия гена eNOS (NOS-3) является одним из важнейших патогенетических механизмов прогрессирования диабетической нефропатии, поскольку в условиях экспериментальной модели, введение животным vorinostat (неселективного ингибитора гистон-деацетилаз класса I и класса II понижало экспрессию данного гена, что способствовало ограничению протеинурии о накопления белков внеклеточного матрикса мезангиальными клетками (Advani A. et al., 2011). Сообщается, что, во-первых, в условиях экспериментальной патологии почек избыточная внутрипочечная продукция оксида азота является важным патогенетическим фактором развития гломерулопаии. Во-вторых, Trichostatin A (TSA) - ингибитор гистон-деацетилаз может способствовать нормализации избыточной продукции NO, как клетками мезангиума, так и индуцибельной iNOS, активируемой некоторыми провоспалительными цитокинами (например, IL-1&beta;) (Van Beneden K. et al., 2013). Возможно, ингибиторы деацетилаз гистонов следует рассматривать в качестве перспективной группы фармакологических препаратов, позволяющих сдерживать целый ряд NO-зависимых патогенетических механизмов прогрессирования почечной недостаточности: воспалительный и просклерозирующий компоненты тубуло-интерстициальных повреждений, блокировать активацию фибробластов почки и апоптоз канальцевого эпителия почки (Jamal A. et al., 2012). Кроме того, ингибиторы деацетилаз гистонов, снижая в почке экспрессию генов iNOS и eNOS, способствуют восстановлению функции почек на фоне ограничения образования &alpha;-SMA, коллагена I, фибронектина, ТФР бета1, а также ограничивают апоптоз в условиях диабетической нефропати (Khan S., Jena G., 2014). Установлено, что гипоксия является одним из наиболее мощных факторов, регулирующих экспрессию гена <em>NOS3</em> эндотелиальной NO-синтазы эндотелиоцитов через снижение и ацетилирования гистоновых белков, и метилирования остатков лизина 4 в гистоне H3 (Fish J.E. et al., 2010). Высказывается мнение о том, что, спровоцированное гипоксией усиление экспрессии индуцибельной iNOS, также может оказывать нефропротекторный эффект при реперфузионных повреждениях органа (Bonventre J.V., Yang L., 2012). Тем не менее, продолжительная стимуляция экспрессии iNOS при токсическом поражении почки рассматривается в качестве неблагоприятного фактора, усугубляющего течение заболевания (Sattarinezhad E. et al., 2017). Необходимо признать, что проблема эпигенетической перестройки внутрипочечной системы оксида азота достаточно многогранна, в частности, в литературе представлены работы, посвященные анализу изменений баланса некоторых гуморальных регуляторов деятельности почки (NO, ангиотензина-II, производных арахидоновой кислоты) в условиях фетального программирования (Tain Y.-L., Joles J.A., 2016). Обсуждаемые вопросы постоянно находятся в поле зрения современной науки, о чем свидетельствуют обзорные публикации, содержащие сведения о фундаментальных эпигенетических механизмах регуляции системы оксида азота (Vasudevan D. et al., 2016; Socco S. et al., 2017). Однако, нельзя исключать возможности наличия органоспецифических, в том числе в ренальной паренхиме, механизмов контроля экспрессии различных изоформ NOS, в частности, iNOS и eNOS (Cerkezkayabekir A. et al., 2017). Суммируя изложенные факты, можно сделать вывод о том, что эпигенетическая трансформация системы оксида азота является важным компонентом патогенетических механизмов нарушения деятельности почек. Имеющиеся в литературе факты указывают, что инициирование данной перестройки, например, гипоксией тканей или под воздействием гормонов и цитокинов, может осуществляться на самых ранних этапах заболевания почек. Кроме того, ренальная система NOS подвергается радикальной модификации по мере прогрессирования почечной недостаточности, что проявляется в уменьшении экспрессии nNOS в корковом веществе почек, неуклонном снижении экспрессии eNOS в эндотелиоцитах и появлении атипичной локализации eNOS в мышечном слое сосудистой стенки, стимуляции экспрессии iNOS. С одной стороны, известно, что NOS почек (главным образом nNOS) принимают участие в регуляции активности внутриорганной РАС, а молекула NO &ndash; один из основных антагонистов ренотропных эффектов ангиотензина-II на сосудистом и канальцевом уровне (Chappell M.C., 2012; Thoonen R. et al., 2013). Следовательно, ослабление внутриорганных регуляторных влияний конститутивных NO-синтаз может являться одной из причин неограниченной активации внутрипочечной РАС и ТФР-бета в условиях прогрессирования почечной недостаточности (Macconi D. et al., 2014). Более того, по мнению некоторых авторов именно активацию митохондриальной NOS допустимо рассматривать в ряду основных патогенетических механизмов эпигенетической активации внутриклеточной РАС (De Mello W.C., 2017). С другой стороны, неадекватная активация экспрессии NOS (eNOS и iNOS) на определенных этапах течения заболевания, в силу специфики физико-химических свойств конечного продукта &ndash; молекулы оксида азота и особенностей функционирования NO-синтазных комплексов, может служить источником активных форм кислорода и азота, внося, таким образом, существенный вклад в усиление патологических процессов (Advani A. et al., 2011; Sattarinezhad E. et al., 2017; Tain Y.-L. et al., 2017). Помимо этого, обсуждение эпигенетических перестроек аргинин-зависимого пути образования NO не исчерпывает всей темы метаболизма оксида азота в почках в норме и при патологии. Известно, что существует механизм ресинтеза молекулы NO, использующий в качестве субстрата химически стабильные продукты окисления NO (нитриты, нитраты и др.) и контролируемый такими белками, как гемоглобин или цитохром Р450. В современных обзорах мы встречаем упоминание аргинин-независимого синтеза NO в связи с эпигенетической модуляций системы оксида азота (Vasudevan D. et al., 2016; Socco S. et al., 2017). Однако, данные механизмы имеют определенную специфику, характерную для обменных и транспортных процессов, протекающих в тканях почек. <strong>3.5. ПАТОФИЗИОЛОГИЧЕСКОЕ ЗНАЧЕНИЕ ЭПИГЕНЕТИЧЕСКОЙ ТРАНСФОРМАЦИИ СИСТЕМЫ ВНУТРИОРГАННОЙ ГУМОРАЛЬНОЙ РЕГУЛЯЦИИ ДЕЯТЕЛЬНОСТИ ПОЧЕК</strong> &nbsp; Результаты проведенного обзора позволяют сделать вывод о том, что эпигенетические механизмы вносят очень важный вклад в перестройку гуморальных систем регуляции деятельности почек в условиях почечной недостаточности, во многом способствуя прогрессирующему сокращению популяции действующих нефронов, непосредственно создавая предпосылки неблагоприятного течения заболевания. При этом, можно выделить несколько общих тенденций, характерных для эпигенетической трансформации внутриренального синтеза и метаболизма физиологически активных веществ. Во-первых, формирование атипичных очагов их продукции, что наиболее явно присутствует в процессах перестройки систем РАС и оксида азота. Во-вторых, гуморальные факторы, осуществляющие в неповрежденной почке координацию физиологических систем контроля гомеостатической деятельности почек, по мере усиления некоторых эпигенетических изменений, все более утрачивают функции регуляции гомеостаза и переключаются на патофизиологический путь индукции ренальных дисфункций и стимуляции прогрессирования почечной недостаточности. В-третьих, эпигенетические изменения, затрагивающие гены белков, выполняющих ключевые функции в синтезе и метаболизме гуморальных факторов регуляции функций почек, могут выполнять роль пускового механизма становления и прогрессирования почечной недостаточности. В дальнейшем, неограниченный синтез этих молекул белковой и небелковой природы приводит к триггерному усилению процесса, в том числе, опять же с привлечением эпигенетической перестройки хроматина. Следовательно, с одной стороны, гены белков, управляющих продукцией гуморальных факторов регуляции функций почек, являются объектом импринтинга. С другой стороны, стимулированная импринтингом модуляция синтеза гуморальных факторов, на последующем витке спирали, содействует дальнейшему углублению эпигенетической модификации хроматина и усилению роста их образования. Наиболее отчетливо указанная закономерность прослеживается в неограниченной активации РААС и системы ТФР-бета почек. В-четвертых, анализируя спровоцированную импринтингом трансформацию баланса ренотропных регуляторных эффектов гуморальных факторов, можно сделать вывод о том, что в этих условиях наблюдается безмерное нарастание вазотонического, просклерозирующего и провоспалительного потенциала в результате неограниченной активации РААС и системы ТФР-бета. На этом фоне происходит неуклонное сокращение регуляторных возможностей оппозиционного вектора контроля, представленного, в частности, системой оксида азота, в первую очередь, конститутивными изоформами eNOS и nNOS. В-пятых, раскрытие эпигенетических процессов в становлении и прогрессировании нефропатий различного генеза не только способствует созданию более прочной теоретической основы патогенеза почечной недостаточности, но и открывает перспективы к разработке принципиально новых фармакологических способов коррекции функции почек. К сожалению, формат рукописи не позволяет уделить данной теме того внимания, которое она безусловно заслуживает. Между тем, важный практический интерес представляют результаты исследования роли эпигенетических механизмов в модуляции систем аргинин-вазопрессина (Murgatroyd C., 2014; Lesse A. et al., 2017), порообразующих белков аквапоринов (Park E.-J., Kwon T.-H., 2015; MacManes M.D., 2017) и атриального натрийуретического пептида (Sergeeva I.A. et al., 2014; 2016). Равно, как и ренотропные эпигенетические эффекты инсулина (Kumar S. et al., 2016; Shiels P.G. et al., 2017), факторов, индуцируемых гипоксией (HIFs семейство протеинов) (Perez-Perri J.I. et al., 2011; Liu J. et al., 2017), продуктов метаболизма арахидоновой кислоты (Yuan H. et al., 2016) и тироксина (Liu X. et al., 2014; Re A. et al., 2016) могут иметь самое непосредственное отношение к обсуждаемым вопросам. По нашему мнению, данная тема может представлять интерес для понимания особенностей патофизиологических механизмов нарушения функции почек. Поскольку, эпигенетическая модификация хроматина играет принципиально важную роль в нарушении баланса внутрипочечного метаболизма гуморальных факторов регуляции функции почек. Средства фармакологической коррекции, разработанные в соответствии с пониманием механизмов импринтинга в значительной степени, могут способствовать в сдерживании хронизации и прогрессирования почечной недостаточности. Напротив, отказ от более современной стратегии сдерживания прогрессирующих нарушений ренальной паренхимы, может иметь последствия в форме изменений процессов синтеза гуморальных факторов под влиянием эпигенетических механизмов, оказыващих дальнейшее влияние на ковалентную модификацию хроматина, а также усиливающих патофизиологические механизмы повреждения ренальной паренхимы. <strong>СПИСОК ЛИТЕРАТУРЫ К ГЛАВЕ </strong>&laquo;<strong>ФАКТОРЫ ВНУТРИОРГАННОЙ ГУМОРАЛЬНОЙ РЕГУЛЯЦИИ ДЕЯТЕЛЬНОСТИ ПОЧЕК. 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J Mol Endocrinol. 2014;52(3):245-254 doi: 10.1530/JME-13-0252 &nbsp; 129.Re A., Nanni S., Aiello A. et al. Anacardic acid and thyroid hormone enhance cardiomyocytes production from undifferentiated mouse ES cells along functionally distinct pathways. Endocrine. 2016;53(3):681-688 doi: 10.1007/s12020-015-0751-2 &nbsp; <strong>ГЛАВА 4. БЕЛКИ РЕНИН-АНГИОТЕНЗИНОВОЙ СИСТЕМЫ ПРИ ОНКОЛОГИЧЕСКИХ ЗАБОЛЕВАНИЯХ </strong> &nbsp; По нашему мнению, обсуждение роли патофизиологических механизмов контроля экспрессии генов белков, принимающих участие в регуляции деятельности почек, предполагает использование в виде иллюстрации какой-либо определенной модели патологии. В качестве такой модели нами были выбраны процессы малигнизации и метастазирования опухолей. С одной стороны, одним из важных маркеров малигнизации клеток, есть изменения в процессах биосинтеза белков, в норме не характерных для данной популяции клеток. С другой стороны, онкологические заболевания, выбранные в качестве примера, на первый взгляд, не имеют прямого отношения к системам контроля водно-солевого гомеостаза. Тем не менее, они, во-первых, демонстрируют некоторые специфические, нельзя сказать, что второстепенные, свойства протеинов, объединяемых общим термином &laquo;компоненты РАС&raquo;. Во-вторых, описываемые закономерности изменения экспрессии белков &mdash; &laquo;компонентов РАС&raquo; при онкологических заболеваниях дают повод оценить широкий спектр функций данной группы протеинов. По данным литературы, компоненты ренин-ангиотензиновой системы (РАС) могут принимать участие в процессах малигнизации тканей, стимулировать рост и метастазирование опухолей (Regulska K. et al., 2013; Gomez R.A., Sequeira-Lopez M.L.S., 2016; Pinter M., Jain R.K., 2017; Pinter M. et al., 2017). Более ранние исследования продемонстрировали диагностическую ценность анализа экспрессии компонентов РАС в онкологии (Rоmer F. K., 1981). Результаты современных исследований подтверждают тезис о диагностической ценности анализа экспрессии компонентов РАС, подчеркивая также их значение в составлении прогноза течения заболевания и выборе способа лечения злокачественных опухолей (Regulska K. et al., 2013; Tawinwung S. et al., 2015; Gomez R.A., Sequeira-Lopez M.L.S., 2016). Наряду с этим, следует указать, что пептиды-компоненты РАС рассматривают в качестве ключевых патогенетических механизмов роста и метастазирования злокачественных опухолей, включая стимуляцию локальной продукции ангиотензина-II (А<strong>-</strong>II), повышение экспрессии рецепторов к А-II, изменение баланса экспрессии ангиотензин-I-превращающих ферментов (АСЕ-1 и АСЕ-2) и уровень образования продуктов их реакции (А-II и А-1-7 соответственно) (Regulska K. et al., 2013; Sobczuk P. et al., 2017; Sun H. et al., 2017). Объектом внимания некоторых исследований является изучение степени риска индукции канцерогенеза ингибиторами РАС (Connolly S. et al., 2011; Azoulay L. et al., 2012; Yang Y. et al., 2015; Sobczuk P. et al., 2017). Вместе с тем, патогенетические механизмы, индуцирующие увеличение экспрессии белков-компонентов РАС в малигнизированных клетках, их роль в процессах роста и метастазирования остаются мало изученными. <strong>4.1. ДИАГНОСТИЧЕСКАЯ ЦЕННОСТЬ АНАЛИЗА ЭКСПРЕССИИ БЕЛКОВ-КОМПОНЕНТОВ РАС В ОНКОЛОГИИ</strong> &nbsp; <strong>4.1.1. Рецепторы к А-</strong><strong>II</strong> &nbsp; А-II оказывает свое влияние через АТ1 и АТ2 популяции рецепторов. Установлено, что в клетках астроцитомы человека частота выявления АТ1 рецепторов у пациентов с высокой степенью злокачественности опухоли (степень III и IV) возрастает до 67% против 10% в группе с низким уровнем злокачественности, что положительно коррелирует с интенсивностью пролиферации клеток и плотностью неоангиогенеза (Arrieta O. et al., 2008). В исследованиях на лабораторных животных, привитых культурой клеток колоректального рака (CRC), было установлено, что А-II через АТ1 и АТ2 рецепторы стимулирует миграцию малигнизированных клеток и их метастазирование в печень (Nguyen L. et al., 2016). Сообщается, что при некоторых онкологических заболеваниях легких раковые клетки, демонстрирующие высокие уровни экспрессии АТ1-рецепторов, обладают резистентностью к воздействию цитостатиков (Cheng Q. et al., 2016). Анализ клинических наблюдений позволяет сделать вывод о том, что повышение экспрессии АТ1 рецепторов малигнизированными клетками свидетельствует о неблагоприятном прогнозе течения заболевания, обусловленном стимуляцией неоангиогенеза, роста и метастазирования опухоли (Keizman D. et al., 2011; Sun H. et al., 2017). Подчеркивается, что активация плейотропных АТ1-зависимых проонкогенных эффектов А-II может затрагивать в том числе лимфоциты и связанные с опухолью макрофаги, что приводит к снижению противоракового иммунитета, изменению продукции интерлейкинов и провоспалительных цитокинов (Coulson R. et al., 2017; Pinter M., Jain R.K., 2017). Значительный прирост АТ1 белка в трансформированных клетках происходит за счет активации гена <em>AGTR</em><em>1</em> (Coulson R. et al., 2017). Возможно, стимуляция неоангиогенеза, реализуемая через АТ1 рецепторы, является одним из универсальных патогенетических механизмов прогрессирования опухолей различного генеза (Osumi H. et al., 2015; Pinter M., Jain R.K., 2017). Приводятся данные о синэргических эффектах систем рецепторов АТ1/А-II и АТ2/А-II в стимуляции неоангиогенеза (Ager E.I. et al., 2011), а также усилении миграции клеток, воспаления формирование внеклеточного матрикса через AT1 и AT2 рецепторы к А-II (Aydiner A. et al., 2015). Показано, что изменения экспрессии АТ1 и АТ2 рецепторов допустимо рассматривать в качестве маркеров малигнизации слизистой желудка, индуцированной патогенными микроорганизмами, например <em>Helicobacter</em><em> </em><em>pylori</em> (Sugimoto M. et al., 2012), а также при прогрессировании плоскоклеточного рака языка (Itinteang T. et al., 2016), прогрессировании колоректального рака и оценке степени риска его метастазирования (Kuniyasu H., 2012; Shimizu Y. et al., 2017), диагностике онкологических заболеваний легких (Gallagher P.E. et al., 2011) и молочной железы (Vinson G.P. et al., 2012). Уровень экспрессии рецепторов А-II рассматривается в качестве прогностического критерия течения плоскоклеточного рака пищевода (Li S.-H. et al., 2016) и светлоклеточного рака почки (Dolley-Hitze T. et al., 2010). Возможно, динамика изменения экспрессии <em>АТ1 и АТ2 </em>может рассматриваться в качестве интегрального индикатора чувствительности малигнизированной ткани к воздействию гуморальных индукторов канцерогенеза (Rhodes D.R. et al., 2009; Vinson G.P. et al., 2012; Sugimoto M. et al., 2012; Regulska K. et al., 2013; Pinter M., Jain R.K., 2017). В ряде обзорных публикаций достаточно подробно изложена оценка результатов исследования особенностей экспрессии АТ1 и АТ2 рецепторов А-II при различных онкологических заболеваниях, их диагностическая и прогностическая ценность. Представлены аргументы с точки зрения их роли в патогенезе заболеваний, прогрессировании и диссеминации опухолей, а также перспективность клинического применения селективных антагонистов рецепторов А-II в целях повышения эффективности химиотерапии, иммунотерапии и ингибиторов неоангиогенеза в онкологии (Vinson G.P. et al., 2012; Regulska K. et al., 2013; Wegman-Ostrosky T. et al., 2015; Sobczuk P. et al., 2017; Pinter M., Jain R.K., 2017). &nbsp; <strong>4.1.2. Ангиотензин-</strong><strong>I</strong><strong>-превращающий фермент (АСЕ-1).