Journal articles on the topic '2Hybrid FRET sample data'

За последние десятилетия концентрация парниковых газов в атмосфере растет беспрецедентными темпами вследствие использования ископаемого топлива и возрастающих темпов сведения лесов. Благодаря своей способности производить большое количество биомассы за короткий промежуток времени ольха серая может рассматриваться как перспективная древесная порода для ведения лесного хозяйства с коротким периодом выращивания в восточных и северных регионах Европы. Ольховые насаждения выполняют почвозащитные, почвоулучшающие, мелиоративные функции, в значительной мере регулируют стоки, предотвращают образование снежных лавин и селевых потоков. Ольха серая обладает рядом существенных лесоводственных преимуществ. Целью наших исследований было получение фактических данных о структуре биомассы ольхи серой, произрастающей в условиях северной тайги Архангельской области, разработка моделей возрастной динамики ее фракций на уровне древостоев и сравнительный анализ полученных результатов с данными о продуктивности ольхи серой в других регионах. По данным перечета деревьев всех пород на 40 пробных площадях по ступеням толщины рассчитаны основные таксационные показатели древостоев в возрасте от 20 до 77 лет, а также запасы биомассы на 1 га с использованием опубликованных аллометрических моделей. Построенные модели таксационных показателей и фракций биомассы, связанные между собой по рекурсивному принципу, адекватны фактическим данным на уровне вероятности p < 0,001. Сопоставление средних значений таксационных показателей и биомассы на 1 га сероольшаников северной и южной тайги показало, что при больших значениях среднего диаметра ствола и средней высоты древостои южной тайги имеют соответственно и большие запасы, чем в северной тайге. Но вследствие специфики структуры массы крон и корней ольшаники южной тайги характеризуются меньшими значениями как надземной, так и общей биомассы. Предложенные модели и таблицы могут быть полезны при оценке углероддепонирующей способности сероольшаников северной и южной тайги. Over the past decades, the concentration of greenhouse gases in the atmosphere has been growing at an unprecedented rate due to the use of fossil fuels and the increasing rate of deforestation. Due to its ability to produce a large amount of biomass in a short period of time, gray alder can be considered as a promising tree species for forestry with a short growing period in the eastern and northern regions of Europe. Alder plantations perform soil-protective, soil-improving, reclamation functions, largely regulate runoff, prevent the formation of snow avalanches and mudfl ows. Gray alder has a number of signifi cant forestry advantages. The purpose of our research was to obtain the data on the biomass structure of gray alder growing in the conditions of the northern taiga of the Arkhangelsk region, to develop models of the age dynamics of its fractions at the level of stands and a comparative analysis of the results obtained with data on the productivity of gray alder in other regions. According to the measurement data of trees of all species on 40 sample plots, the main taxation indicators of stands aged from 20 to 77 years, as well as biomass per 1 ha using published allometric models, were calculated using stem diameter distributions. The models of taxation indicators and biomass fractions, related according to the recursive principle adequate to the actual data at the probability level p < 0.001 are designed. A comparison of the average values of taxation indicators and biomass per 1 ha of gray alder stands of the northern and southern taiga showed that with large values of the average stem diameter and average height, stands of the southern taiga have, respectively, large stem volumes relative to the northern taiga. But due to the specifi c structure of the crown and root masses, the alders of the southern taiga are characterized by lower values of both aboveground and total biomass. The proposed models and tables can be useful in assessing the carbon-depositing capacity of gray alder in the northern and southern taiga subzones.

