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1

EL, IYSAOUY LAHCEN, Mhammed LAHBABI, and Abdelmajid OUMNAD. "Enhancing the Performances of PV Array Configurations Under Partially Shaded Conditions: A Comparative Study." International Journal of Renewable Energy Research 8, no. 3 (2018): 1779–90. https://doi.org/10.5281/zenodo.2639399.

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In this paper, we propose to modelize and analyze the performance of some existing solar photovoltaic (PV) array configurations such as Series Parallel (SP), Bridge Link (BL), Honey Comb (HC), Total Cross Tied (TCT) and proposed configuration Sudoku.  Simulations are based on complex model two diode of solar cell using Matlab and Simulink tools. The PV configurations performance of all these configurations have been compared using P–V characteristics, power loss for different shading patterns. Comprehensive simulations are carried out on these configurations. The proposed configuration, Sudoku, performance have found surpass the other PV configurations for the same shading patterns.
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2

Casamichana, David, Paul S. Bradley, and Julen Castellano. "Influence of the Varied Pitch Shape on Soccer Players Physiological Responses and Time-Motion Characteristics During Small-Sided Games." Journal of Human Kinetics 64, no. 1 (2018): 171–80. http://dx.doi.org/10.1515/hukin-2017-0192.

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Abstract The aim of this study was to investigate the effect of pitch shape modifications on heart rate responses and time-motion characteristics in soccer players during 5-a-side small-sided games (SSGs). Players completed four different SSG dimensions: (1) short narrow pitch (SN; 40 × 25 m), (2) short wide pitch (SW; 66 × 25 m), (3) long narrow pitch (LN; 40 × 50 m), and (4) long wide pitch (LW; 66 × 50 m). Twenty amateur soccer players (age: 21 ± 5 yr; stature: 176.8 ± 1.9 cm; body mass: 72.7 ± 3.7 kg) were monitored using a heart rate monitor and a 10 Hz GPS device. Mean maximum heart rate (%HRmax), rating of perceived exertion (RPE), peak running speed, total distance covered (TD), distance covered in four speed categories, number of moderate and high accelerations (Ac), decelerations (Dc), changes of direction (COD) and player load were recorded. Increasing the pitch length had a greater effect compared to increasing the pitch width especially on RPE (3.8, 6.3, 4.9 and 6.6 AU to SN, LN, SW and LW, respectively) and time-motion characteristics such as TD (101, 127, 108 and 131 m·min-1 to SN, LN, SW and LW, respectively), peak speed (4.8, 6.1, 5.2 and 6.2 m·s-1 to SN, LN, SW and LW, respectively), and the number of accelerations, decelerations, and changes of direction. The data demonstrates that increasing the length rather than the width of 5-a-side SSG has a greater impact on players’ responses in terms of increasing workloads.
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3

Delmore, Kira E., Benjamin M. Van Doren, Greg J. Conway, et al. "Individual variability and versatility in an eco-evolutionary model of avian migration." Proceedings of the Royal Society B: Biological Sciences 287, no. 1938 (2020): 20201339. http://dx.doi.org/10.1098/rspb.2020.1339.

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Seasonal migration is a complex and variable behaviour with the potential to promote reproductive isolation. In Eurasian blackcaps ( Sylvia atricapilla ), a migratory divide in central Europe separating populations with southwest (SW) and southeast (SE) autumn routes may facilitate isolation, and individuals using new wintering areas in Britain show divergence from Mediterranean winterers. We tracked 100 blackcaps in the wild to characterize these strategies. Blackcaps to the west and east of the divide used predominantly SW and SE directions, respectively, but close to the contact zone many individuals took intermediate (S) routes. At 14.0° E, we documented a sharp transition from SW to SE migratory directions across only 27 (10–86) km, implying a strong selection gradient across the divide. Blackcaps wintering in Britain took northwesterly migration routes from continental European breeding grounds. They originated from a surprisingly extensive area, spanning 2000 km of the breeding range. British winterers bred in sympatry with SW-bound migrants but arrived 9.8 days earlier on the breeding grounds, suggesting some potential for assortative mating by timing. Overall, our data reveal complex variation in songbird migration and suggest that selection can maintain variation in migration direction across short distances while enabling the spread of a novel strategy across a wide range.
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Verónica, Romero, Francisco Ruiz, María Luz González-Regalado, et al. "Messinian ostracodes from the western Betic Strait (SW Spain)." Carnets de géologie (Notebooks on geology) 21, no. 8 (2021): 181–92. http://dx.doi.org/10.2110/carnets.2021.2108.

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During the Neogene, the Betic Strait was one of the gateways that connected the Atlantic Ocean and the Mediterranean Sea. In this paper, we have analyzed the ostracod faunas of samples collected from sediments crossed by a long borehole in southwestern Spain. These sediments were deposited in the Betic strait just before the Messinian Salinity Crisis. During the middle Messinian (6.8-6.0 Ma), the scarce and low diversified ostracod assemblages (Krithe, Parakrithe, Henryhowella) are typical of upper bathyal palaeoenvironments (200-400 m water depth). This period includes a short transition (6.26-6.25 Ma) to outer neritic palaeoenvironments, coinciding with a glaciation and characterized by the presence of Acanthocythereis hystrix (Reuss, 1850) and the disappearance of Krithe and Parakrithe. The most abundant species have a wide biostratigraphic distribution, most of them ranging from the Tortonian until the Holocene.
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Bracchi, Valentina Alice, Giulia Piazza, and Daniela Basso. "A stable ultrastructural pattern despite variable cell size in <i>Lithothamnion corallioides</i>." Biogeosciences 18, no. 22 (2021): 6061–76. http://dx.doi.org/10.5194/bg-18-6061-2021.

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Abstract. Recent advances on the mechanism and pattern of calcification in coralline algae led to contradictory conclusions. The evidence of a biologically controlled calcification process, resulting in distinctive patterns at the scale of family, was observed. However, the coralline calcification process has been also interpreted as biologically induced because of the dependency of its elemental composition on environmental variables. To clarify the matter, five collections of Lithothamnion corallioides from the Atlantic Ocean and the Mediterranean Sea, across a wide depth range (12–66 m), have been analyzed for morphology, anatomy and cell wall crystal patterns in both perithallial and epithallial cells to detect possible ultrastructural changes. L. corallioides shows the alternation of tiers of short-squared and long-ovoid/rectangular cells along the perithallus, forming a typical banding. The perithallial cell length decreases according to water depth and growth rate, whereas the diameter remains constant. Our observations confirm that both epithallial and perithallial cells show primary (PW) and secondary (SW) calcite walls. Rectangular tiles, with the long axis parallel to the cell membrane forming a multi-layered structure, characterize the PW. Flattened squared bricks characterize the SW, with roundish outlines enveloping the cell and showing a zigzag and cross orientation. Long and short cells have different thicknesses of PW and SW, increasing in short cells. Epithallial cells are one to three flared cells with the same shape of the PW and SW crystals. Despite the diverse seafloor environments and the variable L. corallioides growth rate, the cell walls maintain a consistent ultrastructural pattern with unaffected crystal shape and arrangement. A comparison with two congeneric species, L. minervae and L. valens, showed similar ultrastructural patterns in the SW but evident differences in the PW crystal shape. Our observations point to a biologically control rather than an induction of the calcification process in coralline algae and suggest a possible new morphological diagnostic tool for species identification, with relevant importance for paleontological applications. Finally, secondary calcite, in the form of dogtooth crystals that fill the cell lumen, has been observed. It represents a form of early alteration in living collections which can have implications in the reliability of climate and paleoclimate studies based on geochemical techniques.
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6

Qin, Lei, Pieter-Ewald Share, Hongrui Qiu, Amir A. Allam, Frank L. Vernon, and Yehuda Ben-Zion. "Internal structure of the San Jacinto fault zone at the Ramona Reservation, north of Anza, California, from dense array seismic data." Geophysical Journal International 224, no. 2 (2020): 1225–41. http://dx.doi.org/10.1093/gji/ggaa482.

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SUMMARY We image the internal structure of the San Jacinto fault zone (SJFZ) near Anza, California, with seismic data recorded by two dense arrays (RA and RR) from ∼42 000 local and ∼180 teleseismic events occurring between 2012 and 2017. The RA linear array has short aperture (∼470 m long with 12 strong motion sensors) and recorded for the entire analysed time window, whereas the RR is a large three-component nodal array (97 geophones across a ∼2.4 km × 1.4 km area) that operated for about a month in September–October 2016. The SJFZ at the site contains three near-parallel surface traces F1, F2 and F3 from SW to NE that have accommodated several Mw > 6 earthquakes in the past 15 000 yr. Waveform changes in the fault normal direction indicate structural discontinuities that are consistent with the three fault surface traces. Relative slowness from local events and delay time analysis of teleseismic arrivals in the fault normal direction suggest a slower SW side than the NE with a core damage zone between F1 and F2. This core damage zone causes ∼0.05 s delay at stations RR26–31 in the teleseismic P arrivals compared with the SW-most station, and generates both P- and S-type fault zone trapped waves. Inversion of S trapped waves indicates the core damaged structure is ∼100 m wide, ∼4 km deep with a Q value of ∼20 and 40 per cent S-wave velocity reduction compared with bounding rocks. Fault zone head waves observed at stations SW of F3 indicate a local bimaterial interface that separates the locally faster NE block from the broad damage zone in the SW at shallow depth and merges with a deep interface that separates the regionally faster NE block from rocks to the SW with slower velocities at greater depth. The multiscale structural components observed at the site are related to the geological and earthquake rupture history at the site, and provide important information on the preferred NW propagation of earthquake ruptures on the San Jacinto fault.
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7

Taylor, Brian. "Reappraisal of the Continental Rifting and Seafloor Spreading That Formed the South China Sea." Geosciences 15, no. 4 (2025): 152. https://doi.org/10.3390/geosciences15040152.

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Recently published marine geophysical and seafloor drilling data permit a substantive reappraisal of the rifting and spreading that formed the South China Sea (SCS). The SCS rifted margins are different from those of the Atlantic type, having higher strain rates, younger orogenic crust, and distributed syn-rift magmatism. Rifting ~66–11 Ma and spreading 30–14 Ma split a Cretaceous Andean arc and forearc, producing >700 km of seafloor spreading in the east and a ~2000-km-wide rifted margin in the west. Luconia Shoals–Dangerous Grounds–Reed Bank–north Palawan–SW Mindoro were separated from China when the SCS opened. Brittle faulting of the upper crust was decoupled from ductile flow and magmatic intrusion of the lower crust, producing wide rifting with thin spots held together by less extended surrounds. Sediments accumulated in inter-montane lakes. Transform faults formed at/after breakup to link offset spreading segments. Spreading in the eastern subbasin from C11n to C5AD was at rates averaging 62 mm/yr, 30–24 Ma, decreasing to 38.5 mm/yr younger than 23 Ma. Spreading reorganization was common as margin segments broke up to the SW and spreading directions changed from ~N-S before 23 Ma to NW-SE after 17 Ma.
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8

Hendrickx, Michel E., and Manuel Ayón-Parente. "A new species of Munidopsis (Anomura, Galatheoidea, Munidopsidae) from the Gulf of California, western Mexico." Crustaceana 86, no. 11 (2013): 1304–15. http://dx.doi.org/10.1163/15685403-00003241.

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A new species ofMunidopsisWhiteaves, 1784 is described from the SW Gulf of California, western Mexico. The new species differs from all other known species ofMunidopsisof the East Pacific by the presence of numerous antero-posteriorly compressed processes on the carapace, many of which are truncate and with crenulated anterior margin, by its oval-shaped carapace, wider in its posterior half, and by the short, subrectangular, narrow rostrum with an upturned distal portion. It also differs from the East Pacific species and other species ofMunidopsisby the densely pitted carapace and appendages.
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9

Sharma, Mona, Smita Pareek, and Kulwant Singh. "Comparative Review and Performance Evaluation of Various Solar PV System Reconfigurations." Asian Journal of Water, Environment and Pollution 19, no. 5 (2022): 31–36. http://dx.doi.org/10.3233/ajw220068.

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In the current context, the most challenging and demanding research is to obtain maximum power from solar photovoltaic (SPV) power systems. Partial shadowing (PS) is a prominent cause of solar photovoltaic panels output power loss. The output decreases proportionally to the shaded area, the placement of shaded panels within the array, panel interconnections, shade pattern, etc. Numerous static and dynamic approaches for reducing the PS effect have been reported in the literature. Of these, the most common is the reconfiguration technique based on the static method, which distributes shaded panels more consistently and increases maximum power generation. The current study investigates the performance of common existing reconfiguration techniques based on static methods, including TCT, Sudoku, odd even (OE), and Latin square (LS) on a 4×4 PV array. The five different shadow patterns: short narrow (SN), short wide (SW), long narrow (LN), long wide (LW), and centre (CN) are implemented in this work. The performance analysis is carried out by comparing the maximum power generated (PM) and the results of panel simulations, as well as their theoretical conclusions, to ensure that these strategies are effectively implemented in hardware configuration.
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10

EL iysaouy, Lahcen, Mhammed LAHBABI, Abdelmajid OUMNAD, et al. "Performance Enhancements and Modeling of Photovoltaic Panel Configurations During Partial Shading Conditions." Journal of Applied Research and Technology 21, no. 5 (2023): 866–77. http://dx.doi.org/10.22201/icat.24486736e.2023.21.5.1506.

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The growing focus on solar energy has led to an expansion of large solar energy projects in the world. Though the most widely adopted Total Cross Tie (TCT) interconnection patterns will reduce the loss of mismatch, the appearance of shades in large-scale photovoltaic arrays drastically decreases the output power as well as several peaks of power in the P-V characteristics. To overcome these problems, the PV panels are organised using either electrical or physical reconfiguration methods. In this respect, it has been established that physical relocation methods are both cost-effective and efficient to disperse the shadow. In this context fits this work, his goal is to study the magic square view (MSV) for the physical rearrangement of the PV module in a TCT scheme. The simulation results reveal the effectiveness of the MSV method efficiently scattering the shade over the whole photovoltaic array. For validation, four types of Partial Shading Conditions (PSCs) patterns are considered and are then compared with the TCT and the recently proved Competence Square (CS) techniques: Short and Wide (SW), Long and Wide (LW), Long and Narrow (LN), and Short and Narrow (SN) shading patterns. Overall, MSV method is important in achieving improved output power enhancement of PV array under shaded conditions. A very clear improvement is obtained in the Long and Wide partial shading pattern.
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11

Janarthanan, L., BR Balakrishnan, V. Karthikeyan, P. Senniappan, BS Venkateswarlu, and G. Anandharaj. "Pharmacognostical Standardization and Phytochemical Studies on the leaves of Solanum torvum Sw." Journal of Drug Delivery and Therapeutics 9, no. 4-A (2019): 290–95. http://dx.doi.org/10.22270/jddt.v9i4-a.3473.

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A genus of plant belongs to the family Solanaceae well distributed in India more than 26 species which are found naturalised in India. Solanum torvum Sw. is a medium sized flowering plant in the Solanaceae family that is found in India, Malaysia. Transverse section of lamina showed the adaxial part has thick, short hump; the midrib and the adaxial hump have thin, angular epidermal cells. The upper part of adaxial hump has a few layers sclerenchyma cells. The inner layer of the adaxial midrib also has few layers of thick walled cells. The ground tissue consists of wide circular thin walled parenchyma cells with narrow inter cellular spaces. The vascular system of the midrib showed bi-collateral structure. Non glandular, profusely branched, thick walled, lignified epidermal trichome occurs as both on the veins and lamina. Physico-chemical standards such as Foreign Matter, Total Ash, Water Soluble Ash, Sulphated Ash, Loss on Drying, Water Soluble Extractive, Alcohol Soluble Extractive and Crude Fiber Content in percentage were estimated. Preliminary phytochemical screening of appropriate solvent extracts showed the presence of Alkaloids, Amino Acids, Carbohydrates, Cellulose, Lignin, Fats & Fixed Oils, Flavonoids, Glycosides, Tannins, Proteins, Starch, Steroids and Triterpenoids and absence of Volatile Oil, Mucilage and Pectin. Microscopic analysis and other parameters were informative and provide valuable information in the identification, standardization of Solanum torvum leaves.
 Keywords: Solanum torvum, Solanaceae, leaf, Microscopical evaluation.
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12

Kozuharova, Ekaterina. "Bumblebees and pollination of endemic Onobrychis pindicola (Fabaceae) in the subalpine habitats of Pirin Mts." Biologica Nyssana, Journal of Biological Sciences 9, no. 2 (2019): 89–101. https://doi.org/10.5281/zenodo.2538600.

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Onobrychis pindicola subsp. urumovii Degen & Dren. is an endemic with restricted distribution on Pirin Mts. and Slavjanka Mts. (Balkan Peninsula, SW Bulgaria). It is obligatorily dependent upon insect vectors for pollen transport, being a self-incompatible plant. The aim of this study was: 1) to identify which bumblebees pollinate Onobrychis pindicola, 2) to identify which plants compete for these pollinators with O. pindicola, 3) to use pollen load analysis to assess bumblebee flower constancy and spatial foraging patterns of bumblebee pollinators of O. pindicola. The nectar is accessible to a wide spectrum of bees including short tongued bumblebees and honey bees. Onobrychis pindicola is pollinated most actively by Bombus pyrenaeus. Other bumblebees visited the flowers more or less actively. These were B. lapidarius, B. terrestris and B. pratorum. The bumblebees tended to utilize plant resources in guilds and follow particular plants for foraging. Observed in the field high flower constancy was confirmed by the pollen analysis.  
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Dimitrovski, Trajche. "Evaluation of the tropical cucurbit chayote Sechium edule (Jacq.) Sw. var. albus spinosum under the temperate climate conditions of North Macedonia." ISPEC Journal of Agricultural Sciences 6, no. 1 (2022): 154–67. http://dx.doi.org/10.46291/ispecjasvol6iss1pp154-167.

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The aim of this study was to evaluate some biological, morphological, productive and quality properties of the tropical cucurbit chayote Sechium edule (Jacq.) Sw. var. albus spinosum grown under temperate climate conditions in the Kochani region, North Macedonia during 2019 and 2020. The short day photoperiod resulted in long vegetative stage (143.25 8.13 days). Flowering started in September, and the generative phase lasted 56.25 4.60 days in average based on the harvest date. Average ripe fruit yield of 28.05 18.67 kg/plant was obtained, with higher yield, number of fruits and fruit weight in 2020. The higher summer temperatures that resulted in a stressed growth, the later development of flowers and higher pest infestation with white fly and aphids may have contributed the lower yield in 2019. According to the ANOVA and LSD test results, 27 to 31 days were enough for fruit maturation. The fruit was 12.14 0.01 cm long, 8,06 0.06 cm wide and 7.30 0.05 cm thick and had an average water content of 94.72 0.13%, total soluble solids of 2.35 0.21 %Brix and pH value of 6.39 0.11.
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14

Merletti, German, Salim Al Hajri, Michael Rabinovich, Russell Farmer, Mohamed Bennis, and Carlos Torres-Verdin. "Assessment of True Formation Resistivity and Water Saturation in Deeply Invaded Tight-Gas Sandstones Based on the Combined Numerical Simulation of Mud-Filtrate Invasion and Resistivity Logs." Petrophysics – The SPWLA Journal of Formation Evaluation and Reservoir Description 64, no. 4 (2023): 502–17. http://dx.doi.org/10.30632/pjv64n4-2023a2.

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The process of mud-filtrate invasion involves immiscible fluid displacement and salt mixing between mud filtrate and formation fluids in porous and permeable rocks. Consequently, the post-invasion spatial distribution of fluids and electrolyte concentration around the borehole affects resistivity measurements with different depths of investigation (DOI). In the presence of deep mud-filtrate invasion, the assessment of water saturation in the uninvaded zone based on the deep resistivity log can be inaccurate. Deep and electrically conductive filtrate invasion coupled with shoulder-bed effects can artificially increase water saturation (Sw) estimations by 20 saturation units (s.u.) in the Barik reservoir, resulting in pessimistic estimates of hydrocarbon pore volume if no corrections are applied. The Barik sandstone reservoir, which is characterized by low porosity ( up to 14%), low-to-medium permeability (up to 40 md), and high residual gas saturation (40 to 50%), exhibits low storage capacity to admit the critical filtrate volume necessary for building an impermeable mudcake. Combined with multiple days of overbalanced exposure to saline water-based mud (WBM), mud-filtrate invasion results in deep and smooth radial transition zones where the uninvaded formation is far beyond the depth of investigation of laterolog tools. Deep resistivity values are, therefore, lower than the true formation resistivity. Additionally, numerical simulations of resistivity logs show that the resistivity reduction by conductive invasion is further aggravated by shoulder-bed effects when individual reservoir thickness falls below 2.5 m. This paper describes the implementation of a compositional fluid-flow simulator to numerically model WBM-filtrate invasion and mudcake buildup in vertical boreholes. The algorithm allows the simulation of physical dispersion and fluid displacement around the borehole in a multilayer model. Time-dependent radial profiles of Sw and salinity are combined with core-calibrated porosity and electrical properties to compute electrical resistivity via Archie’s formulation. Subsequently, numerically simulated logs are generated using vendor-specific forward model processing and compared against field measurements. This workflow was extensively tested in various reservoir intervals with a wide range of petrophysical rock types and drilling conditions. Results show that the deep laterolog exhibits low sensitivity to conductive filtrate invasion when reservoirs’ porosities are lower than 8%. Above that threshold value, invasion length is a nontrivial process involving multiple variables. Even though exposure time to openhole conditions is a key factor leading to deep invasion, certain reservoir characteristics can lead to deeper invasion at short exposure times and significantly increase uncorrected Sw estimates.
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Dutta, A., J. R. Peterson, M. Cianfaglione, and G. Sembroski. "Weak Lensing Analysis of A115, A2219 and A2261: Detection of Galaxy Groups and Filaments Around Clusters." Publications of the Astronomical Society of the Pacific 137, no. 6 (2025): 064102. https://doi.org/10.1088/1538-3873/adcbd5.

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Abstract We present a weak lensing and multi-wavelength analysis of three galaxy clusters: A115, A2219, and A2261. Weak lensing is performed using shape measurements made in short 60 s exposure images obtained using WIYN-ODI. Forced measurement is used to measure low Signal to Noise Ratio sources in individual exposures. We find the weak lensing significance map recovers the galaxy clusters and most galaxy groups in the wide 40 ′ × 40 ′ field. Significant parts of the filamentary structures over this field, as indicated by the galaxy number density map, were also successfully recovered in lensing significance maps. We find the amount of structure recovery depends on both the depth and average seeing of the images. In particular, we detect a >9 Mpc long structure that contains the cluster A2219. We compare our weak lensing maps with Chandra, XMM, and LOFAR observations and find that A115 and A2219 show clear signs of ongoing mergers. In particular, we find a significant separation of hot ICM and the weak lensing contours in A115. On the other hand, while A2261 appears relaxed, based on radio and X-ray analysis, we find that it is likely interacting with a structure 700 kpc SW of the main cluster. We also successfully recovered mass structures in two regions around A2261 indicated by diffuse X-ray emission in XMM images.
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Braga, Francesco. "Leadership in Sustainable Agribusiness, Innovation, and Solar Thermal Renewable Energy: Opportunities for Sustainable Agribusiness." International Journal on Food System Dynamics 7, no. 2 (2016): 143–82. https://doi.org/10.18461/ijfsd.v7i2.727.

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John Hollick, P.Eng, is the President of Conserval Engineering, a private company in Toronto with subsidiaries in Buffalo NY and Paris France. John is a Professional Engineer, an inventor, and a business person. His technology, SolarWall (SW) is one of the top 2% solar technology firms world-wide, as assessed by the US Government. It captures and makes conveniently available a very high proportion of solar radiation, over 90%, dwarfing the efficiency of photo voltaic panels (PV). The original technology is extremely sustainable requiring almost no maintenance, offering long term production with a life expectancy of several decades. Over the years, John has obtained several patents for his inventions, the last one awarded in December 2014. These patents reflect the continuous development of new technologies, new solutions built around the SW. Today SW offers solutions combining PV and thermal energy capture, or systems that may work to provide heating in the cold season and cooling in the warm season. All of the technologies are very cost effective. The case concerns possible agribusiness applications of one or more of the technology solutions developed by Conserval Engineering: the case users have been retained by John – thanks to their knowledge of agribusiness in their own country – to suggest possible applications of these technologies to local agribusiness industry. Mr Hollick has agreed to share a recent proposal (2010) to apply one of these technologies to solve the energy requirements of Food in Italy, while providing a significant carbon footprint reduction. He thinks that the analysis of this proposal and the material presented in the appendices will provide an opportunity to reflect on the potential of his technology and on the complex nature of the decision making process, and on some distortions resulting from public subsidies to some but not all sustainable technologies. That notwithstanding, the reflection on the first part of the case should reflect on the multiple gains that can be achieved with the adoption of these technologies in the specific local context. So, the case users armed with their reflections, their knowledge of agribusiness in their own country, and the short and essential description of the different technologies offered by Conserval Engineering, are asked to provide an illustration of what they consider the most promising applications to their own country’s agribusiness industry. Yes, the case does not provide actual costs of the different technologies, other than for the proposal to Food in Italy. At the same time the case users can certainly provide a qualitative perspective, based on the fact that these are the most advanced and cost effective technologies in the solar thermal industry. Part 1 of the case will primarily be structured around the discussion of these practical application to the local agribusiness.* Part 2 will focus on business sdevelopment. In using the case in teaching, parts 1 and part 2 could be linked to two different days.
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Zhang, Jin-Li, Sheng-Jie Fu, Sun-Jie Chen, et al. "Vestigial mediates the effect of insulin signaling pathway on wing-morph switching in planthoppers." PLOS Genetics 17, no. 2 (2021): e1009312. http://dx.doi.org/10.1371/journal.pgen.1009312.