</strong><strong> </strong> &nbsp; Ангиотензин-I-превращающий фермент (<strong>АСЕ-1</strong>), карбоксипедиптидаза, один из ключевых факторов, осуществляющих превращение ангиотензина-I (А-1) в физиологически активный ангиотензин-II (А-II). Вместе с тем, при патологии, включая онкологические заболевания, роль АСЕ-1 в образовании А-II может изменяться за счет усиления вклада АСЕ-независимого пути конверсии А-1 в А-II в присутствии альфа-химазы и других пептидаз, формируя резистентность опухолевых клеток к современным методам противораковой терапии (Xie G. et al., 2017; Sobczuk P. et al., 2017). Широко известен и тот факт, что АСЕ-1, обладая относительно низкой субстратной специфичностью, может участвовать не только в образовании А-II, но и в метаболизме кининов, а также других физиологически активных молекул, потенциально актуальных для процессов канцерогенеза, роста и диссеминации опухолей (Regulska K. et al., 2013; Sobczuk P. et al., 2017). Привлекают внимание сведения о том, что АСЕ-1, помимо пептидазной активности, может непосредственно участвовать во внутриклеточной передаче сигнала А-II, фактически являясь рецептором октапептида (de Alvarenga E.C. et al., 2016). По мнению авторов цитируемой публикации, механизм АСЕ-зависимой рецепции А-II может выполнять важную роль в управлении миграции и пролиферации раковых клеток. Следовательно, динамика изменения топологии и уровней экспрессии АСЕ при онкологических заболеваниях может служить маркером локализации проонкогенных эффектов А-II и других гуморальных факторов, метаболизм которых связан с функциями компонентов РАС. Например, при онкологических заболеваниях почек наблюдается закономерное изменение активности и топологии экспрессии белков АСЕ (Errarte P. et al., 2017; Sobczuk P. et al., 2017). В норме эпителий корковых сегментов канальцевого отдела нефрона, в частности, эпителий проксимального отдела, демонстрирует высокие показатели экспрессии АСЕ, который отсутствует в клетках светлоклеточного рака почки (CCRCC) и выявляется только в кровеносных сосудах опухоли (Errarte P. et al., 2017). Авторами показано, что уровень экспрессии белка в опухоли и величина его энзиматической активности в плазме крови могут служить маркером CCRCC агрессивности опухоли и является индикатором выживаемости пациентов с CCRCC. С другой стороны, перспектива применения широко известных ингибиторов АСЕ в целях подавления неоангиогенеза в злокачественных новообразованиях рассматривается в качестве одного из основных аргументов к применению препаратов данной группы в онкологии (Shen J. et al., 2016). Показано, что способствующее ускользанию от противоракового иммунитета микроокужение опухолевых клеток мышей, может формироваться макрофагами и связанными с опухолью фибробластами (Nakamura K. et al., 2018). По мнению авторов, резко повышенный в макрофагах уровень экспрессии АСЕ указывает на повышение интенсивности локальной продукции физиологически активных веществ, вызывающих иммуносупрессию: оксид азота, трансфомирующий фактор роста-бета1 и PGE2. В норме экспрессия АСЕ критически важна для формирования специфического микроокружения в процессах цитодифференцировки на стадии эмбрионального развития органа или в некоторых интенсивно пролиферирующих тканях взрослого человека. Однако, чрезмерно высокий уровень экспрессии не только ассоциируется с нарушениями гемопоэза, но и рассматривается, как эффект ACE в онкогематологии (Haznedaroglu I.C., Malkan U.Y., 2016). Существенное повышение экспрессии АСЕ рака гортани свидетельствует о неблагоприятном течении заболевания и высоком риске метастазирования опухоли (Han C., Ge W., 2016). Следовательно, изменение экспрессии АСЕ-1 наряду с изучением полиморфизма гена АСЕ широко используется в современной онкологии в качестве маркера тяжести течения заболевания и его прогноза (Regulska K. et al., 2013). Вместе с тем, уровень экспрессии АСЕ-1 клетками злокачественных опухолей не всегда коррелирует с интенсивностью локального продукции А-II, по причине усиления активности, например, химазы, регулирующей АСЕ-независимый путь образования А-II (Xie G. et al., 2017). Помимо этого, необходимо учитывать, что АСЕ непосредственно принимает участие в регуляции иммунных реакций организма (Haznedaroglu I.C., Malkan U.Y., 2016). &nbsp; <strong>4.1.3. Ангиотензин-</strong><strong>I</strong><strong>-превращающий фермент-2 (АСЕ-2) и ось ACE2/Ang-(1&ndash;7)/M</strong><strong>AS</strong><strong>1.</strong><strong> </strong> &nbsp; АСЕ-2, гомолог АСЕ-1, отвечает за метаболический клиренс А-II, используемого ферментом в качестве субстрата для синтеза ангиотензина-1-7 (А-1-7). В свою очередь, А-1-7, осуществляя регуляторное влияние через МAS1-рецепторы, оказывает оппозиционное действие вазотоническим, провоспалительным и просклерозирующим эффектам А-II (Clarke N.E., Turner A.J., 2012). Снижение уровня экспрессии АСЕ-2 клетками рака молочной железы рассматривается в качестве маркера тяжелой формы течения заболевания с высоким риском метастазирования (Yu С. et al., 2016). По мнению авторов, уровень экспрессии АСЕ-2 отражает степень влияния ACE2/Ang-(1&ndash;7)/MAS1 оси на ограничение трансформации кальций-зависимых путей внутриклеточной передачи сигнала, характерной для процесса малигнизации клеток. Показано, что уровень экспрессии АСЕ-2 отрицательно коррелирует с интенсивностью неоангиогенеза в некоторых опухолях легких и чувствительностью раковых клеток к цитостатикам. Активность оси ACE2/Ang-(1-7)/MAS1 может угнетать секрецию VEGFа и подавлять активности матричных металлопротеиназ MMP-2 и MMP-9, тем самым способствуя ограничению неоангиогенеза, повышению чувствительности опухоли к цитостатикам и снижению риска метестазирования (Feng Y. et al., 2011; Cheng Q. et al., 2016). В ряде публикаций указывается, что гипоксия является признаком солидных опухолей, подчеркивая, что условия гипоксии способствуют усилению проонкогенного влияния АСЕ-1/А-II на фоне снижения эффектов оси ACE-2/Ang-(1-7)/MAS1 (Fan L. et al., 2014). Авторами цитируемой публикации показано, что in vitro в культуре клеток карциномы легких Льюиса гипоксия способствует снижению экспрессии АСЕ-2 на фоне АСЕ-1/А-II-зависимой индукции VEGFа. Приводятся аргументы в пользу перспективности клинического использования А-1-7, как фактора противораковой терапии опухоли груди, клетки которой не экспрессируют рецепторы эстрогенов, рецепторы прогестерона и рецептора-2 эпидермального фактора роста (Luo Y. et al., 2015). Некоторые обзоры также содержат позитивную оценку перспектив ACE-2/Ang-(1-7)/MAS1 оси в противораковой фармакологии (Regulska K. et al., 2013). Наряду с этим, подчеркивается, что характер влияния ACE-2/Ang-(1-7)/MAS1 оси на раковые клетки и прогрессирование опухоли может зависеть от локализации опухоли (Wegman-Ostrosky T. et al., 2015; Haznedaroglu I.C., Malkan U.Y., 2016; Sobczuk P. et al., 2017). В частности, приводятся данные о том, что А-1-7 стимулирует метастазирование почечно-клеточного рака (RCC), индуцирует активацию генов провоспалительных факторов, в целом способствуя прогрессированию заболевания (Sobczuk P. et al., 2017). Принимая к сведению изложенные факты, авторы склоняются к мнению о том, что А-1-7 в отношении RCC обладает, скорее, пронкогенным действием. &nbsp; <strong>4.1.4. Ангиотензиноген.</strong> &nbsp; Ангиотензиноген (<strong>Agt</strong>) является универсальным предшественником А-II и А-1-7. В норме Agt, главным образом, синтезируется в печени. При онкологических заболеваниях, как правило, печень сохраняет роль основного источника Agt (Vinson G.P. et al., 2012). Однако, представляют интерес данные о диагностической ценности локальной продукции Agt, как маркера канцерогенеза. Также привлекают внимание особенности метаболизма Agt раковыми клетками. С одной стороны, локальная продукция Agt рассматривается в качестве одного из наиболее информативных маркеров активности опухолевого неоангиогенеза (Choi J.-H. et al., 2014). С другой стороны, согласно данным цитируемой публикации, доминирующим продуктом конверсии Agt в опухолевых тканях является А-II. При этом, комбинированное влияние HIF-1-альфа и А-II, на фоне более высокой продукции Agt, рассматривается в качестве базового патогенетического механизма стимуляции ростовых факторов (в частности, VEGFа), активирующих опухолевый неоангиогенез. Действительно, результаты клинических исследований показали, что, во-первых, повышенная экспрессия гена Agt у пациентов с глиобластомой, может расцениваться, как маркер резистентности опухоли к противораковой терапии, направленной на угнетение опухолевого неоангиогенеза (Urup T. et al., 2016). Во-вторых, более высокая экспрессия гена Agt опухолевой тканью сопровождается усилением локальной продукции А-II. Тем не менее, в литературе приводятся данные о том, что также Agt обладает способностью угнетать неоангиогенез (Wegman-Ostrosky T. et al., 2015). Обсуждая локальную продукцию Agt, необходимо уточнить, что, по мнению некоторых авторов, стимуляция локальной экспрессии компонентов РАС, включая Agt, рассматривается в качестве центрального индуктора внутриклеточного каскада регуляторных белков, определяющих процессы малигнизации клеток и метастазирования (Sugimoto M. et al., 2012). Более того, опубликованные результаты не исключают возможности активации и перестройки внутриклеточного метаболизма компонентов РАС в раковых клетках (Blanco L.. et al., 2014). Что не противоречит мнению об универсальной патогенетической роли активации внутриклеточной РАС, причастной также и к процессам модуляции экспрессии генов (Ellis B. et al., 2012; De Mello W.C., 2015). Вместе с тем, указывается на тканеспецифические особенности экспрессии Agt, как маркера риска онкологических заболеваний. Например, риск возникновения рака легких ассоциируется со снижением продукции Agt белка (Wang H. et., 2015). По мнению авторов, эпигенетические механизмы снижения экспрессии гена <em>Agt</em> и точечные мутации гена <em>Agt</em> могут рассматриваться в качестве факторов, усиливающих риск онкологических заболеваний легких. Возможно, динамика локальной продукции Agt протеина и его уровни в плазме крови могут по-разному формировать прогноз течения метастазов колоректального рака (Martin P. et al., 2014). Авторами установлено, что повышение уровня Agt протеина в сыворотке крови достоверно ассоциировалось с худшей общей выживаемостью, а эпителиальная экспрессия Agt достоверно ассоциировалась с улучшенной выживаемостью без прогрессирования заболевания. Еще один аспект диагностической ценности локальной продукции Agt протеина опухолевыми тканями может быть обусловлен закономерным изменением экспрессии гена <em>Agt</em> по мере течения заболевания (Vinson G.P. et al., 2012). &nbsp; <strong>4.1.5. (Про)Ренин.</strong> &nbsp; В последнее время молекула (про)ренина и ее рецепторы привлекает все большее внимание не только, в качестве регуляторного фермента РАС, но и как важный элемент механизмов контроля онтогенеза, заживления ран и патогенеза ряда заболеваний (Gomez R.A., Sequeira-Lopez M.L.S., 2016). В некоторых обзорах, посвященных анализу патогенетической роли РАС в онкологических заболеваниях, встречаются сведения о важном влиянии ренина на процессы малигнизации клеток и прогрессирования опухоли (Vinson G.P. et al., 2012; Sugimoto M. et al., 2012). В исследованиях in vitro установлено, что ренин может оказывать стимулирующее влияние на рост культуры клеток почечно-клеточного рака (Hu J. et al., 2015). Экспрессия ренина может рассматриваться, как маркер нормального созревания предшественников клеток крови или их малигнизации (Haznedaroglu I.C., Malkan U.Y., 2016). Авторы цитируемого обзора подчеркивают, что экспрессия ренина была обнаружена в клетках острого миелоидного лейкоза, в клетках хронического миелоидного лейкоза и острого лимфолейкоза. Высказывается мнение о том, что стволовые клетки костного мозга, которые экспрессируют ренин являться источником лимфобластного лейкоза (Belyea B.C. et al., 2014). По данным литературы, экспрессия гена ренина в процессе нормального и малигнизированного емопоэза может регулироваться эпигенетическими механизмами (Belyea B.C. t al., 2014; Haznedaroglu I.C., Malkan U.Y., 2016). В контексте обсуждаемой емы уместно напомнить, что сложно функционирующая, относительно мало зученная, система рецепторов к (про)ренину имеет отношение не только к АС, но и к регуляции экспрессии генов белков-индукторов процессов оспаления и фиброза тканей (Nguyen G., 2011). Дальнейшие исследования одтвердили РАС-независимые эффекты системы рецепторов к (про)ренину, родемонстрировав их роль в регуляции фундаментальных механизмов онтроля гомеостаза клетки (M&uuml;ller D.N. et al., 2012). Также было установлено, то уровни в плазме крови рецепторов к (про)ренину ((P)RR) в группе нкологических пациентов были резко повышены (Shibayama Y. et al., 2015). На сновании анализа динамики экспрессии (P)RR в клетках на различной стадии алигнизации авторами цитируемой публикации делается вывод о том, что P)RR могут быть тесно вовлечены в процессы онкогенез в поджелудочной елезе. Результаты изучения in vitro экспрессии PRR в культивируемых клетках лиомы человека позволяютс сделать вывод о том, что этот рецептор может ыть как прогностическим маркером, так и мишенью в лечении заболевания Kouchi M. et al., 2017). Приводятся данные о том, что изменение экспрессии P)RR в процессе гемопоэза может рассматриваться в качестве перспективного аркера диагностики в онкогематологии (Haznedaroglu I.C., Malkan U.Y., 2016). <strong>4.2. ЭПИГЕНЕТИЧЕСКИЕ МЕХАНИЗМЫ, КАК ВОЗМОЖНЫЕ РЕГУЛЯТОРЫ ЭКСПРЕССИИ ПРОТЕИНОВ-КОМПОНЕНТОВ РАС ПРИ ОНКОЛОГИЧЕСКИХ ЗАБОЛЕВАНИЯХ </strong> &nbsp; Представленная выше краткая информация о динамике экспрессии протеинов-компонентов РАС в опухолевых тканях свидетельствует о том, что, во-первых, этот показатель может существенно повышаться в тканях, для которых в норме не характерны высокие уровни экспрессии данной группы протеинов (Sugimoto M. et al., 2012; Shibayama Y. et al., 2015; Han C., Ge W., 2016; Itinteang T. et al., 2016; Yue Z. et al., 2016). И напротив, при некоторых онкологических заболеваниях клетки постепенно утрачивают присущую им в норме способность экспрессировать белки РАС (Errarte P. et al., 2017; Sobczuk P. et al., 2017). Во-вторых, отмечается закономерное изменение экспрессии и топологии белков РАС в опухолевых тканях в зависимости от стадии течения и степени тяжести заболевания (Vinson G.P. et al., 2012; Haznedaroglu I.C., Malkan U.Y., 2016; Kouchi M. et al., 2017). В ряде публикаций приводятся доказательства ведущей роли эпигенетических механизмов в изменении синтеза белков, способных стимулировать процессы малигнизации, воспаления, фиброза и метастазирования (TsaiY.-P., Wu K.-J., 2012; Tan W. et al., 2014; Harb-De la Rosa A.et al., 2015; Cheng Y. et al., 2016; Haznedaroglu I.C., Malkan U.Y., 2016; Semenza G.L., 2016). При этом, уделяется внимание эпигенетической перестройке экспрессии генов протеинов-компонентов РАС в процессах малигнизации и роста раковых клеток (TsaiY.-P., Wu K.-J., 2012; Han C.-D., Ge W.-S., 2016; Haznedaroglu I.C., Malkan U.Y., 2016; ie G. et al., 2017). Эпигенетическая перестройка экспрессии компонентов РАС тносительно новое и мало изученное направление в онкологии. В норме, л ние пигенетических механизмов на динамику экспрессии генов белков АС про еживается на ранних стадиях гисто- и органогенеза, а также в нтенсивно про ферирующих тканях (Belyea B.C. et al., 2014; Haznedaroglu .C., Malkan U.Y , 016). Сообщается, что одним из универсальных индукторов кспрессии ген в елков-компонентов РАС, по мере прогрессирования локачественных нов бразований, может являться <em>HIF</em><em>-1альфа </em>(TsaiY.-P., Wu .-J., 2012; Choi J.-H. et l., 2014; Xie G. et al., 2017). Установлено, что ряд акторов, соп тствующих ечению сахарного диабета, также может оказывть лияние на экс рессию генов елков РАС, усиливая риск онкологических аболеваний (Ya g X. et al., 2012; eddy M.A. et al., 2012; Reddy M.A., Natarajan., 2015; Weg an-Ostrosky T. et al., 015).<em> Возможно, </em><em>HIF</em><em>-1альфа епосредственно при имает участие в егуляции экспрессии нгиотензиногена (</em>Agt) (Choi J.- . et al., 2014). В свою чередь, локальная родукция Agt критически важна для усиления образования -II, тимулирующего опухолевый неоангиогенез и мет стазирование через Т1-рецепторы. Сообщается, что антагонист АТ1-рецепторов олмесартан ожет ерез активацию синтеза микроРНК-205 инг бировать экспрессию VEGF-а аковыми клетками (Yue Z. et al., 2016). В дан ом случае А-II рассматривается ачестве регулятора процессов тра скрипции. Действительно, спериментальные исследования показывают, что А-II может усиливать дукцию провоспалительных цитокинов (IFN&gamma;, TNF ) и матричных ме аллопротеиназ (MMP2, MMP9), стимулируя адгезию раковых клеток к эндотелиальным клеткам, а также активируя их трансэндотелиальную миграцию и миграцию опухолевых клеток через белки внеклеточного матрикса (Rodrigues-Ferreira S., et al. 2012). Наряду с этим, высказывается мнение об универсальности эпигенетической перестройки экспрессии компонентов РАС в патогенезе, в том числе, и онкологических заболеваний (Kemp J.R. et al., 2014; Reddy M.A., NatarajanR., 2015). С другой стороны, приводятся сведения об эпигенетических эффектах А-1-7, направленных на ограничение подвижности раковых клеток и их способности к метастазированию (de Oliveira da Silva B. et al., 2016). С этой точки зрения особое значение приобретают данные о способности АСЕ-1 участвовать в механизмах внутриклеточной передачи сигнала А-II (de Alvarenga E.C. et al., 2016). Не менее актуальной является информация о важности системы (про)ренин &mdash; рецепторы к (про)ренину в управлении экспрессии генов, независимо от состояния активности РАС (Nguyen G., 2011; M&uuml;ller D.N. et al., 2012). Результаты дальнейших исследований подтверждают тезис о том, что система (про)ренин &mdash; рецепторы к (про)ренину могут выполнять важную функцию в патогенезе и течении онкологических заболеваний (Shibayama Y. et al., 2015; Wang C. et al., 2016; Kaneko K. et al., 2017). <strong>4.3. ОНКОЛОГИЧЕСКИЕ АСПЕКТЫ ЭКСПРЕССИИ КОМПОНЕНТОВ РАС И ЛОКАЛЬНАЯ РЕНИН-АНГИОТЕНЗИНОВАЯ СИСТЕМА</strong> &nbsp; Само понятие &laquo;локальная РАС&raquo; формировалось, как представление об элементе внутриорганного гуморального комплекса контроля гомеостатических функций данного органа. На примере локальной РАС почек эту мысль можно проиллюстрировать следующим примером. В норме, адекватным стимулом активации внутриренальной РАС есть два различных механизма, обусловленных внутриорганными изменениями кровяного давления и интенсивности транспорта хлорида натрия в области macula densa. В результате активации системы происходит усиление секреции клетками юкста-гломерулярного аппарата регуляторного фермента РАС &mdash; ренина и повышение продукции А-II. Основные ренотропные физиологические эффекты А-II реализуются, в основном, в отношении параметров внутрипочечной гемодинамики, процессов фильтрации и канальцевого транспорта веществ (г.