В статье представлены результаты изучения ресурсов дикорастущих плодовых растений подлеска сосновых и берёзовых насаждений южно-уральской таёжной зоны Свердловской области. В качестве основного показателя характеристики запасов было выбрано количество экземпляров на гектаре (густота). Кроме того, определялась текущая биологическая урожайность плодов в год наблюдения. Данные были получены на 28 пробных площадях в пяти наиболее распространённых в районе исследования типах леса. Для размещения пробных площадей подбирались наиболее типичные для района исследования насаждения различного возраста, происхождения, состава древостоя, относительной полноты и других таксационных показателей. Плодовые растения подлеска представлены 8 видами. Это рябина обыкновенная Sorbus aucupatia L., малина обыкновенная Rubus idaeus L., черёмуха обыкновенная Pronus padus L., облепиха крушиновидная Hippophae rhamnoides L., яблоня Malus P. Mill, боярышник кроваво-красный Crataegus sanguinea Pall. и ирга овальная Melanchier rotundifolia Dum. Cours. Лесопокрытые площади района исследования характеризуются низкими запасами плодов дикорастущих растений подлеска. Они непригодны для организации промышленного сбора. Совокупная урожайность плодовых растений подлеска всех видов на заложенных ПП не превышает 36,0 кг/га. The article presents results of studying of resources of wild fruit plants of undergrowth of pine and birch plantations of the south ural taiga zone of the Sverdlovsk region. The number of specimens per hectare (density) was chosen as the main indicator of stock characteristics. In addition, the current biological yield of fruits in the year of observation was determined. Data were obtained on 28 test areas inve most common types of forest in area of study. To place sample areas, most typical plantings of various age, origin, composition of the stand, relative completeness and other taxation indicators were selected for study area. The fruit plants of understory are represented by 8 species: mountain ash Sorbus aucupatia L., raspberry Rubus idaeus L., bird cherry Pronus padus L., sea buckthorn buckthorn Hippophae rhamnoides L., apple Malus P. Mill, hawthorn Crataegus sanguinea Pall. and irga oval Melanchier rotundifolia Dum.Cours. The forested areas of the research area are characterized by low stocks of fruits of wild plants of the understory. They are not suitable for the organization of industrial collection. The total yield of fruit plants of undergrowth of all types on the laid PP does not exceed 36.0 kg/ha.

Copper is essential for life, and beyond its well-established ability to serve as a tightly bound, redox-active active site cofactor for enzyme function, emerging data suggest that cellular copper also exists in labile pools, defined as loosely bound to low-molecular-weight ligands, which can regulate diverse transition metal signaling processes spanning neural communication and olfaction, lipolysis, rest–activity cycles, and kinase pathways critical for oncogenic signaling. To help decipher this growing biology, we report a first-generation ratiometric fluorescence resonance energy transfer (FRET) copper probe, FCP-1, for activity-based sensing of labile Cu(I) pools in live cells. FCP-1 links fluorescein and rhodamine dyes through a Tris[(2-pyridyl)methyl]amine bridge. Bioinspired Cu(I)-induced oxidative cleavage decreases FRET between fluorescein donor and rhodamine acceptor. FCP-1 responds to Cu(I) with high metal selectivity and oxidation-state specificity and facilitates ratiometric measurements that minimize potential interferences arising from variations in sample thickness, dye concentration, and light intensity. FCP-1 enables imaging of dynamic changes in labile Cu(I) pools in live cells in response to copper supplementation/depletion, differential expression of the copper importer CTR1, and redox stress induced by manipulating intracellular glutathione levels and reduced/oxidized glutathione (GSH/GSSG) ratios. FCP-1 imaging reveals a labile Cu(I) deficiency induced by oncogene-driven cellular transformation that promotes fluctuations in glutathione metabolism, where lower GSH/GSSG ratios decrease labile Cu(I) availability without affecting total copper levels. By connecting copper dysregulation and glutathione stress in cancer, this work provides a valuable starting point to study broader cross-talk between metal and redox pathways in health and disease with activity-based probes.

SummaryRecent data from animal models indicate that the eNOS null mice present a phenotype that resemble the human metabolic syndrome (hypertension, insulin resistance and hypertriglyceridemia). In this work, we have studied whether NOS3 gene, previously related to endothelial dysfunction, might have a role in metabolic syndrome susceptibility in hypertensive patients. To carry out the study, we genotyped 105 hypertensive patients ≤ 60 years old with two polymorphisms of NOS3 gene: 1132 T>C and 7164 G>T (GeneBank:AF519768.1).To check the allelic frequency of these polymorphisms in our geographical area, we also genotyped 94 unselected healthy controls (control group). To perform sample genotyping, we designed a novel FRET system coupled to real time PCR. There were no differences in genotypic distribution or allelic frequency between hypertensive patients and the control group. However, we observed that 786CC genotype was significantly more frequent in hypertensive patients with metabolic syndrome than in those without the syndrome (p=0.0022). When both polymorphisms were analyzed, we identified the 786C894G as the risk haplotype for metabolic syndrome susceptibility (p=0.011). These data suggest a role of the NOS3 gene in the pathogenesis of metabolic syndrome in hypertensive patients.