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Wing polymorphism is an evolutionary feature found in a wide variety of insects, which offers a model system for studying the evolutionary significance of dispersal. In the wing-dimorphic planthopper Nilaparvata lugens, the insulin/insulin-like growth factor signaling (IIS) pathway acts as a ‘master signal’ that directs the development of either long-winged (LW) or short-winged (SW) morphs via regulation of the activity of Forkhead transcription factor subgroup O (NlFoxO). However, downstream effectors of the IIS–FoxO signaling cascade that mediate alternative wing morphs are unclear. Here we found that vestigial (Nlvg), a key wing-patterning gene, is selectively and temporally regulated by the IIS–FoxO signaling cascade during the wing-morph decision stage (fifth-instar stage). RNA interference (RNAi)-mediated silencing of Nlfoxo increase Nlvg expression in the fifth-instar stage (the last nymphal stage), thereby inducing LW development. Conversely, silencing of Nlvg can antagonize the effects of IIS activity on LW development, redirecting wing commitment from LW to the morph with intermediate wing size. In vitro and in vivo binding assays indicated that NlFoxO protein may suppress Nlvg expression by directly binding to the first intron region of the Nlvg locus. Our findings provide a first glimpse of the link connecting the IIS pathway to the wing-patterning network on the developmental plasticity of wings in insects, and help us understanding how phenotypic diversity is generated by the modification of a common set of pattern elements.
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18

Greenhill, L. J., C. Goddi, C. J. Chandler, E. M. L. Humphreys, and L. D. Matthews. "Dynamical detection of a magnetocentrifugal wind driven by a 20 M⊙ YSO." Proceedings of the International Astronomical Union 8, S287 (2012): 166–70. http://dx.doi.org/10.1017/s1743921312006850.

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AbstractWe have tracked the proper motions of ground-state λ7mm SiO maser emission excited by radio Source I in the Orion BN/KL region. Based on dynamical arguments, Source I is believed to be a hard 20 M⊙ binary. The SiO masers trace a linear bipolar outflow (NE-SW) 100 to 1000 AU from the binary. The median 3D velocity is 18 km s−1. An overlying distribution of 1.3 cm H2O masers betrays similar characteristics. The outflow is aligned with the rotation axis of an edge-on disk and wide angle flow known inside 100 AU. Gas dynamics and emission morphology traced by masers around Source I provide dynamical evidence of a magnetocentrifugal disk-wind around this massive YSO, notably a measured gradient in line-of-sight velocity perpendicular to the flow axis, in the same direction as the disk rotation and with comparable speed. The linearity of the flow, despite the high proper motion of Source I and the proximity of dense gas associated with the Orion Hot Core, is also more readily explained for a magnetized flow. The extended arcs of ground-state maser emission bracketing Source I are a striking feature, in particular since dust formation occurs at smaller radii. We propose that the arcs mark two C-type shocks at the transition radius to super-Alfvénic flow.
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BLAKE, JAMES A. "Redescription of Capitella capitata (Fabricius) from West Greenland and designation of a neotype (Polychaeta, Capitellidae)." Zoosymposia 2, no. 1 (2009): 55–80. http://dx.doi.org/10.11646/zoosymposia.2.1.7.

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Capitella capitata (Fabricius, 1780), the type species of the genus, was originally described from the SW coast of Greenland; however, current definitions of the species are largely based on Eisig (1887), who described specimens from Naples on the Mediterranean coast. Eisig’s excellent account of the morphology of a sexually dimorphic species has subsequently been applied to specimens collected from worldwide locations. C. capitata was considered cosmopolitan in its distribution until J.P. Grassle and others identified numerous sibling species in laboratory culture. A large collection of Capitella from locations along the W, SW, and SE coasts of Greenland was provided by the Zoological Museum, University of Copenhagen (ZMUC). The collections included about 680 specimens with distinctive prostomial, thoracic, and setal morphology that differs from the typical depiction of the species in most faunal accounts. A neotype was selected from an excellent collection of approximately 250 specimens from Mârmorilik on the Affarlikassâ Fjord on the inner Uummannaq Fjord system, north of Disko Bay. All available collections from the ZMUC are believed to belong to the same species, which is here considered to be Arctic and subarctic in distribution. Additional material from the Alaskan Arctic supports this hypothesis. The Greenland species has a short, cup-shaped, wider-than-long prostomium joined to a narrow peristomial ring that is retracted slightly into setiger 1. In most specimens, the first four thoracic setigers gradually increase in size, with setiger 4 being the largest thoracic segment, and then decrease over the next five setigers, with setiger 9 being smaller than subsequent anterior abdominal segments. The thoracic region is thus markedly distinct visually from the abdominal region. Setigers 8–9 typically lack fascicles of notosetae, but 80% of the specimens examined have genital spines and most have copulatory organs that are secretory in nature: they contain numerous small secretion granules but no sperm. Many of these specimens have eggs and larvae within their tubes, suggesting functional hermaphroditism. The present redescription of C. capitata serves to establish a baseline against which the many known but undescribed species can be compared. Previous accounts of C. capitata based on the Eisig concept and largely from boreal and temperate localities should be reevaluated.
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Schilirò, L., C. Esposito, and G. Scarascia Mugnozza. "Evaluation of shallow landslide-triggering scenarios through a physically based approach: an example of application in the southern Messina area (northeastern Sicily, Italy)." Natural Hazards and Earth System Sciences 15, no. 9 (2015): 2091–109. http://dx.doi.org/10.5194/nhess-15-2091-2015.

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Abstract. Rainfall-induced shallow landslides are a widespread phenomenon that frequently causes substantial damage to property, as well as numerous casualties. In recent~years a wide range of physically based models have been developed to analyze the triggering process of these events. Specifically, in this paper we propose an approach for the evaluation of different shallow landslide-triggering scenarios by means of the TRIGRS (transient rainfall infiltration and grid-based slope stability) numerical model. For the validation of the model, a back analysis of the landslide event that occurred in the study area (located SW of Messina, northeastern Sicily, Italy) on 1 October 2009 was performed, by using different methods and techniques for the definition of the input parameters. After evaluating the reliability of the model through comparison with the 2009 landslide inventory, different triggering scenarios were defined using rainfall values derived from the rainfall probability curves, reconstructed on the basis of daily and hourly historical rainfall data. The results emphasize how these phenomena are likely to occur in the area, given that even short-duration (1–3 h) rainfall events with a relatively low return period (e.g., 10–20~years) can trigger numerous slope failures. Furthermore, for the same rainfall amount, the daily simulations underestimate the instability conditions. The high susceptibility of this area to shallow landslides is testified by the high number of landslide/flood events that have occurred in the past and are summarized in this paper by means of archival research. Considering the main features of the proposed approach, the authors suggest that this methodology could be applied to different areas, even for the development of landslide early warning systems.
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Schilirò, L., C. Esposito, and G. Scarascia Mugnozza. "Evaluation of shallow landslide triggering scenarios through a physically-based approach: an example of application in the southern Messina area (north-eastern Sicily, Italy)." Natural Hazards and Earth System Sciences Discussions 3, no. 5 (2015): 2975–3022. http://dx.doi.org/10.5194/nhessd-3-2975-2015.

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Abstract. Rainfall-induced shallow landslides are a widespread phenomenon that frequently causes substantial damage to property, as well as numerous casualties. In recent years a wide range of physically-based models has been developed to analyze the triggering process of these events. Specifically, in this paper we propose an approach for the evaluation of different shallow landslide triggering scenarios by means of TRIGRS numerical model. For the calibration of the model, a back-analysis of the landslide event occurred in the study area (located SW of Messina, north-eastern Sicily, Italy) on 1 October 2009 was performed, by using different methods and techniques for the definition of the input parameters. After evaluating the reliability of the model through the comparison with the 2009 landslide inventory, different triggering scenarios were defined using rainfall values derived from the rainfall probability curves, reconstructed on the basis of daily and hourly historical rainfall data. The results emphasize how these phenomena are likely to occur in the area, given that even short-duration (3–6 h) rainfall events having a relatively low return period (e.g. 10 years) can trigger numerous slope failures. On the contrary, for the same rainfall amount, the daily simulations overestimate the instability conditions. The tendency of shallow landslides to trigger in this area agrees with the high number of landslide/flood events occurred in the past and summarized in this paper by means of archival researches. Considering the main features of the proposed approach, the authors suggest that this methodology could be applied to different areas, even for the development of landslide early warning systems.
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22

Chandra, Naveen, Sachiko Hayashida, Tazu Saeki, and Prabir K. Patra. "What controls the seasonal cycle of columnar methane observed by GOSAT over different regions in India?" Atmospheric Chemistry and Physics 17, no. 20 (2017): 12633–43. http://dx.doi.org/10.5194/acp-17-12633-2017.

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Abstract. Methane (CH4) is one of the most important short-lived climate forcers for its critical roles in greenhouse warming and air pollution chemistry in the troposphere, and the water vapor budget in the stratosphere. It is estimated that up to about 8 % of global CH4 emissions occur from South Asia, covering less than 1 % of the global land. With the availability of satellite observations from space, variability in CH4 has been captured for most parts of the global land with major emissions, which were otherwise not covered by the surface observation network. The satellite observation of the columnar dry-air mole fractions of methane (XCH4) is an integrated measure of CH4 densities at all altitudes from the surface to the top of the atmosphere. Here, we present an analysis of XCH4 variability over different parts of India and the surrounding cleaner oceanic regions as measured by the Greenhouse gases Observation SATellite (GOSAT) and simulated by an atmospheric chemistry-transport model (ACTM). Distinct seasonal variations of XCH4 have been observed over the northern (north of 15° N) and southern (south of 15° N) parts of India, corresponding to the peak during the southwestern monsoon (July–September) and early autumn (October–December) seasons, respectively. Analysis of the transport, emission, and chemistry contributions to XCH4 using ACTM suggests that a distinct XCH4 seasonal cycle over northern and southern regions of India is governed by both the heterogeneous distributions of surface emissions and a contribution of the partial CH4 column in the upper troposphere. Over most of the northern Indian Gangetic Plain regions, up to 40 % of the peak-to-trough amplitude during the southwestern (SW) monsoon season is attributed to the lower troposphere ( ∼ 1000–600 hPa), and ∼ 40 % to uplifted high-CH4 air masses in the upper troposphere ( ∼ 600–200 hPa). In contrast, the XCH4 seasonal enhancement over semi-arid western India is attributed mainly ( ∼ 70 %) to the upper troposphere. The lower tropospheric region contributes up to 60 % in the XCH4 seasonal enhancement over the Southern Peninsula and oceanic region. These differences arise due to the complex atmospheric transport mechanisms caused by the seasonally varying monsoon. The CH4 enriched air mass is uplifted from a high-emission region of the Gangetic Plain by the SW monsoon circulation and deep cumulus convection and then confined by anticyclonic wind in the upper tropospheric heights ( ∼ 200 hPa). The anticyclonic confinement of surface emission over a wider South Asia region leads to a strong contribution of the upper troposphere in the formation of the XCH4 peak over northern India, including the semi-arid regions with extremely low CH4 emissions. Based on this analysis, we suggest that a link between surface emissions and higher levels of XCH4 is not always valid over Asian monsoon regions, although there is often a fair correlation between surface emissions and XCH4. The overall validity of ACTM simulation for capturing GOSAT observed seasonal and spatial XCH4 variability will allow us to perform inverse modeling of XCH4 emissions in the future using XCH4 data.
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Rahiman, Tariq I. H., and Jarg R. Pettinga. "Fracture lineaments, fault mesh formation and seismicity." Bulletin of the New Zealand Society for Earthquake Engineering 42, no. 1 (2009): 63–72. http://dx.doi.org/10.5459/bnzsee.42.1.63-72.

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Viti Levu, the main island of Fiji, is located in a seismically active area within the Fiji Platform, a remnant island arc that lies in a diffuse plate boundary zone between the Pacific and Australian tectonic plates in the SW Pacific. The upper crust of Viti Levu is dissected by numerous intersecting fault/lineament zones mapped from remote sensing imagery of the land surface (topography, radar and aerial photos) and basement (magnetic) and have been subject to rigorous statistical tests of reproducibility and verification with field mapped fault data. Lineaments on the various imagery correlate with faults mapped in the field, and show spatial continuity between and beyond mapped faults, thereby providing a fuller coverage of regional structural patterns than previously known. Some fault/lineaments zones extend beyond the coastline to the offshore area from the SE Viti Levu study area. Multibeam bathymetry and seismic reflection data show the fault zones occur along and exert control on the location of a number of submarine canyons on the SE slope of Viti Levu. Evidence for Late Quaternary fault activity is only rarely observed in onshore SE Viti Levu (e.g. by displaced shoreline features), and in seismic reflection profiles from offshore.
 The principal fault sets in Viti Levu represent generations of regional tectonic faulting that pervade the Fiji Platform during and after the disruption of the proto Fijian arc in the Middle to Late Miocene (~15Ma). These fault sets combine to form a complex network of interlocking faults creating a fault mesh that divides the upper crust into a number of fault blocks ranging from ~2-30 km wide. It is inferred that the fault mesh evolved throughout the Neogene as a response to the anticlockwise rotation of the Fiji Platform through progressive development of different fault sets and intervening crustal block rotations. Regional tectonic deformation is presently accommodated in a distributed manner through the entire fault mesh. Low magnitude earthquakes (<M4) occur regularly and may represent ruptures along short linking segments of the fault mesh, while infrequent larger earthquakes (>M4) may result from complex rupture propagation through several linking fault segments of the mesh that lie close to optimum stress orientations. The interpreted model of distributed deformation through the fault mesh for the study area in SE Viti Levu is inferred to be characteristic of the style of active deformation that occurs throughout the entire Fiji Platform.
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24

Swartz, William, Steven Lorentz, Stergios Papadakis, et al. "RAVAN: CubeSat Demonstration for Multi-Point Earth Radiation Budget Measurements." Remote Sensing 11, no. 7 (2019): 796. http://dx.doi.org/10.3390/rs11070796.

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The Radiometer Assessment using Vertically Aligned Nanotubes (RAVAN) 3U CubeSat mission is a pathfinder to demonstrate technologies for the measurement of Earth’s radiation budget, the quantification of which is critical for predicting the future course of climate change. A specific motivation is the need for lower-cost technology alternatives that could be used for multi-point constellation measurements of Earth outgoing radiation. RAVAN launched 11 November 2016, into a nearly 600-km, Sun-synchronous orbit, and collected data for over 20 months. RAVAN successfully demonstrates two key technologies. The first is the use of vertically aligned carbon nanotubes (VACNTs) as absorbers in broadband radiometers for measuring Earth’s outgoing radiation and the total solar irradiance. VACNT forests are arguably the blackest material known and have an extremely flat spectral response over a wide wavelength range, from the ultraviolet to the far infrared. As radiometer absorbers, they have greater sensitivity for a given time constant and are more compact than traditional cavity absorbers. The second technology demonstrated is a pair of gallium phase-change black body cells that are used as a stable reference to monitor the degradation of RAVAN’s radiometer sensors on orbit. Four radiometers (two VACNT, two cavity), the pair of gallium black bodies, and associated electronics are accommodated in the payload of an agile 3U CubeSat bus that allows for routine solar and deep-space attitude maneuvers, which are essential for calibrating the Earth irradiance measurements. The radiometers show excellent long-term stability over the course of the mission and a high correlation between the VACNT and cavity radiometer technologies. Short-term variability—at greater than the tenths-of-a-Watt/m2 needed for climate accuracy—is a challenge that remains, consistent with insufficient thermal knowledge and control on a 3U CubeSat. There are also VACNT–cavity biases of 3% and 6% in the Total and SW channels, respectively, which would have to be overcome in a future mission. Although one of the black bodies failed after four months, the other provided a repeatable standard for the duration of the project. We present representative measurements from the mission and demonstrate how the radiometer time series can be used to reconstruct outgoing radiation spatial information. Improvements to the technology and approach that would lead to better performance and greater accuracy in future missions are discussed.
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25

Mulatu Alemu, Mitike, Andargachew Gedebo, Amsalu Gobena Roro, and Tatek Tamiru Geletu. "Effect of Moisture Stress on Physiological and Yield Responses of Common Bean Varieties at Lath House Condition, Hawassa University, Southern Ethiopia." International Journal of Agronomy 2023 (January 14, 2023): 1–15. http://dx.doi.org/10.1155/2023/2626225.

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Common bean is one of the most traditional foods, with social and economic importance, and is suitable for food security due to its short growing cycle and adaptability to different cropping systems. However, various constraints like biotic and abiotic factors reported for the low productivity of the crop. Therefore, the study was designed to identify the common bean varieties’ response on physiological properties and yield performance under different irrigation intervals in Lath House condition at Hawassa University, Ethiopia, from June, 2020, to September, 2020. The experiment was laid out in a completely randomized design with a factorial combination of seven common bean varieties (Awash-1, Awash-2, Dursitu, Fedis, Gofta, Nasir, and Tinike) and three moisture levels, eight day irrigation interval, four day irrigation interval, and daily watering with three replication. There was a significant main and interaction effects among the measured parameters. From those significantly affected parameters, the highest stomata numbers (25) were recorded from the Dursitu variety with a daily irrigation interval. The longest stomata aperture was observed with Gofta (133.76 nm) and Fedis varieties (131.13 nm) to daily irrigation and four day irrigation intervals, respectively. A wider stomata aperture was found in the Awash-1(12.6 nm) variety on a daily irrigation interval, and maximum stomata conductance (193.67) was obtained in the Tinike variety treated with a four day irrigation interval. The maximum relative leaf moisture content (96%) was found on the Awash-1 variety at a four day irrigation interval. Maximum water use efficiency was obtained from the Awash-1 (3.95) variety, which is an insignificant variation with the Awash-2 (3.85) and Gofta varieties (3.85) at a four day irrigation interval. Among the varieties, the highest photosynthesis rate (10.43 μmol·m−2·s−1) and transpiration rate (5.42 mmol·m−2·s−1) were obtained in the Gofta variety treated by a four day irrigation interval. In the photosynthetic pigment analysis, the highest leaf chl-a, chl-b, and chl-a + b concentration was recorded at 2.4 μg/ml, 1.53 g/ml, and 3.93 μg/ml, respectively, in the Gofta variety irrigated at a four days interval. The highest (0.586) chlorophyll fluorescence was obtained in the eight day irrigation interval. The highest proline content was found from the Awash-1 (0.78) variety regarding to drought stress condition. The highest pods plant−1 (9) and seed pod−1 (5) was found in the Awash-2 and Awash-1 varieties, respectively, at a four days irrigation interval and the highest main effect of 100 SW (39.48 g), (28.6 g) was found in the Fedis variety and at a four day irrigating interval, respectively. The maximum yield was obtained from the varieties Awash-1 (7.8 g plant−1) and Gofta (7.36 g plant−1) with a four day irrigated interval and they are also superior at drought condition and Awash-2 varieties are enhanced at waterlogging condition as compared to the other varieties.
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de Graciansky, Pierre-Charles, and Thierry Jacquin. "Structural and palaeogeographical evolution of the Paris Basin during the Mid-Cimmerian events." Bulletin de la Société Géologique de France 174, no. 1 (2003): 3–17. http://dx.doi.org/10.2113/174.1.3.

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Abstract Presentation. The deposition of unconformable condensed layers associated with frequent sedimentary gaps record the Mid-Cimmerian events in the Paris Basin, dated as Aalenian to lowermost Bajocian. In order to document the Mid-Cimmerian episodes, a set of 250 commercial wells (fig. 1) together with several outcropping sections located on the southern and southeastern borders of the Paris Basin have been interpreted in terms of sequence stratigraphy. Seventeen third-order depositional sequences sensu Vail et al. [1991] have been identified and correlated. These are numbered as To1 to To7, Aa1 and Aa2, Bj1 to Bj5, Bt1 to Bt3 (fig. 2). They have been mapped out from subsurface data and from a few available outcrop data. Isopach maps corresponding to the depositional sequences illustrate both the role of synsedimentary faults and the evolution with time of depocenters (figs. 10 to 17). One purpose is to compare through time the depocentres location with the fault pattern (fig. 1). The well-known Jurassic Paris Basin faults make a three-branch system that converges towards the centre of the Paris Basin. These are as follows: NW-SE trending faults. These include the Pays de Bray, Somme River and Lower-Seine River faults. They intersect the northwestern border of the Paris Basin. They acted several times as major extensional faults from Permian through early Cretaceous times ; the NE-SW trending Luxembourg, Metz and other faults. They were typically extensional faults as a remote effect of the Tethyan rifting episodes. They have been active during late Toarcian through Bajocian times in Burgundy as shown from outcrop data ; the N-S trending group, including the Saint Martin de Bossenay, Sens, Loire and Sennely fault lines, are mainly located at the southern side of the Bray fault system. They probably correspond to the reactivation of an old Hercynian lineament such as the sillon houiller in the Massif Central. They have been documented also from outcrops in Burgundy and in the Macon area. Development of the main Cimmerian events in the Paris Basin During the overall regressive phase dated as Middle to Late Toarcian (fig. 3), the central Paris Basin and the northern Champagne are characterised by relatively thick deposits (fig. 11 to 14). Then, the onset of mid-Cimmerian events is marked by stratigraphic gaps and condensed layers dated as latermost Toarcian through Aalenian in the same areas (fig. 5). The correlative long-term maximum regression dated as close to the Aalenian/Bajocian boundary is expressed by maximum progradation of the carbonate platforms towards the Swabische Jura and the western Sub-Alps, i.e. the nascent Tethyan margin. At the same time the sedimentary accumulation was considerably attenuated within the whole Paris Basin during Aalenian times (fig. 9). This illustrates the structural high position of the Paris Basin during the Cimmerian events. Nevertheless, the central Paris Basin has been episodically flooded as a consequence of two, short duration and wide amplitude, transgressive events dated as middle and late Aalenian, related to Murchisonae and Concavum ammonite zones respectively (fig. 2). Bajocian times are marked by the onset of the Middle Jurassic transgression (fig. 7). Some faults active during Triassic and Liassic times are concealed by the Bajocian layers (fig. 8). This is accompanied by a new distribution of the depocentres location with respect to the Toarcian situation (compare figs 11–13 to fig. 7). The structural control of the Paris Basin development during the Mid-Cimmerian events can be illustrated on both (2D) isopach maps (figs. 5, 7 and 10 to 19) and 3D diagrams (fig. 19). They both document the evolution with time of the main groups of synsedimentary fault lines. Third-order and second-order cycles and bounding unconformities have been found to be coeval from the North Sea to the Tethyan margins including North Africa. This documents the partly eustatic control of the Mid-Cimmerian events.
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Videt, Blaise, and Didier Néraudeau. "Palaeoecology of Cenomanian oysters of the northern margin of the Aquitaine Basin." Bulletin de la Société Géologique de France 178, no. 1 (2007): 39–50. http://dx.doi.org/10.2113/gssgfbull.178.1.39.