о. натрия) на уровне проксимального отдела нефрона. В том числе через инициацию эффектов, контролирующих транскрипцию транспортных и регуляторных белков в проксимальных нефроцитах (Li X.C. et al., 2012; Satou R., Gonzalez-Villalobos R.A., 2012). В физиологических условиях, индукция секреции ренина (Sparks M.A. et al., 2014) и А-II-зависимая стимуляция транскрипции транспортных белков канальцевого эпителия (Shao W. et al., 2013) адекватны текущему состоянию водно-солевого баланса организма. Например, вызванная гипонатриевой диетой физиологическая стимуляция РАС не сопряжена с повышением ренальных потерь Agt или с развитием повреждений ренальной паренхимы (Shao W. et al., 2013). Следовательно, конечным результатом деятельности сложного комплекса гуморальных регуляторов внутрипочечной системы контроля гомеостаза является поддержание стабильных параметров кровяного давления, ионного гомеостаза, кислотно-основного равновесия, постоянства объема внеклеточной жидкости организма (Satou R., Gonzalez-Villalobos R.A., 2012; Zhuo J.L. et al., 2013; Sparks M.A. et al., 2014; Ferr&atilde;o F.M. et al., 2014). При этом, топология основной массы ренин-секретирующих клеток почки в области ЮГА, скорее всего, имеет принципиально важное значение. Поскольку данная популяция клеток непосредственно получает информацию о параметрах гемодинамики и о состоянии транспорта ионов хлора и натрия в дистальных извитых сегментах нефрона. Напротив, появление эктопических очагов секреции, например, ренина за пределами ЮГА (в канальцевом эпителии, клетках мезангиума) лишает клетки-продуценты адекватной стимуляции. По нашему мнению, такие события, в совокупности с локализацией продукции всех белков-компонентов РАС в одной клетке может создавать предпосылки для неограниченной активации сформировавшихся эктопических очагов РАС, нацеленных на инициацию и каскадное усиление патологических изменений в ренальной паренхиме. С другой стороны, гипоксия, оксидативный стресс, высокий уровень глюкозы в крови и другие неблагоприятные факторы способны индуцировать эпигенетические механизмы активации фиброза и воспаления ренальной паренхимы, в том числе и изменений топологии компонентов РАС через механизмы контроля экспрессии генов (Macconi D.et al., 2014; Reddy M.A., NatarajanR. , 2015, Nangaku M. et al., 2017). Одним из важных результатов указанных событий есть усиление секреции ренина в паренхиме мозгового слоя почки (ZhuoJ.L., 2011), в гладкомышечных клетках артериол, мезангиальных клетках и в интерстиции через эпигенетические механизмы контроля экспрессии генов (Sparks M.A. et al., 2014; De Mello W.C., 2015). Ренин &mdash; регуляторный фермент РАС, детерминирующий интенсивность дальнейшей продукции А-II. Накопление активных форм кислорода (ROS) в почечной ткани способствует усилению экскреции Agt почками, усиливая цепь обратной связи между активацией ROS и дальнейшей активацией ROS синтеза Agt (Nguyen M.T.X. et al., 2015). Показано, что инфузия животным А-II существенно активирует биосинтез Agt в проксимальных нефроцитах, приводя к дальнейшему росту канальцевой продукции А-II и повышению его патогенетического влияния (Ramkumar N. et al., 2016). В целом, аккумуляция компонентов РАС в проксимальных нефроцитах, усиление внутриклеточной продукции А-II и его эффектов на процессы транскрипции, функции митохондрий, усиление продукции ROS &mdash; один из базовых патогенетических механизмов нарушений гомеостатических функций почек (Navar L.G. et al., 2011; Ellis B. et al., 2012; Li X.C., Zhuo J.L., 2016). В качестве диагностического критерия патологической трансформации внутрипочечной РАС рекомендуется использование ренальной экскреции ангиотензиногена (Kobori H.et al., 2002; Navar L.G. et al., 2011; Alge J.L. et al., 2013). Таким образом, патологическая трансформация внутрипочечной РАС осуществляется: 1.Под контролем эпигенетических механизмов, изменяющих процессы транскрипции, в том числе белков-компонентов РАС. 2.В результате формирования эктопических очагов биосинтеза ключевых белков-компонентов РАС. 3.В результате усиления внутриклеточной продукции А-II и усиления влияния октапептида на транскрипцию белков. 4.Ослаблением экспрессии АСЕ-2, снижением продукции А-1-7, эффекты которого носят оппозиционный характер по отношению к А-II. В результате, в отличии от физиологических условий, патологическое изменение топологии и уровня экспрессии компонентов РАС приводит: 1.К появлению эктопических очагов РАС, в т.ч. усилению внутриклеточной РАС канальцевого эпителия.2.К образованию эктопических очагов РАС (ренин, ангиотензиноген) способствующих ускользанию пусковых механизмов активации РАС от базовых егуляторных стимулов параметров водно-солевого баланса организма и гемодинамики. 3.К изменению вектора регуляторных эффектов эктопических очагов РАС, которые больше не направлены на поддержание гомеостаза. Сформировавшиеся эктопические очаги экспрессии компонентов РАС обеспечивает дальнейшую неограниченную индукцию фиброза, воспаления и гипертрофии клеток ренальной паренхимы. 4.К изменению внутриклеточных систем передачи сигнала (Satou R., Gonzalez-Villalobos R.A., 2012) и баланса регуляторного влияния А-II и А-1-7, в сторону усиления активности АСЕ-1 и альфа-химазы на фоне снижения экспрессии АСЕ-2 (Sparks M.A. et al., 2014).Отметим, что подобные изменения происходят и в результате перестройки экспрессии компонентов РАС в клетках злокачественных опухолей, индуцирующих малигнизацию клеток, фиброз и воспаление ткани, неоангиогенез, метастазирование и иммуносупрессию (Regulska K. et al., 2013; Pinter M., Jain R.K., 2017; Sobczuk P. et al., 2017). Также, как в патогенезе и прогрессировании почечной недостаточности, компоненты РАС раковых клеток не причастны к выполнению гомеостатических функций. Следовательно, по нашему мнению, локальной РАС не являются. С точки зрения интересов практической медицины речь идет о целесообразности использования блокаторов РАС в лечении онкологических заболеваний. По нашему мнению, в этом вопросе можно выделить несколько аспектов. С одной стороны, усиление экспрессии эктопических очагов РАС клетками злокачественной опухоли, на первый взгляд, дает основание ожидать результативности использования ингибиторов АСЕ и антагонистов рецепторов А-II в лечении онкологических заболеваний. В действительности, монотерапия блокаторами РАС онкологических заболеваний может демонстрировать достаточно умеренный терапевтический результат. Сообщается о путях усиления противораковых эффектов ингибиторов РАС в комплексе с мероприятиями иммунотерапии и химиотерапии (Pinter M., Jain R.K., 2017). Наряду с этим, указывается на потенциальные риски, связанные с использованием определенных блокаторов РАС в лечении конкретных онкологических заболеваний (Sobczuk P. et al., 2017), вплоть до полной нецелесообразности их применения (S&oslash;rensen G.V. et al., 2013; Chae Y.K. et al., 2014; Nakai Y. et. al., 2016). С другой стороны, на основе анализа природы экспрессии эктопических очагов РАС, предполагаются качественно иные способы их фармакологической коррекции, основанные на модулировании эпигенетических механизмов подавления активности эктопической РАС (Zhong Y. et al., 2013; Reddy M.A. et al., 2014). Высказывается мнение об универсальной роли эпигенетических механизмов в патогенезе формирования эктопической РАС при онкологических и неонкологических заболеваниях (Kemp J.R. et al., 2014; Tang J., Zhuang S., 2015; Reddy M.A., NatarajanR., 2015). Анализируются перспективные способы лечения онкологических заболеваний, полностью базирующиеся на управлении эпигенетическими механизмами при помощи синтетических microRNA (Tan W. et al., 2014; Felipe A.V. et al., 2014). Новые перспективы использования селективных модуляторов эпигенетических процессов в практической медицине, представляющих интерес и для онкологии, подтверждаются сведениями о готовности применения данной группы фармакологических препаратов (ингибиторы деацетилаз) в доклинических испытаниях (Van Beneden K. et al., 2013). Таким образом, проведенный обзор литературы показал, что изменение экспрессии компонентов РАС тесно связано с патогенезом малигнизации клеток, прогрессированием раковых опухолей, а также стимуляцией процессов метастазирования. Сведения о состоянии экспрессии белков компонентов РАС способствуют пониманию механизмов канцерогенеза и диссеминации клеток опухоли. Эти данные позволяют использовать качественные и количественные параметры экспрессии компонентов РАС в качестве маркеров тяжести течения онкологического заболевания. Тесная вовлеченность компонентов РАС вканцерогенез послужила основой для использования ингибиторов РАС (ингибиторов АСЕ-1 и антагонистов рецепторов А-II) в терапии онкологических заболеваний. Вместе с тем, анализ причин изменения экспрессии белков РАС в клетках опухоли позволил выявить, что весьма значимая функция в этих процессах принадлежит эпигенетическим механизмам регуляции экспрессии генов. Благодаря исследованиям состояния эпигенетических механизмов при онкологических заболеваниях были разработаны принципиально новые методы их коррекции, основанные на применении селективных регуляторов систем ковалентной модификации белков-гистонов (например, ингибитор деацетилаз) и технология синтеза микро РНК. 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Hypoxia-regulated target genes implicated in tumor metastasis. J Biomed Sci. 2012; 19(1): 102. doi: 10.1186/1423-0127-19-102 &nbsp; 61.Tan W., Li Y., Lim S.-G., Tan T.M.C. <em>miR-106b-25/miR-17-92</em> clusters: Polycistrons with oncogenic roles in hepatocellular carcinoma. World J Gastroenterol. 2014; 20(20): 5962&ndash;5972 doi:10.3748/wjg.v20.i20.5962 &nbsp; 62.Cheng Y., Guo Y., Zhang Y. et al. MicroRNA-106b is involved in transforming growth factor &beta;1&ndash;induced cell migration by targeting disabled homolog 2 in cervical carcinoma. J Exp Clin Cancer Res. 2016; 35: 11 doi: 10.1186/s13046-016-0290-6 &nbsp; 63.Harb-De la Rosa A., Acker M., Swain S., Manoharan M. The role of epigenetics in kidney malignancies. Cent European J Urol. 2015; 68(2): 157&ndash;164 doi:10.5173/ceju.2015.453 &nbsp; 64.Semenza G.L. The Hypoxic Tumor Microenvironment: A Driving Force for Breast Cancer Progression. Biochim Biophys Acta. 2016; 1863(3): 382&ndash;391 doi:10.1016/j.bbamcr.2015.05.036 &nbsp; 65.Han C.-D., Ge W.-S. Up-Regulation of Angiotensin-Converting Enzyme (<em>ACE</em>) Enhances Cell Proliferation and Predicts Poor Prognosis in Laryngeal Cancer. Med Sci Monit. 2016; 22: 4132&ndash;4138 doi: 10.12659/MSM.896933 &nbsp; 66.Choi J.-H., Nguyen M.-P., Lee D. et al. Hypoxia-Induced Endothelial Progenitor Cell Function Is Blunted in Angiotensinogen Knockout Mice. Mol Cells. 2014; 37(6): 487&ndash;496 doi: 10.14348/molcells.2014.0119 &nbsp; 67.Yang X., So W.Y., Ma R.C. et al. Diabetes and cancer: the mechanistic implications of epidemiological analyses from the Hong Kong Diabetes Registry. Diabetes Metab Res Rev. 2012;28(5):379-387 doi: 10.1002/dmrr.2287 &nbsp; 68.Reddy M.A., Park J.T., NatarajanR. Epigenetic modifications and diabetic nephropathy. Kidney Res Clin Pract. 2012; 31(3): 139&ndash;150 doi:10.1016/j.krcp.2012.07.004 &nbsp; 69.Reddy M.A., NatarajanR. Recent Developments in Epigenetics of Acute and Chronic Kidney Diseases. Kidney Int. 2015; 88(2): 250&ndash;261 doi:10.1038/ki.2015.148 &nbsp; 70.Rodrigues-Ferreira S., Abdelkarim M., Dillenburg-Pilla P. et al. Angiotensin II Facilitates Breast Cancer Cell Migration and Metastasis. PLoS One. 2012; 7(4): e35667 doi: 10.1371/journal.pone.0035667 &nbsp; 71.Kemp J.R., Unal H., Desnoyer R. et al. Angiotensin II-regulated microRNA 483-3p directly targets multiple components of the Renin-Angiotensin System. J Mol Cell Cardiol. 2014; 75: 25&ndash;39 doi: 10.1016/j.yjmcc.2014.06.008 &nbsp; 72.de Oliveira da Silva B., Furtado Lima K., Gon&ccedil;alves L. et al. MicroRNA Profiling of the Effect of the Heptapeptide Angiotensin-(1-7) in A549 Lung Tumor Cells Reveals a Role for miRNA149-3p in Cellular Migration Processes. PLoSOne. 2016; 11(9): e0162094 doi: 10.1371/journal.pone.0162094 &nbsp; 73.Wang C., Guo D., Wang Q. et al. Aliskiren targets multiple systems to alleviate cancer cachexia. Oncol Rep. 2016;36(5):3014-3022 doi: 10.3892/or.2016.5118 74.Kaneko K., Ohba K., Hirose T. et al. Expression of (Pro)renin Receptor During Rapamycin-Induced Erythropoiesis in K562 Erythroleukemia Cells and Its Possible Dual Actions on Erythropoiesis. Tohoku J. Exp. Med. 2017; 241:35-43 doi: 10.1620/tjem.241.35 &nbsp; 75.Li X.C., Hopfer U., Zhuo J.L. Novel signaling mechanisms of intracellular angiotensin II-induced NHE3 expression and activation in mouse proximal tubule cells. Am J Physiol Renal Physiol. 2012; 303(12): F1617&ndash;F1628 doi: 10.1152/ajprenal.00219.2012 76.Satou R., Gonzalez-Villalobos R.A.The role of the JAK-STAT pathway in blood pressure and intrarenal renin-angiotensin system regulation. JAKSTAT. 2012; 1(4): 250&ndash;256 doi: 10.4161/jkst.22729 &nbsp; Shao W., Seth D.M., Prieto M.C. et al. Activation of the renin-angiotensin system by a low-salt diet does not augment intratubular angiotensinogen and angiotensin II in rats. Am J Physiol Renal Physiol. 2013; 304(5): F505&ndash;F514 doi: 10.1152/ajprenal.00587.2012 Sparks M.A., Crowley S.D., Gurley S.B. et al. Classical Renin-Angiotensin System in Kidney Physiology. Compr Physiol. 2014; 4(3): 1201&ndash;1228 doi: 10.1002/cphy.c130040 Ferr&atilde;o F.M., Lara L.S., Lowe J. Renin-angiotensin system in the kidney: What is new? World J Nephrol. 2014; 3(3): 64&ndash;76 doi: 10.5527/wjn.v3.i3.64 Zhuo J.L., Ferrao F.M., Zheng Y., Li X.C. New Frontiers in the Intrarenal Renin-Angiotensin System: A Critical Review of Classical and New Paradigms. Front Endocrinol (Lausanne). 2013; 4: 166 doi: 10.3389/fendo.2013.00166 &nbsp; Macconi D., Remuzzi G., Benigni A. Key fibrogenic mediators: old players. Renin&ndash;angiotensin system. Kidney Int Supp l. 2014; 4(1): 58&ndash;64 doi: 10.1038/kisup.2014.11 &nbsp; Nangaku M., Hirakawa Y., Mimura I. et al. Epigenetic Changes in the Acute Kidney Injury-to-Chronic Kidney Disease Transition. Nephron. 2017;137:256&ndash;259 doi.org/10.1159/000476078 &nbsp; ZhuoJ.L. Augmented intratubular renin and prorenin expression in the medullary collecting ducts of the kidney as a novel mechanism of angiotensin II-induced hypertension. Am J Physiol Renal Physiol. 2011; 301(6): F1193&ndash;F1194 doi: 10.1152/ajprenal.00555.2011 &nbsp; Nguyen M.T.X., Han J., Ralph D.L. et al. Short-term nonpressor angiotensin II infusion stimulates sodium transporters in proximal tubule and distal nephron. Physiol Rep. 2015; 3(9): e12496 doi: 10.14814/phy2.12496 Ramkumar N.,Stuart D.,Calquin M. et al. Possible role for nephron‐derived angiotensinogen in angiotensin‐II dependent hypertension. Physiol Rep. 2016; 4(1): e12675 doi: 10.14814/phy2.12675 Navar L.G., Kobori H., Prieto M.C., Gonzalez-Villalobos R.A. Intratubular renin-angiotensin system in hypertension. Hypertension. 2011; 57(3): 355&ndash;362 doi: 10.1161/HYPERTENSIONAHA.110.163519 &nbsp; Ellis B., Li X.C., Miguel-Qin E. et al. <strong>Review:</strong> Evidence for a functional intracellular angiotensin system in the proximal tubule of the kidney. Am J Physiol Regul Integr Comp Physiol.2012;302(5):R494&ndash;R509 doi:10.1152/ajpregu.00487.2011 &nbsp; Li X.C., Zhuo J.L. Recent Updates on the Proximal Tubule Renin-Angiotensin System in Angiotensin II-Dependent Hypertension. Curr Hypertens Rep. 2016; 18(8): 63 doi:10.1007/s11906-016-0668-z &nbsp; Kobori H., Harrison-Bernard L.M., Navar L.G. Urinary excretion of angiotensinogen reflects intrarenal angiotensinogen production. Kidney Int. 2002; 61(2): 579&ndash;585 doi:10.1046/j.1523-1755.2002.00155.x &nbsp; Alge J.L., Karakala N., Neely B.A. et al. Urinary Angiotensinogen and Risk of Severe AKI. Clin J Am Soc Nephrol. 2013; 8(2): 184&ndash;193 doi:10.2215/CJN.06280612 &nbsp; S&oslash;rensen G.V., Ganz P.A., Cole S.W. et al. Use of &beta;-Blockers, Angiotensin-Converting Enzyme Inhibitors, Angiotensin II Receptor Blockers, and Risk of Breast Cancer Recurrence: A Danish Nationwide Prospective Cohort Study. J.Clin Oncol. 2013;31(18):2265&ndash;2272 doi:10.1200/JCO.2012.43.9190 &nbsp; Chae Y.K.,, Dimou A., Pierce S. et al. The effect of calcium channel blockers on the outcome of acute myeloid leukemia. Leuk. Lymphoma. 2014;55(12): 2822&ndash;2829 doi:10.3109/10428194.2014.901513 &nbsp; Nakai Y., Isayama H., Sasaki T. et al. No Survival Benefit from the Inhibition of Renin&ndash;Angiotensin System in Biliary Tract Cancer. Anticancer research. 2016; 36: 4965-4970 doi:10.21873/anticanres.11065 Zhong Y., Chen E.Y., Liu R. et al. Renoprotective Effect of Combined Inhibition of Angiotensin-Converting Enzyme and Histone Deacetylase. J Am Soc Nephrol. 