Abstract INTRODUCTION: Many cases of idiopathic thrombotic thrombocytopenic purpura (TTP) are characterized by very low levels of the metalloprotease ADAMTS13, presumably resulting in dysregulation of platelet/vWF interactions and microvascular thrombosis. Treatment for TTP begins with therapeutic plasma exchange (TPE), allowing replacement of ADAMTS13 and removal of autoantibody, when present. Fresh frozen plasma (FFP) and plasma cryoprecipitate reduced (also known as cryoprecipitate poor plasma [CPP]) have been used as replacement fluids. While current AABB standards allow thawed FFP and CPP components to be stored at 1–6°C for up to 5 days before use, the amount of ADAMTS13 activity in these products has not previously been measured nor has its stability been evaluated over the 5 days. Given the labor-intensive nature of our standard collagen-binding assay for ADAMTS13 activity, we chose to analyze ADAMTS13 activity in FFP and CPP components using a modified FRETS-VWF73 assay recently developed in our laboratory. Preliminary data indicate that the two assays correlate well. METHODS: Ten whole blood (WB) units (5 group O and 5 group A) were centrifuged within 8 hours of collection. Prior to freezing of the FFP, an aliquot of plasma was obtained from each component. The aliquots and FFP components were frozen at <-18°C. Following AABB guidelines, each FFP unit was processed into cryoprecipitated AHF and CPP components. The FFP aliquots and their related CPP components were stored frozen at <-18°C for 46 days and then thawed per AABB standards. Once thawed, aliquots and components were placed in a 1–6°C monitored refrigerator for 30 minutes. Each FFP aliquot (approximately 6 ml) was divided in half; one tube (day 0) was frozen at <-70°C, while the other was stored at 1–6°C until expiration of the product (day 5) and then frozen at <-70°C. Samples were sterilely collected from each CPP unit (mean volume 235 ml) 30 minutes after thaw (day 0), and then every 24 hours until expiration (days 1–5). Immediately after sampling, all CPP samples were stored at <-70°C until assayed. Day 0 and Day 5 FFP and CPP samples derived from the same WB units were analyzed for ADAMTS13 activity using a modified VWF73 FRET-labeled peptide assay (Kokame, 2005). RESULTS: Using the 2-sample t-test assuming equal variances, no statistical difference in ADAMTS13 activity was observed between the FFP and CPP products, nor was there a statistical difference observed between day 0 and day 5 samples (Table). Mean levels of ADAMTS13 activity were statistically similar to 20 normal donor (Precision BioLogic) samples (127 ±27 v. 125 ±24, p=NS). However, the mean activity level in group O units was statistically higher when compared to group A units (149 ±13 v. 104 ±16, p <0.00001). ADAMTS13 Activity* Plasma Type (n) Day 0 Day 5 p *Mean ± SD Total FFP (10) 127 ± 24 122 ± 28 NS FFP A (5) 106 ± 12 108 ± 23 NS FFP O (5) 148 ± 14 144 ± 22 NS Total CPP (5) 126 ± 29 123 ± 25 NS CPP A (5) 102 ± 19 102 ± 16 NS CPP O (5) 151 ± 12 145 ± 11 NS CONCLUSION: Our data indicate that both FFP and CPP products have similar ADAMTS13 activity, suggesting that both products should be equally effective in restoring ADAMTS13 activity in TTP patients. The data also indicate that ADAMTS13 is stable at 1–6°C, showing consistent efficacy of thawed FFP and CPP products for ADAMTS13 replacement throughout the refrigerated shelf life of the thawed product. These results reinforce previously reported data that there is a difference in ADAMTS13 activity between blood group O and A individuals.

This file contains all images and preprocessed data tables from both instruments (confocal and imaging plate reader). These raw and pre-processed data sets correspond to the presented binding curves and thus contain images and data tables of the entire reference system provided.

Abstract Förster Resonance Energy Transfer combined with Fluorescence Lifetime Imaging Microscopy (FRET-FLIM) is revolutionizing plant biology, by enabling the study of protein–protein interactions (PPIs) within live cells. This manuscript describes the principles of FRET and the practical application of FRET acceptor photobleaching (FRET-APB) in exploring PPIs in vivo. It mainly focuses on the superior characteristics of FRET-FLIM and details the materials and methods for implementing this technique in plants. It provides a profound overview about the required instruments, protocols for sample preparation, methods for calibration and acquisition, and pipelines for data analyses including novel analyses for binding and FRET efficiencies. Furthermore, it discusses the potential pitfalls and challenges related to the sample autofluorescence, protein expression heterogeneity, and acquisition photodamage or bleaching. This works aims to highlight the great prospects of FRET-FLIM in advancing our understanding of PPIs in living plant cells.