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Abstract The purpose of this paper is to understand which parameters control the palaeoenvironmental distribution of Middle Cretaceous oysters. To reach this objective, the following two step analysis has been carried out. First, ten major Upper Albian to Lower Turonian outcrops from the northern part of the Aquitain Basin (SW France) (fig. 1) were analysed with respect to their sedimentological and palaeontological features (see fig. 2 for oyster distribution). They represent a time interval corresponding to a 2nd order transgression [Hardenbol et al., 1997; Néraudeau et al., 1997], characterised in the Charentes (North Aquitaine Basin) by a great variety of depositional environments and very rich in oyster assemblages [Videt, 2004]. According to previous authors [Moreau, 1993; Néraudeau et al., 1997; Platel, 1989, 1996], this series can be divided into seven lithological units, A to G, four units (A, B, C and G) being subdivided into two or three subunits (A1 and A2, B1 to B3, C1 to C4, G1 and G2). Apart from the sub-units A1 and Tu, which correspond to the Late Albian and Early Turonian respectively, all the lithological sub-units A2 to G2 correspond to the Cenomanian series. A and B belong to the lower Cenomanian, C1 to C3 to the middle Cenomanian, and C4 to G2 to the Upper Cenomanian. In terms of palaeoenvironments, unit A is considered as deposits of a sandy estuary (with local lignite layers) [Néraudeau et al., 2002, 2003; Perrichot, 2003], and unit B as shallow subtidal sand dunes [Vullo et al., 2003]. Unit C corresponds to the optimal development of a carbonate platform with rudists [Chéreau et al., 1997], unit D to a marly open shelf marly facies, unit E to an oyster bank mainly composed of Pycnodonte biauriculata [Dhondt, 1984], unit F to a moderately deep bioclastic facies colonised by rudist Ichthyosarcolites triangularis and, unit G, which forms progressively marly up-section, to progressive platform flooding at the Cenomanian-Turonian boundary. From the analysis of these different lithological units and subunits, the North Aquitaine Basin can be considered as a mixed siliciclastic-carbonate platform. Two main depositional systems have been identified, namely an open one and a closed one. The synthetic distribution of twelve kinds of oysters biofacies (bf1 to bf12) described in these units and subunits is summed up in figure 3 according to depositional type, lithology and depth. Based on the palaeoenvironmental distribution of Middle Cretaceous oysters in the northern part of the Aquitain Basin, the palaeoecological affinities of the nine marine species are discussed regarding seven main parameters i.e., oxygenation, water turbulence, salinity, turbidity, bathymetry, grain size, and substrate consistency (i.e. “hardground” VS “softground”) (fig. 4). Acutostrea aff. incurva (Nilson, 1827) (figs 4, 5h) and Curvostrea rouvillei (Coquand, 1862) (figs 4, 5i) are very rare species with a distribution that is still ambiguous. Apparently they preferred soft substrates and seem to have tolerated lowered oxygen levels. In addition, they are encountered in quiet, deep environments, i.e., the lower infralittoral to circalittoral zones sensu Néraudeau et al. [2001]. Ceratostreon flabellatum (Goldfuss, 1833) (figs 4, 5e) is not a prolific species but was widely distributed all over the carbonate platform. Nevertheless it is mainly marine and located in the infralittoral zone [sensu Néraudeau et al., 2001]. Gyrostrea delettrei (Coquand, 1862) (figs 4, 5g) might have been very widespread but is very rare. It was most abundant in marginal marine environments where it was the sole oyster that tolerated brackish water conditions. Pycnodonte biauriculata (Lamarck, 1819) (figs 4, 5d), in spite of its very short stratigraphic range (Naviculare Ammonite Zone), colonised a wide variety of environments. It is mainly a relatively medium water species (regarding to other species) [Stenzel, 1971; Harry, 1985; Freneix and Viaud, 1986], from the lower infralittoral zone [sensu Néraudeau et al., 2001] but it needed food-laden currents. Pycnodonte vesicularis (Lamarck, 1819) (figs 4, 5f) is also an ubiquitous species. However, in contrast to Pycnodonte biauriculata, it preferred deep, soft substrates (circalittoral and deeper ones? [Néraudeau and Villier, 1997]). Rastellum carinatum (Lamarck, 1806) (figs 4, 5c) and Rastellum diluvianum (Linne, 1767) (figs 4, 5b) exhibit an identical distribution pattern in spite of the fact that R. diluvianum is more selective than Rastellum carinatum. Carter [1968], Jablonsky and Lutz [1980] and Freneix and Viaud [1986] have already demonstrate that these species do not tolerate turbulent conditions but are particularly adapted to quiet water and soft substrates. The two species also do not tolerate salinity variations. Rhynchostreon suborbiculatum (Lamarck, 1801) (figs 4, 5a) is the most ubiquitous species in the Cenomanian of the Aquitain Basin. Videt and Néraudeau [2003] and Videt [2004] have already defined the parameters that affected its shape and its abundance. As the species does not occur in brackish water deposits, salinity seems to have been a major factor limiting its distribution.
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Wojnicki, Mirosław, Renata Cicha-Szot, Helena Cygnar, et al. "Perspectives of the offshore CCS development in the polish EEZ on a Baltic Sea: insights from ongoing preliminary research for pilot CO2 injection." Baltic Carbon Forum 2 (October 13, 2023): 4–5. http://dx.doi.org/10.21595/bcf.2023.23574.

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The key to effectively combatting progressive climate change lies in promptly reducing greenhouse gas (GHG) emissions, particularly carbon dioxide (CO2), whose concentration continues to rise due to human activities. The European Union (EU) has established legally binding targets, including achieving climate neutrality by 2050, with an intermediate goal of reducing GHG emissions by 55 % by 2030 compared to 1990 levels. Poland, one of the major CO2 emitters in Europe, also possesses significant storage potential in terms of projected CO2 capacity within its sedimentary basins. Considering this, the implementation of Carbon Capture and Storage (CCS) technology could play a crucial role in Poland's efforts to decarbonize its economy. For several years, the Oil and Gas Institute – National Research Institute (INiG – PIB) has been at the forefront of domestic research activities concerning underground CO2 injection. The involvement dates back to 1996 by pioneering the development of a concept, design, and implementation of one of Europe's earliest industrial installations for reinjecting acid gas, consisting of approximately 80 % CO2 and 20 % H2S, into the reservoir water underlying a productive natural gas field. This facility in Borzęcin operated by PGNiG (now part of ORLEN GROUP), is a unique testing ground where the injection process has been running continuously for 27 years. The research conducted at INiG – PIB has played an important role in identifying appropriate geological structures in Poland with a total storage capacity of 10-15 Gt CO2. Around 90-93 % of the CO2 storage capacity is found in saline aquifers, with a significant portion of approximately 7-10 % identified within mature hydrocarbon fields.Despite recent progress in managing industrial CO2 emissions, the peace of CCS development falls short of meeting the objectives set by the Paris Agreement. The main hindrance lies in the absence of a suitable regulatory framework for CO2 transport and storage infrastructure. Recognizing this challenge, the national offshore operator, Lotos Petrobaltic (LPB), presented in 2021 a Green Paper on CCS development in Poland. This document outlines a set of recommendations for legislative alternations to facilitate the initiation of large-scale, commercial CCS projects within the country. Given the current national regulations and assumptions regarding low social barriers, the most expeditious approach to implementing a First-of-a-Kind (FOAK) large-scale CCS project in Poland seems to involve deploying depleted hydrocarbon reservoirs located at the Polish Exclusive Economic Zone (EEZ) within the Baltic Sea. The LPB, with research and scientific support from INiG – PIB, has launched a program aimed at conducting a preliminary assessment of CO2 injection in Middle Cambrian sandstones. The project is scheduled to begin with a pilot injection into the well-identified, depleted structure of the B3 oil reservoir. Subsequently, it may be expanded to include adjacent hydrocarbon reservoirs and could ultimately encompass the entire Cambrian aquifer.The Cambrian aquifer is characterized by complex tectonics, comprising several blocks separated by fault zones. These fault zones may act as barriers to the propagation of reservoir fluids, as demonstrated by the presence of hydrocarbon traps in the vicinity of some fault zones.The B3 oil field, covering an area of 36,2 km2, is an elongated SW-NE, asymmetric anticline, cut on the west side by an inverted fault zone. The reservoir interval, with an average depth of 1450 meters below sea level comprises of sandstones of the Paradoxides Paradoxissimus (Middle Cambrian Zone) horizon showing a monoclinal dip towards the south-east. The reservoir formation exhibits heterogeneity in both within the vertical profile and the horizontal direction, characterized by a wide range of petrophysical parameters values. According to preliminary assessments, the CO2 storage capacity of the B3 site is estimated to be around 7 Mt CO2. The expansion of CO2 storage to the remaining hydrocarbon fields and the overarching Cambrian aquifer megastructure could potentially increase the total storage capacity to more than 150 Mt. The envisioned CO2 sources include emitters from the chemical industry, where CO2 is a by-product of the fertilizer production process. During the project’s pilot phase, the predicted CO2 injection rate is expected to be between 25-50 kt CO2 per year. As the project transitions to the upscaled phase, the injection rate is projected to increase significantly, reaching approximately 2 Mt CO2 per year. The transportation of CO2 for full-scale injection is planned through multimodal means, considering potential synergies with the multimodal CO2 Terminal in Gdansk, which is being planned under the ECO2CEE project (formerly EU CCS Interconnector). A pipeline connection between the offshore storage area and the CO2 Terminal is under consideration.The main research problems addressed within the INiG – PIB and LPB joint initiative include determining the sequestration potential of the Cm2pp aquifer, assessing the feasibility of the CO2-EOR process, recognizing the effects of injected CO2/reservoir rock/caprock interactions on injectivity, geomechanical parameters, and sealing integrity, as well as investigating CO2-induced corrosion of steel components in the transport and injection plant.
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Goričan, Špela, Martin Đaković, Peter O. Baumgartner, et al. "Mesozoic basins on the Adriatic continental margin – a cross-section through the Dinarides in Montenegro / Mezozojski bazeni na kontinentalnem robu Jadranske plošče – presek čez Dinaride v Črni gori." Folia biologica et geologica 63, no. 2 (2022): 85–150. http://dx.doi.org/10.3986/fbg0099.

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The Dinarides, together with the Albanides and Hellenides, preserve stratigraphic successions derived from the eastern margin of the Adriatic microplate and remnants of ophiolites obducted from the Maliac-Vardar branch of the Neotethys Ocean. The main stages in the Mesozoic geodynamic history are: 1) rifting leading to opening of the Maliac Ocean in the Late Anisian, 2) onset of an east-dipping intra-oceanic subduction in the Early-Middle Jurassic and sea-floor spreading in a supra-subduction setting (Vardar Ocean), 3) formation of ophiolitic mélanges in trench-like basins, westward obduction of young supra-subduction ophiolites in the Middle-Late Jurassic and accumulation of flysch-type deposits in foreland basins in the latest Jurassic to Early Cretaceous, 4) subaerial exposure of the newly formed nappes followed by middle to Late Cretaceous transgression, and 5) continental collision in the Maastrichtian and Paleogene. On the continental margin, the Middle Triassic to Early Jurassic extension created a complex horst-and-graben geometry that is apparent in the stratigraphic record. The present day NW-SE striking tectonic units are in rough accordance with the Mesozoic paleogeography. Hence, the inferred configuration for the most complete SW to NE transect through Montenegro and Serbia is as follows: The Dalmatian Carbonate Platform, the Budva Basin, the High Karst Carbonate Platform, the Bosnian Basin, the Durmitor High, the Lim Basin, the Drina-Ivanjica High, and the deep-marine distal continental-margin domain.
 We present a short description of the stratigraphy for these tectonic/paleogeographic units and discuss their possible connection with other units of the Dinarides and Hellenides. The field guide focuses on deep-water deposits, in which radiolarians are the crucial tool for dating. We describe the complete Mesozoic succession of the Budva Zone, the Middle Triassic pelagic episode of the High Karst Zone, the Upper Triassic and Jurassic pelagic rocks of the Lim Zone and two localities with radiolarites associated with ophiolites.
 The largest part of the guide is devoted to the Budva Zone, a deeply rifted trough in the continuation of the Pindos Basin. The Budva Zone with its external location in the Dinaric orogen was a site of continuous pelagic sedimentation from the Middle Triassic to the end Cretaceous. Radiolarites characterize the Middle Triassic, Hettangian–Sinemurian, Aalenian to Tithonian, and Hauterivian–Barremian to lower Turonian; pelagic limestones prevail in the Upper Triassic, Berriasian–Valanginian and upper Turonian to Maastrichtian. Calcareous turbidites from the adjacent High Karst Carbonate Platform are interstratified in all units and completely replace radiolarites in the Pliensbachian.
 Pelagic sequences also occur in the High Karst Zone, but are confined to the Middle Triassic syn- and early post-rift deposits. A 20 m thick unit of Middle Triassic nodular limestone and radiolarite within shallow-water carbonates is a typical example.
 More internally, the western Ćehotina Subzone of the Lim Zone records pelagic sedimentation from the Middle Triassic to early Cretaceous, when synorogenic mixed carbonate-siliciclastic deposition began. This zone has been less investigated than the Budva Zone. A 100 m thick Norian to Rhaetian succession of limestone with chert nodules is dated with conodonts. A Callovian-early Oxfordian age of lime-free cherts is determined with radiolarians. The Mihajlovići Subzone that may have been part of the Drina-Ivanjica paleogeographic unit shows Triassic shallow-water carbonates and a Jurassic deepening upward sequence ending with Oxfordian radiolarites.
 The last two field-trip stops show upper Bathonian-lower Callovian radiolarites in an ophiolitic mélange and upper Anisian radiolarites in direct contact with basalt. These ages, obtained in the south-westernmost ophiolite remnants of the Dinarides, agree with previously documented ophiolite ages in the wider region.
 In comparison with the Southern Alps and the Apennines, pelagic deposits of the Dinarides are characterized by an earlier onset and considerably higher proportions of silica with respect to carbonate throughout the Mesozoic. The Dinaric basins were connected with the central Neotethys, where the high fertility of surface waters enabled radiolarite formation since the oceanisation (Anisian or earlier) until the early Late Cretaceous, when planktonic foraminifera and calcareous nannoplankton began to dominate worldwide.
 Key words: Dinarides, Neotethys, radiolarites, continental margin, ophiolitic mélange
 
 IZVLEČEK
 V Dinaridih, Albanidih in Helenidih so ohranjena stratigrafska zaporedja vzhodnega roba Jadranske mikroplošče in ostanki ofiolitov, narinjenih na kontinent iz oceana Maliak-Vardar, ki je bil del Neotetide. Glavne stopnje v mezozojski geodinamični evoluciji tega ozemlja so bile: 1) rifting, ki je v zgornjem aniziju privedel do odprtja oceana Maliak, 2) v spodnji do srednji juri začetek intraoceanske subdukcije in raztezanje oceanskega dna v suprasubdukcijskem okolju Vardarskega oceana, 3) v srednji do zgornji juri formacija ofiolitnega melanža v jarkom podobnih bazenih in obdukcija mladih suprasubdukcijskih ofiolitov proti zahodu ter na koncu jure in v spodnji kredi akumulacija flišnih sedimentov v predgornih bazenih, 4) emerzija novo nastalih pokrovov in nato transgresija v srednji do zgornji kredi, 5) kolizija kontinentov v maastrichtiju in paleogenu. Kontinentalni rob se je med ekstenzijo od srednjega triasa do spodnje jure diferenciral na horste in grabne, kar se odraža v stratigrafskem zapisu. Današnje NW-SE usmerjene tektonske enote v Dinaridih se v grobem ujemajo z mezozojsko paleogeografijo, iz česar sklepamo, da je bila konfiguracija kontinentalnega roba v prečnem preseku čez Črno goro in Srbijo naslednja: Dalmatinska karbonatna platforma, Budvanski bazen, karbonatna platforma Visokega Krasa, Bosanski bazen, Durmitorski prag, Limski bazen, prag Drina-Ivanjica in globokomorski distalni kontinentalni rob.
 V članku je najprej na kratko opisan stratigrafski razvoj tektonskih oziroma paleogeografskih enot tega preseka in domnevna povezava z drugimi enotami v Dinaridih in Helenidih. V nadaljevanju so opisane ogledne točke ekskurzije s poudarkom na globokomorskih sedimentnih kamninah, ker so za določanje starosti teh kamnin radiolariji najpomembnejši in pogosto edini fosili. Podrobno predstavljamo celotno mezozojsko zaporedje Budvanske cone, srednjetriasno pelagično epizodo v coni Visokega Krasa, zgornjetriasne in jurske pelagične kamnine Limske cone in dve lokaliteti z radiolariti v ofiolitih.
 Največji del vodnika je posvečen Budvanski coni v Zunanjih Dinaridih. V mezozoiku je bila ta cona globokomorski jarek v nadaljevanju bazena Pindos s kontinuirano pelagično sedimentacijo od srednjega triasa do konca krede. Radiolariti so značilni za obdobje srednjega triasa, hettangija in sinemurija, aalenija do tithonija ter hauterivija-barremija do spodnjega turonija. Pelagični apnenci prevladujejo v zgornjem triasu, berriasiju in valanginiju ter od zgornjega turonija do maastrichtija. Karbonatni turbiditi, prinešeni s sosednje karbonatne platforme Visokega Krasa, so interstratificirani v vseh formacijah, v pliensbachiju pa prevladujejo in popolnoma izpodrinejo pelagične sedimente.
 Pelagična zaporedja v coni Visokega Krasa so omejena na sinriftne in zgodnje postriftne sedimente. Kot tipičen primer predstavljamo 20 m debelo zaporedje srednjetriasnih gomoljastih apnencev in radiolaritov znotraj plitvovodnih karbonatov.
 V bolj interni Limski coni je za podcono Ćehotina značilna pelagična sedimentacija od srednjega triasa do začetka krede, ko so se začeli odlagati sinorogeni mešani karbonatno-siliciklastični sedimenti. Stratigrafsko zaporedje te podcone do sedaj ni bilo podrobneje proučeno. V članku je prvič datiran 100 m debel profil apnencev z gomolji roženca, ki smo ga s konodonti uvrstili v norij in retij. Z radiolariji smo dokazali callovijsko do spodnjeoksfordijsko starost plastovitih rožencev brez karbonata. V podconi Mihajlovići je stratigrafski razvoj podoben kot v enoti Drina-Ivanjica. Triasnim plitvovodnim karbonatom sledijo jurski apnenci, ki kažejo na postopno poglabljanje sedimentacijskega okolja. Zaporedje se konča z oxfordijskimi radiolariti.
 V Črni gori so ohranjeni najbolj jugozahodno ležeči ostanki ofiolitov v Dinaridih. Zadnji dve točki prikazujeta bathonijske do spodnjecallovijske radiolarite v ofiolitnem melanžu in zgornjeanizijske radiolarite v kontaktu z bazaltom. Te starosti se ujemajo z do sedaj znanimi datacijami v ofiolitih širše regije.
 V primerjavi z Južnimi Alpami in Apenini je za pelagične sedimente Dinaridov značilno, da so se začeli odlagati prej in da so skozi ves mezozoik vsebovali znatno višji delež kremenice glede na karbonat. Dinarski bazeni so bili povezani s centralno Neotetido, kjer je visoka produktivnost površinskih voda omogočala nastanek radiolaritov od oceanizacije (v aniziju ali še prej) do sredine zgornje krede, ko so po vsem svetu začeli prevladovati foraminifere in kalcitni nanoplankton.
 Ključne besede: Dinaridi, Neotetida, radiolariti, kontinentalni rob, ofiolitni melanž
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30

Rasmussen, Uffe. "Lystrup Østergård – En værkstedsplads fra yngre stenalder." Kuml 61, no. 61 (2012): 9–73. http://dx.doi.org/10.7146/kuml.v61i61.24496.