2013; 24(5):801&ndash;811 doi:10.1681/ASN.2012060590 &nbsp; Reddy M.A., Sumanth P., Lanting L. et al. Losartan reverses permissive epigenetic changes in renal glomeruli of diabetic db/db mice. Kidney Int. 2014; 85(2): 362&ndash;373 doi:10.1038/ki.2013.387 &nbsp; Felipe A.V., de Oliveira J., Chang P.Y. et al. RNA Interference: a Promising Therapy for Gastric Cancer. Asian Pac J Cancer Prev. 2014; 15(14):5509-5515 doi.org/10.7314/APJCP.2014.15.14.5509 &nbsp; Tang J., Zhuang S. Epigenetics in acute kidney injury. Curr Opin Nephrol Hypertens. 2015;24(4):351&ndash;358 doi:10.1097/MNH.0000000000000140 &nbsp; Van Beneden K., Mannaerts I., Pauwels M. et al. HDAC inhibitors in experimental liver and kidney fibrosis. Fibrogenesis Tissue Repair. 2013; 6: 1 doi:10.1186/1755-1536-6-1 &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; <strong>Dolomatov S.I., Zukow W. </strong><strong>Эпигенетика почек</strong><strong> = Kidneys epigenetics</strong><strong>. </strong><strong>RSW. Radom,</strong><strong> 144 </strong><strong>p. 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Introduction The short film Noah (2013) depicts the contemporary story of an adolescent relationship breakdown and its aftermath. The film tells the story by showing events entirely as they unfold on the computer screen of Noah, the film’s teenaged protagonist. All of the characters, including Noah, appear on film solely via technological mediation.Although it is a fictional representation, Noah has garnered a lot of acclaim within an online public for the authenticity and realism of its portrayal of computer-mediated life (Berkowitz; Hornyak; Knibbs; Warren). Judging by the tenor of a lot of this commentary, the film has keyed in to a larger cultural anxiety around issues of communication and relationships online. Many reviewers and interested commentators have expressed concern at how closely Noah’s distracted, frenetic and problematic multitasking resembles their own computer usage (Beggs; Berkowitz; Trumbore). They frequently express the belief that it was this kind of behaviour that led to the relationship breakdown depicted in the film, as Noah proves to be “a lot better at opening tabs than at honest communication” (Knibbs para. 2).I believe that the cultural resonance of the film stems from the way in which the film is an implicit attempt to assess the nature of contemporary online persona. By understanding online persona as a particular kind of “hybrid object” or “quasi-object”—a combination of both human and technological creation (Latour We Have)—the sense of the overall problems, as well as the potential, of online persona as it currently exists, is traceable through the interactions depicted within the film. By understanding social relationships as constituted through dynamic interaction (Schutz), I understand the drama of Noah to stem principally from a tension in the operation of online persona between a) the technological automation of presentation that forms a core part of the nature of contemporary online persona, and b) the need for interaction in effective relationship development. However, any attempt to blame this tension on an inherent tendency in technology is itself problematised by the film’s presentation of an alternative type of online persona, in a Chatroulette conversation depicted in the film’s second half.Persona and Performance, Mediation and DelegationMarshall (“Persona Studies” 163) describes persona as “a new social construction of identity and public display.” This new type of social construction has become increasingly common due to a combination of “changes in work, transformation of our forms of social connection and networking via new technologies, and consequent new affective clusters and micropublics” (Marshall “Persona Studies” 166). New forms of “presentational” media play a key role in the construction of persona by providing the resources through which identity is “performed, produced and exhibited by the individual or other collectives” (Marshall “Persona Studies” 160).In this formulation of persona, it is not clear how performance and presentation interlink with the related concepts of production and exhibition. Marshall’s concept of “intercommunication” suggests a classificatory scheme for these multiple registers of media and communication that are possible in the contemporary media environment. However, Marshall’s primary focus has so far been on the relationship between existing mediated communication forms, and their historical transformation (Marshall “Intercommunication”). Marshall has not as yet made clear the theoretical link between performance, presentation, production and exhibition. Actor-Network Theory (ANT) can provide this theoretical link, and a way of understanding persona as it operates in an online context: as online persona.In ANT, everything that exists is an object. Objects are performative actors—the associations between objects produce the identity of objects and the way they perform. The performative actions of objects, equally, produce the nature of the associations between them (Latour Reassembling). Neither objects nor associations have a prior existence outside of their relationship to each other (Law).For Latour, the semiotic distinction between “human” and “non-human” is itself an outcome of the performances of objects and their associations. There are also objects, which Latour calls “quasi-objects” or “hybrids,” that do not fit neatly on one side of the human/non-human divide or the other (Latour We Have). Online persona is an example of such a hybrid or quasi-object: it is a combination of both human creation and technological mediation.Two concepts formulated by Latour provide some qualitative detail about the nature of the operation of Actor-Networks. Firstly, Latour emphasises that actors are also “mediators.” This name emphasises that when an actor acts to create a connection between two or more other objects, it actively transforms the way that objects encounter the performance of other objects (Latour Reassembling). This notion of mediation resembles Hassan’s definition of “media” as an active agent of transferral (Hassan). But Latour emphasises that all objects, not just communication technologies, act as mediators. Secondly, Latour describes how an actor can take on the actions originally performed by another actor. He refers to this process as “delegation.” Delegation, especially delegation of human action to a technological delegate, can render action more efficient in two ways. It can reduce the effort needed for action, causing “the transformation of a major effort into a minor one.” It can also reduce the time needed to exert effort in performing an action: the effort need not be ongoing, but can be “concentrated at the time of installation” (Latour “Masses” 229-31).Online persona, in the terminology of ANT, is a constructed, performative presentation of identity. It is constituted through a combination of human action, ongoing mediation of present human action, and the automation, through technological delegation, of previous actions. The action of the film Noah is driven by the changes in expected and actual interaction that these various aspects of persona encourage.The Problems and Potential of Online PersonaBy relaying the action entirely via a computer screen, the film Noah is itself a testament to how encounters with others solely via technological mediation can be genuinely meaningful. Relaying the action in this way is in fact creatively productive, providing new ways of communicating details about characters and relationships through the layout of the screen. For instance, the film introduces the character of Amy, Noah’s girlfriend, and establishes her importance to Noah through her visual presence as part of a photo on his desktop background at the start of the film. The film later communicates the end of the relationship when the computer boots up again, but this time with Amy’s photo notably absent from the background.However, the film deviates from a “pure” representation of a computer screen in a number of ways. Most notably, the camera frame is not static, and moves around the screen in order to give the viewer the sense that the camera is simulating Noah’s eye focus. According to the directors, the camera needed to show viewers where the focus of the action was as the story progressed. Without this indication of where to focus, it was hard to keep viewers engaged and interested in the story (Paulas).Within the story of the film itself, the sense of drama surrounding Noah’s actions similarly stem from the exploration of the various aspects of what it is and is not possible to achieve in the performance of persona – both the positive and the negative consequences. At the start of the film, Noah engages in a Skype conversation with his girlfriend Amy. While Noah is indeed “approximating being present” (Berkowitz para. 3) for the initial part of this conversation, once Noah hears an implication that Amy may want to break up with him, the audience sees his eye movements darting between Amy’s visible face in Skype and Amy’s Facebook profile, and nowhere else.It would be a mistake to think that this double focus means Noah is not fully engaging with Amy. Rather, he is engaging with two dimensions of Amy’s available persona: her Facebook profile, and her Skype presence. Noah is fully focusing on Amy at this point of the film, but the unitary persona he experiences as “Amy” is constructed from multiple media channels—one dynamic and real-time, the other comparatively stable and static. Noah’s experience of Amy is multiplexed, a unitary experience constructed from multiple channels of communication. This may actually enhance Noah’s affective involvement with Amy.It is true that at the very start of the Skype call, Noah is focusing on several unrelated activities, not just on Amy. The available technological mediators enable this division of attention. But more than that, the available technological mediators also assume in their functioning that the user’s attention can be and should be divided. Thus some of the distractions Noah experiences at this time are of his own making (e.g. the simple game he plays in a browser window), while others are to some degree configured by the available opportunity to divide one’s attention, and the assumption of others that the user will do so. One of the distractions faced by Noah comes in the form of repeated requests from his friend “Kanye East” to play the game Call of Duty. How socially obligated is Noah to respond to these requests as promptly as possible, regardless of what other important things (that his friend doesn’t know about) he may be doing?Unfortunately, and for reasons which the audience never learns, the Skype call terminates abruptly before Noah can fully articulate his concerns to Amy. With a keen eye, the audience can see that the image of Amy froze not long after Noah started talking to her in earnest. She did indeed appear to be having problems with her Skype, as her later text message suggested. But there’s no indication why Amy decided, as described in the same text message, to postpone the conversation after the Skype call failed.This is a fairly obvious example of the relatively common situation in which one actor unexpectedly refuses to co-operate with the purposes of another (Callon). Noah’s uncertainty at how to address this non-cooperation leads to the penultimate act of the film when he logs in to Amy’s Facebook account. In order to fully consider the ethical issues involved, a performative understanding of the self and of relationships is insufficient. Phenomenological understandings of the self and social relationships are more suited to ethical considerations.Online Persona and Social RelationshipsIn the “phenomenological sociology” of Alfred Schutz, consciousness is inescapably temporal, constantly undergoing slight modification by the very process of progressing through time. The constitution of a social relationship, for Schutz, occurs when two (and only two) individuals share a community of space and time, simultaneously experiencing the same external phenomena. More importantly, it also requires that these two individuals have an ongoing, mutual and simultaneous awareness of each other’s progress and development through time. Finally, it requires that the individuals be mutually aware of the very fact that they are aware of each other in this ongoing, mutual and simultaneous way (Schutz).Schutz refers to this ideal-typical relationship state as the “We-relationship,” and the communal experience that constitutes it as “growing older together.” The ongoing awareness of constantly generated new information about the other is what constitutes a social relationship, according to Schutz. Accordingly, a lack of such information exchange will lead to a weaker social bond. In situations where direct interaction does not occur, Schutz claimed that individuals would construct their knowledge of the other through “typification”: pre-learned schemas of identity of greater or lesser generality, affixed to the other based on whatever limited information may be available.In the film, when Amy is no longer available via Skype, an aspect of her persona is still available for interrogation. After the failed Skype call, Noah repeatedly refreshes Amy’s Facebook profile, almost obsessively checking her relationship status to see if it has changed from reading “in a relationship.” In the process he discovers that, not long after their aborted Skype conversation, Amy has changed her profile picture—from one that had an image of the two of them together, to one that contains an image of Amy only. He also in the process discovers that someone he does not know named “Dylan Ramshaw” has commented on all of Amy’s current and previous profile pictures. Dylan’s Facebook profile proves resistant to interrogation—Noah’s repeated, frustrated attempts to click on Dylan’s profile picture to bring up more detail yields no results. In the absence of an aspect of persona that undergoes constant temporal change, any new information attained—a profile picture changed, a not-previously noticed regular commenter discovered—seems to gain heightened significance in defining not just the current relationship status with another, but the trajectory which that relationship is taking. The “typification” that Noah constructs of Amy is that of a guilty, cheating girlfriend.The penultimate act of the film occurs when Noah chooses to log in to Amy’s Facebook account using her password (which he knows), “just to check for sketchy shit,” or so he initially claims to Kanye East. His suspicions appear to be confirmed when he discovers that private exchanges between Amy and Dylan which indicate that they had been meeting together without Noah’s knowledge. The suggestion to covertly read Amy’s private Facebook messages comes originally from Kanye East, when he asks Noah “have you lurked [covertly read] her texts or anything?” Noah’s response strongly suggests the normative uncertainty that the teenaged protagonist feels at the idea; his initial response to Kanye East reads “is that the thing to do now?” The operation of Facebook in this instance has two, somewhat contradictory, delegated tasks: let others feel connected to Amy and what she’s doing, but also protect Amy’s privacy. The success of the second goal interferes with Noah’s desire to achieve the first. And so he violates her privacy.The times that Noah’s mouse hovers and circles around a button that would send a message from Amy’s account or update Amy’s Facebook profile are probably the film’s most cringe-inducing moments. Ultimately Noah decides to update Amy’s relationship status to single. The feedback he receives to Amy’s account immediately afterwards seems to confirm his suspicions that this was what was going to happen anyway: one friend of Amy’s says “finally” in a private message, and the suspicious “Dylan” offers up a shoulder to cry on. Apparently believing that this reflects the reality of their relationship, Noah leaves the status on Amy’s Facebook profile as “single.”The tragedy of the film is that Noah’s assumptions were quite incorrect. Rather than reflecting their updated relationship status, the change revealed to Amy that he had violated her privacy. Dylan’s supposedly over-familiar messages were perfectly acceptable on the basis that Dylan was not actually heterosexual (and therefore a threat to Noah’s role as boyfriend), but gay.The Role of Technology: “It’s Complicated”One way to interpret the film would be to blame Noah’s issues on technology per se. This is far too easy. Rather, the film suggests that Facebook was to some degree responsible for Noah’s relationship issues and the problematic way in which he tried to address them. In the second half of the film, Noah engages in a very different form of online interaction via the communication service known as Chatroulette. This interaction stands in sharp contrast to the interactions that occurred via Facebook.Chatroulette is a video service that pairs strangers around the globe for a chat session. In the film, Noah experiences a fairly meaningful moment on Chatroulette with an unnamed girl on the service, who dismisses Facebook as “weird and creepy”. The sheer normative power of Facebook comes across when Noah initially refuses to believe the unnamed Chatroulette girl when she says she does not have a Facebook profile. She suggests, somewhat ironically, that the only way to have a real, honest conversation with someone is “with a stranger, in the middle of the night”, as just occurred on Chatroulette.Besides the explicit comparison between Facebook and Chatroulette in the dialogue, this scene also provides an implicit comparison between online persona as it is found on Facebook and as it is found