Abstract Fluorescence lifetime imaging microscopy (FLIM) combined with Förster resonance energy transfer (FRET) is a powerful tool for studying conformational changes of biosensors, protein-protein interactions and the dynamics of molecular processes in living cells. This approach enables measurements of molecular distances at the nanometer scale, with high spatial and temporal resolution. FLIM-FRET relies only on donor and acceptor lifetime changes induced by energy transfer thus avoiding possible artifacts of pure intensity-based FRET measurements. This tutorial provides a step-by-step workflow of widefield FLIM measurements of immune competent cells, including instrument setup, sample preparation, data acquisition and analysis. Practical guidelines are offered to optimize experimental conditions and troubleshoot some common issues. A case study highlights the application of FLIM-FRET to investigate protein conformational changes during T-cell receptor activation, demonstrating the technique’s sensitivity and versatility in the context of cellular signaling.

In multicellular organisms, metabolic coordination across multiple tissues and cell types is essential to satisfy regionalized energetic requirements and respond coherently to changing environmental conditions. However, most metabolic assays require the destruction of the biological sample, with a concomitant loss of spatial information. Fluorescent metabolic sensors and probes are among the most user-friendly techniques for collecting metabolic information with spatial resolution. In a previous work, we have adapted to an animal system, Drosophila melanogaster, genetically encoded metabolic FRET-based sensors that had been previously developed in single-cell systems. These sensors provide semi-quantitative data on the stationary concentrations of key metabolites of the bioenergetic metabolism: lactate, pyruvate, and 2-oxoglutarate. The use of these sensors in intact organs required the development of an image processing method that minimizes the contribution of spatially complex autofluorescence patterns, that would obscure the FRET signals. In this article, we show step by step how to design FRET-based sensor experiments and how to process the fluorescence signal to obtain reliable FRET values.

In order to study the spatial distribution of Na, K-ATPase alpha and beta subunits in the plasma membrane of epithelial cells we have developed sample preparation protocols based on genetic code expansion, click chemistry and expansion microscopy. HEK 293T cells were transiently transfected with constructs containing the coding sequence for Na, K-ATPase subunits (alpha1 and beta1) carrying a premature amber or ochre stop codon. Two bioorthogonal tRNA and tRNA synthetase pairs were also transfected for stop codon suppression and site-specific insertion of two unnatural amino acids. The unnatural amino acids were then labelled with small organic dyes using CuAAC and SPIEDAC click reactions. Care was taken to keep the transfection and expression levels low.In living cells, we used Fluorescence Correlation Spectroscopy (FCS) and FRET to assess the density of click-labeled and endogenous [SE1] Na, K-ATPase in the plasma membrane. The dyes Alexa488 and Alexa647 were used as a FRET sensor for homo- and heterodimer analysis and, using FRET-FCS, we could detect the oligomerization of alpha and beta in a complex containing two alpha and two beta subunits in a heterodimer.FCS measurements in cells where both the alpha and beta subunits were labeled was used to calculate the absolute density of endogenous and exogenous Na, K-ATPase in the plasma membrane. To detect oligomerization, we used cells where the beta subunit was labeled with a 50/50 mix of Alexa488 and Alexa647. We successfully detected FRET signals and an analysis of the FRET-FCS data revealed a high density of oligomers. FRET-FCS is, compared to conventional cross-correlation FCCS, one to two orders of magnitude more sensitive for the detection of oligomers. FRET-FCS is also inherently insensitive to heterogenous labeling efficiencies, which is a great advantage during live cell measurementsExpansion microscopy was performed using a modified MAP protocol, reaching an isotropic expansion of approximately 4.5-fold. Confocal airyscan microscopy of the expanded cells with <50nm resolution revealed local variations in Na, K-ATPase density and enabled detailed cluster analysis.In conclusion we demonstrate that the density of Na, K-ATPase has large local variations and that the Na, K-ATPase oligomerizes in the plasma membrane. We propose that this oligomerization can have a regulatory function for the net efficiency of Na, K-ATPase in the cell. VR 2020-05347, VR 2019-00217 (Swedish Research Council) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Intravital microscopy can provide unique insights into the function of biological processes in a native context. However, physiological motion caused by peristalsis, respiration and the heartbeat can present a significant challenge, particularly for functional readouts such as fluorescence lifetime imaging (FLIM), which require longer acquisition times to obtain a quantitative readout. Here, we present and benchmark Galene, a versatile multi-platform software tool for image-based correction of sample motion blurring in both time resolved and conventional laser scanning fluorescence microscopy data in two and three dimensions. We show that Galene is able to resolve intravital FLIM-FRET images of intra-abdominal organs in murine models and NADH autofluorescence of human dermal tissue imaging subject to a wide range of physiological motions. Thus, Galene can enable FLIM imaging in situations where a stable imaging platform is not always possible and rescue previously discarded quantitative imaging data.