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Lystrup ØstergårdA Neolithic workshopIn 2007 a remarkable small site dating from the later part of the Early Neolithic Funnel Beaker culture was discovered at Lystrup, north of Aarhus in Eastern Jutland. Careful total excavation of the site revealed a well-defined cultural deposit with dense concentrations of flint debitage and implements lying in situ in a shallow hollow resulting from a group of windthrows. Via a series of analyses, the distribution of the finds relative to the individual features which were demonstrated, including a central hearth, has made it possible to reconstruct the events which took place, thereby permitting a detailed characterisation of the site. It can be perceived as a workshop which lay isolated in the landscape, at some distance from the actual settlement areas.The finds primarily reflect two activities at the site: The production of blanks for thin-butted flint axes, where the raw material was obtained from the local moraine clay, and an activity which probably involved the working of bone or antler, judging from the remarkable number of burins which were recovered. Flake scrapers, normally the commonest tool type at settlements of this period, were virtually absent. The marked occurrence of burins and the site’s potential with respect to finds-distribution analysis together constitute a situation rarely encountered in a Neolithic context. Through identification of the various sequences of events, the activities acquire the character of brief targeted incidents.Analyses of small Neolithic sites and the identification of specialised workshops can make a significant contribution to our understanding of the period’s patterns of ­settlement and activity – and prompt a critical examination of the settlement models used for the period to date.In a series of regional investigations of Funnel Beaker culture settlement over the last three decades, a model has been applied whereby the sites are divided up into base settlements and hunting stations, respectively. Associated with these were sites related to the ritual sphere: offerings, graves and causewayed enclosures. Collectively, these reveal the general organisation of the population in the landscape. But this model is constructed on the basis of a general consideration that has, in particular, demonstrated changes in the settlement development through time. If we take a closer look at the individual sites in order to obtain a better understanding of the dynamics of Neolithic settlement, the pattern of ‘base settlements and hunting stations’ becomes too rigid to work with – and perhaps even misleading.LandscapeThe site lies within the broad Egå valley, formed at the end of the Ice Age by glacial erosion and melt water. In Atlantic times a 5.5 km long and 1.5 km wide fjord, Egå Fjord, extended inland from the Bay of Aarhus. In the Early Sub-Boreal, when the site was active, the fjord was partially closed at its mouth by beach ridges and constituted a sheltered, shallow brackish water environment. The site lay on a low undulating moraine surface at the foot of the hilly northern side of the valley, 0.8 km from the shore of the fjord (fig. 1).The siteOn the edge of a slight elevation running down towards a narrow, peat-filled depression, the Neolithic finds extended over an area of 47 m2 – 12 m in length and 5.5 m in width – within a shallow hollow (figs. 2-4). The hollow was characterised by two crescent-shaped features (A16, A41), together with a further oval feature to the north (A8) (see figs. 5-6). The crescent-shaped features were able to shed some light on the formation of the hollow. Their form and stratigraphy revealed that they resulted from windthrows. In an archaeological context this phenomenon is often connected with disturbances that have disrupted the stratigraphy of archaeological deposits. But in this case the trees had been blown over prior to formation of the archaeological deposits and the shallow root pits functioned as an actual surface for the activities.The archaeological deposits had an average thickness of 5 cm and comprised dark, charcoal-rich sandy clay within which there was an even spread of dense finds concentrations. These lay directly over the heavy, stony yellow moraine clay. Intervening layers, for example earlier vegetation horizons, were not encountered. In certain areas, the finds, first and foremost flint debitage, a number of flint tools and a small quantity of potsherds, lay densely concentrated in up to three layers, one above the other. In some instances, heaps of homogeneous flint and axe flakes and chips could be readily distinguished, giving the impression of relatively undisturbed episodes of flint working.In spite of a careful search, no traces of post-built dwellings were found associated with the archaeological deposits. A central hearth was, however, revealed as well as possible traces of a fence or a flimsy hut wall (figs. 9-10). The distribution and composition of the finds around the hearth revealed this to be the hub of the site’s structure, where various activities had taken place. Close to the hearth were two large stones which could have served as seats or work surfaces. An elongated flat-bottomed pit of uncertain function located directly north of the hearth should perhaps, together with the discovery of a polygonal axe, be seen as an indication of ritual activities.The orientation of the windthrow pits shows that the trees fell away from each other, resulting in the formation at the site of a small sheltered hollow with exposed moraine clay (figs.7-8). The site stratigraphy suggests that only a short period of time elapsed before the finds were deposited within this hollow. This observation prompts the article’s hypothesis that the windthrow pits gave access to the moraine clay’s rich content of flint, which was then worked in situ.The artefactsThe artefacts are predominantly of flint. In their manufacture, use was made of local moraine-deposited flint which in this area is of particularly good quality and varied type. The flint tools and flint debitage have a total weight of 74.1 kg; the tools number 295 examples, while the debitage is estimated to include c. 10,000 pieces.The distribution of flint and stone artefacts is given in tables 1-2. Almost half the flint debitage can be linked to the production of axes of thin-butted type. In addition to large quantities of various axe flakes/chips, there are seven discarded blanks/rough-outs and 20 hammerstones (fig. 16).In the tool inventory, special attention should be drawn to the 120 burins (40.7%), an unusual feature in a Neolithic context (figs. 11-14), in addition to 38 core and flake drills (12.9%) and 35 knives (11.9%) (fig. 15). Further to these are 52 small tools in the form of blades or flakes with retouch or visible use-wear (17.6%). The burins were produced on simple robust flakes that appear to have been specially produced for the purpose. Transverse burins on retouch are in the majority, followed by edge burins (table 3). One find stands out from the rest, namely half of a finely-worked polygonal axe of basalt (fig. 17). This was not made at the site.A small, poorly-preserved assemblage of pottery (1.8 kg) lay deposited in concentrations around the site. In terms of vessel forms, the presence has been demonstrated of funnel beakers, a lugged beaker and a bowl. The decoration is characterised by simple rim ornamentation, vertical belly stripes and the use of twisted cord (fig. 18). The minimum number of vessels represented in the assemblage is calculated to be seven.DatingThe typological date for the site is based on the pottery, the flint axes, the polygonal axe, denticulates, a single ‘disk knife’ and, to a certain extent, the burins. The vessel form and decoration of the pottery corresponds to the Funnel Beaker culture’s phase TN II. There are close parallels in the pottery recovered from the palisade ditch at Sarup I, which is linked to the Fuchsberg group (note 38). This date is also supported by the flint and stone tools, although these also open up the possibility of a component from the subsequent MNA I.A radiocarbon analysis of a charred seed coat from the archaeological deposits near the hearth shows, with a probability of 95.4% (±2 standard deviations), a double peak with an 8.6% probability of a date of between 3630 and 3580 BC and a 86.8% probability for 3530-3360 BC. The greatest part of the curve corresponds, accordingly, with the radiocarbon dates for Sarup I.With a possible small component from MNA I, the date for the archaeological deposits falls within the Funnel Beaker culture’s TN II phase with links to the Fuchsberg group.SubsistenceThe conditions for preservation of bone at the site were unfortunately very poor. The humus content of the archaeological deposits does, however, bear witness to the presence of a certain amount of degraded organic material. The animal remains comprise two badly-preserved teeth of, respectively, a young domestic cow and a large ruminant. Further to these, 11 small bones were found by fine sieving, of which three are fish bones, probably cod.Soil samples processed by flotation yielded 23 charred cereal grains, of which 11 were of barley and one of wheat, while the others were unidentifiable. Charred hazelnut shells featured in several samples and a single charred apple pip was recorded.A strange component of small water-rolled stones found in the deposits could possibly originate from seaweed, bladder wrack, gathered on the coast. The function of the seaweed is unclear, but there are a number of possibilities, e.g. a soft underlay, fuel, animal fodder or manure; it could also have constituted human food.Activities and activity areasThe natural sources of good raw flint in Eastern Jutland are the coastal cliffs and potentially also the banks of streams and rivers, where the flint is exposed naturally and can be gathered directly. On the forest floor of the interior, flint would have been rarely encountered. It seems therefore very likely that the hollow created by the windfalls gave very welcome access to the flint in the moraine deposits, which could then have been the subject of more systematic searches and collection. Several of the flint nodules found in the archaeological deposits have only one or a few scars resulting from blows, probably resulting from testing of the flint quality. One very large block (42 kg) was found in four pieces scattered around the site, with a few missing pieces that could have been worked further (fig. 19).The debitage from the axe production has been analysed with the aim of discovering the types and number of axes produced at the site. Several definite axe-knapping episodes have been distinguished on the basis of in situ concentrations, identification of debitage from the same flint nodules and with the aid of refitting (figs. 21-23, table 4). The flint flakes have been classified according to the use of hard and soft knapping techniques, i.e. the employment of, respectively, hammerstones and fabricators of antler, in order to discover the number of stages in the production of the four-sided axes present at the site (figs. 20, 24-27). In the course of this analysis the character and extent of the material was compared with related finds and the results of modern experiments (note 60).Large flakes retaining the original cortex of the flint show that some pieces were produced in situ from raw unworked flint nodules (stage I), whereas other examples appear to have been brought to the site as roughly-worked axe blanks (stage II). The aim of the production was the manufacture of axes up to stage III. No clear traces of stage IV, the last trimming of the axe sides and edges, or of the final polishing, stage V, could be demonstrated. A total of about 15 individual axes were worked at the site, of which about half were abandoned and discarded at the site as failures, while the finished examples were taken away to another workshop or a base settlement to be given their final finish. Through comparisons with modern experiments, the total time expenditure for the axe production is estimated as a maximum of 12 hours. If production was continuous, then all that was involved was a single day’s work for two flint knappers. The quality of the work is considered to be fully on a par with the general level in the Funnel Beaker culture.The other activity that characterises the site is apparent from the large number of burins in the assemblage. Burins are associated with the working of hard materials such as antler and bone, and this was confirmed by wear analysis of 13 pieces from the site. The activity could well have involved other elements of the inventory such as drills, knives and diverse tools with retouch. The activities took place in particular in the vicinity of the hearth, but a particularly high concentration of burins and burin spalls was found on the eastern periphery, in the deeper part of the hollow, behind a possible fence (fig. 28). This could represent the deposition of burin waste or the existence of a small isolated work place.Even though burins rarely occur in large numbers at the settlements of the period, they are occasionally present and in a few cases they are seen in large numbers as for example at the site of Grønvang 2, near Kalundborg in Western Zealand.The items which were produced could have been antler axes, chisels, bodkins or harpoons. A close relationship with the production of flint axes is also conceivable in the form of the manufacture of antler fabricators. This is, however, not supported by evidence from other flint axe workshops, where burins have never been recorded in the tool inventory.The settlement around Egå Fjord in TN II (- MNA I)The area around the site and along the northern side of the fjord has, over the course of the past 12 years, been subjected to extensive and comprehensive archaeological investigation in connection with road construction and development of building land. It is therefore now possible to see the site in a wider settlement-related perspective for the period TN II - MNA I (fig. 29). The nearest settlement-like finds have been located 325 m ENE of the workshop site, but these are difficult to evaluate in detail due to disturbance later in prehistory. Possible base settlements with the remains of houses were encountered 2.1 km north and 7 km west of the site, respectively. In addition, possible hunting stations were demonstrated on the nearby shore of the fjord. Four other sites within a 2 km radius bear witness to ritual activities; these comprise two isolated system-ditch complexes and two dolmen sites.The area within a radius of 300 m of the site has been investigated via field-walking and trial excavations, and these did not reveal the existence of any contemporaneous settlement traces here. It can therefore be reasonably securely concluded that the workshop lay at a distance from the settlement sites. It is possible that it was located on the edge of recently-established arable fields. Clearance of the primeval forest would have given the wind easy access to the old forest trees which then, at the woodland edge, became easy victims for storms.Workshop sites of the Funnel Beaker cultureDuring the Funnel Beaker culture, workshops were often associated with flint quarrying and flint-knapping sites and several of these were specifically oriented towards axe production, for example that at Hastrup Vænget in Eastern Zealand.Apart from axe production, specialised workshop activities have rarely been recognised in the Funnel Beaker culture. The above-mentioned Grønvang 2 on Zealand resembles Lystrup Østergård with respect to its size and a large content of burins. Another site with a specialised activity is Studeli Klit in Northern Jutland, characterised by a huge number of flake drills.Neolithic sites that were not actual ordinary settlements but sites for special workshop activities are possibly under-represented in the overall archaeological record, either because they are small and easily overlooked during archaeological investigations or because their uniform and more specific site circumstances are more vulnerable to repeated and possibly also changing use of the localities. Several of the sites we perceive as base settlements could possibly represent the accumulated remains of more specialised activities. An important feature type relative to so-called base settlements is the house! Investigations of Scandinavian house remains from the period have demonstrated a clear tendency for houses, activity areas and refuse deposits not to be located in the same place; there may possibly have been rules with respect to cleanliness around settlement areas. This tendency has subsequently been demonstrated in connection with new archaeological investigations in Scania and in the Sarup area in SW Funen.Consequently, we must see settlement and activities in the early agricultural society as a more widespread and dynamic use of the landscape. In future regional investigations it will be important to look critically at the term ‘settlement’ and distinguish to a greater degree between sites for activities, refuse deposition and habitation. During excavations we should be aware of the minor find complexes and focus on their possible unique features – and remember that houses are to be looked for at some distance from the find-rich areas.Uffe RasmussenMoesgård Museum
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"Neurological Etiologies Necessitating Elective Tracheostomy in Pediatric Age Group: A 100 Patient Experience." International Journal of Medical Science in Clinical Research and Review 05, no. 05 (2022): 645–51. https://doi.org/10.5281/zenodo.7105759.

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<strong>Original Research Paper</strong> <strong>Neurological Etiologies Necessitating Elective Tracheostomy in Pediatric Age Group: A 100 Patient Experience</strong> <strong>Authors:</strong> <strong><sup>1</sup></strong><strong>Anju Benny, <sup>2</sup>Muhammad Faraz Shamshad, <sup>3</sup>Soumyadeep Sikdar, <sup>4</sup>Shreshtha Singh, <sup>5</sup>Amir Hassan, <sup>6</sup>Yi Sin Leong, <sup>7</sup>Areeba Siddiqui, <sup>8</sup>Fazila Imtiaz,<sup>9</sup> Hafsa Ashraf, <sup>10</sup>Misha Anam</strong> <em><sup>1</sup></em><em>ACS Medical College and Hospital(India)</em> <em><sup>2</sup></em><em>Karachi Medical &amp; Dental College, Karachi (Pakistan)</em> <em><sup>3</sup></em><em>Institute of Medical Sciences, Benaras Hindu University (India)</em> <em><sup>4</sup></em><em>Synergy Hospital, Dehradun, Uttarakhand (India)</em> <em><sup>5</sup></em><em>Rehamn Medical Institute (RMI), Peshawar (Pakistan)</em> <em><sup>6</sup></em><em>Universiti Kebangsaan Malaysia Medical Centre (Malaysia)</em> <em><sup>7</sup></em><em>Dow Medical College (Pakistan)</em> <em><sup>8</sup></em><em>Lahore Medical &amp; Dental College, Lahore (Pakistan)</em> <em><sup>9</sup></em><em>Shalamar Hospital, Lahore (Pakistan)</em> <em><sup>10</sup></em><em>Children Hospital &amp; The Institute of Child Health, Lahore (Pakistan)</em> Corresponding Author: Misha Anam, Children Hospital &amp; The Institute of Child Health, Lahore (Pakistan) <strong>Article Received:</strong>&nbsp; 10-08-2022&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; <strong>Revised:</strong>&nbsp; 31-08-2022&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; <strong>Accepted:</strong> 21-09-2022 <strong>ABSTRACT</strong><strong>:</strong> <strong>Background: </strong>Tracheostomy is a life-saving procedure and is indicated in a variety of peripheral or central conditions leading to respiratory compromise. Mainly, tracheostomy is done for three reasons; to bypass obstructed upper airway, to remove secretions and keep the airway patent; and to effectively ventilate the patient.<strong> Objective:</strong> The aim of this study is to quantify the frequency of different neurological disorders which indicate elective tracheostomy to avoid respiratory compromise and its associated complications.<strong> Materials and Methods:</strong> In this Prospective, observational study, 100 pediatric patients with confirmed neurological diagnosis and associated severe respiratory impairment requiring invasive mechanical ventilation were enrolled. The study was conducted over the period of 3 months, from May 2022 to July 2022, at Children Hospital and Institute of Child Health, Lahore.<strong> Results: </strong>According to the collected data, prevalence of male patients was almost twice as that of females (2.9:1). The main indication of tracheostomy was the requirement of prolonged mechanical ventilation due to neurological condition (n= 36, p&lt;0.02). Among all the neurological etiologies, the CNS infections with serious respiratory manifestations were most common including Guillain Barre Syndrome (n=26, p=&lt;0.01), Diphtheria Polyneuropathy (n=8, p=0.06), Hypoxic-Ischemic Encephalopathy (n=5, p&lt;0.08), Varicella Zoster Infection (n=3, p=0.09), Poliomyelitis / Post-Polio Syndrome (n=2, p&lt;0.1), and Tetanus (n=1, p=0.15).<strong> Conclusion: </strong>Infectious neurological diseases such as GBS, Diphtheria, and polio, causing respiratory failure are still prevalent in developing countries like Pakistan &amp; India and are the main causes of tracheostomy in pediatric age group. Prolonged mechanical ventilation was the main indication for tracheostomy. <strong><em>Keywords: tracheostomy, pediatrics, infections, GBS, ETT, neurological intensive care</em></strong> &nbsp; &nbsp; <strong>INTRODUCTION</strong><strong>:</strong> Tracheostomy is one of the ancient surgical procedures to bypass upper airway tract obstructions. About 40 years ago, pediatric tracheostomy was only indicated in the cases of infectious diseases of upper airway such as Diphtheria, Epiglottitis etc. [1]. Over the time, the indications and applications of tracheostomy were revised and evolved, considerably. With the administration of vaccines, the incidence of infectious diseases caused by <em>Cornybacterium Diphtheria</em> and <em>Hemophillus Influenzae</em> has decreased dramatically [2]. Hence, the number of pediatric emergency tracheostomies has fallen, globally. However, with the introduction of other indications of tracheostomy, the overall number of tracheostomies has increased over time [3]. Worldwide, the pediatric tracheostomy is performed in tertiary care facilities only, in children with acute or chronic, congenital, or acquired respiratory and/or neurological impairment, and in children with long term dependency on invasive mechanical ventilation [4]. There are a number of neurological etiologies that can result in severe respiratory impairments. Emergent Endotracheal Tube Placement with mechanical ventilation can save the patient temporarily but if the long term invasive mechanical ventilation is required, ETT must be replaced by tracheostomy [5]. According to the experts, if patient needs ETT and mechanical ventilation for 7 days or more, then tracheostomy should be performed for sustainable and secured airway [6]. Although in some cases of severe neurological impairment, patients need ETT assisted mechanical ventilation for a longer period of time, for example in case of a brain injury with poor GCS and unconsciousness, tracheostomy cannot protect the airway against secretions and gastric aspiration as ETT does [7]. Tracheostomy decreases the work required for breathing but that is not an issue for the patients with brain injury. Prolonged ETT intubation is associated with high risk of nosocomial pneumonia and prolonged ventilator dependency which can be significantly reduced by tracheostomy. Prolonged ETT intubation is also associated with a number of tracheal complications especially in the patients with severe brain trauma. If patient is intubated for more than 14 days, there is a high risk of development of serious complications such as laryngeal injury including laryngeal edema and inflammation, vocal cord injury, ulceration, paresis, paralysis, granuloma, and stenosis. So, it is important to weigh the pros and cons after regular and careful clinical evaluations of the patients [8]. There are a variety of neurological disorders which require tracheostomy as a part of an active or long-term management plan. These diseases/disorders include congenital/chromosomal/neurometabolic, idiopathic, traumatic, infective, autoimmune, and inflammatory disorders [9]. The incidence of infective neurological causes has decreased worldwide over the time, by the virtue of effective immunization. Nevertheless, these are still prevalent in developing countries due to poor vaccination coverage and healthcare provision. There are several neurological infections that cause acute flaccid paralysis and hence the respiratory compromise. The mechanism of respiratory disability may vary slightly, but the ones that are mentioned in the article required tracheostomy at some point in the management of the disease [9, 10]. Among all the acute neurological infections, Guillian Barre Syndrome (GBS) is the most common cause of peripheral, motor neuropathy with presumed antecedent infection and autoimmune etiology. Usually, the infection caused by <em>Campylobacter Jejuni</em> precedes the GBS with rapidly progressive axonal injury, ascending paralysis, respiratory failure, severe residual disability and slow recovery [11]. Globally, the incidence of GBS ranges from 1.1 to 1.8 per 100,000 people, annually [12]. The overall incidence of GBS increases with the age; peaks after 50 years. In Pakistan, the exact incidence and prevalence is not known. Electrophysiological studies for early detection, definitive diagnosis, and classification of GBS play an important role in significant reduction of morbidity and disability [13]. Poliomyelitis or Post Polio Syndrome is caused by highly infective, Enterovirus via feco-oral route [14]. It is a type of acute flaccid paralysis associated with prolonged or permanent flaccid muscle paralysis, respiratory failure, and death. Due to combined efforts, this sporadic, highly contagious infection is successfully eradicated from the world, except some areas of Sub-Saharan Africa and South Asia where it is still an endemic. In 95% of the infectious cases, the patients either remain asymptomatic or have a flu-like illness [15]. Despite effective preventive and management policies, some patients develop late functional complications after a long period of complete stability and recovery. This condition is called Post-Polio Syndrome. The bulbar form of Poliomyelitis involves high morbidity and mortality due to vasomotor, circulatory, and autonomic dysfunction along with respiratory failure. The acute respiratory failure needs emergent intubation and prolonged mechanical ventilation which can later indicate elective tracheostomy [16]. Diphtheritic Polyneuropathy (DP) is a serious bulbar, respiratory, and circulatory complication. It is a direct and acute indication for intubation and tracheostomy [17]. The etiology causes a decrease in lung&rsquo;s vital capacity (&lt;16mL/kg body weight), paralytic collapse of layrngeal muscles, and multiple cranial nerves impairment causing peripheral motor disturbances. Diphtheritic Polyneuropathy is also a late complication of diphtheria infection, so special attention should be given from the fourth to seventh week of DP [18]. The objective of this study is to determine the current neurological indications of tracheostomy in the pediatric age group and to assess the burden of infectious causes with central involvement in developing countries like Pakistan, despite the discovery and implementation of effective immunization all around the globe. <strong>MATERIAL &amp; METHODS</strong><strong>:</strong> In this prospective, observational study, 100 patients were recruited by following the inclusion and exclusion criteria. The study was conducted over the period of 3 months, from May 2022 to July 2022 at a tertiary care hospital. The Qualitative and Quantitative data was collected for 100 selected patients who were registered through the CH &amp; ICH, Lahore. Multi-departmental Collaboration was ensured. Verbally explained, written informed consent was taken from the guardians, mostly from the parents of the children. The whole purpose and procedure of this study was explained to the attendants. Few parents refused to take part in the study, their rights were respected, and they were not included in our study. By any means, this study does not adversely affect the rights and welfare of the subjects. All the patients had some neurological manifestation as a primary cause for elective tracheostomy. Those patients were also not included in the study which needed tracheostomy due to some other primary cause while the neurological condition co-existed. <strong>INCLUSION CRITERIA:</strong> Patients diagnosed with neurological impairments that were compromising the respiration by any means, i.e. Upper airway obstruction, Vocal cord paralysis/paresis, respiratory muscle paralysis/paresis, altered sensorium, skeletal/laryngeal muscle weakness, prolonged mechanical ventilation, etc. Patients who were on mechanical ventilation for &gt; 7 days Patients who required prolonged mechanical ventilation or on whom, ventilator weaning off attempts were performed but failed <strong>EXCLUSION CRITERIA:</strong> Patients of age more than 14 years Patients who needed tracheostomy for causes other than neurological anomaly Patients with incomplete previous medical record Patients with underlying diseases (other than neurological issues) such as hypothyroidism, diabetes, hepatic or renal dysfunction, vasculitis, metastasis, intoxication, etc. <strong>RESULTS</strong><strong>:</strong> According to the collected data, there were 67 males (p &lt;0.02) and 23 females (&lt;0.04) with male to female ratio of 2.9:1. The peak incidence of tracheostomy is seen at the ages of 2-5 years (n=29, p &lt;0.05) and 6-12 months (n=21, p&lt;0.05). P value less than 0.05 is considered significant. The demographic characteristics including age group and gender are described in Table 1. &nbsp; <strong>Demographic Characteristics</strong> <strong>Frequency (%age)</strong> <strong>P-value</strong> <strong>Age</strong> 0-6 months 16 (16%) Not Significant 6-12 months 21 (21%) &lt;0.05 1-2 years 11 (11%) Not Significant 2-5 years 29 (29%) &lt;0.05 5-10 years 10 (10%) Not Significant 10-14 years 13 (13%) Not Significant <strong>Gender</strong> Male 67 (67%) &lt;0.02 Female 23 (23%) &lt;0.04 <strong>Table 1 Demographic Characteristics of selected children (n=100)</strong> &nbsp; Eight different indications of elective tracheostomy in pediatric population were documented in this study. In descending order, the 3 main indications are prolonged invasive mechanical ventilation (n=36, p=&lt;0.02), Upper airway obstruction (n=20, p=&lt;0.04), and Metabolic/ Genetic/ Chromosomal anomalies (n=16. p=&lt;0.05). &nbsp; &nbsp; &nbsp; <strong>Indications of Tracheostomy</strong> <strong>Frequency (%age)</strong> <strong>P-value</strong> Upper Airway Obstruction 20 (20%) &lt;0.04 Prolonged Invasive Mechanical Ventilation 36 (36%) &lt;0.02 Hypoxic Brain Injury 8 (8%) Not Significant Metabolic / Genetic / Chromosomal 16 (16%) &lt;0.05 Cerebral Hemorrhage 4 (4%) Not Significant CNS Infection 9 (9%) Not Significant Demyelinating Disorder 5 (5%) Not Significant Brain Tumor 2 (2%) Not Significant <strong>Table 2 Indications of Elective Tracheostomy in children with neurological disease associated respiratory compromise</strong> &nbsp; Among the 100 selected patients, 16 different neurological conditions were documented, all leading to the respiratory failure by different mechanisms. Surprisingly, 45% of the total patients had neurological manifestation due to infectious etiology. The infectious causes of neurological conditions include (in descending order) Guillain Barre Syndrome (n=26, p=&lt;0.01), Diphtheria Polyneuropathy (n=8, p=0.06), Hypoxic-Ischemic Encephalopathy (n=5, p&lt;0.08), Varicella Zoster Infection (n=3, p=0.09), Poliomyelitis / Post-Polio Syndrome (n=2, p&lt;0.1), and Tetanus (n=1, p=0.15). &nbsp; <strong>Neurological Conditions</strong> <strong>Frequency (%age)</strong> <strong>P- value</strong> Poliomyelitis / Post-Polio Syndrome 2 (2%) Not Significant Guillain Barre Syndrome 26 (26%) &lt;0.01 Myasthenia Gravis 14 (14%) &lt;0.03 Diphtheria Polyneuropathy 8 (8%) Not Significant Varicella Zoster Infection 3 (3%) Not Significant Duchenne Muscular Dystrophy &nbsp;5 (5%) Not Significant Hypoxic-Ischemic Encephalopathy 5 (5%) Not Significant Cerebral Palsy 8 (8%) &lt;0.05 Spinal Muscular Atrophy (SMA) 3 (3%) Not Significant Neurofibromatosis 4 (4%) Not Significant Brain Tumors 6 (6%) Not Significant Kernicterus 5 (5%) Not Significant Leukodystrophy 2 (2%) Not Significant Amyotrophic Lateral Sclerosis 3 (3%) Not Significant Traumatic Brain Injury 5 (5%) Not Significant Tetanus 1 (1%) Not Significant <strong>Table 3 Spectrum and frequency of various neurological conditions necessitating elective tracheostomy</strong> &nbsp; During the period of 3 months of this study, no tracheostomy related mortality was documented. None of the patients underwent decannulation. The limitations of this study are that the duration of three months period did not allow the proper follow up of the patient and the prognosis, duration, and late complications of tracheostomy could not be assessed. <strong>DISCUSSION</strong><strong>:</strong> Tracheostomy is an operative procedure that creates a surgical opening on the anterior aspect of cervical trachea, mainly for effective ventilation [1, 2]. Patients with tracheostomy are a heterogeneous group of population with various indications and associated conditions. In this study, we only included the patients from pediatric age group requiring elective tracheostomy due to any congenital or acquired neurological condition [3, 4]. Children with congenital or acquired neurological disorders are prone to have respiratory co-morbidities [5]. There are multiple neurological manifestations that cause respiratory compromise such as pharyngeal/ laryngeal muscle weakness leading to Vocal cords dysfunction, respiratory muscle weakness or paralysis causing hypoventilation, skeletal muscle weakness i.e. SMA associated with prolonged immobility, pooling of respiratory secretions in respiratory tract, recurrent lung infections, and irreversible destruction of lung parenchyma [6, 7]. In patients with Cerebral Palsy and other neuromuscular disorders, pharyngeal and laryngeal muscles fail to maintain the patency of upper airway tract against inspiratory negative pressure [8, 9]. The hypotonic pharynx tends to remain collapsed causing upper airway narrowing or obstruction. Whenever, the patient develops acute upper or lower respiratory tract infection, the condition worsens with additional mucosal swelling and pooling of respiratory secretions due to ineffective clearing of sputum [10]. Moreover, the muscular weakness also results in disrupted swallowing mechanism causing GERD (Gastro-Esophageal Reflux Disease) and micro aspirations due to prolonged immobility and loss of protective cough reflex. All these factors make the patient prone to develop permanent lung damage via recurrent bronchitis, bronchiectasis, and pneumonia [11]. Tracheostomy has several therapeutic as well as palliative applications in patients with neurological diseases. As discussed in this article, tracheostomy provides secure and sustainable pathway for short or long-term invasive mechanical ventilation. It secures the breathing pathway in case of upper respiratory tract obstruction. It reduces the physiological dead space and significantly reduces the work required for breathing [12]. It provides effective, less traumatic and easily accessible route for airway toileting via sputum suctioning which is essential, especially during acute respiratory tract infections. In the case of emergency positive pressure ventilation, tracheostomy provides a quick, accessible route and significantly decreases the morbidity and mortality caused by delayed or unsuccessful attempts of endotracheal tube placement. Although noninvasive ventilation is an alternative emergency maneuver, but in the patients with neurological diseases, the invasive ventilation is preferred due to the high risks of secretions and gastric content aspiration. It also provides a safe route for diagnostic as well as therapeutic endoscopy, reduces the duration of procedure, and significantly minimizes the procedure associated risks [13, 14]. In most of the conditions, early tracheostomy proves to be highly beneficial in terms of decreasing morbidity and disability [23]. For example, in GBS, early tracheostomy can end the dependency on mechanical ventilator, providing more comfort and a chance of commencement of early oral nutrition with adequate oral hygiene that can speed up the weaning process [24]. It also adds to the parent&rsquo;s satisfaction and patient&rsquo;s motivation for the quick recovery. But the decision of tracheostomy is often critical, and it needs expert&rsquo;s opinion and evaluation to assess the pros and cons of tracheostomy. In the neurological cases where there is an increased risk of aspiration, ETT intubation with inflated cuff provides more protection and safer airway. However, ETT is not a sustainable way to maintain airway and is associated with various early and late complications [25-27]. In the literature, the indications of tracheostomy are not found consistent throughout the world. In developed countries, tracheostomy is mainly indicated in traumatic, chromosomal, autoimmune, or inflammatory conditions [15-18]. Whereas, in developing countries, the major causes requiring tracheostomy are infections and their peripheral and central ramifications. In Pakistan, the vaccination rate in children is considerably lower than that of developing countries due to ineffective surveillance and lack of general public awareness [19, 20]. However, the unvaccinated or partially vaccinated are not the only ones susceptible to infectious diseases. The severe neurological infectious diseases such as GBS, Diphtheria etc., are also found common in fully immunized children. In that case, there are multiple reasons to consider, but the experts speculate that the main reason for this poor coverage is low quality vaccination. In developing countries like Pakistan, less immunogenic vaccinations are prevalent because of limited resources and cost effectiveness [21, 22] <strong>CONCLUSION</strong> This study demonstrates the frequency of different neurological conditions with significant respiratory complications that necessitate surgical tracheostomy. All over the world, the infectious causes of tracheostomy have almost diminished but these are still prevalent in our setup. According to this study, prolonged mechanical ventilation was the most common indication and GBS was the most common neurological condition requiring elective tracheostomy for better prognosis in children. <strong>REFERENCES</strong><strong>:</strong> Monteiro S, Farias TP, Camargo Millen MD, Locio RV. The History of Tracheostomy. InTracheostomy 2018 (pp. 1-9). Springer, Cham. Noorulla KA, Kumar MK, Talat M, Rajesh P. EVALUATION OF THE INDICATIONS AND OUTCOMES OF TRACHEOSTOMY DONE IN ICU: A CLINICO-OBSERVATION STUDY. 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Respiratory care. 2021 Jan 1;66(1):144-55. Swain SK, Sahu MC, Choudhury J, Bhattacharyya B. Tracheostomy among paediatric patients: Our experiences at a tertiary care teaching hospital in Eastern India. Pediatria Polska-Polish Journal of Paediatrics. 2018;93(4):312-7. Bontempo LJ, Manning SL. Tracheostomy emergencies. Emergency Medicine Clinics. 2019 Feb 1;37(1):109-19. Chia AZ, Ng ZM, Pang YX, Ang AH, Chow CC, Teoh OH, Lee JH. Epidemiology of pediatric tracheostomy and risk factors for poor outcomes: an 11-year single-center experience. Otolaryngology&ndash;Head and Neck Surgery. 2020 Jan;162(1):121-8. Veder LL, Joosten KF, Zondag MD, Pullens B. Indications and clinical outcome in pediatric tracheostomy: Lessons learned. International Journal of Pediatric Otorhinolaryngology. 2021 Dec 1;151:110927. Rees JH, Soudain SE, Gregson NA, et al. Campylobacter jejuni infection and Guillain&ndash;Barr&eacute; syndrome. New Engl J Med. 1995;333:1374-9. McGrogan A, Madle GC, Seaman HE, et al. The epidemiology of Guillain-Barr&eacute; syndrome worldwide. Neuroepidemiology. 2009;32:150-63. Yakoob MY, Rahman A, Jamil B, et al. Characteristics of patients with Guillain Barre Syndrome at a tertiary care centre in Pakistan during 1995-2003. J Pakistan Med Assoc. 2005;55:493. World Health Organization.&nbsp;<em>Geneva declaration for the eradication of poliomyelitis</em>. Geneva: WHO, 2004. Lancet T. Poliomyelitis-eradication initiative&#39;s wider lessons. The Lancet. 2004 Jan 10;363(9403):93. Sejvar JJ. West Nile virus and &ldquo;poliomyelitis&rdquo;. Neurology. 2004 Jul 27;63(2):206-7. Piradov MA, Pirogov VN, Popova LM, Avdunina IA. Diphtheritic Polyneuropathy:&nbsp;Clinical Analysis of Severe Forms.&nbsp;<em>Arch Neurol.</em>&nbsp;2001;58(9):1438&ndash;1442. doi:10.1001/archneur.58.9.1438 Gampa M, Karna PN, Reddy KV, Priyadarshini T. Study of Diphtheria and Its Complications: A Retrospective Study from a Tertiary Care Hospital. Pediatric Infectious Disease. 2021 Oct;3(4):141. Lancet T. Poliomyelitis-eradication initiative&#39;s wider lessons. The Lancet. 2004 Jan 10;363(9403):93. Iqbal W, Sayed TM, Wali W, Ahmed N, Butt AW, Gill ZA. Is Guillain-Barr&eacute; syndrome different in Pakistan?. PAFMJ. 2018 Feb 28;68(1):119-24. Papri N, Islam Z, Leonhard SE, Mohammad QD, Endtz HP, Jacobs BC. Guillain&ndash;Barr&eacute; syndrome in low-income and middle-income countries: challenges and prospects. Nature Reviews Neurology. 2021 May;17(5):285-96. Samar SS, Ahmed SI, Bareeqa SB. Guillain&ndash;Barr&eacute; syndrome in Pakistan: A short review of literature. J Neurol Neurorehabil Res. 2018; 3 (1): 34-35. J Neurol Neurorehabil Res 2018 Volume 3 Issue. 2018;1. Sachdev A, Chaudhari ND, Singh BP, Sharma N, Gupta D, Gupta N, Gupta S, Chugh P. Tracheostomy in Pediatric Intensive Care Unit&mdash;A Two Decades of Experience. Indian Journal of Critical Care Medicine: Peer-reviewed, Official Publication of Indian Society of Critical Care Medicine. 2021 Jul;25(7):803. Gobatto AL, Besen BA, Tierno PF, Mendes PV, Cadamuro F, Joelsons D, Melro L, Park M, Malbouisson LM. Comparison between ultrasound-and bronchoscopy-guided percutaneous dilational tracheostomy in critically ill patients: a retrospective cohort study. Journal of Critical Care. 2015 Feb 1;30(1):220-e13. Chew JY, Cantrell RW. Tracheostomy: complications and their management. Archives of Otolaryngology. 1972 Dec 1;96(6):538-45. McClelland RM. Complications of tracheostomy. British Medical Journal. 1965 Sep 9;2(5461):567. Dane TE, King EG. A prospective study of complications after tracheostomy for assisted ventilation. Chest. 1975 Apr 1;67(4):398-404.
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Ritter, Joachim R. R., Yvonne Fröhlich, Yasmin Sanz Alonso, and Michael Grund. "Short-scale laterally varying SK(K)S shear wave splitting at BFO, Germany — implications for the determination of anisotropic structures." Journal of Seismology, October 6, 2022. http://dx.doi.org/10.1007/s10950-022-10112-w.

