The increase in coverage is substantial compared to the first version of the dataset, which included 21,060 polygons extending over 57,277 km2. The polygons cover open cuts, tailings dams, waste rock dumps, water ponds, processing plants, and other ground features related to the mining activities. The dataset is available for download from 10.1594/PANGAEA.942325 and visualisation at www.fineprint.global/viewer.The growing demand for minerals has pushed mining activities into new areas increasingly affecting biodiversity-rich natural biomes. Mapping the land use of the global mining sector is, therefore, a prerequisite for quantifying, understanding and mitigating adverse impacts caused by mineral extraction. This paper updates our previous work mapping mining sites worldwide. Using visual interpretation of Sentinel-2 images for 2019, we inspected more than 34,000 mining locations across the globe. The result is a global-scale dataset containing 44,929 polygon features covering 101,583   Mining can cause a wide range of adverse impacts during mining operation and after closure, e.g. fragmenting the landscape and polluting soils and water with effects on human settlements, agriculture plantations, and natural ecosystems5. Mapping the global mining areas is increasingly important for quantifying pressures of mineral extraction on biodiversity9, land-use modelling10, estimating the impacts of global supply chains and sustainable resource use13, for risk assessments of major environmental disasters on mining areas15, and planning and reinforcing mine reclamation16.Driven by the growing global demand for raw materials18. However, automatically mapping mining areas on a global scale is challenging because they are composed of a set of heterogeneous land cover types17. Mining areas are used for various purposes, including the mine itself , waste dumps , water ponds, and industrial processing facilities. Additionally, different minerals , extraction and processing methods, and landscape characteristics also increase intraclass variability, challenging automated mapping approaches using Earth observation data on a large scale.The increasing availability of high-resolution Earth observation data and new machine learning approaches has allowed mapping and monitoring of mining land use and its related environmental impacts on a local or regional scalekm2 19. The second data source mapped a total area of 31,396 km2 including active and inactive mining sites20 and the third dataset, described in our previous article21, covered 6,201 active mining sites that add to 57,277 km2. These three datasets are not comparable because they were derived using different satellite data sources acquired at different times and with distinct spatial resolutions. In addition, each dataset covered a different subset of mining locations, which can lead to underestimating the global mining land use because subnational mining activities are usually underreported compared to national accounts22.Visual interpretation of high-resolution satellite images has been used as an alternative to producing three global mining land use datasets. The first dataset mapped the 295 major mine sites worldwide, adding a total area of 3,633 23. The data update includes 44,929 polygon features covering 101,583 km2 of large-scale mining (LSM) as well as artisanal and small-scale mining (ASM). We followed a similar methodology based on visual interpretation to map all 34,820 mining coordinates reported in the SNL Metals & Mining database24. Compared to the first version, this is a substantial expansion, which covered only 6,201 coordinates of mines reported as active in the SNL database. As in the previous version, we mapped all land cover types related to mining without distinguishing them within the polygons. Although significantly expanded, our dataset still does not cover all existing mines worldwide, as we only inspected areas within a 10 km buffer around the coordinates from SNL24. However, to date, our updated dataset provides the most comprehensive information on global mining land use, including openly available georeferenced mining locations.Here we present a new dataset that improves global mining land use accounting by significantly expanding our previous global-scale dataset of mining sites23 and follows a similar methodology. We updated the areas in the first version using satellite images from 2019 and added new areas not included in the previous version. We inspected all 34,820 coordinates reported in the SNL database, substantially expanding the coverage compared to Version 1, which covered only 6,201 coordinates of mines reported with the status “active” or having any reported production between 2000 and 2017 by SNL21. We inspected all SNL coordinates in the second version because several SNL locations with “inactive” status and no reported production have clear ongoing mining activities visible in satellite images. Therefore, inspecting all SNL coordinates independently from their reported status was critical to provide a more comprehensive overview of the global mining land use. This data update also improved the coverage of ASM areas, which were almost absent from the first version because most ASM activities do not report production or activity in the SNL database, although their approximate coordinates are reported.Version 2 of the global-scale mining area dataset builds on the polygons from the first data releasekm buffer around the coordinates in the SNL database. Based on our previous experience21, this buffer size is sufficient to cover large mining sites expanding over several kilometres and also takes into account the imprecision in the SNL coordinates that can be up to 3 km distant from the actual mining sites8. We mapped all mines identified inside or intersecting the buffers’ borders, including areas that start inside the buffer and extend beyond its limits. This protocol was adopted to make sure mines that extend over long distances would be well captured, e.g. ASM mining following deposits on rivers and streams.To make the visual interpretation of images viable on a global scale, we limited the area of inspection to a 10 We defined mining areas as all land used by the mining sector at any step in extraction and processing at the mining site. Our mining areas