Abstract Study question Is there a difference in endometrial immune cell expression at the time of embryo transfer in fresh vs. frozen transfer cycles? Summary answer Our study is the first to compare the endometrial immune cell profile at the time of the embryo transfer between fresh and frozen cycles. What is known already Endometrial receptivity plays a crucial role in implantation success for both natural conception and assisted reproductive technology cycles; however, limited information is available about the endometrial environment during the actual implantation window. Although some data suggest a difference in pregnancy rates between fresh and frozen embryo transfers, the underlying mechanisms remain unclear. This study aims to address this gap by analyzing the endometrial milieu using an innovative approach that examines immune cells collected at the time of embryo transfer in both fresh and frozen transfer cycles. Study design, size, duration This study was approved by Institutional Review Board (# 49174). The study was designed as a prospective observational cohort study at a single academic fertility center. Fifty-two participants with embryos available for transfer were recruited, from which 42 underwent frozen embryo transfer (FET) and 10 underwent fresh embryo transfer (FrET). IVF protocols for FrET consisted of antagonist protocols with gonadotropin dose adjusted based on ovarian reserve, while FET cycles used hormone replacement protocols. Participants/materials, setting, methods All embryo transfers were performed under transabdominal ultrasound guidance. Upon completion of transfer and catheter verification, the transfer catheter tip was rinsed in IMDM medium containing 10% FBS. After centrifugation, pelleted cells were stained for the following surface markers: CD45, CD3, CD19, CD4, CD8, gamma delta TCR, CD25, CD127, CD56, CD14, and CD66b. The samples were acquired on Sony SP6800 Spectral Analyzer. Mann-Whitney U test was used to test statistical differences between groups. Main results and the role of chance A statistically significant difference in the immune milieu was observed in patients undergoing FET compared to FrET cycles. In patients that received a FrET, there was a significantly higher expression of total immune cells (CD45+), 20.50%, compared to FET cycles, 6.60%, within all live cells (p = 0.0008). In addition, FrET cycles had an increase in the endometrial expression of total T cells (CD3+), 17.75%, compared to 2.70% T cells in the endometrium from patients undergoing FET cycles (p = 0.0051). Furthermore, patients undergoing FET cycles, had significantly greater endometrial expression of CD8+ T cells compared to FrET cycles, 20.5% vs. 2.30%, respectively (p = 0.0058). Finally, FET cycles were noted to have increased endometrial expression of B cells (CD19+) and NK cells (CD56+), while the endometrium for patients undergoing FrET cycles demonstrated greater GDT+ and T regulatory cells (CD4+CD25+CD127-), these differences did not reach statistical significance. There were similar endometrial expressions of T cells (CD4+), macrophages (CD14+), and neutrophils (CD66b+) among both types of cycles. Limitations, reasons for caution Limitations of our study include the small sample size undergoing FrET. In addition, we are planning to compare the immune cell profiles between the two groups in relation to cycle outcomes. Furthermore, multicolor flow cytometry may provide additional immune cell composition of the immune milieu during the window of implantation. Wider implications of the findings The endometrial milieu in general, and the immune environment in particular, differ between fresh and frozen cycles, and this could play a critical role in implantation. Identifying cycle specific immune cell profiles may reveal factors that optimize implantation, paving the way for diagnostic and therapeutic innovations to improve IVF outcomes. Trial registration number No

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current ‘state of the art’ from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of ‘soft recommendations’ about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage ‘open science’ practices.