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AbstractThe splitting of SK(K)S phases is an important observational constraint to study past and present geodynamic processes in the Earth based on seismic anisotropy. The uniqueness of the derived models is unclear in most cases, because the azimuthal data coverage is often limited due to recordings from only a few backazimuthal directions. Here, we analyze an exceptional dataset from the permanent broadband seismological recording station Black Forest Observatory (BFO) in SW Germany with a very good backazimuthal coverage. This dataset well represents the potential teleseismic ray paths, which can be observed at Central European stations. Our results indicate that averaging splitting parameters over a wide or the whole backazimuthal range can blur both vertical and lateral variations of anisotropy. Within the narrow backazimuthal interval of 30–100°, we observe a complete flip of the fast polarization direction. Such a splitting pattern can be caused by two layers with about NW–SE (lower layer) and NE-SW (upper layer) fast polarization directions for shear wave propagation. However, the possible model parameters have quite a large scatter and represent only the structure to the northeast of BFO. In contrast, within the wide backazimuthal range 155–335°, we prevailingly determine null splits, hence, no signs for anisotropy. This null anomaly cannot be explained satisfactorily yet and is partly different to published regional anisotropy models. Our findings demonstrate that there is significant small-scale lateral variation of upper mantle anisotropy below SW Germany. Furthermore, even low-noise long-term recording over 25 years cannot properly resolve these anisotropic structural variations.
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Misch, Jonathan, Sawsan Abu‐Reyal, Danyal Lohana, et al. "Combined Effect of Abutment Height and Restoration Emergence Angle on Peri‐Implant Bone Loss Progression: A Retrospective Analysis." Clinical Oral Implants Research, February 10, 2025. https://doi.org/10.1111/clr.14408.

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ABSTRACTIntroductionThis study aimed to investigate the combined effect of trans‐mucosal abutment height (TmAH) and restorative emergence angle (REA) on marginal bone loss (MBL) around bone‐level implants.MethodsImplant radiographs 12–18 months after crown placement (T0) and at least one year later (T1) were retrospectively analyzed. Sites were separated into four groups: Long/Narrow‐Angle (LN) with TmAH &gt; 2 mm and REA &lt; 30°, Long/Wide‐Angle (LW) with TmAH &gt; 2 mm and REA ≥ 30°, Short/Narrow‐Angle (SN) with TmAH &lt; 2 mm and REA &lt; 30°, and Short/Wide‐Angle (SW) with TmAH &lt; 2 mm and REA ≥ 30°. MBL was calculated, and multiple linear regression analysis was performed to control for patient‐level and implant/prosthesis‐level factors.Results192 implants pertaining to 119 patients were included. Group significantly influenced MBL experience (p &lt; 0.001). Group SW experienced on average 0.48 mm (95% CI: 0.25–0.71, p &lt; 0.001), 0.43 mm (95% CI: 0.18–0.68, p = 0.001), and 0.25 mm (95% CI: 0.00–0.45, p = 0.013) greater MBL compared to Groups LN, LW, and SN, respectively. Group was also a significant factor impacting the development of peri‐implantitis (p = 0.041), with Group SW displaying a roughly 4× greater likelihood of having peri‐implantitis (PI) diagnosed compared to Groups LN (OR: 4.04; p = 0.091) and LW (OR: 4.19; p = 0.013). Every 1 mm increase in TmAH significantly decreased the likelihood of MBL &gt; 0 mm (OR = 0.63; p = 0.003).ConclusionsAbutment height &gt; 2 mm may play a role in reducing PI and MBL related to ≥ 30° REA around bone‐level implants. REA was found to only be a significant factor when TmAH is less than 2 mm.
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Donkers, Alex, Bauke de Vries, and Dujuan Yang. "Creating occupant-centered digital twins using the Occupant Feedback Ontology implemented in a smartwatch app." Semantic Web, November 8, 2022, 1–26. http://dx.doi.org/10.3233/sw-223254.

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Occupant feedback enables building managers to improve occupants’ health, comfort, and satisfaction. However, acquiring continuous occupant feedback and integrating this feedback with other building information is challenging. This paper presents a scalable method to acquire continuous occupant feedback and directly integrate this with other building information. Semantic web technologies were applied to solve data interoperability issues. The Occupant Feedback Ontology was developed to describe feedback semantically. Next to this, a smartwatch app – Mintal – was developed to acquire continuous feedback on indoor environmental quality. The app gathers location, medical information, and answers on short micro surveys. Mintal applied the Occupant Feedback Ontology to directly integrate the feedback with linked building data. A case study was performed to evaluate this method. A semantic digital twin was created by integrating linked building data, sensor data, and occupant feedback. Results from SPARQL queries gave more insight into an occupant’s perceived comfort levels in the Open Flat. The case study shows how integrating feedback with building information allows for more occupant-centric decision support tools. The approach presented in this paper can be used in a wide range of use cases, both within and without the architecture, building, and construction domain.
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Chan, Lilian, Mattie Adams, Tomas S. Aleman, and Steven M. Bloom. "Intrafamilial Variability of IMPG1-Associated Vitelliform Dystrophy." RETINAL Cases & Brief Reports, April 25, 2025. https://doi.org/10.1097/icb.0000000000001772.

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Purpose: To describe the clinical and multimodal imaging findings of two siblings with different vitelliform phenotypes associated with a novel IMPG1 gene deletion. Methods: Case series of two siblings. Patients underwent standard optical coherence tomography (OCT), fundus color and short-wavelength fundus autofluorescence (SW-FAF) imaging, fluorescein angiography, and genetic testing for a panel of inherited retinal disorders. Results: A 43-year-old brother and his 41-year-old sister were found to have bilateral vitelliform lesions. The brother presented with single bilateral foveal lesions whereas his asymptomatic sister had multifocal extrafoveal lesions. OCT imaging demonstrated classic subretinal vitelliform lesions in various stages. SW-FAF confirmed that the lesions were hyperautofluorescent. Genetic testing identified an identical heterozygous exon 7 deletion of IMPG1 in both patients. Conclusion: Segregation of a novel deletion in exon 7 of IMPG1 in a family with a vitelliform macular dystrophy, together with a previously reported similar phenotype in patients with heterozygous nucleotide changes within this region of the gene supports the pathogenicity of this variant. Intrafamilial variability in the topography of the lesions with multifocality in one of the siblings suggests retina-wide abnormalities with individual modifiers modulating disease expression.
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Hosseini Beghaeiraveri, Seyed Amir, Jose Emilio Labra Gayo, Andra Waagmeester, et al. "Wikidata subsetting: Approaches, tools, and evaluation." Semantic Web, December 27, 2023, 1–27. http://dx.doi.org/10.3233/sw-233491.

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Wikidata is a massive Knowledge Graph (KG), including more than 100 million data items and nearly 1.5 billion statements, covering a wide range of topics such as geography, history, scholarly articles, and life science data. The large volume of Wikidata is difficult to handle for research purposes; many researchers cannot afford the costs of hosting 100 GB of data. While Wikidata provides a public SPARQL endpoint, it can only be used for short-running queries. Often, researchers only require a limited range of data from Wikidata focusing on a particular topic for their use case. Subsetting is the process of defining and extracting the required data range from the KG; this process has received increasing attention in recent years. Specific tools and several approaches have been developed for subsetting, which have not been evaluated yet. In this paper, we survey the available subsetting approaches, introducing their general strengths and weaknesses, and evaluate four practical tools specific for Wikidata subsetting – WDSub, KGTK, WDumper, and WDF – in terms of execution performance, extraction accuracy, and flexibility in defining the subsets. Results show that all four tools have a minimum of 99.96% accuracy in extracting defined items and 99.25% in extracting statements. The fastest tool in extraction is WDF, while the most flexible tool is WDSub. During the experiments, multiple subset use cases have been defined and the extracted subsets have been analyzed, obtaining valuable information about the variety and quality of Wikidata, which would otherwise not be possible through the public Wikidata SPARQL endpoint.
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S. C. Stiros. "Late Holocene relative sea level changes in SW Crete: evidence of an unusual earthquake cycle." Annals of Geophysics 39, no. 3 (1996). http://dx.doi.org/10.4401/ag-3999.

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Coastal challenges ill West Crete ill the last 4000 years can be described as a series of 11 relatively small (25 cm on the average) land subsidences alternating with short (150-250 year long) relatively still stands of the sea level. At 1500 B.P. an up to 9 m episodic relative land uplift and tilting of this part of the island occurred, but since then no significant coastal changes have been identified. There is strong evidence that these Late Holocene coastal changes are not a product of fluctuations of sea level, but reflect palaeoseismic events. The sequence of the latter is at variance with models of seismic deformation deduced from a wide range of observations in different tectonic environments, including coastal uplifts near major trenches: according to these models, strain buildup and release through earthquakes is described as a cyclic and rather uniform process, the earthquake cycle. In this process, the permanent seismic deformation accumulates after each earthquake to produce geological features, while the long-term deformation rate is approximately equal to the short term one. Obviously this is not the case with West Crete. The unusual pattern of seismic deformation in this island has been observed in other cases as well, but its explanation is not easy. The juxtaposition of different earthquake cycles, variations in the source and rate of stress or internal deformation of the uplifted hanging wall of a thrust in the pre-seismic period are some possible explanations for this unusual pattern of earthquake cycle in Greece.
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Sadaquat, Ali, Lahcen El Iysaouy, Mhammed Lahbabi, et al. "A matlab-based modelling to study and enhance the performance of photovoltaic panel configurations during partial shading conditions." Frontiers in Energy Research 11 (October 3, 2023). http://dx.doi.org/10.3389/fenrg.2023.1169172.

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Introduction: The utilization of solar energy in large-scale photovoltaic arrays has gained immense popularity on a global scale. However, shadows in the array lead to significant reductions in power output and create multiple power peaks in the P-V characteristics. To address this issue, the Total Cross Tie (TCT) interconnection pattern is commonly employed to minimize mismatch loss. Additionally, physical relocation methods have proven effective in dispersing shadows.Method: In this context, the Magic Square View (MSV) offers a physical rearrangement of PV modules within a TCT scheme, effectively scattering shadows across the entire photovoltaic array.Results: Simulation results confirm the MSV efficacy in enhancing the PV array’s output power under various Partial Shading Conditions (PSCs) patterns. Four PSCs patterns (Short and Wide, Long and Wide, Long and Narrow, and Short and Narrow) are considered and compared to the TCT and the recently validated Competence Square (CS) techniques. The MSV method is vital in improving the PV array’s power output, especially when confronted with Long and Wide shading patterns. The outcomes demonstrate that adopting the MSV configuration leads to a substantial increase of 33.78% and 29.83% in power output for LW and SW shading patterns, respectively, compared to the TCT setup. Even under LN and SN shading patterns, there is a notable power enhancement, achieving a remarkable 25.15% increase for the LW shading pattern compared to the TCT, surpassing enhancements achieved by SuDoKu, DS, and CS methods, which improved by 20.5%, 18.2%, and 21.6%, respectively. Overall, the MSV configuration presents a promising solution for enhancing the performance of photovoltaic arrays under shading conditions.
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Raiskaya, Yulia Timoshok Evgeny. "Ecological peculiarities of Cypripedium guttatum Sw. in the Southern Evenkia («Tungusskiy» reserve)." October 4, 2022. https://doi.org/10.5281/zenodo.7141581.

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Cypripedium guttatum Sw. is a species of the orchids, which had a wide range (East Europe. Asia, North&nbsp; America) but this species are rare overall. Human impact on nature affects number of existing popula - tions, so studies on ecology of the species have meaning in sense of conservation of this orchid. We&nbsp; studied how this species fit to the conditions of Evenkia - cold region of the globe with long cold winter (about&nbsp; 225 days) and short and hot summer. Studies covered 39 model coenopopulations located in 12 sampling areas. We have fetched out the species&nbsp; occurs in larch-fir, larch, fir, pine and pine-larch forests with grass - dwarf shrubs -moss and dwarf shrub - moss&nbsp; - lichen types of land cover. Population density varies from 0,1 to 46,1 (mean 12.2) individuals per sq.m.&nbsp; Analysis of standard ecological scales of I, Tsatsenkin made by the E. Prokopiev method allowed us to&nbsp; fetch out the tolerancy ranges of Cypripedium guttatum are 15 steps (58 to 73) by humidity scale and 5 steps (6&nbsp; to 11) by active soil richness scale. Range of ecological factors intensity was studied by the method of phytoin- dication of Ramensky-Tsatsenkin. All habitats of where Cypripedium guttatum was present lied in range of&nbsp; 63,1 to 68 by humidity scale and 7,9 to 9,4, Maximal abundance of the species (about 5%) was reached in habi- tats with hunitiy of 63-66 steps and active soil richness of 8-9 scales. Our studies allowed to understand the area of the reserve is optima for the Cypripedium guttatum l by the&nbsp; factors of humidity and soil reachness. Populations here are stable and plants are capable to reproduction. It&nbsp; make the reserve a key element of the species stability in conditions of global climate changes and could serve&nbsp; as a point of future dispersion of Cypripedium guttatum seeds in case if surrounding populations will be lost&nbsp; because of natural or human-caused impacts.
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40

Mao, Xingpeng, Ju Li, Heyue Huang, Yiming Wang, and Junjie Lang. "A short wave radar beam sharpening method based on generalised oblique projection operator with flexible parameter." IET Radar, Sonar & Navigation, March 12, 2024. http://dx.doi.org/10.1049/rsn2.12551.

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AbstractBeamforming is an effective way of resolving target direction and anti‐jamming in short wave (SW) radar systems. In conventional beamforming (CBF) at a certain frequency, to get high resolution, the array aperture should be increased, and this is often not allowed in practical applications. A new narrow beam forming (NBF) method for beam sharpening based on the generalised oblique projection (GOP) filter with a flexible parameter is proposed. This method uses a GOP filter bank to form deep nulls in the undesired azimuth range on the pattern and utilises the logic product process to synthesise the GOP filters’ outputs and thus obtains a narrow beam. Compared to traditional beamforming methods, the result of NBF has the characteristics of narrower beam width and bigger side lobe suppression ratio (SLSR). Especially, a narrower beam can be obtained in the case of a small array aperture, which is valuable for practical applications. Experimental results of the range‐Doppler spectrum of short wave radar show that this narrow beam forming method can achieve super resolution of targets within a wide beam and greatly suppress clutter. Therefore, NBF can improve the azimuth resolution and achieve interference suppression in a conventional beam.
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Deckers, Jef, and Bernd Rombaut. "Tournaisian Tectonic Phase With Major Carbonate Buildup Structures in the Campine Basin (Northeastern Belgium)." Basin Research 37, no. 2 (2025). https://doi.org/10.1111/bre.70023.

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ABSTRACTBy means of seismic interpretations, this study provides improved constraints on a major Tournaisian (lowermost Carboniferous) tectonic phase with faulting across the Campine Basin, northeastern Belgium. Faults are normal with throws below 100 m, except for some larger intra‐rift horst and graben structures with throws up to 300 m. In an asymmetric graben structure in the southern study area, an estimated average of 1000 m of Tournaisian sediments accumulated. Outside the graben, Tournaisian thicknesses are in the order of 300–500 m, which agrees with the limited available well data outside the study area of the Campine Basin. There is an uncertainty on fault strikes since the individual fault segments are short compared to the spacing between the seismic lines, but we estimate it to vary between SW–NE and WNW–ESE. The wide range of fault strikes can be related to the reactivation of pre‐existing faults in the Cambro‐Silurian basement. The SW–NE and WNW–ESE directions of the Tournaisian fault strikes have been identified as lineaments on gravimetric and aeromagnetic maps of the lower Palaeozoic Brabant Massif to the southeast and southwest of the study area, respectively. Such fault strikes imply a roughly NNW–SSE to N–S extensional stress field prevailing in the area during the Tournaisian. The range of fault strikes is very similar to the strike of contemporaneous faults in Ireland and the United Kingdom, which suggests that the NNW–SSE to N–S extensional stress field occurred throughout much of northwestern Europe. The Tournaisian succession of the Campine Basin includes numerous mound‐shaped complexes, interpreted as buildup structures. We show examples of major buildup complexes that developed in graben structures. One of them reaches a height of 750 m and is 3 km wide. Given the similarity in timing of formation and size of the buildup complexes in the Campine Basin with buildup complexes in southern Belgium and Ireland, we consider it likely that the buildup complexes in the Campine Basin represent Waulsortian mudmounds.
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Malvé, Mariano Ezequiel, Nicolás Battini, Georgina Cordone, et al. "Potential impacts and priority areas of research of the on-going invasion of green crabs along the SW Atlantic." Environmental Reviews, December 3, 2024. https://doi.org/10.1139/er-2024-0093.