on Chatroulette. The style of interaction on each service is starkly different. On Facebook, users largely present themselves and perform to a “micro-public” of their “friends.” They largely engage in static self-presentations, often “interacting” only through interrogating the largely static self-presentations of others. On Chatroulette, users interact with strangers chosen randomly by an algorithm. Users predominantly engage in dialogue one-on-one, and interaction tends to be a mutual, dynamic affair, much like “real life” conversation.Yet while the “real-time” dialogue possible on Chatroulette may seem more conducive to facilitating Schutz’ idea of “growing older together,” the service also has its issues. The randomness of connection with others is problematic, as the film frankly acknowledges in the uncensored shots of frontal male nudity that Noah experiences in his search for a chat partner. Also, the problematic lack of a permanent means of staying in contact with each other is illustrated by a further tragic moment in the film when the session with the unnamed girl ends, with Noah having no means of ever being able to find her again.ConclusionIt is tempting to dismiss the problems that Noah encounters while interacting via mediated communication with the exhortation to “just go out and live [… ] life in the real world” (Trumbore para. 4), but this is also over-simplistic. Rather, what we can take away from the film is that there are trade-offs to be had in the technological mediation of self-presentation and communication. The questions that we need to address are: what prompts the choice of one form of technological mediation over another? And what are the consequences of this choice? Contemporary persona, as conceived by David Marshall, is motivated by the commodification of the self, and by increased importance of affect in relationships (Marshall “Persona Studies”). In the realm of Facebook, the commodification of the self has to some degree flattened the available interactivity of the online self, in favour of what the unnamed Chatroulette girl derogatorily refers to as “a popularity contest.”The short film Noah is to some degree a cultural critique of dominant trends in contemporary online persona, notably of the “commodification of the self” instantiated on Facebook. By conceiving of online persona in the terms of ANT outlined here, it becomes possible to envision alternatives to this dominant form of persona, including a concept of persona as commodification. Further, it is possible to do this in a way that avoids the trap of blaming technology for all problems, and that recognises both the advantages and disadvantages of different ways of constructing online persona. The analysis of Noah presented here can therefore provide a guide for more sophisticated and systematic examinations of the hybrid-object “online persona.”References Beggs, Scott. “Short Film: The Very Cool ‘Noah’ Plays Out Madly on a Teenager’s Computer Screen.” Film School Rejects 11 Sep. 2013. 3 Mar. 2014. Callon, M. “Some Elements of a Sociology of Translation: Domestication of the Scallops and the Fishermen of St Brieuc Bay.” Power, Action and Belief: A New Sociology of Knowledge? Ed. John Law. London, UK: Routledge &amp; Kegan Paul, 1986. 196–223. Berkowitz, Joe. “You Need to See This 17-Minute Film Set Entirely on a Teen’s Computer Screen.” Fast Company 10 Sep. 2013. 1 Mar. 2014. Hassan, Robert. Media, Politics and the Network Society. Maidenhead: Open University Press, 2004. Hornyak, Tim. “Short Film ‘Noah’ Will Make You Think Twice about Facebook—CNET.” CNET 19 Sep. 2013. 2 Mar. 2014. Knibbs, Kate. “‘Have You Lurked Her Texts?’: How the Directors of ‘Noah’ Captured the Pain of Facebook-Era Dating.” Digital Trends 14 Sep. 2013. 9 Feb. 2014. Latour, Bruno. Reassembling the Social: An Introduction to Actor-Network Theory. Oxford University Press, 2005. Latour, Bruno. We Have Never Been Modern. Cambridge, Mass: Harvard University Press, 1993. Latour, Bruno. “Where Are the Missing Masses? The Sociology of a Few Mundane Artifacts.” Shaping Technology/Building Society: Studies in Sociotechnical Change. Ed. Wiebe E. Bijker and John Law. Cambridge, MA: MIT Press, 1992. 225–58. Law, John. “After ANT: Complexity, Naming and Topology.” Actor-Network Theory and After. Ed. John Law and John Hassard. Oxford: Blackwell Publishers, 1999. 1–14. Marshall, P. David. “Persona Studies: Mapping the Proliferation of the Public Self.” Journalism 15.2 (2014): 153–170. Marshall, P. David. “The Intercommunication Challenge: Developing a New Lexicon of Concepts for a Transformed Era of Communication.” ICA 2011: Proceedings of the 61st Annual ICA Conference. Boston, MA: Intrenational Communication Association, 2011. 1–25. Paulas, Rick. “Step inside the Computer Screen of ‘Noah.’” VICE 18 Jan. 2014. 8 Feb. 2014. Schutz, Alfred. The Phenomenology of the Social World. Trans. George Walsh and Frederick Lehnert. London, UK: Heinemann, 1972. Trumbore, Dave. “Indie Spotlight: NOAH - A 17-Minute Short Film from Patrick Cederberg and Walter Woodman.” Collider 2013. 2 Apr. 2014. Warren, Christina. “The Short Film That Takes Place Entirely inside a Computer.” Mashable 13 Sep.2013. 9 Feb. 2014. Woodman, Walter, and Patrick Cederberg. Noah. 2013.

Introduction Most developed countries, including Australia, have a strong focus on national, state and local strategies for emergency management and response in the face of disasters and crises. This framework can include coping with catastrophic dislocation, service disruption, injury or loss of life in the face of natural disasters such as major fires, floods, earthquakes or other large-impact natural events, as well as dealing with similar catastrophes resulting from human actions such as bombs, biological agents, cyber-attacks targeting essential services such as communications networks, or other crises affecting large populations. Emergency management frameworks for crisis and disaster response are distinguished by their focus on the domestic context for such events; that is, how to manage and assist the ways in which civilian populations, who are for the most part inexperienced and untrained in dealing with crises and disasters, are able to respond and behave in such situations so as to minimise the impacts of a catastrophic event. Even in countries like Australia that demonstrate a strong public commitment to cultural pluralism and social cohesion, ethno-cultural diversity can be seen as a risk or threat to national security and values at times of political, natural, economic and/or social tensions and crises. Australian government policymakers have recently focused, with increasing intensity, on “community resilience” as a key element in countering extremism and enhancing emergency preparedness and response. In some sense, this is the result of a tacit acknowledgement by government agencies that there are limits to what they can do for domestic communities should such a catastrophic event occur, and accordingly, the focus in recent times has shifted to how governments can best help people to help themselves in such situations, a key element of the contemporary “resilience” approach. Yet despite the robustly multicultural nature of Australian society, explicit engagement with Australia’s cultural diversity flickers only fleetingly on this agenda, which continues to pursue approaches to community resilience in the absence of understandings about how these terms and formations may themselves need to be diversified to maximise engagement by all citizens in a multicultural polity. There have been some recent efforts in Australia to move in this direction, for example the Australian Emergency Management Institute (AEMI)’s recent suite of projects with culturally and linguistically diverse (CALD) communities (2006-2010) and the current Australia-New Zealand Counter-Terrorism Committee-supported project on “Harnessing Resilience Capital in Culturally Diverse Communities to Counter Violent Extremism” (Grossman and Tahiri), which I discuss in a longer forthcoming version of this essay (Grossman). Yet the understanding of ethno-cultural identity and difference that underlies much policy thinking on resilience remains problematic for the way in which it invests in a view of the cultural dimensions of community resilience as relic rather than resource – valorising the preservation of and respect for cultural norms and traditions, but silent on what different ethno-cultural communities might contribute toward expanded definitions of both “community” and “resilience” by virtue of the transformative potential and existing cultural capital they bring with them into new national and also translocal settings. For example, a primary conclusion of the joint program between AEMI and the Australian Multicultural Commission is that CALD communities are largely “vulnerable” in the context of disasters and emergency management and need to be better integrated into majority-culture models of theorising and embedding community resilience. This focus on stronger national integration and the “vulnerability” of culturally diverse ethno-cultural communities in the Australian context echoes the work of scholars beyond Australia such as McGhee, Mouritsen (Reflections, Citizenship) and Joppke. They argue that the “civic turn” in debates around resurgent contemporary nationalism and multicultural immigration policies privileges civic integration over genuine two-way multiculturalism. This approach sidesteps the transculturational (Ortiz; Welsch; Mignolo; Bennesaieh; Robins; Stein) aspects of contemporary social identities and exchange by paying lip-service to cultural diversity while affirming a neo-liberal construct of civic values and principles as a universalising goal of Western democratic states within a global market economy. It also suggests a superficial tribute to cultural diversity that does not embed diversity comprehensively at the levels of either conceptualising or resourcing different elements of Australian transcultural communities within the generalised framework of “community resilience.” And by emphasising cultural difference as vulnerability rather than as resource or asset, it fails to acknowledge the varieties of resilience capital that many culturally diverse individuals and communities may bring with them when they resettle in new environments, by ignoring the question of what “resilience” actually means to those from culturally diverse communities. In so doing, it also avoids the critical task of incorporating intercultural definitional diversity around the concepts of both “community” and “resilience” used to promote social cohesion and the capacity to recover from disasters and crises. How we might do differently in thinking about the broader challenges for multiculturalism itself as a resilient transnational concept and practice? The Concept of Resilience The meanings of resilience vary by disciplinary perspective. While there is no universally accepted definition of the concept, it is widely acknowledged that resilience refers to the capacity of an individual to do well in spite of exposure to acute trauma or sustained adversity (Liebenberg 219). Originating in the Latin word resilio, meaning ‘to jump back’, there is general consensus that resilience pertains to an individual’s, community’s or system’s ability to adapt to and ‘bounce back’ from a disruptive event (Mohaupt 63, Longstaff et al. 3). Over the past decade there has been a dramatic rise in interest in the clinical, community and family sciences concerning resilience to a broad range of adversities (Weine 62). While debate continues over which discipline can be credited with first employing resilience as a concept, Mohaupt argues that most of the literature on resilience cites social psychology and psychiatry as the origin for the concept beginning in the mid-20th century. The pioneer researchers of what became known as resilience research studied the impact on children living in dysfunctional families. For example, the findings of work by Garmezy, Werner and Smith and Rutter showed that about one third of children in these studies were coping very well despite considerable adversities and traumas. In asking what it was that prevented the children in their research from being negatively influenced by their home environments, such research provided the basis for future research on resilience. Such work was also ground-breaking for identifying the so-called ‘protective factors’ or resources that individuals can operationalise when dealing with adversity. In essence, protective factors are those conditions in the individual that protect them from the risk of dysfunction and enable recovery from trauma. They mitigate the effects of stressors or risk factors, that is, those conditions that predispose one to harm (Hajek 15). Protective factors include the inborn traits or qualities within an individual, those defining an individual’s environment, and also the interaction between the two. Together, these factors give people the strength, skills and motivation to cope in difficult situations and re-establish (a version of) ‘normal’ life (Gunnestad). Identifying protective factors is important in terms of understanding the particular resources a given sociocultural group has at its disposal, but it is also vital to consider the interconnections between various protective mechanisms, how they might influence each other, and to what degree. An individual, for instance, might display resilience or adaptive functioning in a particular domain (e.g. emotional functioning) but experience significant deficits in another (e.g. academic achievement) (Hunter 2). It is also essential to scrutinise how the interaction between protective factors and risk factors creates patterns of resilience. Finally, a comprehensive understanding of the interrelated nature of protective mechanisms and risk factors is imperative for designing effective interventions and tailored preventive strategies (Weine 65). In short, contemporary thinking about resilience suggests it is neither entirely personal nor strictly social, but an interactive and iterative combination of the two. It is a quality of the environment as much as the individual. For Ungar, resilience is the complex entanglements between “individuals and their social ecologies [that] will determine the degree of positive outcomes experienced” (3). Thinking about resilience as context-dependent is important because research that is too trait-based or actor-centred risks ignoring any structural or institutional forces. A more ecological interpretation of resilience, one that takes into a person’s context and environment into account, is vital in order to avoid blaming the victim for any hardships they face, or relieving state and institutional structures from their responsibilities in addressing social adversity, which can “emphasise self-help in line with a neo-conservative agenda instead of stimulating state responsibility” (Mohaupt 67). Nevertheless, Ungar posits that a coherent definition of resilience has yet to be developed that adequately ‘captures the dual focus of the individual and the individual’s social ecology and how the two must both be accounted for when determining the criteria for judging outcomes and discerning processes associated with resilience’ (7). Recent resilience research has consequently prompted a shift away from vulnerability towards protective processes — a shift that highlights the sustained capabilities of individuals and communities under threat or at risk. Locating ‘Culture’ in the Literature on Resilience However, an understanding of the role of culture has remained elusive or marginalised within this trend; there has been comparatively little sustained investigation into the applicability of resilience constructs to non-western cultures, or how the resources available for survival might differ from those accessible to western populations (Ungar 4). As such, a growing body of researchers is calling for more rigorous inquiry into culturally determined outcomes that might be associated with resilience in non-western or multicultural cultures and contexts, for example where Indigenous and minority immigrant communities live side by side with their ‘mainstream’ neighbours in western settings (Ungar 2). ‘Cultural resilience’ considers the role that cultural background plays in determining the ability of individuals and communities to be resilient in the face of adversity. For Clauss-Ehlers, the term describes the degree to which the strengths of one’s culture promote the development of coping (198). Culturally-focused resilience suggests that people can manage and overcome stress and trauma based not on individual characteristics alone, but also from the support of broader sociocultural factors (culture, cultural values, language, customs, norms) (Clauss-Ehlers 324). The innate cultural strengths of a culture may or may not differ from the strengths of other cultures; the emphasis here is not so much comparatively inter-cultural as intensively intra-cultural (VanBreda 215). A culturally focused resilience model thus involves “a dynamic, interactive process in which the individual negotiates stress through a combination of character traits, cultural background, cultural values, and facilitating factors in the sociocultural environment” (Clauss-Ehlers 199). In understanding ways of ‘coping and hoping, surviving and thriving’, it is thus crucial to consider how culturally and linguistically diverse minorities navigate the cultural understandings and assumptions of both their countries of origin and those of their current domicile (Ungar 12). Gunnestad claims that people who master the rules and norms of their new culture without abandoning their own language, values and social support are more resilient than those who tenaciously maintain their own culture