Abstract Encapsulation of enhanced green fluorescent protein (EGFP) in complex coacervate core micelles (C3Ms) can be established by mixing EGFP with diblock polymers at equal charge ratio. It has previously been shown that this encapsulation system is highly dynamic, implying existence of different populations; GFP free in solution or complexed with polymers (small complexes) and EGFP encapsulated in C3Ms. We performed time resolved fluorescence anisotropy experiments to determine the relative populations of EGFP encapsulated in C3Ms using three different fluorescence anisotropy decay analysis methods. First, Maximum Entropy Method (MEM) data analysis was employed for five different EGFP concentrations in C3Ms that were mixed with dark fluorescent proteins (10, 20, 30, 40 and 50% EGFP, respectively). In all cases, correlation-time distributions between 0.1 and 100 ns (on a logarithmic timescale) are clearly visible showing bimodal distribution. The distribution between 0.1 and 2.0 ns is due to homo-FRET between EGFP molecules packed in micelles and the distribution between 8 and 30 ns coincides with the correlation-time distribution of free EGFP in solution. The fraction of homo-FRET distribution linearly increases with increase of relative micellar EGFP concentrations. These MEM results were corroborated by two different analysis methods: global population analysis of all five fluorescence anisotropy decays arising from EGFP in micelles together with the one of free EGFP (direct analysis of anisotropies) and global associative population analysis of anisotropies by fitting parallel and perpendicular fluorescence decay components. In contrast to global analyses approaches, the MEM method directly reveals distributions of correlation times without any prior information about the sample. However, global associative analysis of anisotropies by fitting parallel and perpendicular fluorescence decay components is the only method that allows to estimate accurately fractions of free fluorophores in solution and encapsulated fluorophores.

Abstract:Our highly perceived form of word is the distance that reads these small traders fret. If retail business grows year by year in the absence of prevailing regulations then it is with conventional market which will also reduce revenue from the conventional market itself, because the adjacent distance is certainly cheaper than traditional market. The purpose of this research is to know and analyze comparison of consumer perception about store atmosphere in modern retail (Indomaret) and conventional retail (Toko Dedy). The data used is primary data. This research method using purposive sampling, with the terms and conditions applicable. Samples studied were 91 respondents. Number of variables studied is 1 ie store atmosphere with 4 indicators. In this research using Paired Sample T-Test analysis by comparing the average value in modern retail (Indomaret) and conventional retail (Toko Dedy). From the results of the study stated that the first hypothesis statement in this study which states "that there are significant differences in consumer perceptions of store atmosphere in modern retail and conventional retail" received. This is reinforced by the result of Paired Sample T-Test obtained by tcount equal to 37,919 which is bigger than ttable that is 1,9867 with probability (Sig.) 0.000 which is also smaller than 0,05. Then a statement from the second hypothesis stating that "the difference in consumer perception of store atmosphere in modern retail is more dominant than conventional retail" is accepted. It is proved by comparison result of mean value of Paired Sample Statistics obtained by mean value in modern retail (indomaret) equal to 58,5275 while mean value in conventional retail (Toko Dedy) is smaller that is equal to 29,7692. Keywords: Store Atmosphere, Modern Retail, Conventional Retail.

The common severe Z mutation (E342K) of α1-antitrypsin forms intracellular polymers that are associated with liver cirrhosis. The native fold of this protein is well-established and models have been proposed from crystallographic and biophysical data for the stable inter-molecular configuration that terminates the polymerization pathway. Despite these molecular ‘snapshots’, the details of the transition between monomer and polymer remain only partially understood. We surveyed the RCL (reactive centre loop) of α1-antitrypsin to identify sites important for progression, through intermediate states, to polymer. Mutations at P14P12 and P4, but not P10P8 or P2P1′, resulted in a decrease in detectable polymer in a cell model that recapitulates the intracellular polymerization of the Z variant, consistent with polymerization from a near-native conformation. We have developed a FRET (Förster resonance energy transfer)-based assay to monitor polymerization in small sample volumes. An in vitro assessment revealed the position-specific effects on the unimolecular and multimolecular phases of polymerization: the P14P12 region self-inserts early during activation, while the interaction between P6P4 and β-sheet A presents a kinetic barrier late in the polymerization pathway. Correspondingly, mutations at P6P4, but not P14P12, yield an increase in the overall apparent activation energy of association from ~360 to 550 kJ mol−1.