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The European green crab (Carcinus maenas) is one of the most extensively studied decapod species worldwide and it currently inhabits every continent except Antarctica. Its effects are observed across various spatio-temporal scales, impacting a wide range of taxa and environments. While extensive research has been conducted in the Northern Hemisphere, populations in the Southern Hemisphere (e.g., Argentina, Australia, and South Africa) have not been thoroughly investigated. This study has three main goals: 1) summarise and contextualise the invasion history of green crabs in the Southwest Atlantic since their initial detection in 2000, 2) present nine case studies identifying the potential ecological and economic impacts of green crabs on coastal ecosystems and to highlight priority research areas, and 3) discuss appropriate management actions in response to the species' rapid spread in the region. Our findings suggest that green crabs are likely to impact foundation species along rocky shores, alter the physical characteristics of soft-bottom environments, and affect infaunal organisms in sandy shores. Most of these impacts are either occurring or expected to occur in numerous marine protected areas. We also examine green crab interactions with other key species, highlighting its dual role as both an invasive predator and prey for native species, thus serving as a novel food resource. Furthermore, we consider their effects on commercially important species, tourism, and implications for threatened native species. Finally, we recommend prioritising prevention and rapid response strategies for managing green crab invasions, emphasising the importance of early detection and prompt action to address new incursions.
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43

Starostenko, V., T. Janik, W. Czuba, et al. "The SHIELD’21 deep seismic experiment." Geofizicheskiy Zhurnal 45, no. 1 (2023). http://dx.doi.org/10.24028/gj.v45i1.275126.

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The wide-angle reflection and refraction (WARR) SHIELD’21 profile carried out in 2021 crosses from SW to NE the main tectonic of Ukraine. The SHIELD’21 targeted the structure of the Earth’s crust and upper mantle of the southwestern margin of the East European Craton with overlying Neogene Carpathian Foredeep and Vendian-Paleozoic Volyn-Podolian Monocline, Archaean and Paleoproterozoic segments of Ukrainian Shield and Late Paleozoic Dnieper-Donets Basin. The ~650 km long profile is an extension of realized in 2014 RomUkrSeis line in Romania and Ukraine from Apuseni Mountains to southwestern Ukrainian Shield. The field work performed in 2021, included the deployment of autonomous seismic stations and drilling-explosive works. A total of 264 seismic receivers were deployed (160 DATA-CUBE and 104 TEXAN stations) with the average spacing between the observation points about 2.65 km. The sampling interval for all stations was 0.01 s. Seismic energy was generated by 10 shot points with ~50 km of distance between them and total charge in all wells 5775 kg. The SHIELD’21 experiment using TEXAN and DATA-CUBE short-period seismic stations provided high-quality seismic records. The main recorded seismic waves are the refractions of P- and S-waves in sediments, basement, crust and upper-most mantle, and reflections from crustal boundaries, Moho interface and boundaries in the uppermost mantle. Correlation of travel time arrivals of seismic waves provides calculation of the velocity model for both P- and S-waves. The main objective of the SHIELD’21 project is to get new seismic data that increase our knowledge on the lithosphere structure as well as geodynamics of the oil-and-gas-bearing and ore regions of Ukraine.
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Sharkey, Camilla R., Jorge Blanco, Nathan P. Lord, and Trevor J. Wardill. "Jewel beetle opsin duplication and divergence is the mechanism for diverse spectral sensitivities." Molecular Biology and Evolution, January 31, 2023. http://dx.doi.org/10.1093/molbev/msad023.

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Abstract The evolutionary history of visual genes in Coleoptera differs from other well-studied insect orders, such as Lepidoptera and Diptera, as beetles have lost the widely conserved short-wavelength (SW) insect opsin gene that typically underpins sensitivity to blue light (∼ 440 nm). Duplications of the ancestral ultraviolet (UV) and long-wavelength (LW) opsins have occurred in many beetle lineages and have been proposed as an evolutionary route for expanded spectral sensitivity. The jewel beetles (Buprestidae) are a highly ecologically diverse and colorful family of beetles that use color cues for mate and host detection. In addition, there is evidence that buprestids have complex spectral sensitivity with up to five photoreceptor classes. Previous work suggested that opsin duplication and subfunctionalization of the two ancestral buprestid opsins, UV and LW, has expanded sensitivity to different regions of the light spectrum, but this has not yet been tested. We show that both duplications are likely unique to Buprestidae or the wider superfamily of Buprestoidea. To directly test photopigment sensitivity, we expressed buprestid opsins from two Chrysochroa species in Drosophila melanogaster and functionally characterized each photopigment type as UV- (356 - 357 nm), blue- (431 - 442 nm), green- (507 - 509 nm) and orange-sensitive (572 - 584 nm). As these novel opsin duplicates result in significantly shifted spectral sensitivities from the ancestral copies, we explored spectral tuning at four candidate sites using site-directed mutagenesis. This is the first study to directly test opsin spectral tuning mechanisms in the diverse and specious beetles.
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sprotocols. "Design and Cloning of an shRNA into a Lentiviral Silencing Vector: Version A." December 30, 2014. https://doi.org/10.5281/zenodo.13615.

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Authors: Gustavo Tiscornia, Oded Singer and Inder M. Verma This protocol was adapted from “Development of Lentiviral Vectors Expressing siRNA,” Chapter 3, in [*Gene Transfer: Delivery and Expression of DNA and RNA*](http://www.cshlpress.com/link/genetrnp.htm) (eds. Friedmann and Rossi). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2007. ### INTRODUCTION This protocol combines the specificity of small interfering RNA (siRNA)-mediated silencing cassettes with the versatility of lentiviral vectors to stably transduce a wide range of cell types. A short hairpin RNA (shRNA) designed against a given target is cloned into a plasmid containing the pol III promoter. The design uses a 5′ forward primer upstream of the pol III promoter and a 3′ reverse primer that includes the entire shRNA sequence (i.e., sense, loop, and antisense sequences followed by five Ts), followed by 22 bases complementary to the last 22 bp upstream of the +1 transcriptional start site of the pol III promoter. An NheI-compatible restriction site is included at the 5′ end of both forward and reverse primers. A single round of PCR is used to amplify this template. The resulting DNA fragment contains an shRNA expression cassette that can be cloned into a simple cloning vector, tested, and then transferred to the lentiviral vector, or cloned into the lentiviral vector directly. This procedure uses a unique restriction site in the 3′ long terminal repeat (LTR). During integration, the 5′ LTR of the provirus is copied from the 3′ LTR, cloning the H1-driven shRNA into the 3′ LTR, resulting in duplication of the silencing cassette. This strategy maximizes the silencing power of the lentiviral vector. The combination of the lentiviral and siRNA technologies provides a powerful tool to achieve long-term down-regulation of specific target genes both in vitro and in vivo. ### RELATED INFORMATION Figure 1 presents a schematic representation of the protocol. One undesirable consequence of this procedure is that the siRNA target sequence is also present in the mRNA expressing the marker gene, resulting in somewhat lower expression of the marker. In an alternative method, [Design and Cloning of an shRNA into a Lentiviral Silencing Vector: Version B](http://cshprotocols.cshlp.org/lookup/doi/10.1101/pdb.prot5010) (Tiscornia et al. 2008), the position of the silencing cassette is upstream of the marker expression cassette, thus avoiding down-regulation of the marker. Because the silencing cassette is not in the 3′ LTR, only one copy of the silencing cassette is delivered per viral particle. ![Figure 1](https://i.imgur.com/xdgtACe.gif &quot;Figure 1&quot;) **Figure 1**. The siRNA expression cassette consists of a nucleotide sense sequence (identical to the target sequence in the mRNA to be down-regulated), followed by a 9-bp loop, an antisense sequence, and a stretch of five Ts (T5) as a pol III transcriptional termination signal downstream from an H1 promoter. After PCR amplification, this is packaged into the NheI site of a transfer vector containing all the cis-acting elements required for replication and packaging of RNA into viral particles, including a Rev-responsive element (RRE) to enhance nuclear export of unspliced viral genomic RNA, woodchuck hepatitis virus regulatory element (WPRE) to enhance expression of the transgene, and a central polypurine tract (cPPT) purported to increase efficiency of nuclear import of the preintegration complex. Green fluorescent protein (GFP) is used as a marker. A number of algorithms have been developed to predict effective siRNA sequences (e.g., [http://www.ambion.com](http://www.ambion.com/)/ or [http://sfold.wadsworth.org/](http://sfold.wadsworth.org/)). A database search is recommended to filter out candidate targets that are present in other genes to avoid silencing of these loci. ### MATERIALS **Reagents** 1. Advantage GC 2 polymerase mix (Clontech) - 2X BES-buffered saline (BBS) - 2.5 M CaCl2 (10X stock) - *Store 1.5-mL aliquots at -20°C*. - Cells (293T human embryonic kidney) (Invitrogen) - *Cells should be of low-passage number and should not be used after passage 20 or if growth is slow.* - Dimethyl sulfoxide (7% [v/v]) (DMSO) - Dulbecco’s modified Eagle’s medium (DMEM) with 2% and 10% fetal bovine serum (FBS) - *Certain brands of FBS do not support efficient transfection and can result in low viral titers*. - Enzyme-linked immunosorbent assay (ELISA) Kit, p24 (PerkinElmer) (optional; see Step 20) - Gels for purifying lentivector plasmid and insert - H1 promoter cloned into pGEM-T (Promega) - Hank’s balanced salt solution (HBSS; Invitrogen) - Phosphate-buffered saline (PBS) containing 0.001% (w/v) poly-L-lysine (Sigma-Aldrich) - *Filter-sterilize and store at -20°C*. - Plasmids: pMDL (Gag-Pol), pREV, and pVSV-G - *For plasmid preparation, use QIAGEN plasmid maxipreps at 1 μg/μL*. - Primer, 5′ forward - *The 5′ forward primer must contain an XbaI site*. - Primer, 3′ reverse - *This primer should be designed to contain 22 nucleotides from the 3′ end of the pol III promoter, and a 5′ tail including the entire shRNA loop against the gene to be silenced, the transcriptional stop signal (T5), and XbaI site sequences*. - Restriction endonucleases: NheI, SspI, XbaI - Sucrose (20% [w/v] in HBSS) ### Equipment 1. Centrifuge - Dishes (tissue-culture, 15-cm) - Dishes (tissue-culture, six-well) - Filters (0.22- or 0.45-μm) - Incubators preset to 37°C (3% and 10% CO2) - Microcentrifuge - PCR machine - Rotors (SW 28 and SW 55) (Beckman) - Tubes (50-mL) - Tubes (centrifuge, polyallomer, 5-mL) (Beckman) - Tubes (centrifuge, polyallomer, 30-mL) (Beckman) - Tubes (microcentrifuge) - Vortexer ### METHOD **Design and Cloning of shRNAs** 1. Select a target within the gene to be silenced and design primers to amplify the silencing cassette. For GFP, the target would be: GCAAGCTGACCCTGAAGTTC (Tiscornia et al. 2003). The 3′ reverse primer would be: 5′ CTGTCTAGACAAAAAGCAAGCTGACCCTGAAGTTCTCTCTTGAAGAACTTCAGGGTCAGCTTGCGGGGATCTGTGGTCTCATACA3′, where the H1 sequence is in italic bold, XbaI (NheI compatible) is underlined, and the loop is bold underlined. - Mix 10 ng of the pGEM-T plasmid containing the H1 promoter (the template) and 10 μM (final concentration) each of the 5′ forward primer and the 3′ reverse primer, with the Advantage GC 2 polymerase mix, according to the manufacturer’s instructions. Add 7% (v/v) DMSO or a similar agent to a regular Taq polymerase reaction to prevent hairpin formation. - *Use the GC-melt additive as 10X*. - Amplify using the parameters listed below: ![Table 1](https://i.imgur.com/NM8AYRK.png &quot;Table 1&quot;) *This results in an amplified fragment of ~400 bp that can be cloned in an A/T vector for sequencing or directly cloned in the lentivector plasmid*. 4.Digest the insert with XbaI and gel-purify. 5.Digest the lentivector plasmid with NheI, gel-purify, and then dephosphorylate. 6.Validate the cloned shRNA cassettes by transfecting or transducing (as lentiviral particles) to a cell line that expresses the target gene. - *Typically, 50 ng of vector are ligated to 100 ng of insert and transformed into competent bacteria. Plasmid DNA from the resulting colonies can be screened by digestion with SspI. The parental vector should have only one SspI site, whereas the vector containing the insert will acquire an additional SspI site located in the H1 promoter. It is important to verify the integrity of the hairpin by sequencing using the H1-F primer 5′-TGGCAGGAAGATGGCTGTGA-3′, because mutations in the hairpin can significantly reduce the efficiency of down-regulation*. 7.Alternatively, coexpress a tagged cDNA of the target gene together with shRNA silencing cassettes in an easily transfected cell line (e.g., 293T). - *This is useful when target mRNA is restricted to certain cell types or a specific antibody against the target is unavailable. Typically, transfect 200 ng of target cDNA plasmid plus 500-1000 ng of the plasmid containing the silencing cassette per well (six-well cluster) and harvest the cells for immunoblot analysis 48-72 h after transfection*. **Preparation of Lentiviral Vectors** 8.Twenty-four hours before transfection, prepare plates and cells: - i. To increase cell adherence, precoat twelve 15-cm dishes with 10 mL of PBS containing 0.001% (w/v) poly-L-lysine. Incubate for 15 min at room temperature. Aspirate the liquid. - ii. Immediately seed the 293T cells (from two 15-cm plates of confluent cells) to the twelve 15-cm plates in DMEM with 10% FBS. - *Addition of 1% antibiotic-antimycotic solution does not interfere with transfection*. - iii. Grow the cells overnight. - Make sure that the cells are 70%-80% confluent and evenly distributed at the time of transfection to optimize viral titer. 9.Transfect the plasmid mix into the cells using the CaPO4 precipitation method: - i. Aliquot the four plasmids into a 50-mL tube. For each set of twelve 15-cm dishes, use: - 270 μg of lentivector - 176 μg of pMDL (Gag-Pol) - 95 μg of pVSV-G - 68 μg of pREV - ii. Prepare 13.5 mL of a 0.25 M solution of CaCl2 from a 10X stock solution of CaCl2. Add to the plasmid mix. - iii. Add 13.5 mL of 2X BBS to the mixture from Step 9.ii. Mix gently by inversion. Incubate for 15 min at room temperature. - iv. Add 2.25 mL of the transfection mixture from Step 9.iii dropwise to each plate. Swirl the plates gently to distribute. Incubate in a 3% CO2 atmosphere overnight at 37°C. 10.Approximately 16-20 h after transfection, remove the media. Add 15 mL of fresh DMEM with 2% FBS to each plate. Incubate in a 10% CO2 atmosphere overnight at 37°C. 11.Collect the supernatant from the plates. Filter through 0.22- or 0.45-μm filters. Add 15 mL of fresh medium to each plate and incubate overnight. - *Filtered supernatants can be stored for several days at 4°C*. 12.Collect media from the plates and filter as in Step 11. 13.Pool collected supernatants from Steps 11 and 12. Transfer to 30-mL centrifuge tubes, using 25-29 mL per tube. Concentrate the viral particles by centrifuging in an SW 28 rotor at 19,400 rpm for 2 h at 20°C. 14.Resuspend all pellets in a total of 1 mL of HBSS. Wash tubes a second time with 1 mL of HBSS. 15.Add HBSS to the resuspended pellets and tube washing fluid to a final total volume of 3 mL. 16.Prepare a 1.5-mL cushion of 20% sucrose in HBSS in 5-mL centrifuge tubes. Layer the resuspended pellets on the sucrose cushion. Centrifuge using an SW 55 rotor at 21,000 rpm for 1.5 h at 20°C. 17.Resuspend the pellet in 100 μL of HBSS. Wash the tube with an additional 100 μL of HBSS. 18.Shake the resuspended viral preparation on a low-speed vortexer for 15-30 min. 19.Centrifuge for 10 sec to remove debris. Aliquot the cleared viral solution. - *The solution can be stored at -80°C for many months. Avoid repeated freeze-thaw cycles*. 20.Titrate the viral preparations by quantitating levels of the capsid protein p24 using a p24 ELISA kit (or by biological titration if an adequate marker is contained in the lentivector). - Titers normally range between 10e9 and 10e10 viral particles/mL but can be lower if transfection efficiency of packaging plasmids is suboptimal. ### REFERENCES 1. Tiscornia G., Singer O., Ikawa M., Verma I.M. (2003) [A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA.](http://cshprotocols.cshlp.org/cgi/ijlink?linkType=ABST&amp;journalCode;=pnas&amp;resid;=100/4/1844) *Proc. Natl. Acad. Sci*. 100:1844–1848. - Tiscornia G., Singer O., Verma I.M. (2008) [Design and cloning of an shRNA into a lentiviral silencing vector: Version B.](http://cshprotocols.cshlp.org/cgi/ijlink?linkType=ABST&amp;journalCode;=protocols&amp;resid;=2008/8/pdb.prot5010) *CSH Protocols* doi:10.1101/pdb.prot5010.
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46

Yu, Colburn. "Policies Affecting Pregnant Women with Substance Use Disorder." Voices in Bioethics 9 (April 22, 2023). http://dx.doi.org/10.52214/vib.v9i.10723.