at the expense of adjusting to their new environment. They are also more resilient than those who forego their own culture and assimilate with the host society (14). Accordingly, if the combination of both valuing one’s culture as well as learning about the culture of the new system produces greater resilience and adaptive capacities, serious problems can arise when a majority tries to acculturate a minority to the mainstream by taking away or not recognising important parts of the minority culture. In terms of resilience, if cultural factors are denied or diminished in accounting for and strengthening resilience – in other words, if people are stripped of what they possess by way of resilience built through cultural knowledge, disposition and networks – they do in fact become vulnerable, because ‘they do not automatically gain those cultural strengths that the majority has acquired over generations’ (Gunnestad 14). Mobilising ‘Culture’ in Australian Approaches to Community Resilience The realpolitik of how concepts of resilience and culture are mobilised is highly relevant here. As noted above, when ethnocultural difference is positioned as a risk or a threat to national identity, security and values, this is precisely the moment when vigorously, even aggressively, nationalised definitions of ‘community’ and ‘identity’ that minoritise or disavow cultural diversities come to the fore in public discourse. The Australian evocation of nationalism and national identity, particularly in the way it has framed policy discussion on managing national responses to disasters and threats, has arguably been more muted than some of the European hysteria witnessed recently around cultural diversity and national life. Yet we still struggle with the idea that newcomers to Australia might fall on the surplus rather than the deficit side of the ledger when it comes to identifying and harnessing resilience capital. A brief example of this trend is explored here. From 2006 to 2010, the Australian Emergency Management Institute embarked on an ambitious government-funded four-year program devoted to strengthening community resilience in relation to disasters with specific reference to engaging CALD communities across Australia. The program, Inclusive Emergency Management with CALD Communities, was part of a wider Australian National Action Plan to Build Social Cohesion, Harmony and Security in the wake of the London terrorist bombings in July 2005. Involving CALD community organisations as well as various emergency and disaster management agencies, the program ran various workshops and agency-community partnership pilots, developed national school education resources, and commissioned an evaluation of the program’s effectiveness (Farrow et al.). While my critique here is certainly not aimed at emergency management or disaster response agencies and personnel themselves – dedicated professionals who often achieve remarkable results in emergency and disaster response under extraordinarily difficult circumstances – it is nevertheless important to highlight how the assumptions underlying elements of AEMI’s experience and outcomes reflect the persistent ways in which ethnocultural diversity is rendered as a problem to be surmounted or a liability to be redressed, rather than as an asset to be built upon or a resource to be valued and mobilised. AEMI’s explicit effort to engage with CALD communities in building overall community resilience was important in its tacit acknowledgement that emergency and disaster services were (and often remain) under-resourced and under-prepared in dealing with the complexities of cultural diversity in emergency situations. Despite these good intentions, however, while the program produced some positive outcomes and contributed to crucial relationship building between CALD communities and emergency services within various jurisdictions, it also continued to frame the challenge of working with cultural diversity as a problem of increased vulnerability during disasters for recently arrived and refugee background CALD individuals and communities. This highlights a common feature in community resilience-building initiatives, which is to focus on those who are already ‘robust’ versus those who are ‘vulnerable’ in relation to resilience indicators, and whose needs may require different or additional resources in order to be met. At one level, this is a pragmatic resourcing issue: national agencies understandably want to put their people, energy and dollars where they are most needed in pursuit of a steady-state unified national response at times of crisis. Nor should it be argued that at least some CALD groups, particularly those from new arrival and refugee communities, are not vulnerable in at least some of the ways and for some of the reasons suggested in the program evaluation. However, the consistent focus on CALD communities as ‘vulnerable’ and ‘in need’ is problematic, as well as partial. It casts members of these communities as structurally and inherently less able and less resilient in the context of disasters and emergencies: in some sense, as those who, already ‘victims’ of chronic social deficits such as low English proficiency, social isolation and a mysterious unidentified set of ‘cultural factors’, can become doubly victimised in acute crisis and disaster scenarios. In what is by now a familiar trope, the description of CALD communities as ‘vulnerable’ precludes asking questions about what they do have, what they do know, and what they do or can contribute to how we respond to disaster and emergency events in our communities. A more profound problem in this sphere revolves around working out how best to engage CALD communities and individuals within existing approaches to disaster and emergency preparedness and response. This reflects a fundamental but unavoidable limitation of disaster preparedness models: they are innately spatially and geographically bounded, and consequently understand ‘communities’ in these terms, rather than expanding definitions of ‘community’ to include the dimensions of community-as-social-relations. While some good engagement outcomes were achieved locally around cross-cultural knowledge for emergency services workers, the AEMI program fell short of asking some of the harder questions about how emergency and disaster service scaffolding and resilience-building approaches might themselves need to change or transform, using a cross-cutting model of ‘communities’ as both geographic places and multicultural spaces (Bartowiak-Théron and Crehan) in order to be more effective in national scenarios in which cultural diversity should be taken for granted. Toward Acknowledgement of Resilience Capital Most significantly, the AEMI program did not produce any recognition of the ways in which CALD communities already possess resilience capital, or consider how this might be drawn on in formulating stronger community initiatives around disaster and threats preparedness for the future. Of course, not all individuals within such communities, nor all communities across varying circumstances, will demonstrate resilience, and we need to be careful of either overgeneralising or romanticising the kinds and degrees of ‘resilience capital’ that may exist within them. Nevertheless, at least some have developed ways of withstanding crises and adapting to new conditions of living. This is particularly so in connection with individual and group behaviours around resource sharing, care-giving and social responsibility under adverse circumstances (Grossman and Tahiri) – all of which are directly relevant to emergency and disaster response. While some of these resilient behaviours may have been nurtured or enhanced by particular experiences and environments, they can, as the discussion of recent literature above suggests, also be rooted more deeply in cultural norms, habits and beliefs. Whatever their origins, for culturally diverse societies to achieve genuine resilience in the face of both natural and human-made disasters, it is critical to call on the ‘social memory’ (Folke et al.) of communities faced with responding to emergencies and crises. Such wellsprings of social memory ‘come from the diversity of individuals and institutions that draw on reservoirs of practices, knowledge, values, and worldviews and is crucial for preparing the system for change, building resilience, and for coping with surprise’ (Adger et al.). Consequently, if we accept the challenge of mapping an approach to cultural diversity as resource rather than relic into our thinking around strengthening community resilience, there are significant gains to be made. For a whole range of reasons, no diversity-sensitive model or measure of resilience should invest in static understandings of ethnicities and cultures; all around the world, ethnocultural identities and communities are in a constant and sometimes accelerated state of dynamism, reconfiguration and flux. But to ignore the resilience capital and potential protective factors that ethnocultural diversity can offer to the strengthening of community resilience more broadly is to miss important opportunities that can help suture the existing disconnects between proactive approaches to intercultural connectedness and social inclusion on the one hand, and reactive approaches to threats, national security and disaster response on the other, undermining the effort to advance effectively on either front. This means that dominant social institutions and structures must be willing to contemplate their own transformation as the result of transcultural engagement, rather than merely insisting, as is often the case, that ‘other’ cultures and communities conform to existing hegemonic paradigms of being and of living. In many ways, this is the most critical step of all. A resilience model and strategy that questions its own culturally informed yet taken-for-granted assumptions and premises, goes out into communities to test and refine these, and returns to redesign its approach based on the new knowledge it acquires, would reflect genuine progress toward an effective transculturational approach to community resilience in culturally diverse contexts.References Adger, W. Neil, Terry P. Hughes, Carl Folke, Stephen R. 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Erasmuskloof: South African Military Health Service, Military Psychological Institute, Social Work Research &amp; Development, 2001. Weine, Stevan. “Building Resilience to Violent Extremism in Muslim Diaspora Communities in the United States.” Dynamics of Asymmetric Conflict 5.1 (2012): 60-73. Welsch, Wolfgang. “Transculturality: The Puzzling Form of Cultures Today.” Spaces of Culture: City, Nation World. Eds. M. Featherstone and S. Lash. London: Sage, 1999. 194-213. Werner, Emmy E., and Ruth S. Smith. Vulnerable But Invincible: A Longitudinal Study of\ Resilience and Youth. New York: McGraw Hill, 1982. NotesThe concept of ‘resilience capital’ I offer here is in line with one strand of contemporary theorising around resilience – that of resilience as social or socio-ecological capital – but moves beyond the idea of enhancing general social connectedness and community cohesion by emphasising the ways in which culturally diverse communities may already be robustly networked and resourceful within micro-communal settings, with new resources and knowledge both to draw on and to offer other communities or the ‘national community’ at large. In effect, ‘resilience capital’ speaks to the importance of finding ‘the communities within the community’ (Bartowiak-Théron and Crehan 11) and recognising their capacity to contribute to broad-scale resilience and recovery.I am indebted for the discussion of the literature on resilience here to Dr Peta Stephenson, Centre for Cultural Diversity and Wellbeing, Victoria University, who is working on a related project (M. Grossman and H. Tahiri, Harnessing Resilience Capital in Culturally Diverse Communities to Counter Violent Extremism, forthcoming 2014).

Introduction This paper offers a survey of familiar popular music performers and songwriters who reference coffee in their work. It examines three areas of discourse: the psychoactive effects of caffeine, coffee and courtship rituals, and the politics of coffee consumption. I claim that coffee carries a cultural and musicological significance comparable to that of the chemical stimulants and consumer goods more readily associated with popular music. Songs about coffee may not be as potent as those featuring drugs and alcohol (Primack; Schapiro), or as common as those referencing commodities like clothes and cars (Englis; McCracken), but they do feature across a wide range of genres, some of which enjoy archetypal associations with this beverage. m.o.m.m.y. Needs c.o.f.f.e.e.: The Psychoactive Effect of Coffee The act of performing and listening to popular music involves psychological elements comparable to the overwhelming sensory experience of drug taking: altered perceptions, repetitive grooves, improvisation, self-expression, and psychological empathy—such as that between musician and audience (Curry). Most popular music genres are, as a result, culturally and sociologically identified with the consumption of at least one mind-altering substance (Lyttle; Primack; Schapiro). While the analysis of lyrics referring to this theme has hitherto focused on illegal drugs and alcoholic beverages (Cooper), coffee and its psychoactive ingredient caffeine have been almost entirely overlooked (Summer). The most recent study of drugs in popular music, for example, defined substance use as “tobacco, alcohol, marijuana, cocaine and other stimulants, heroin and other opiates, hallucinogens, inhalants, prescription drugs, over-the-counter drugs, and nonspecific substances” (Primack 172), thereby ignoring a chemical stimulant consumed by 90 per cent of adult Americans every day (Lovett). The wide availability of coffee and the comparatively mild effect of caffeine means that its consumption rarely causes harm. One researcher has described it as a ubiquitous and unobtrusive “generalised public activity […] ‘invisible’ to analysts seeking distinctive social events” (Cooper 92). Coffee may provide only a relatively mild “buzz”—but it is now accepted that caffeine is an addictive substance (Juliano) and, due to its universal legality, coffee is also the world’s most extensively traded and enthusiastically consumed psychoactive consumer product (Juliano 1). The musical genre of jazz has a longstanding relationship with marijuana and narcotics (Curry; Singer; Tolson; Winick). Unsurprisingly, given its Round Midnight connotations, jazz standards also celebrate the restorative impact of coffee. Exemplary compositions include Burke/Webster’s insomniac torch song Black Coffee, which provided hits for Sarah Vaughan (1949), Ella Fitzgerald (1953), and Peggy Lee (1960); and Frank Sinatra’s recordings of Hilliard/Dick’s The Coffee Song (1946, 1960), which satirised the coffee surplus in Brazil at a time when this nation enjoyed a near monopoly on production. Sinatra joked that this ubiquitous drink was that country’s only means of liquid refreshment, in a refrain that has since become a headline writer’s phrasal template: “There’s an Awful Lot of Coffee in Vietnam,” “An Awful Lot of Coffee in the Bin,” and “There’s an Awful Lot of Taxes in Brazil.” Ethnographer Aaron Fox has shown how country music gives expression to the lived social experience of blue-collar and agrarian workers (Real 29). Coffee’s role in energising working class America (Cooper) is featured in such recordings as Dolly Parton’s Nine To Five (1980), which describes her morning routine using a memorable “kitchen/cup of ambition” rhyme, and Don't Forget the Coffee Billy Joe (1973) by Tom T. Hall which laments the hardship of unemployment, hunger, cold, and lack of healthcare. Country music’s “tired truck driver” is the most enduring blue-collar trope celebrating coffee’s analeptic powers. Versions include Truck Drivin' Man by Buck Owens (1964), host of the country TV show Hee Haw and pioneer of the Bakersfield sound, and Driving My Life Away from pop-country crossover star Eddie Rabbitt (1980). Both feature characteristically gendered stereotypes of male truck drivers pushing on through the night with the help of a truck stop waitress who has fuelled them with caffeine. Johnny Cash’s A Cup of Coffee (1966), recorded at the nadir of his addiction to pills and alcohol, has an incoherent improvised lyric on this subject; while Jerry Reed even prescribed amphetamines to keep drivers awake in Caffein [sic], Nicotine, Benzedrine (And Wish Me Luck) (1980). Doye O’Dell’s Diesel Smoke, Dangerous Curves (1952) is the archetypal “truck drivin’ country” song and the most exciting track of its type. It subsequently became a hit for the doyen of the subgenre, Red Simpson (1966). An exhausted driver, having spent the night with a woman whose name he cannot now recall, is fighting fatigue and wrestling his hot-rod low-loader around hairpin mountain curves in an attempt to rendezvous with a pretty truck stop waitress. The song’s palpable energy comes from its frenetic guitar picking and the danger implicit in trailing a heavy load downhill while falling asleep at the wheel. Tommy Faile’s Phantom 309, a hit for Red Sovine (1967) that was later covered by Tom Waits (Big Joe and the Phantom 309, 1975), elevates the “tired truck driver” narrative to gothic literary form. Reflecting country music’s moral code of citizenship and its culture of performative storytelling (Fox, Real 23), it tells of a drenched and exhausted young hitchhiker picked up by Big Joe—the driver of a handsome eighteen-wheeler. On arriving at a truck stop, Joe drops the traveller off, giving him money for a restorative coffee. The diner falls silent as the hitchhiker orders up his “cup of mud”. Big Joe, it