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Photo by 14825144 © Alita Xander | Dreamstime.com ABSTRACT The US government's approach to the War on Drugs has created laws to deter people from using illicit drugs through negative punishment. These laws have not controlled illicit drug use, nor has it stopped the opioid pandemic from growing. Instead, these laws have created a negative bias surrounding addiction and have negatively affected particularly vulnerable patient populations, including pregnant women with substance use disorder and newborns with neonatal abstinence syndrome. This article highlights some misconceptions and underscores the challenges they face as they navigate the justice and healthcare systems while also providing possible solutions to address their underlying addiction. INTRODUCTION Pregnant women with substance use disorder require treatment that is arguably for the benefit of both the mother and the fetus. Some suggest that addiction is a choice; therefore, those who misuse substances should not receive treatment. Proponents of this argument emphasize social and environmental factors that lead to addiction but fail to appreciate how chronic substance use alters the brain’s chemistry and changes how it responds to stress, reward, self-control, and pain. The medical community has long recognized that substance use disorder is not simply a character flaw or social deviance, but a complex condition that requires adequate medical attention. Unfortunately, the lasting consequences of the War on Drugs have created a stigma around addiction medicine, leading to significant treatment barriers. There is still a pervasive societal bias toward punitive rather than rehabilitative approaches to addiction. For example, many women with substance use disorder lose custody of their baby or face criminal penalties, including fines and jail time.[1] These punitive measures may cause patients to lose trust in their physicians, ultimately leading to high-risk pregnancies without prenatal care, untreated substance misuse, and potential lifelong disabilities for their newborns.[2] As a medical student, I have observed the importance of a rehabilitative approach to addiction medicine. Incentivizing pregnant women with substance use disorder to safely address their chronic health issues is essential for minimizing negative short-term and long-term outcomes for women and their newborns. This approach requires an open mind and supportive perspective, recognizing that substance use disorder is truly a medical condition that requires just as much attention as any other medical diagnosis.[3] BACKGROUND The War on Drugs was a government-led initiative launched in 1970 by President Richard M. Nixon with the aim of curtailing illegal drug use, distribution, and trade by imposing harsher prison sentences and punishments.[4] However, it is worth noting that one can trace the roots of this initiative back further. In 1914, Congress enacted the Harrison Narcotics Tax Act to target the recreational use of drugs such as morphine and opium.[5] Despite being in effect for over four decades, the War on Drugs failed to achieve its intended goals. In 2011, the Global Commission on Drug Policy released a report that concluded that the initiative had been futile, as “arresting and incarcerating tens of millions of these people in recent decades has filled prisons and destroyed lives and families without reducing the availability of illicit drugs or the power of criminal organizations.”[6] One study published in the International Journal of Drug Policy in the same year found that funding drug law enforcement paradoxically contributed to increasing gun violence and homicide rates.[7] The Commission recommended that drug policies focus on reducing harm caused by drug use rather than solely on reducing drug markets. Recognizing that many drug policies were of political opinion, it called for drug policies that were grounded in scientific evidence, health, security, and human rights.[8] Unfortunately, policy makers did not heed these recommendations. In 2014, Tennessee’s legislature passed a “Fetal Assault Law,” which made it possible to prosecute pregnant women for drug use during pregnancy. If found guilty, pregnant women could face up to 15 years in prison and lose custody of their child. Instead of deterring drug use, the law discouraged pregnant women with substance use disorder from seeking prenatal care. This law required medical professionals to report drug use to authorities, thereby compromising the confidentiality of the patient-physician relationship. Some avoided arrest by delivering their babies in other states or at home, while others opted for abortions or attempted to go through an unsafe withdrawal prior to receiving medical care, sacrificing the mother's and fetus's wellbeing. The law had a sunset provision and expired in 2016. During the two years this law was in effect, officials arrested 124 women.[9] The fear that this law instilled in pregnant women with substance use disorder can still be seen across the US today. Many pregnant women with substance use disorders stated that they feared testing positive for drugs. Due to mandatory reporting, they were not confident that physicians would protect them from the law.[10] And if a woman tried to stop using drugs before seeking care to avoid detection, she often ended up delaying or avoiding care.[11] The American College of Obstetricians and Gynecologists (ACOG) recognizes the fear those with substance use disorders face when seeking appropriate medical care and emphasizes that “obstetric–gynecologic care should not expose a woman to criminal or civil penalties, such as incarceration, involuntary commitment, loss of custody of her children, or loss of housing.”[12] Mandatory reporting strains the patient-physician relationship, driving a wedge between the doctor and patient. Thus, laws intended to deter people from using substances through various punishments and incarceration may be doing more harm than good. County hospitals that mainly serve lower socioeconomic patients encounter more patients without consistent health care access and those with substance use disorders.[13] These hospitals are facing the consequences of the worsening opioid pandemic. At one county hospital where I recently worked, there has been a dramatic increase in newborns with neonatal abstinence syndrome born to mothers with untreated substance use disorders during pregnancy. Infants exposed to drugs prenatally have an increased risk of complications, stillbirth, and life-altering developmental disabilities. At the hospital, I witnessed Child Protective Services removing two newborns with neonatal abstinence syndrome from their mother’s custody. Four similar cases had occurred in the preceding month. In the days leading up to their placement with a foster family, I saw both newborns go through an uncomfortable drug withdrawal. No baby should be welcomed into this world by suffering like that. Yet I felt for the new mothers and realized that heart-wrenching custody loss is not the best approach. During this period, I saw a teenager brought to the pediatric floor due to worsening psychiatric symptoms. He was born with neonatal abstinence syndrome that neither the residential program nor his foster family could manage. His past psychiatric disorders included attention deficit disorder, conduct disorder, major depressive disorder, anxiety disorder, disruptive mood dysregulation disorder, intellectual developmental disorder, and more. During his hospitalization, he was so violent towards healthcare providers that security had to intervene. And his attitude toward his foster parents was so volatile that we were never sure if having them visit was comforting or agitating. Throughout his hospital course, it was difficult for me to converse with him, and I left every interview with him feeling lost in terms of providing an adequate short- and long-term assessment of his psychological and medical requirements. What was clear, however, was that his intellectual and emotional levels did not match his age and that he was born into a society that was ill-equipped to accommodate his needs. Just a few feet away from his room, behind the nurses’ station, were the two newborns feeling the same withdrawal symptoms that this teenager likely experienced in the first few hours of his life. I wondered how similar their paths would be and if they would exhibit similar developmental delays in a few years or if their circumstance may follow the cases hyped about in the media of the 1980s and 1990s regarding “crack babies.” Many of these infants who experienced withdrawal symptoms eventually led normal lives.[14] Nonetheless, many studies have demonstrated that drug use during pregnancy can adversely impact fetal development. Excessive alcohol consumption can result in fetal alcohol syndrome, characterized by growth deficiency, facial structure abnormalities, and a wide range of neurological deficiencies.[15] Smoking can impede the development of the lungs and brain and lead to preterm deliveries or sudden infant death syndrome.[16] Stimulants like methamphetamine can also cause preterm delivery, delayed motor development, attention impairments, and a wide range of cognitive and behavioral issues.[17] Opioid use, such as oxycodone, morphine, fentanyl, and heroin, may result in neonatal opioid withdrawal syndrome, in which a newborn may exhibit tremors, irritability, sleeping problems, poor feeding, loose stools, and increased sweating within 72 hours of life.[18] In 2014, the American Association of Pediatrics (AAP) reported that one newborn was diagnosed with neonatal abstinence syndrome every 15 minutes, equating to approximately 32,000 newborns annually, a five-fold increase from 2004.[19] The AAP found that the cost of neonatal abstinence syndrome covered by Medicaid increased from $65.4 million to $462 million from 2004 to 2014.[20] In 2020, the CDC published a paper that showed an increase in hospital costs from $316 million in 2012 to $572.7 million in 2016.[21] Currently, the impact of the COVID-19 pandemic on the prevalence of newborns with neonatal abstinence syndrome is unknown. I predict that the increase in opioid and polysubstance use during the pandemic will increase the number of newborns with neonatal abstinence syndrome, thereby significantly increasing the public burden and cost.[22] In the 1990s, concerns arose about the potentially irreparable damage caused by intrauterine exposure to cocaine on the development of infants, which led to the popularization of the term “crack babies.”[23] Although no strong longitudinal studies supported this claim at the time, it was not without merit. The Maternal Lifestyle Study (NCT00059540) was a prospective longitudinal observational study that compared the outcomes of newborns exposed to cocaine in-utero to those without.[24] One of its studies revealed one month old newborns with cocaine exposure had “lower arousal, poorer quality of movements and self-regulation, higher excitability, more hypertonia, and more nonoptimal reflexes.”[25] Another study showed that at one month old, heavy cocaine exposure affected neural transmission from the ear to the brain.[26] Long-term follow up from the study showed that at seven years old, children with high intrauterine cocaine exposure were more likely to have externalizing behavior problems such as aggressive behavior, temper tantrums, and destructive acts.[27] While I have witnessed this behavior in the teenage patient during my pediatrics rotation, not all newborns with intrauterine drug exposure are inevitably bound to have psychiatric and behavioral issues later in life. NPR recorded a podcast in 2010 highlighting a mother who used substances during pregnancy and, with early intervention, had positive outcomes. After being arrested 50 times within five years, she went through STEP: Self-Taught Empowerment and Pride, a public program that allowed her to complete her GED and provided guidance and encouragement for a more meaningful life during her time in jail. Her daughter, who was exposed to cocaine before birth, had a normal childhood and ended up going to college.[28] From a public health standpoint, more needs to be done to prevent the complications of substance misuse during pregnancy. Some states consider substance misuse (and even prescribed use) during pregnancy child abuse. Officials have prosecuted countless women across 45 states for exposing their unborn children to drugs.[29] With opioid and polysubstance use on the rise, the efficacy of laws that result in punitive measures seems questionable.[30] So far, laws are not associated with a decrease in the misuse of drugs during pregnancy. Millions of dollars are being poured into managing neonatal abstinence syndrome, including prosecuting women and taking their children away. Rather than policing and criminalizing substance use, pregnant women should get the appropriate care they need and deserve. I. Misconception One: Mothers with Substance Use Disorder Can Get an Abortion If an unplanned pregnancy occurs, one course of action could be to terminate the pregnancy. On the surface, this solution seems like a quick fix. However, the reality is that obtaining an abortion can be challenging due to two significant barriers: accessibility and mandated reporting. Abortion laws vary by state, and in Tennessee, for instance, abortions are banned after six weeks of gestation, typically when fetal heart rhythms are detected. An exception to this is in cases where the mother's life is at risk.[31] Unfortunately, many women with substance use disorders are from lower socioeconomic backgrounds and cannot access pregnancy tests, which could indicate they are pregnant before the six-week cutoff. If a Tennessee woman with substance use disorder decides to seek an abortion after six weeks, she may need to travel to a neighboring state. However, this is not always a feasible option, as the surrounding states (WV, MO, AR, MI, AL, and GA) also have restrictive laws that either prohibit abortions entirely or ban them after six weeks. Moreover, she may be hesitant to visit an obstetrician for an abortion, as some states require physicians by law to report their patients' substance use during pregnancy. For example, Virginia considers substance use during pregnancy child abuse and mandates that healthcare providers report it. This would ultimately limit her to North Carolina if she wants to remain in a nearby state, but she must go before 20 weeks gestation.[32] For someone who may or may not have access to reliable transportation, traveling to another state might be impossible. Without resources or means, these restrictive laws have made it incredibly difficult to obtain the medical care they need. II. Misconception Two: Mothers with SUD are Not Fit to Care for Children If a woman cannot take care of herself, one might wonder how she can take care of another human being. Mothers with substance use disorders often face many adversities, including lack of economic opportunity, trauma from abuse, history of poverty, and mental illness.[33] Fortunately, studies suggest keeping mother and baby together has many benefits. Breastfeeding, for example, helps the baby develop a strong immune system while reducing the mother’s risk of cancer and high blood pressure.[34] Additionally, newborns with neonatal abstinence syndrome who are breastfed by mothers receiving methadone or buprenorphine require less pharmacological treatment, have lower withdrawal scores, and experience shorter hospital stays.[35] Opioid concentration in breastmilk is minimal and does not pose a risk to newborns.[36] Moreover, oxytocin, the hormone responsible for mother-baby bonding, is increased in breastfeeding mothers, reducing withdrawal symptoms and stress-induced reactivity and cravings while also increasing protective maternal instincts.[37] Removing an infant from their mother’s care immediately after birth would result in the loss of all these positive benefits for both the mother and her newborn. The newborns I observed during my pediatrics rotation probably could have benefited from breastfeeding rather than bottle feeding and being passed around from one nurse to the next. They probably would have cried less and suffered fewer withdrawal symptoms had they been given the opportunity to breastfeed. And even if the mothers were lethargic and unresponsive while going through withdrawal, it would still have been possible to breastfeed with proper support. Unfortunately, many believe mothers with substance use disorder cannot adequately care for their children. This pervasive societal bias sets them up for failure from the beginning and greatly inhibits their willingness to change and mend their relationship with their providers. It is a healthcare provider’s duty to provide non-judgmental care that prioritizes the patient’s well-being. They must treat these mothers with the same empathy and respect as any other patient, even if they are experiencing withdrawal. III. Safe Harbor and Medication-Assisted Treatment Addiction is like any other disease and society should regard treatment without stigma. There is no simple fix to this problem, given that it involves the political, legal, and healthcare systems. Punitive policies push pregnant women away from receiving healthcare and prevent them from receiving beneficial interventions. States need to enact laws that protect these women from being reported to authorities. Montana, for example, passed a law in 2019 that provides women with substance use disorders safe harbor from prosecution if they seek treatment for their condition.[38] Medication-assisted treatment with methadone or buprenorphine is the first line treatment option and should be available to all pregnant women regardless of their ability to pay for medical care.[39] To promote continuity of care, health officials could include financial incentives to motivate new mothers to go to follow-up appointments. For example, vouchers for groceries or enrollment in the Special Supplemental Nutrition Program for Women, Infants, and Children (WIC) may offset financial burdens and allow a mother to focus on taking care of her child and her recovery. IV. Mandated Substance Abuse Programs Although the number of people sentenced to state prisons for drug related crimes has been declining, it is still alarming that there were 171,300 sentencings in 2019.[40] Only 11 percent of the 65 percent of our nation’s inmates with substance use disorder receive treatment, implying that the other 89 percent were left without much-needed support to overcome their addiction.[41] It is erroneous to assume that their substance use disorder would disappear after a period without substance use while behind bars. After withdrawal, those struggling with substance use disorder may still have cravings and the likelihood of relapsing remains high without proper medical intervention. Even if they are abstinent for some time during incarceration, the underlying problem persists, and the cycle inevitably continues upon release from custody. In line with the recommendations by Global Commission on Drug Policy and the lessons learned from the failed War on Drugs, one proposed change in our criminal justice system would be to require enrollment and participation in assisted alcohol cessation programs before legal punishment. Policy makers must place emphasis on the safety of the patient and baby rather than the cessation of substance use. This would incentivize people to actively seek medical care, restore the patient-physician relationship, and ensure that they take rehabilitation programs seriously. If the patient or baby is unsafe, a caregiver could intervene while the patient re-enrolls in the program. Those currently serving sentences in prisons and jails can treat their substance use disorder through medication assisted treatment, cognitive behavioral therapy, and programs like Self Taught Empowerment and Pride (STEP). Medication assisted treatment under the supervision of medical professionals can help inmates achieve and maintain sobriety in a healthy and safe way. Furthermore, cognitive behavioral therapy can help to identify triggers and teach healthier coping mechanisms to prepare for stressors outside of jail. Finally, multimodal empowerment programs can connect people to jobs, education, and support upon release. People often leave prisons and jail without a sense of purpose, which can lead to relapse and reincarceration. Structured programs have been shown to decrease drug use and criminal behavior by helping reintegrate productive individuals into society.[42] V. Medical Education: Narcotic Treatment Programs and Suboxone Clinics Another proactive approach could be to have medical residency programs register with the Drug Enforcement Administration (DEA) as Narcotic Treatment Programs and incorporate suboxone clinics into their education and rotations. Rather than family medicine, OB/GYN, or emergency medicine healthcare workers having to refer their patients to an addiction specialist, they could treat patients with methadone for maintenance or detoxification where they would deliver their baby. Not only would this educate and prepare the future generation of physicians to handle the opioid crisis, but it would allow pregnant women to develop strong patient-physician relationships. CONCLUSION Society needs to change from the mindset of tackling a problem after it occurs to taking a proactive approach by addressing upstream factors, thereby preventing those problems from occurring in the first place. Emphasizing public health measures and adequate medical care can prevent complications and developmental issues in newborns and pregnant women with substance use disorders. Decriminalizing drug use and encouraging good health habits during pregnancy is essential, as is access to prenatal care, especially for lower socioeconomic patients. Many of the current laws and regulations that policy makers initially created due to naïve political opinion and unfounded bias to serve the War on Drugs need to be changed to provide these opportunities. To progress as a society, physicians and interprofessional teams must work together to truly understand the needs of patients with substance use disorders and provide support from prenatal to postnatal care. There should be advocation for legislative change, not by providing an opinion but by highlighting the facts and conclusions of scientific studies grounded in scientific evidence, health, security, and human rights. There can be no significant change if society continues to view those with substance use disorders as underserving of care. Only when the perspective shifts to compassion can these mothers and children receive adequate care that rehabilitates and supports their future and empowers them to raise their children. - [1] NIDA. 2023, February 15. Pregnant People with Substance Use Disorders Need Treatment, Not Criminalization. https://nida.nih.gov/about-nida/noras-blog/2023/02/pregnant-people-substance-use-disorders-need-treatment-not-criminalization [2] Substance Use Disorder Hurts Moms and Babies. National Partnership for Women and Families. June 2021 [3] All stories have been fictionalized and anonymized. [4] A History of the Drug War. Drug Policy Alliance. https://drugpolicy.org/issues/brief-history-drug-war [5] The Harrison Narcotic Act (1914) https://www.druglibrary.org/Schaffer/library/studies/cu/cu8.html [6] The War on Drugs. The Global Commission on Drug Policy. Published June 2011. https://www.globalcommissionondrugs.org/reports/the-war-on-drugs [7] Werb D, Rowell G, Guyatt G, Kerr T, Montaner J, Wood E. Effect of drug law enforcement on drug market violence: A systematic review. Int J Drug Policy. 2011;22(2):87-94. doi:10.1016/j.drugpo.2011.02.002 [8] Global Commission on Drug Policy, 2011 [9] Women NA for P. Tennessee’s Fetal Assault Law: Understanding its impact on marginalized women - New York. Pregnancy Justice. Published December 14, 2020. https://www.pregnancyjusticeus.org/tennessees-fetal-assault-law-understanding-its-impact-on-marginalized-women/ [10] Roberts SCM, Nuru-Jeter A. Women’s perspectives on screening for alcohol and drug use in prenatal care. Womens Health Issues Off Publ Jacobs Inst Womens Health. 2010;20(3):193-200. doi:10.1016/j.whi.2010.02.003 [11] Klaman SL, Isaacs K, Leopold A, et al. Treating Women Who Are Pregnant and Parenting for Opioid Use Disorder and the Concurrent Care of Their Infants and Children: Literature Review to Support National Guidance. J Addict Med. 2017;11(3):178-190. doi:10.1097/ADM.0000000000000308 [12] Substance Abuse Reporting and Pregnancy: The Role of the Obstetrician–Gynecologist. https://www.acog.org/en/clinical/clinical-guidance/committee-opinion/articles/2011/01/substance-abuse-reporting-and-pregnancy-the-role-of-the-obstetrician-gynecologist [13] R. Ghertner, G Lincoln The Opioid Crisis and Economic Opportunity: Geographic and Economic Trends. ASPE. Office of Assistant Secretary for Planning and Evaluation. DHHS Revised September 11, 2018 https://aspe.hhs.gov/reports/economic-opportunity-opioid-crisis-geographic-economic-trends [14] Midon, M. Z., Gerzon, L. R., &amp; de Almeida, C. S. (2021). Crack and motor development of babies living in an assistance shelter. ABCS Health Sciences, 46, e021215-e021215. And for example, see Crack Babies: Twenty Years Later : NPR https://www.npr.org/templates/story/story.php?storyId=126478643 [15] Williams JF, Smith VC, the Committee on Substance Abuse. Fetal Alcohol Spectrum Disorders. Pediatrics. 2015;136(5):e20153113. doi:10.1542/peds.2015-3113 [16] CDC Tobacco Free. Smoking During Pregnancy. Centers for Disease Control and Prevention. Published April 11, 2022. https://www.cdc.gov/tobacco/basic_information/health_effects/pregnancy/index.htm [17] Abuse NI on D. What are the risks of methamphetamine misuse during pregnancy? National Institute on Drug Abuse. https://nida.nih.gov/publications/research-reports/methamphetamine/what-are-risks-methamphetamine-misuse-during-pregnancy [18] CDC. Basics About Opioid Use During Pregnancy | CDC. Centers for Disease Control and Prevention. Published July 21, 2021. https://www.cdc.gov/pregnancy/opioids/basics.html [19] Honein MA, Boyle C, Redfield RR. Public Health Surveillance of Prenatal Opioid Exposure in Mothers and Infants. Pediatrics. 2019;143(3):e20183801. doi:10.1542/peds.2018-3801 [20] Winkelman TNA, Villapiano N, Kozhimannil KB, Davis MM, Patrick SW. Incidence and Costs of Neonatal Abstinence Syndrome Among Infants with Medicaid: 2004–2014. Pediatrics. 2018;141(4):e20173520. doi:10.1542/peds.2017-3520 [21] Strahan AE, Guy GP Jr, Bohm M, Frey M, Ko JY. Neonatal Abstinence Syndrome Incidence and Health Care Costs in the United States, 2016. JAMA Pediatr. 2020;174(2):200-202. doi:10.1001/jamapediatrics.2019.4791 [22] Ghose R, Forati AM, Mantsch JR. Impact of the COVID-19 Pandemic on Opioid Overdose Deaths: a Spatiotemporal Analysis. J Urban Health Bull N Y Acad Med. 2022;99(2):316-327. doi:10.1007/s11524-022-00610-0 [23] Mayes LC, Granger RH, Bornstein MH, Zuckerman B. The Problem of Prenatal Cocaine Exposure: A Rush to Judgment. JAMA. 1992;267(3):406-408. doi:10.1001/jama.1992.03480030084043 [24] NICHD Neonatal Research Network. The Maternal Lifestyle Study. clinicaltrials.gov; 2016. https://clinicaltrials.gov/ct2/show/study/NCT00059540 [25] Lester BM, Tronick EZ, LaGasse L, et al. The maternal lifestyle study: effects of substance exposure during pregnancy on neurodevelopmental outcome in 1-month-old infants. Pediatrics. 2002;110(6):1182-1192. doi:10.1542/peds.110.6.1182 [26] Lester BM, Lagasse L, Seifer R, et al. The Maternal Lifestyle Study (MLS): effects of prenatal cocaine and/or opiate exposure on auditory brain response at one month. J Pediatr. 2003;142(3):279-285. doi:10.1067/mpd.2003.112 [27] Bada HS, Bann CM, Bauer CR, et al. Preadolescent behavior problems after prenatal cocaine exposure: Relationship between teacher and caretaker ratings (Maternal Lifestyle Study). Neurotoxicol Teratol. 2011;33(1):78-87. doi:10.1016/j.ntt.2010.06.005 [28] N, P, R. Crack Babies: Twenty Years Later. NPR. Published May 3, 2010. https://www.npr.org/templates/story/story.php?storyId=126478643 [29] Miranda L, Dixon V, September CRP on, 30, 2015. How States Handle Drug Use During Pregnancy http://projects.propublica.org/graphics/maternity-drug-policies-by-state [30] NCDAS: Substance Abuse and Addiction Statistics [2023]. NCDAS. https://drugabusestatistics.org/ [31] (Tenn. Code Ann. § 39-15-216). [32] Institute G. Interactive Map: US Abortion Policies and Access After Roe. https://states.guttmacher.org/policies/ [33] Whitesell M, Bachand A, Peel J, Brown M. Familial, Social, and Individual Factors Contributing to Risk for Adolescent Substance Use. J Addict. 2013;2013:579310. doi:10.1155/2013/579310 [34] CDC. Five Great Benefits of Breastfeeding. Centers for Disease Control and Prevention. Published July 27, 2021. https://www.cdc.gov/nccdphp/dnpao/features/breastfeeding-benefits/index.html [35] Welle-Strand GK, Skurtveit S, Jansson LM, Bakstad B, Bjarkø L, Ravndal E. Breastfeeding reduces the need for withdrawal treatment in opioid-exposed infants. Acta Paediatr. 2013;102(11):1060-1066. doi:10.1111/apa.12378 [36] Ilett KF, Hackett LP, Gower S, Doherty DA, Hamilton D, Bartu AE. Estimated dose exposure of the neonate to buprenorphine and its metabolite norbuprenorphine via breastmilk during maternal buprenorphine substitution treatment. Breastfeed Med Off J Acad Breastfeed Med. 2012;7:269-274. doi:10.1089/bfm.2011.0096 [37] Pedersen CA, Smedley KL, Leserman J, et al. Intranasal Oxytocin Blocks Alcohol Withdrawal in Human Subjects. Alcohol Clin Exp Res. 2013;37(3):484-489. doi:10.1111/j.1530-0277.2012.01958.x [38] Montana SB0289. https://leg.mt.gov/bills/2019/billhtml/SB0289.htm [39] Mullins N, Galvin SL, Ramage M, Gannon M, Lorenz K, Sager B, Coulson CC. Buprenorphine and Naloxone Versus Buprenorphine for Opioid Use Disorder in Pregnancy: A Cohort Study. J Addict Med. 2020 May/Jun;14(3):185-192. doi: 10.1097/ADM.0000000000000562. PMID: 31567599. [40] Drug Related Crime Statistics [2023]: Offenses Involving Drug Use. NCDAS. https://drugabusestatistics.org/drug-related-crime-statistics/ [41] Association APH. Online only: Report finds most U.S. inmates suffer from substance abuse or addiction. Nations Health. 2010;40(3):E11-E11. [42] Principles of Drug Addiction Treatment: A Research-Based Guide (Third Edition) | NIDA Archives. Published January 17, 2018. http://archives.nida.nih.gov/publications/principles-drug-addiction-treatment-research-based-guide-third-edition
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47

sprotocols. "High Resolution Melting Analysis for fast and cheap polymorphism screening of marine populations." January 14, 2015. https://doi.org/10.5281/zenodo.13940.

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Abstract:
Authors: Anne-Leila Meistertzheim, Isabelle Calves, Sébastien Artigaud, Carolyn S. Friedman, Christine Paillard, Jean Laroche &amp; Claude Ferec ### Abstract This protocol permits the mutation scanning of PCR products by high-resolution DNA melting analysis requiring the inclusion of a saturating intercalating dye in the PCR mix without labelled probe. During a scanning process, fluorescent melting curves of PCR amplicons are analyzed. Mutations modifying melting curve shapes, are allowed to be further characterized by sequencing because melting is not destructive. The method detects on small amplicons (120pb), 100% of heterozygous and 75% of homozygous variants in a single step. Homozygous variants are detected in a second time at 100% by adding wild-type reference DNA in the tube. For mitochondrial haplotypes, homozygous variants are discriminated by increased the size of the amplicons (700pb). HRMA can be conducted without knowledge on number of mutations, small insertions or deletions of the studied sequence in non-model species. The method accomplishes simultaneous gene scanning in a fraction of the time required when using traditional methods, while maintaining a closed-tube environment. The PCR requires &lt;1h20 (96- or 384-well plates) and melting acquisition takes 10 min per plate. ### Introduction Melting curve analysis in combination with real-time PCR was introduced in 1997 (1,2) and is a natural extension of continuously monitored PCR within each cycle (3). During High resolution DNA melting analysis (HRM or HRMA), melting curves are produced using dyes that fluoresce in the presence of double-stranded DNA (dsDNA). Using specialized instruments designed to monitor fluorescence during heating; as the temperature increases, the fluorescence decreases, producing a characteristic melting profile (4). Each PCR product is identified by its temperature melting (Tm) corresponding to the temperature at which half of the double strand DNA is denatured. When the PCR product sequence is altered, duplex stability is changed, leading to different melting behavior. When the change is homozygous, a shift in melting temperature is usually observed (5). When the change is heterozygous, four duplexes are formed following PCR: two heteroduplexes and two homoduplexes. Each duplex will have differing stabilities, the sum of which can be observed by high-resolution melting analysis (see Fig. 1), enabling sequence variations to be detected. HRM analysis detects single nucleotide polymorphisms (SNPs) and small insertions or deletions in a fragment of amplified DNA by comparing the fluorescence as a function of the temperature. Several years after its first introduction, HRMA appears as a rapid method for genotyping known variants or scanning unknown variants (6,7), with its applications recently reviewed (8-11). Variations of HRMA have been developed to enhance resolution in small amplicons, the 3’ ends of a primer set placed at a very short distance from the informative SNP (12). Alternatively, unlabelled probe can be used when multiple informative SNPs are present within a longer stretch of sequence, but also when the GC content is low or when the presence of polymorphisms prevents the placement of HRMA primers for small amplicons. In this latter case, the Tm of the unlabelled probe, and not of the entire amplicon, is used for genotyping (13-15). However, the software available on HRMA instruments is an important element determining the ultimate sensitivity achieved (16), and not all packages yet facilitate the use of temperature calibration probes. HRMA presents a specificity of 98.8% with an overall sensitivity of 99.3% of the samples with one or more heterozygous loci distinguished from wild type10. Although HRMA heterozygote detection is excellent with a rate of 100% (17), successful discrimination between homozygous variants that melt in a single domain depends mostly on their Tm with an average detection rate of 75% from 6 prior studies using constitutional human variants (8,17,18). The ability to use absolute temperatures differences for genotyping depends on the temperature precision of the instrument (19) and the consistency (ionic strength) of compared samples (20). Interestingly, best homozygous variants detection rates of 93% and 96.5% were reported for human BRCA1 and mitochondrial DNA (21,22). In both cases, PCR products were longer than 500 pb. These results suggest that longer PCR products may be preferred for homozygote detection, while for heterozygote detection shorter PCR products showed better results (10,11). Another technique consists in creating an artificial heteroduplex by mixing wild type DNA with homozygous variants, converting homozygous variants into heterozygotes with an overall detection sensitivity of 96.9% (10,23). HRMA is simple, rapid, and inexpensive but depends strongly on the quality of the PCR, instruments and dyes. Different DNA isolation methods did not influence scanning accuracy (21). Although different reconstitution buffers can affect absolute Tms (20) also modified by salt, MgCl2 and dye concentrations used for the amplification. DNA concentrations could vary at least 4-fold without affecting melting results (20), but identical concentrations must be favored. Saturating DNA dyes are not required for some HRMA applications like methylation analysis (24). However, scanning and genotyping are entirely dependent on heteroduplex identification and different dyes are variably effective. For example, LCGreen® Plus detects heterozygotes better than SYTO® 9, which is better than EvaGreen®, which is in turn better than SYBR Green I (8). Pricing of the dyes differs significantly; all have slightly different characteristics, and they often demand slightly different PCR buffers and conditions. Best PCR product length was also studied on the HRTM-1(25) and LightScanner® Instruments (11), revealing more errors as the length increases above 400 bp. However the optimal number of melting domains remains controversial between authors who argue between one domain melting (18) and two or more domains (22). As the precision of qPCR and melting further improves with time, the number of replicates required decreases, making the approach even more accessible. Simultaneous scanning and genotyping allows better differentiation of multiple variants, the sequencing burden concerning only the different variants in large-scale screening projects. Firstly confined to clinical and diagnostic studies (10), this method appeared recently in wild population studies of fish populations (26,27), symbionts from the genus Symbiodinium (28) or Wolbachia using reference genotypes (29). As a closed-tube system, HRMA represents a sensitive inexpensive and fast technique comparatively to new generation sequencing (30) or other modern gene scanning methods such as single-strand conformational polymorphism analysis in capillary sequencer (31), denaturing high-performance liquid chromatography (32) or temperature gradient capillary electrophoresis (33). Each of these methods requires application of the PCR products onto a matrix to separate and detect the heteroduplexes. In contrast, closed-tube systems eliminate the need for automation, greatly decrease the risk of laboratory contamination from open PCR product and significantly reduce analysis time. Based on all these attributes, a wide use of HRMA would be expected; however, a literature review as of May 2012 revealed no hits of this technique in non-model species despite the increasing importance of SNPs in this field, except on four recent populations studies focused on a a salmon (27), swordfish (26), a dipter (34) and a plant (35). Here we used HRMA to genotype alleles in fish and shellfish populations. The entire procedure we propose of HRMA in combination with qPCR is completed within 1h20 as a single closed-tube assay in non-model species without SNPs knowledge required. In the same time this procedure allows the real-time monitoring of PCR quality and the scanning of 384 or 96 samples on 384- or 96-well plates on a LightCycler® 480 Instrument. Screened polymorphism of different genes is compared between the best dyes LCGreen® Plus dye in LightScanner® Instrument previsouly described (15) with the new Resolight® in LightCycler® 480 Instrument on two different marine species: an invertebrate, the abalone Haliotis tuberculata and a vertebrate, the fish Platichthys flesus. Experimental design There are several experimental design considerations that should be considered before initiating mutation screening using HRM, explained further below. DNA extraction. The DNA extraction method should be similar for all the DNA templates, as salts in the DNA template can influence the melting curve in the HRM analysis. This can be overcome to some extent by adding unlabelled probe as internal temperature standards (12), but this is both a laborious and to some degree expensive step. Regarding the quality and quantity of DNA, it is also better to add the exact amount of template DNA to the PCR before amplification, as the dynamic range of HRMA is closed (see FIRST RESULTS). The quantity of the DNA can be measured by OD measurements or by using commercially available kits for measuring double-stranded DNA (e.g., PICOgreen) or directly by Nanodrop technology. The quality of DNA must be assessed by Agilent or migration on 1% agarose gel. The DNA samples were further diluted with PCR grade water to a concentration of 15 ng/μL for use in qPCR. PCR design and optimization. For nuclear gene, PCR primers should be designed to amplify fragments of 150–400 bp and each amplicon should ideally span an exon with one or more melting domains identified by software such as Poland server (36). To PCR-amplify larger exons, it may be necessary to design primers that amplify several overlapping amplicons. As a significant loss of sensitivity has been shown for heterozygous variants detection when using long PCR amplicons, we therefore do not recommend the amplification of PCR products longer than 400 bp. Long products commonly have multiple melting domains and the ability to detect all the variants decrease with the number of domains. Short PCR products have usually only one domain and homozygous variants result in little if any shape change. However, we find it critical for high sensitivity that the melt profile contains not more than one or two melt domains. When assays are designed to type specific variants (SNP typing) we recommend fragment sizes of 80–100 bp. On the contrary, for mitochondrial genome screening, we recommend amplicons longer than 600 bp to identify homozygous variants (22) containing more than one domain and with mutations located at different parts of melt domains. The primers should hybridize a region without any known SNP (use appropriate SNP databases) and the subsequent PCR amplicon could include more than one SNP. Primers were designed to flank the coding regions, without second structure (hairpin or dimer formation) and to be annealed at 60°C using Primer Express software (Applied Biosystems, Foster City, CA). All primers should be HPLC-purified before use to ensure the correct size and quality of the primers. PCRs should be optimized using a temperature gradient to determinate the optimal annealing temperature for primer sets at which a specific and robust amplicon is obtained. As the primer Tms are around 60, first PCR should consist on a touchdown temperature from 65 to 53°C. Controls. At least one normal control must be used for comparison of melting curve patterns when unknown samples are analyzed (e.g., if a specimen is screened for mutations in several exons of a gene, one wild sample should be screened in the same exons for comparison). It should be noted that when larger series of sample are screened for the presence of different mutations, we recommend using representative known samples as references in each PCR to standardize the calibration between samples. Each sample with a peak pattern that differs from the normal controls should be sequenced to verify the presence of a mutation and to determine the exact nucleotide change. A negative ‘no-template’ control may be included in the PCR to detect potential DNA contamination. However, false-positive or false-negative results due to PCR carryover are usually not a problem in this assay, because each sample contains same amount of genomic DNA as starting material. Instrumentation and dyes. Different sets of fluorescent dyes are commercially available, al in the same spectral emission: SYTO® 9, LC Green® and more recently the Resolight® included in HRM Master Mix (Roche, Indianapolis, IN). This protocol describes HRM analysis using two different types of dyes and their corresponding instrument base procedures. The LC Green® on the LightScanner® Instrument (Roche, Indianapolis, IN) and the Resolight® on real time PCR, the LightCycler® 480 Instrument (Roche, Indianapolis, IN). These Instruments generate fluorescence data from 45–95°C at a temperature transition rate of 0.1 to 0.02°C/sec, and 22 to 25 acquisitions per °C for the LightScanner® and the LightCycler® 480 Instruments, respectively. The uses of these two dyes are slightly different. Resolight® presents the advantage to be less concentrated than LCGreen® PLUS dye that prevents the inhibitory effect of the intercalating fluorescent dye during PCR amplification. Thus, LightCycler® 480 Instrument allowed the visualization of the real-time amplification additionally to the melting curve analysis. In principle, only thermocycler intended for real-time PCR with more than 22 images per second can be used. Some alterations may be necessary when adapting the protocol to a different instrument. Data analysis. This is the most time-consuming part of the procedure and also the part that requires some practice. Data can be analyzed using a program designed for melting curves analysis which depends on the instrument used: data from LightCycler® 480 Instrument can be analyzed by LightCycler® 480 Gene Scanning Software SW 1.5.0 and also LightScanner® Software version 2.0 Call-IT (v2.0.0.1331;Idaho Technology) using the melt calibration module, previously adapted only for LightScanner® Instrument data. The normalization of the melting curve required to choose two ranges of temperatures corresponding to 100% and 0 % values of fluorescence (Fig. 2 A). In the area chosen, parallel curves must be observed. Another horizontal calibration called temperature shift can be done between all the samples (threshold of 5 per default, Fig. 2 B1). This normalization is made to compensate the dispersion between samples from same group and improve homogeneity of the group (Fig. 2 C1). Differential efficiency of the amplification between the samples in the 96-well plate could create a Tm shift of 1.5 °C between same variants. In the case of all the samples are similar (same DNA concentration and quality) and similar PCR amplification efficiency, this normalization is not required because disturbed the analysis of homozygous variants corresponding to peaks at different Tms. However, to use the temperature shift without loss of information identified homozygote samples can be mixed with wild type reference in a second time. Data are then compared to reference samples identified as standards and then converted to difference plots (6) (Fig. 2 C2). The temperature window for those specific genotyping tests is ranged from 50°C to 95°C. ### Reagents **DNA extraction** 1. Sodium chloride (NaCl; Merck, cat. no. 567441) - Cethyl trimethyl amonium bromide (CTAB; Amersco, cat. no. 0833) - Chloroform:isoamyl alcohol (24:1) (Uptima, cat. no. UP899255) ! CAUTION Irritant - Tris-HCl (Uptima, cat. no. UP091549) ! CAUTION Irritant - Tris Biotech.Grade (Uptima, cat. no. 031657) - Boric acid (Uptima, cat. co. UP070440) - EDTA (Sigma, cat. no. ED4SS) ! CAUTION Irritant. - Sodium acetate (NaOAC; Merck, cat. no. 567418) - Ethanol (Merck, cat. no. 1.00983.1000) - proteinase K, 10 % (wt/vol) (Sigma, T2308 500mg) - β-Mercaptoethanol (Merck, cat. no. 444203) ! CAUTION Hazardous. **DNA migration electrophoresis and purification for HRM by LightScannerTM products** 1. Ethidium bromide (Roth, cat. no. 2218.1) ! CAUTION Mutagenic properties. DNA ladder 100pb with loading buffer (Gena Biosciences, cat. no. M-214) - Agarose (Interchim, cat. no. 31292L) - 50X TAE buffer (Eppendorf, cat. no. 955155335) **High Resolution Melting** 1. HPLC-purified PCR primers - (1) LightScanner® Master Mix (Idaho Technology, cat. no. HRLS-ASY-003) and Mineral Oil Light (Sigma, cat. no. M5904) - or (2) LightCycler®480 High Resolution Melting Master (Roche, cat. no. 04 909 631 001) **REAGENT SETUP** - Δ CRITICAL. Prepare solutions in DNase-free glassware using autoclaved - DNA extraction buffer Mix 100mM Tris-HCl ph8, 20 mM EDTA pH8, 1.4 M NaCl, 2% CTAB, stable up to 1 month at RT. - Agarose gel Mix 1%(wt/vol) agarose dissolved in 0.6 X electrophoresis buffer. - Electrophoresis buffer (TBE 10X) Mix 89 mM Tris base, 89 mM Boric acid and 2 mM EDTA (pH 8.3); store at RT. ### Equipment 1. 2ml microcentrifuge tubes - Pipetman (Gilson, P-20, P-200 and P1000) - Pipette tips (Rainin) - Bath incubator - Thermo-fast ® 96, semi-skirted (Thermo scientific, cat. no. AB0990) - Thermocycler Gene Amp PCR System 7500 (ABI) - Gel tank for electrophoresis (Maxicell® EC360M) - UV light, camera (Vilber Lourmat) - LightScanner® Instrument (Roche) - LightCycler® 480 Instrument (Roche) - Nanodrop (MD-1000, Spectrophotometer) - Centrifuge with rotor for microtiter plates (e.g., Eppendorf 5804) ### Procedure **DNA extraction** ● TIMING 45 min to 4 h (depending on the number of samples) 1.Extract DNA from the tissue/organism of interest (e.g., muscle, gills or fin) using an appropriate DNA extraction method. We routinely use the CTAB (37) method for marine mollusks muscle, and the CCDB (38) method for the fish. - Δ CRITICAL STEP Any other kits/standard methods of DNA extraction could alternatively be used (see Experimental design). Nevertheless, when samples have to be compared with each other or with a control, all of them should be extracted using the same extraction method. 2.Resuspend the DNA in water. Determine DNA quantity and quality by the A260/A280 ratio using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE). For reliable results, and following the HRM manufacturer´s recommendations, a ratio of 1.8-2 was required. Adjust concentrations of DNA to 15ng/µL. **PCR amplification and HRM analysis** ● TIMING ~ 2 h 3.Perform PCR amplification using any conventional PCR amplification protocol appropriate for the chosen primer pair according to manufacture protocols. Include a negative control where no template DNA is added (optional) and positive control with known genotype. In the case of homozygous variants, template DNA is constituted by half of wild type reference and half of the unknown samples. For example, as in a standard PCR protocol for amplicons in the size range between 150-400 bps (10 µl reaction), combine the following reagents using option A or option B depending on the instrument used: 3.A.Using LightScanner Instrument: 2µL template DNA, 4 µL of 2.5X LightScanner® Master Mix, 10 pmol of each primer (HPLC-purified) and water up to 10 µL. Load PCR-Plate with 15µL mineral oil, seal with film and centrifuge briefly 1min at 2500 g. Use the following amplification protocol: 95°C 2 min, 40-45 cycles of 94°C 30 s, 60 °C 30 s, 72°C 40 s and a terminal cycle for heteroduplex formation 94°C 30s, 25-28°C 30s. - PAUSE POINT Products may be stored at this point at -20°C for at least 2 weeks. If plates are refrigerated, spin for 30 s at 1600g before analysis. Following PCRs, insert the 96-well plate into a LightScanner® Instrument to melt the samples with a continuous increase temperature starting at 50°C to 95°C with a continuous acquisition mode. Following the melt, use the LightScanner 2.0 software to manage and analyze the data. 3.B.Using LightCycler®480 Instrument To obtain LightCycler® 480 data: 2µL template DNA, 2.5 µL of 2X LightCycler® 480 High Resolution Melting Master, 1µL of primer mix at 4µM (HPLC-purified), 3mM MgCl2 and water up to 10 µL. Seal with film and centrifuge briefly at 2500 rpm. Use the following amplification protocol: 95°C 10 min, 45 cycles of 95°C 15 s, 60°C (or 65°C – 0.5°C per cycle until 53°C) 30 s, 72°C 30 s. High resolution melting protocol consists of heteroduplex formation 95°C 1 min, 40°C 1min following by a continuous increase temperature starting at 50°C to 95°C with a continuous acquisition mode. - PAUSE POINT Products may be stored at this point at -20°C for at least 2 weeks before sequencing. Following the melt, use the LightCycler® 480 Software release 1.5.0. with the Gene Scanning module to manage and analyze the data. ? TROUBLESHOOTING **Melting analysis** ● TIMING ~ 2-10 min 4.Verify the amplification efficiency and removed of the data any sample without amplification before analysis. For LightCycler® 480 data, samples presenting unusual or abnormal amplification curve can also be removed (Fig. 3). Standard amplification curves presents crossing point (Cp) or cycle threshold, Cp &lt; 30 cycles or Cpsample =+/- 1,5 Cpwild type. ? TROUBLESHOOTING 5.Calibration (see Fig. 2) : Select two ranges of 1°C temperatures corresponding to 100% and 0 % fluorescence values when curves of all samples are parallel, before and after the fall curves (Fig. 2 A). Temperature shift: let the threshold per default at 5 (Fig. 2 B1). 6.Compare samples data to reference samples identified as standards (Fig. 2 C1) then convert to differences plots (Fig. 2 C2). Different resolution levels can be used to group the different variants: modify the level starting per default at 0.3. Melting peaks allowed visualizing different Tms of homozygous variants with the temperature shift engaged (Fig. 2 B2). For nuclear gene, homozygous variants can be discriminated from each other by adding wild type reference DNA to sample DNA in a second step and start over the HRMA (step 3). For mitochondrial genes, large amplicons can be directly discriminated by their melting peaks when ΔTm&gt;0.2°C. ? THROUBLESHOOTING **Agarose gel electrophoresis (optional for LightCycler® 480 treatment)** ● TIMING ~ 1 h 7.Prepare agarose gel (1% (w/v) agarose in 1X TAE buffer). Add one drop of ethidium bromide 0.625 mg. mL-1 for every 50 ml gel. 8.Perform agarose gel electrophoresis to verify successful PCR amplification as follows: mix 5 µl PCR product with 1 µl 6X loading buffer and perform the electrophoresis for 20 min at 120 V. Visualize the DNA bands using a UV-transilluminator. Distinct single bands should be visible for each PCR amplification. - PAUSE POINT Products may be stored at this point at -20°C for at least 2 weeks. ? THROUBLESHOOTING **Purification (and/or cloning) of PCR products for variants sequencing** 9.To determine the exact nature of the genetic variation, purify PCR products directly after PCR for LightCycler® 480 procedure or from DNA bands after electrophoresis of the products for LightScanner procedure and subsequently perform DNA double pass-sequencing analysis for mitochondrial gene. For nuclear marker, purify qPCR products, tail with the *Escherichia coli* poly A polymerase (New England Biolabs, Ipswich, MA, USA) for LightCycler® 480 procedure, clone and perform DNA sequencing analysis of several clones to separate alleles. Alleles can also be separated by a Single Strand Conformation Polymorphism (SSCP) starting from PCR products. ### Troubleshooting Troubleshooting advice can be found in Table3. ### Anticipated Results Examples of results used for a genetic study of marine populations39 from a standard HRM assay using the LightScannerTM Instrument and the LightCycler® 480 Instruments are shown in Figure 3 and 4. Variants of nuclear genes like exon 1 of gene encoding ferritin from the abalone *Haliotis tuberculata* correspond to small size amplicons (A-B), whereas variants of exon 2 of gene encoding myoadenylate deaminase from the teleost fish *Platichthys flesus* correspond to large sequences with multiple melting domains (C-D). Melting peaks allow visual differentiations of curves comforted with normalized and temp-shifted difference plots representing the data normalized by reference samples. Homozygote sample usually produces only one narrow melting peak corresponding to only one double-stranded DNA fragment (see Fig. 3 A1-B1), and single narrow peaks at different Tms could correspond to homozygous variants (green and blue melting peaks, Fig. 3 A1-B1). In contrast, heterozygous variants display broad double melting peaks (red line, Fig. 3 A1-B1), similar to heteroduplex forms in the case of mixed homozygote with wild type DNA. During scanning process 100% of heterozygous and 75% of homozygous variants are expected to be detected. Homozygous variants are detected in a second time at 100% by adding wild type reference DNA in the tube. The peak patterns of mixed of DNA amplification (homozygote with wild type) are reproducible. The location of more than one SNP within the same PCR product usually displays a complex variation of peak patterns across a series of samples (see Fig. 3 C1-D1). However, some distinct peaks display very close Tms (&lt;0.2°C) that could correspond to only one mutation (A/T; Table 1). Peak heights depend on the concentration of the DNA used for the PCR. Variable amplification efficiencies of a single sequence modify the Tm of the peaks in a range of 0.5°C, a level which discriminated supplementary variants for a single SNP (blue and yellow melting peaks Fig. 3 A1-B1). Homogeneity among variant groups on small size amplicons is similar between LightCycler® 480 and LightScanner data, whereas more advantages clearly appear when mutation scanning is realized using LightCycler® 480 Instrument and chemistry (see Fig. 3). Firstly, the Resolight© dye allows the amplification of amplicons with more than two melting-domains compared to the LC Green©. Amplification of particular long sequences with several SNPs permits to differentiate mitochondrial sequences without heteroduplexes formation as observed for a 689 bp region of the Cytochrome C Oxidase sub-unit 1 (CO1) gene with 3 SNPs in P. flesus (see Fig. 4)39. This chemistry increased also the Δfluorescence between the normalized and Temp-shifted difference plots (Fig. 3 A2-B2). On LightCycler® 480 data, plots representing relative signal differences lower than 4 could be grouped (Fig. 3 B2). Secondly, primer-dimers or unspecific priming in the PCR may occasionally result in additional background peaks which are reduced when using Resolight©. Thirdly, amplification and HRMA steps are performed on a single step, without opening the tube. Finally on the LightCycler® 480 Instrument, variable amplification efficiencies between variants are directly assessed during the real-time PCR and negative samples are identified (Fig. 5). We recommend considering two samples represented by their melting peaks with Tm shift of 0.2°C as two distinct variants, whatever the instrument and chemistry. All variants identified in 96-well plates have to be sequenced to determine the exact nature of the genetic variation. We also recommend sequencing samples with aberrant peaks. In marine populations, number of sequences is reduced from 250 and 290 samples to only 10 and 18 variants per marker for a vertebrate and invertebrate species, respectively. In conclusion, HRMA allows the efficient scanning of 96 (up to 384 for the LightCycler® 480 Instrument) samples and reduces drastically the number of samples to sequence in a cheap, fast and reproducible manner compared to other modern gene scanning methods (Table 2). ### References 1. Ririe, K.M., Rasmussen, R.P., and Wittwer, C.T. Product Differentiation by Analysis of DNA Melting Curves during the Polymerase Chain Reaction. *Anal. 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Legal Med*. 124 (4), 277-285 (2010). - Mader, E., Lohwasser, U., Borner, A., and Novak, J. Population structures of genebank accessions of Salvia officinalis L. (Lamiaceae) revealed by high resolution melting analysis. *Biochem. Syst. Ecol*. 38 (2), 178-186 (2010). - Poland. Biophysics Department University, Duesseldorf, available at http://www.biophys.uni-duesseldorf.de/local/POLAND/poland.html. - Doyle, J.J. &amp; Doyle, J.L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical bulletin 19 (11) (1987). - Ivanova, N.V., Dewaard, J.R., and Hebert, P.D.N. An inexpensive, automation-friendly protocol for recovering high-quality DNA. *Mol. Ecol*. Notes 6 (4), 998-1002 (2006). - Calves, I. et al. Genetic structure of the European flounder (Platichthys flesus) considering the southern limit of the species’ range and the potential impact of chemical stress. *Mar. Ecol. Prog. Ser*. Doi:10.3354/meps09797 (2012). - Venter, J.C. et al. The Sequence of the Human Genome. *Science* 291 (5507), 1304-1351 (2001). ### Acknowledgements The authors declare no competing financial interests. This research program was financially supported by (1) the European program SUDEVAB (Grant Agreement no 222 156), (2) a CPER – FEDER program for the sequencing part, and (3) by the EVOLFISH Program (financed by the National Agency for Research-VMCS, Paris, France). ### Figures **Figure 1.: Schematic principle of HRMA**. ![Fig 1](http://i.imgur.com/LIcUNTY.png &quot;Fig 1&quot;) *A. DNA fragments are amplified using fluorescently intercalating DNA dye, heat-denatured and cooled. Heterozygote (W/U) variant formed after denaturation and rehybridization, two homoduplexes (W/W and U/U) and two heteroduplexes (W/U). B. The results are illustrated as denaturation melting curves (fluorescence normalized or derivative fluorescence in function of temperature). HRMA detects mutations in DNA fragments due to temperature shift of the melting curve caused by variation of the amplicon Tms or variation of the curve shapes in heteroduplexes presence*. **Figure 2.: Step by step of the HRMA data analysis**. ![Fig 2](http://i.imgur.com/PPmrPMo.png &quot;Fig 2&quot;) *A. HRMA Melting curves of heterozygous (red) and homozygous variants (blue, yellow and green) are obtained after a continuous denaturation of amplicons with a progressive liberation of the intercalated fluorescent dyes. B1. Normalized melting curves are obtained after determination of 100% and 0% fluorescence values. B2. Normalized melting peaks are the derivative of the normalized melting curves. C1. Normalized and shifted melting curves were obtained according to a horizontal normalization by the temperature shift at a threshold of 3 (red line in B1). C2. Normalized and Temp-shifted difference plots are obtained by comparison of the curves to a reference sample (blue line). He. Heterozygote. Ho. Homozygote* **Figure 3.: Examples of HRMA results on nuclear marker**. ![Fig 3](http://i.imgur.com/DZ916P8.png &quot;Fig 3&quot;) *HRMA results obtained by LightScanner® (A-C) and LightCycler® 480 (B-D) Instruments on exon 1 of ferritin gene from the abalone Haliotis tuberculata (A-B) and exon 2 of myoadenylate deaminase gene from the teleost fish Platichthys flesus (C-D). Normalized and Temp-shifted difference plots are obtained by comparison of the melting curves to reference one. Homozygous genotypes are distinguished by Tm, whereas heterozygous by melting curve shape. Details and localizations of SNPs are presented for each variant regrouped by color*. **Figure 4.: Examples of HRMA on mitochondrial marker**. ![Fig 4](http://i.imgur.com/CeENTe7.png &quot;Fig 4&quot;) *HRMA results obtained by LightCycler® 480 Instruments on partial mitochondrial gene (689 pb) coding for Cytochrome C Oxidase sub-unit 1 (CO1) from the teleost fish Platichthys flesus. Temp-shifted difference plots are obtained by comparison of the melting curves to reference one. Homozygous genotypes are distinguished by Tm and melting curve shape. Details and localizations of SNPs are presented for each variant regrouped by color*. **Figure 5.: Amplification curves obtained only on LightCycler® 480 Instrument**. ![Fig 5](http://i.imgur.com/wGgyJKb.png &quot;Fig 5&quot;) *Amplification of positive samples is observed before 30 cycles. Samples with negative or abnormal amplification can be directly identified and removed of the analysis*. **Table 1: SNP classes as defined in the human genome** [Download Table 1](http://www.nature.com/protocolexchange/system/uploads/2125/original/Table_1.doc?1337031099) **Table 2. : Comparison of High Resolution Melting Analysis to some other modern gene scanning methods** [Download Table 2.](http://www.nature.com/protocolexchange/system/uploads/2126/original/Table_2.doc?1337031153) **Table 3. : Troubleshooting table**. [Download Table 3](http://www.nature.com/protocolexchange/system/uploads/2127/original/Table_3.doc?1337031233). ### Associated Publications [Genetic structure of the European flounder (Platichthys flesus) considering the southern limit of the species’ range and the potential impact of chemical stress](http://www.nature.com/protocolexchange/protocols/2383/publications/1290) ### Author information **Anne-Leila Meistertzheim**, Centre de Formation et de Recherche sur les Environnements Méditerranéens (CEFREM) **Isabelle Calves, Sébastien Artigaud, Christine Paillard &amp; Jean Laroche**, Laboratoire des Sciences de l'Environnement Marin, UMR CNRS/UBO/IRD 6539, Institut Universitaire Européen de la Mer, Université de Bretagne Occidentale, 29280 Plouzané, France **Carolyn S. Friedman**, School of Aquatic and Fishery Sciences, University of Washington, Box 355020, Seattle, 98195 Washington, United States **Claude Ferec**, Laboratoire de génétique moléculaire et d'histocompatibilité. CHU Morvan UBO, EFS-Bretagne BP 454 29275 Brest Cedex, France Correspondence to: Anne-Leila Meistertzheim (leila.meistertzheim@gmail.com) *Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2383) (2012) doi:10.1038/protex.2012.015. Originally published online 15 May 2012*.
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