transpires, is a phantom trucker. After running off the road to avoid a school bus, his distinctive ghost rig now only reappears to rescue stranded travellers. Punk rock, a genre closely associated with recreational amphetamines (McNeil 76, 87), also features a number of caffeine-as-stimulant songs. Californian punk band, Descendents, identified caffeine as their drug of choice in two 1996 releases, Coffee Mug and Kids on Coffee. These songs describe chugging the drink with much the same relish and energy that others might pull at the neck of a beer bottle, and vividly compare the effects of the drug to the intense rush of speed. The host of “New Music News” (a segment of MTV’s 120 Minutes) references this correlation in 1986 while introducing the band’s video—in which they literally bounce off the walls: “You know, while everybody is cracking down on crack, what about that most respectable of toxic substances or stimulants, the good old cup of coffee? That is the preferred high, actually, of California’s own Descendents—it is also the subject of their brand new video” (“New Music News”). Descendents’s Sessions EP (1997) featured an overflowing cup of coffee on the sleeve, while punk’s caffeine-as-amphetamine trope is also promulgated by Hellbender (Caffeinated 1996), Lagwagon (Mr. Coffee 1997), and Regatta 69 (Addicted to Coffee 2005). Coffee in the Morning and Kisses in the Night: Coffee and Courtship Coffee as romantic metaphor in song corroborates the findings of early researchers who examined courtship rituals in popular music. Donald Horton’s 1957 study found that hit songs codified the socially constructed self-image and limited life expectations of young people during the 1950s by depicting conservative, idealised, and traditional relationship scenarios. He summarised these as initial courtship, honeymoon period, uncertainty, and parting (570-4). Eleven years after this landmark analysis, James Carey replicated Horton’s method. His results revealed that pop lyrics had become more realistic and less bound by convention during the 1960s. They incorporated a wider variety of discourse including the temporariness of romantic commitment, the importance of individual autonomy in relationships, more liberal attitudes, and increasingly unconventional courtship behaviours (725). Socially conservative coffee songs include Coffee in the Morning and Kisses in the Night by The Boswell Sisters (1933) in which the protagonist swears fidelity to her partner on condition that this desire is expressed strictly in the appropriate social context of marriage. It encapsulates the restrictions Horton identified on courtship discourse in popular song prior to the arrival of rock and roll. The Henderson/DeSylva/Brown composition You're the Cream in My Coffee, recorded by Annette Hanshaw (1928) and by Nat King Cole (1946), also celebrates the social ideal of monogamous devotion. The persistence of such idealised traditional themes continued into the 1960s. American pop singer Don Cherry had a hit with Then You Can Tell Me Goodbye (1962) that used coffee as a metaphor for undying and everlasting love. Otis Redding’s version of Butler/Thomas/Walker’s Cigarettes and Coffee (1966)—arguably soul music’s exemplary romantic coffee song—carries a similar message as a couple proclaim their devotion in a late night conversation over coffee. Like much of the Stax catalogue, Cigarettes and Coffee, has a distinctly “down home” feel and timbre. The lovers are simply content with each other; they don’t need “cream” or “sugar.” Horton found 1950s blues and R&amp;B lyrics much more sexually explicit than pop songs (567). Dawson (1994) subsequently characterised black popular music as a distinct public sphere, and Squires (2002) argued that it displayed elements of what she defined as “enclave” and “counterpublic” traits. Lawson (2010) has argued that marginalised and/or subversive blues artists offered a form of countercultural resistance against prevailing social norms. Indeed, several blues and R&amp;B coffee songs disregard established courtship ideals and associate the product with non-normative and even transgressive relationship circumstances—including infidelity, divorce, and domestic violence. Lightnin’ Hopkins’s Coffee Blues (1950) references child neglect and spousal abuse, while the narrative of Muddy Waters’s scorching Iodine in my Coffee (1952) tells of an attempted poisoning by his Waters’s partner. In 40 Cups of Coffee (1953) Ella Mae Morse is waiting for her husband to return home, fuelling her anger and anxiety with caffeine. This song does eventually comply with traditional courtship ideals: when her lover eventually returns home at five in the morning, he is greeted with a relieved kiss. In Keep That Coffee Hot (1955), Scatman Crothers supplies a counterpoint to Morse’s late-night-abandonment narrative, asking his partner to keep his favourite drink warm during his adulterous absence. Brook Benton’s Another Cup of Coffee (1964) expresses acute feelings of regret and loneliness after a failed relationship. More obliquely, in Coffee Blues (1966) Mississippi John Hurt sings affectionately about his favourite brand, a “lovin’ spoonful” of Maxwell House. In this, he bequeathed the moniker of folk-rock band The Lovin’ Spoonful, whose hits included Do You Believe in Magic (1965) and Summer in the City (1966). However, an alternative reading of Hurt’s lyric suggests that this particular phrase is a metaphorical device proclaiming the author’s sexual potency. Hurt’s “lovin’ spoonful” may actually be a portion of his seminal emission. In the 1950s, Horton identified country as particularly “doleful” (570), and coffee provides a common metaphor for failed romance in a genre dominated by “metanarratives of loss and desire” (Fox, Jukebox 54). Claude Gray’s I'll Have Another Cup of Coffee (Then I’ll Go) (1961) tells of a protagonist delivering child support payments according to his divorce lawyer’s instructions. The couple share late night coffee as their children sleep through the conversation. This song was subsequently recorded by seventeen-year-old Bob Marley (One Cup of Coffee, 1962) under the pseudonym Bobby Martell, a decade prior to his breakthrough as an international reggae star. Marley’s youngest son Damian has also performed the track while, interestingly in the context of this discussion, his older sibling Rohan co-founded Marley Coffee, an organic farm in the Jamaican Blue Mountains. Following Carey’s demonstration of mainstream pop’s increasingly realistic depiction of courtship behaviours during the 1960s, songwriters continued to draw on coffee as a metaphor for failed romance. In Carly Simon’s You’re So Vain (1972), she dreams of clouds in her coffee while contemplating an ostentatious ex-lover. Squeeze’s Black Coffee In Bed (1982) uses a coffee stain metaphor to describe the end of what appears to be yet another dead-end relationship for the protagonist. Sarah Harmer’s Coffee Stain (1998) expands on this device by reworking the familiar “lipstick on your collar” trope, while Sexsmith &amp; Kerr’s duet Raindrops in my Coffee (2005) superimposes teardrops in coffee and raindrops on the pavement with compelling effect. Kate Bush’s Coffee Homeground (1978) provides the most extreme narrative of relationship breakdown: the true story of Cora Henrietta Crippin’s poisoning. Researchers who replicated Horton’s and Carey’s methodology in the late 1970s (Bridges; Denisoff) were surprised to find their results dominated by traditional courtship ideals. The new liberal values unearthed by Carey in the late 1960s simply failed to materialise in subsequent decades. In this context, it is interesting to observe how romantic coffee songs in contemporary soul and jazz continue to disavow the post-1960s trend towards realistic social narratives, adopting instead a conspicuously consumerist outlook accompanied by smooth musical timbres. This phenomenon possibly betrays the influence of contemporary coffee advertising. From the 1980s, television commercials have sought to establish coffee as a desirable high end product, enjoyed by bohemian lovers in a conspicuously up-market environment (Werder). All Saints’s Black Coffee (2000) and Lebrado’s Coffee (2006) identify strongly with the culture industry’s image of coffee as a luxurious beverage whose consumption signifies prominent social status. All Saints’s promotional video is set in a opulent location (although its visuals emphasise the lyric’s romantic disharmony), while Natalie Cole’s Coffee Time (2008) might have been itself written as a commercial. Busting Up a Starbucks: The Politics of Coffee Politics and coffee meet most palpably at the coffee shop. This conjunction has a well-documented history beginning with the establishment of coffee houses in Europe and the birth of the public sphere (Habermas; Love; Pincus). The first popular songs to reference coffee shops include Jaybird Coleman’s Coffee Grinder Blues (1930), which boasts of skills that precede the contemporary notion of a barista by four decades; and Let's Have Another Cup of Coffee (1932) from Irving Berlin’s depression-era musical Face The Music, where the protagonists decide to stay in a restaurant drinking coffee and eating pie until the economy improves. Coffee in a Cardboard Cup (1971) from the Broadway musical 70 Girls 70 is an unambiguous condemnation of consumerism, however, it was written, recorded and produced a generation before Starbucks’ aggressive expansion and rapid dominance of the coffee house market during the 1990s. The growth of this company caused significant criticism and protest against what seemed to be a ruthless homogenising force that sought to overwhelm local competition (Holt; Thomson). In response, Starbucks has sought to be defined as a more responsive and interactive brand that encourages “glocalisation” (de Larios; Thompson). Koller, however, has characterised glocalisation as the manipulative fabrication of an “imagined community”—whose heterogeneity is in fact maintained by the aesthetics and purchasing choices of consumers who make distinctive and conscious anti-brand statements (114). Neat Capitalism is a more useful concept here, one that intercedes between corporate ideology and postmodern cultural logic, where such notions as community relations and customer satisfaction are deliberately and perhaps somewhat cynically conflated with the goal of profit maximisation (Rojek). As the world’s largest chain of coffee houses with over 19,400 stores in March 2012 (Loxcel), Starbucks is an exemplar of this phenomenon. Their apparent commitment to environmental stewardship, community relations, and ethical sourcing is outlined in the company’s annual “Global Responsibility Report” (Vimac). It is also demonstrated in their engagement with charitable and environmental non-governmental organisations such as Fairtrade and Co-operative for Assistance and Relief Everywhere (CARE). By emphasising this, Starbucks are able to interpellate (that is, “call forth”, “summon”, or “hail” in Althusserian terms) those consumers who value environmental protection, social justice and ethical business practices (Rojek 117). Bob Dylan and Sheryl Crow provide interesting case studies of the persuasive cultural influence evoked by Neat Capitalism. Dylan’s 1962 song Talkin’ New York satirised his formative experiences as an impoverished performer in Greenwich Village’s coffee houses. In 1995, however, his decision to distribute the Bob Dylan: Live At The Gaslight 1962 CD exclusively via Starbucks generated significant media controversy. Prominent commentators expressed their disapproval (Wilson Harris) and HMV Canada withdrew Dylan’s product from their shelves (Lynskey). Despite this, the success of this and other projects resulted in the launch of Starbucks’s in-house record company, Hear Music, which released entirely new recordings from major artists such as Ray Charles, Paul McCartney, Joni Mitchell, Carly Simon and Elvis Costello—although the company has recently announced a restructuring of their involvement in this venture (O’Neil). Sheryl Crow disparaged her former life as a waitress in Coffee Shop (1995), a song recorded for her second album. “Yes, I was a waitress. I was a waitress not so long ago; then I won a Grammy” she affirmed in a YouTube clip of a live performance from the same year. More recently, however, Crow has become an avowed self-proclaimed “Starbucks groupie” (Tickle), releasing an Artist’s Choice (2003) compilation album exclusively via Hear Music and performing at the company’s 2010 Annual Shareholders’s Meeting. Songs voicing more unequivocal dissatisfaction with Starbucks’s particular variant of Neat Capitalism include Busting Up a Starbucks (Mike Doughty, 2005), and Starbucks Takes All My Money (KJ-52, 2008). The most successful of these is undoubtedly Ron Sexsmith’s Jazz at the Bookstore (2006). Sexsmith bemoans the irony of intense original blues artists such as Leadbelly being drowned out by the cacophony of coffee grinding machines while customers queue up to purchase expensive coffees whose names they can’t pronounce. In this, he juxtaposes the progressive patina of corporate culture against the circumstances of African-American labour conditions in the deep South, the shocking incongruity of which eventually cause the old bluesman to turn in his grave. Fredric Jameson may have good reason to lament the depthless a-historical pastiche of postmodern popular culture, but this is no “nostalgia film”: Sexsmith articulates an artfully framed set of subtle, sensitive, and carefully contextualised observations. Songs about coffee also intersect with politics via lyrics that play on the mid-brown colour of the beverage, by employing it as a metaphor for the sociological meta-narratives of acculturation and assimilation. First popularised in Israel Zangwill’s 1905 stage play, The Melting Pot, this term is more commonly associated with Americanisation rather than miscegenation in the United States—a nuanced distinction that British band Blue Mink failed to grasp with their memorable invocation of “coffee-coloured people” in Melting Pot (1969). Re-titled in the US as People Are Together (Mickey Murray, 1970) the song was considered too extreme for mainstream radio airplay (Thompson). Ike and Tina Turner’s Black Coffee (1972) provided a more accomplished articulation of coffee as a signifier of racial identity; first by associating it with the history of slavery and the post-Civil Rights discourse of African-American autonomy, then by celebrating its role as an energising force for African-American workers seeking economic self-determination. Anyone familiar with the re-casting of black popular music in an industry dominated by Caucasian interests and aesthetics (Cashmore; Garofalo) will be unsurprised to find British super-group Humble Pie’s (1973) version of this song more recognisable. Conclusion Coffee-flavoured popular songs celebrate the stimulant effects of caffeine, provide metaphors for courtship rituals, and offer critiques of Neat Capitalism. Harold Love and Guthrie Ramsey have each argued (from different perspectives) that the cultural micro-narratives of small social groups allow us to identify important “ethnographic truths” (Ramsey 22). Aesthetically satisfying and intellectually stimulating coffee songs are found where these micro-narratives intersect with the ethnographic truths of coffee culture. 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I. First Brexit, then Trump: Has the past year or so ushered in a “wave” (Weisberg), a “barrage” (Desmond-Harris) or a “deluge” (Sidahmed) of that notoriously noxious affect, hate? It certainly feels that way to those of us identified with progressive social and political causes—those of us troubled, not just by Trump’s recent electoral victory, but by the far-right forces to which that victory has given voice. And yet the questions still hanging over efforts to quantify emotional or affective states leaves the claim that there has been a clear spike in hate moot (Ngai 26; Massumi 136-7; Ahmed, Promise 3-8). So let’s try asking a different question. Has this same period seen a rise, across liberal media platforms, in the rhetorical work of “hate-attribution”? Here, at least, an answer seems in readier reach. For no one given to scrolling distractedly through liberal Anglophone media outlets, from The New York Times, to The Guardian, to Slate, will be unfamiliar with a species of journalism that, in reporting the appalling activities associated with what has become known as the “alt-right” (Main; Wallace-Wells; Gourarie), articulates those activities in the rubric of a calculable uptick in hate itself.Before the U.S. Presidential election, this fledgling journalistic genre was already testing its wings, its first shudderings felt everywhere from Univision anchor Jorge Ramos’s widely publicized documentary, Hate Rising (2016), which explores the rise of white supremacist movements across the South-West U.S, to an edition of Slate’s Trumpcast entitled “The Alt-Right and a Deluge of Hate,” which broached the torment-by-Twitter of left-wing journalist David French. In the wake of the election, and the appalling acts of harassment and intimidation it seemed to authorize, the genre gained further momentum—leading to the New Yorker’s “Hate Is on the Rise After Trump’s Election,” to The Guardian’s “Trump’s Election led to Barrage of Hate,” and to Vox’s “The Wave of Post-Election Hate Reportedly Sweeping the Nation, Explained.” And it still has traction today, judging not just by James King’s recent year-in-review column, “The Year in Hate: From Donald Trump to the Rise of the Alt-Right,” but by Salon’s “A Short History of Hate” which tracks the alt-right’s meteoric 2016 rise to prominence, and the New York Times’ recently launched hate-speech aggregator, “This Week in Hate.”As should already be clear from these brisk, thumbnail accounts of the texts in question, the phenomena alluded to by the titular term “hate” are not instances of hate per se, but rather instances of “hate-speech.” The word “hate,” in other words, is being deployed here not literally, to refer to an emotional state, but metonymically, as a shorthand for “hate-speech”—a by-now widely conventionalized and legally codified parlance originating with the U.N. Declaration to describe “violent or violence-inciting speech or acts that “aim or intend to inflict injury, or incite prejudice or hatred, against persons of groups” because of their ethnic, religious, sexual or social affiliation. And there is no doubt that, beyond the headlines, these articles do incredibly important work, drawing connections between, and drawing attention to, a host of harmful activities associated with the so-called “alt-right”—from a pair of mangled, pretzel-shaped swastikas graffiti-ed in a children’s playground, to acts of harassment, intimidation and violence against women, African-Americans, Latinos, Muslims, Jews, and LGBTQ people, to Trump’s own racist, xenophobic and misogynistic tweets. Yet the fact that an emotion-term like hate is being mobilized across these texts as a metonym for the “alt-right” is no oratorical curio. Rather, it perpetuates a pervasive way of thinking about the relationship between the alt-right (a political phenomenon) and hate (an emotional phenomenon) that should give pause to those of us committed to mining that vein of cultural symptomatology now consigned, across the social sciences and critical humanities, to affect theory. Specifically, these headlines inscribe, in miniature, a kind of micro-assessment, a micro-geography and micro-theory of hate. First, they suggest that, even prior to its incarnation in specific, and dangerous, forms of speech or action, hate is in and of itself anathema, a phenomenon so unquestioningly dangerous that a putative “rise” or “spike” in its net presence provides ample pretext for a news headline. Second, they propose that hate may be localized to a particular social or political group—a group subsisting, unsurprisingly, on that peculiarly contested frontier between the ideological alt-right and the American Midwest. And third, they imply that hate is so indubitably the single most significant source of the xenophobic, racist and sexist activities they go on to describe that it may be casually used as these activities’ lexical proxy. What is crystallizing here, I suggest, is what scholars of rhetoric dub a rhetorical “constellation” (Campbell and Jamieson 332)—a constellation from which hate emerges as, a) inherently problematic, b) localizable to the “alt-right,” and, c) the primary engine of the various activities and expressions we associate with them. This constellation of conventions for thinking about hate and its relationship to the activities of right-wing extremist movement has coalesced into a “genre” we might dub the genre of “hate-attribution.” Yet while it’s far from clear that the genre is an effective one in a political landscape that’s fast becoming a political battleground, it hasn’t appeared by chance. Treating “hate,” then, less as a descriptive “grid of analysis” (Sedgwick 152), than as a rhetorical projectile, this essay opens by interrogating the “hate-attribution” genre’s logic and querying its efficacy. Having done so, it approaches the concept of “alternatives” by asking: how might calling time on the genre help us think differently about both hate itself and about the forces catalyzing, and catalyzed by, Trump’s presidential campaign? II.The rhetorical power of the genre of hate-attribution, of course, isn’t too difficult to pin down. An emotion so thoroughly discredited that its assignment is now in and of itself a term of abuse (see, for example, the O.E.D’s freshly-expanded definition of the noun “hater”), hate is an emotion the Judeo-Christian tradition deems not just responsible for but practically akin to murder (John 3:1). In part as a result of this tradition, hate has proven thoroughly resistant to efforts to elevate it from the status of an expression of a subject’s pestiferous inner life to the status of a polemical response to an object in the world. Indeed, while a great deal of the critical energy amassing under the rubric of “affect theory” has recently been put into recuperating the strategic or diagnostic value of emotions long scorned as irrelevant to oppositional struggle—from irritation and envy, to depression, anger and shame (Ngai; Cvetkovich; Gould; Love)—hate has notably not been among them. In fact, those rare scholarly accounts of affect that do address “hate,” notably Ahmed’s excellent work on right-wing extremist groups in the United Kingdom, display an understandable reluctance to rehabilitate it for progressive thought (Cultural Politics). It should come as no surprise, then, that the genre of “hate-attribution” has a rare rhetorical power. In identifying “hate” as the source of a particular position, gesture or speech-act, we effectively drain said position, gesture or speech-act of political agency or representational power—reducing it from an at-least-potentially polemical action in or response to the world, to the histrionic expression of a reprehensible personhood. Yet because hate’s near-taboo status holds across the ideological and political spectrum, what is less clear is why the genre of hate-attribution has achieved such cachet in the liberal media in particular. The answer, I would argue, lies in the fact that the work of hate-attribution dovetails all too neatly with liberal political theory’s longstanding tendency to laminate its social and civic ideals to affective ideals like “love,” “sympathy,” “compassion,” and, when in a less demonstrative humor, “tolerance”. As Martha Nussbaum’s Political Emotions has recently shown, this tradition has an impressive philosophical pedigree, running from Aristotle’s philia (16), John Locke’s “toleration” and David Hume’s “sympathy” (69-75), to the twentieth century’s Universal Declaration of Human Rights, with its promotion of “tolerance and friendship among all nations, racial or religious groups.” And while the labour of what Lauren Berlant calls “liberal sentimentality” (“Poor Eliza”, 636) has never quite died away, it does seem to have found new strength with the emergence of the “intimate public sphere” (Berlant, Queen)—from its recent popular apotheosis in the Clinton campaign’s notorious “Love Trumps Hate” (a slogan in which “love,” unfortunately, came to look a lot like resigned technocratic quietism in the face of ongoing economic and environmental crisis [Zizek]), to its revival as a philosophical project among progressive scholars, many of them under the sway of the so-called “affective turn” (Nussbaum; Hardt; Sandoval; hooks). No surprise, then, that liberalism’s struggle to yoke itself to “love” should have as its eerie double a struggle to locate among its ideological and political enemies an increasingly reified “hate”. And while the examples of this project we’ve touched on so far have hailed from popular media, this set of protocols for thinking about hate and its relationship to the activities of right-wing extremist movements is not unique to media circles. It’s there in political discourse, as in ex-DNC chair Debbie Wasserman Schultz’s announcement, on MSNBC, that “Americans will unite against [Trump’s] hatred.” And it’s there, too, in academic media studies, from FLOW journal’s November 2016 call for papers inviting respondents to comment, among other things, on “the violence and hatred epitomized by Trump and his supporters,” to the SCMS conference’s invitation to members to participate in a pop-up panel entitled “Responding to Hate, Disenfranchisement and the Loss of the Commons.” Yet while the labor of hate-attribution to which many progressive forces have become attached carries an indisputable rhetorical force, it also has some profound rhetorical flaws. The very same stigma, after all, that makes “hate” such a powerful explanatory grenade to throw also makes it an incredibly tough one to land. As Ahmed’s analysis of the online rhetoric of white supremacist organizations should remind us (Cultural Politics), most groups structured around inciting and promoting violence against women and minorities identify, perversely, not as hate groups, but as movements propelled by the love of race and nation. And while left-wing pundits pronounce “hate” the signature emotion of a racist, misogynist Trump-voting right, supporters of Trump ascribe it, just as routinely, to the so-called “liberal elite,” a group whose mythical avatars—from the so-called “Social Justice Warrior” or “SJW,” to the supercilious Washington politico—are said to brand “ordinary [white, male] Americans” indiscriminately as racist, misogynistic, homophobic buffoons. Thus, for example, The Washington Post’s uncanny, far-right journalistic alter-ego, The Washington Times, dubs the SPLC a “liberal hate group”; the Wikipedia mirror-site, Conservapedia, recasts liberal objections to gun violence as “liberal hate speech” driven by an “irrational aversion to weapons”; while one blood-curdling sub-genre of reportage on Steve Bannon’s crypto-fascist soapbox, Breitbart News, is devoted to denouncing what it calls “ ‘anti-White Racism.’” It’s easy enough, of course, to defend the hate-attribution genre’s liberal incarnations while dismissing its right-wing variants as cynical, opportunistic shams, as Ahmed does (Cultural Politics)—thereby re-establishing the wellspring of hate where we are most comfortable locating it: among our political others. Yet to do so seems, in some sense, to perpetuate a familiar volley of hate-attribution. And to the extent that, as many media scholars have shown (Philips; Reed; Tett; Turow), our digital, networked political landscape is in danger of being reduced to a silo-ed discursive battleground, the ritual exchange of terminological grenades that everyone seems eager to propel across ideological lines, but that no one, understandably, seems willing to pick up, seems counter-productive to say the least.Even beyond the genre’s ultimate ineffectiveness, what should strike anyone used to reflecting on affect is how little justice it does to the ubiquity and intricacy of “hate” as an affective phenomenon. Hate is not and cannot be the exclusive property or preserve of one side of the political spectrum. One doesn’t have to stretch one’s critical faculties too far to see the extent to which the genre of hate-attribution participates in the emotional ballistics it condemns or seeks to redress. While trafficking in a relatively simple hate-paradigm (as a subjective emotional state that may be isolated to a particular person or group), the genre itself incarnates a more complex, socially dynamic model of hate in which the emotion operates through logics of projection perhaps best outlined by Freud. In the “hate-attribution” genre, that is, hate—like those equally abjected categories “sentimentality,” “worldliness” or “knowingness” broached by Sedgwick in her bravura analyses of “scapegoating attribution” (150-158)—finds its clearest expression in and through the labor of its own adscription. And it should come as no surprise that an emotion so widely devalued, where it is not openly prohibited, might also find expression in less overt form.Yet to say as much is by no means to discredit the genre. As legal scholar Jeremy Waldron has recently pointed out, there’s no particular reason why “the passions and emotions that lie behind a particular speech act” (34)—even up to and including hate—should devalue the speech acts they rouse. On the contrary, to pin the despicable and damaging activities of the so-called “alt right” on “hate” is, if anything, to do an injustice to a rich and complex emotion that can be as generative as it can be destructive. As Freud suggests in “Group Psychology and the Analysis of the Ego,” for example, hate may be the very seed of love, since the forms of “social feeling” (121) celebrated under the liberal rubric of “tolerance,” “love,” and “compassion,” are grounded in “the reversal of what was first a hostile feeling into a positively-toned tie in the nature of an identification” (121; italics mine). Indeed, Freud projects this same argument across a larger, historical canvas in Civilization and its Discontents, which contends that it is in our very struggle to combat our “aggressive instincts” that human communities have developed “methods intended to incite people into identifications and aim-inhibited relationships of love” (31). For Freud, that is, the practice of love is a function of ongoing efforts to see hate harnessed, commuted and transformed. III.What might it mean, then, to call time on this round of hate-attribution? What sort of “alternatives” might emerge when we abandon the assumption that political engagement entails a “struggle over who has the right to declare themselves as acting out of love” (Ahmed, Cultural Politics 131), and thus, by that same token, a struggle over the exact location and source of hate? One boon, I suggest, is the license it gives those of us on the progressive left to simply own our own hate. There’s little doubt that reframing the dangerous and destructive forms of speech fomented by Trump’s campaign, not as eruptions of hate, or even as “hate-speech,” but as speech we hate would be more consistent with what once seemed affect theory’s first commandment: to take our own affective temperature before launching headlong into critical analysis. After all, when Lauren Berlant (“Trump”) takes a stab at economist Paul Krugman’s cautions against “the Danger of Political Emotions” with the timely reminder that “all the messages are emotional,” the “messages” she’s pointing to aren’t just those of our political others, they’re ours; and the “emotions” she’s pointing to aren’t just the evacuated, insouciant versions of love championed by the Clinton campaign, they’re of the messier, or as Ngai might put it, “uglier” (2) variety—from shame, depression and anger, to, yes, I want to insist, hate.By way of jump-starting this program of hate-avowal, then, let me just say it: this essay was animated, in part, by a certain kind of hate. The social critic in me hates the breathtaking simplification of the complex social, economic and emotional forces animating Trump voters that seem to actuate some liberal commentary; the psychologist in me hates the self-mystification palpable in the left’s insistence on projecting and thus disowning its own (often very well justified) aggressions; and the human being in me, hating the kind of toxic speech to which Trump’s campaign has given rise, wishes to be able to openly declare that hatred. Among its other effects, hate is characterized by hypervigilance for lapses or failings in an object it deems problematic, a hypervigilance that—sometimes—animates analysis (Zeki and Romoya). In this sense, “hate” seems entitled to a comfortable place in the ranks of what Nick Salvato has recently dubbed criticism’s creative “obstructions”—phenomena that, while “routinely identified as detriments” to critical inquiry, may also “form the basis for … critical thinking” (1).Yet while one boon associated with this disclosure might be a welcome intellectual honesty, a more significant boon, I’d argue, is what getting this disclosure out of the way might leave room for. Opting out of the game of hurling “hate” back and forth across a super-charged political arena, that is, we might devote our column inches and Facebook posts to the less sensational but more productive task of systematically challenging the specious claims, and documenting the damaging effects, of a species of utterance (Butler; Matsuda; Waldron) we’ve grown used to simply descrying as pure, distilled “hate”. And we also might do something else. Relieved of the confident conviction that we can track “Trumpism” to a spontaneous outbreak of a single, localizable emotion, we might be able to offer a fuller account of the economic, social, political and affective forces that energize it. Certainly, hate plays a part here—although the process by which, as Isabelle Stengers puts it, affect “make[s] present, vivid and mattering … a worldly world” (371) demands that we scrutinize that hate as a syndrome, rather than simply moralize it as a sin, addressing its mainsprings in a moment marked by the nerve-fraying and life-fraying effects of what has become known across the social sciences and critical humanities as conditions of social and economic “precarity” (Muehlebach; Neil and Rossiter; Stewart).But perhaps hate’s not the only emotion tucked away under the hood. Here’s something affect theory knows today: affect moves not, as more traditional theorists of political emotion have it, “unambiguously and predictably from one’s cognitive processing,” but in ways that are messy, muddled and indirect (Gould 24). That form of speech is speech we hate. But it may not be “hate speech.” That crime is a crime we hate. But it may not be a “hate-crime.” One of the critical tactics we might crib from Berlant’s work in Cruel Optimism is that of decoding and decrypting, in even the most hateful acts, an instance of what Berlant, herself optimistically, calls “optimism.” For Berlant, after all, optimism is very often cruel, attaching itself, as it seems to have done in 2016, to scenes, objects and people that, while ultimately destined to “imped[e] the aim that brought [it to them] initially,” nevertheless came to seem, to a good portion of the electorate, the only available exponent of that classic good-life genre, “the change that’s gonna come” (“Trump” 1-2) at a moment when the Democratic party’s primary campaign promise was more of the free-market same. 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