also cover all 111 different commodities reported in the SNL database, including primary and companion commodities  and polygons with persistent invalid geometries, finally producing a consistent set of polygons simple features29.The delineated mining areas produced a raw data collection of polygons, which were checked and corrected by geoprocessing operations in R using the packages sf30. After that, a spatial join query acquired country names and ISO 3166-1 alpha-3 codes from the country’s administrative units geometries available from EUROSTAT31. The final set of polygons thus includes the geometries (polygons) covering the mining areas, their respective areas in square kilometres, country name, and ISO 3166-1 alpha-3 code of the corresponding country.We then calculated the area of each feature and added information on the country in which each polygon is located. We calculated the area in square kilometres using spherical geometryarcsecond, 5 arcminute and 30 arcminute spatial resolution . This is useful as many modelling applications require regular grid data32. The 30 arcsecond grid was derived from the percentage of the polygons’ area intersecting each cell. The percentages were rounded to zero decimal digits to reduce the size of the dataset. Therefore, the percentage of mining area covering a cell should be greater than 0.5% to be considered, i.e., approximately 0.5 ha at the equator. To obtain the gridded mining area, we estimated the area of each cell in square kilometres and multiplied it with the percentage of mining cover per cell, resulting in a 30 arcsecond global grid indicating the mining area within each cell. The other two grid levels, 5 arcminute and 30 arcminute, were resampled from the 30 arcsecond grid. The scripts used in the geoprocessing of data records are available with our open-source web application tool27.Similarly to Version 1, we also derived global grid datasets with the mining area at 30 km2 of mining areas worldwide33. It more than doubles the number of polygons compared to Version 1  and nearly doubles the mapped area, previously 57,277 km2 21. The number of countries covered also increased from 121 to 145. Besides the polygons, grid data provides a ready-to-use dataset for modelling with the mining area in square kilometres per grid cell provided at 30 arcsecond, 5 arcminute, and 30 arcminute spatial resolution. All data records were deposited to PANGAEA  and are available from 10.1594/PANGAEA.942325. The data is also available for visualisation from our platform www.fineprint.global/viewer. In what follows, we present a few examples to illustrate the data and provide an overview of the global mining land use compared to the first version of the data.The new dataset consists of 44,929 polygon features covering 101,583 34. The same spatial pattern can be found in other areas worldwide, such as in Ghana35. In the bottom right of Fig. www.fineprint.global/viewer.The maps in Fig. km grid to facilitate visualisation. Except for Antarctica, mining spreads across all continents with some hot-spot regions, for example, in northern Chile mainly due to copper extraction, northeastern Australia and East Kalimantan in Indonesia because of coal mining, and in the Amazon rain forest primarily due to small-scale gold mining.Figure A summary of our data aggregated by country shows that 52% of the mapped mining area is concentrated in only six countries: Russia, China, Australia, the United States, Indonesia, and Brazil. Another 21 countries account for 39%, and the remaining 118 countries add up to only 9% of the total mapped mining area  and omission errors (mine areas not mapped as mines). Besides that, the borders of the mines are not always evident in the images, creating another source of uncertainty.38. We drew a set of 1,220 random points stratified between the area mapped as mine and those not mapped as mine (no-mine) within the region of interest . These validation points were inspected independently by experts that did not participate in the delineation of the mines. They classified these validation points as mine or no-mine based on the satellite data without information on whether the points were mapped as part of a mining area. The validation points are also available from the data record33.We performed an independent classification of random points to assess these mapping errors. We followed the best practices on map accuracy assessment and sample design for overall accuracy, user’s accuracy (or commission error), and producer’s accuracy (or omission error)39. The Kappa index was 0.77 and Matthews correlation coefficient (MCC) 0.78 (Kappa and MCC range from −1 to 140). Negative values imply that the agreement is worse than random; 1 presents a complete agreement, while 0 is the expected value for a random classification). Our dataset also had an 89.7% probability of correctly distinguishing mining from non-mining areas according to the area under the curve (AUC) of the Receiver Operating Characteristic (ROC) curve41. We also derived the user’s and producer’s accuracy along with the error matrix . However, the mapped mining areas had 97.2% user’s accuracy, i.e. the mapped mining areas have a high probability of being correctly mapped as mining (less than 3% incorrectly mapped as mining).Based on these control points, we provide a range of assessment metrics. The overall accuracy shows that 88.3% of the control points were correctly classified, and the high F1 score of 0.87 indicates a low penalisation for false negativesm from the borders of the delineated polygons. On the other hand, only 16% of points with an agreement are located closer than 50 m to the polygons’ borders. These results indicate that higher uncertainty lies closer to the borders of the mapped areas. Additionally, it indicates high confidence in the existence of mines within the mapped polygons.We also investigated whether the proximity to the borders of the mines has affected the accuracy. We found that 54.5% of the control points with disagreement are located less than 50 Creative Commons Attribution-ShareAlike 4.0 International (CC-BY-SA) license. The data records include the same resources as the previous data release23 – the mining polygons, validation points, mining area grid, and a summary per country’s mining area.GeoPackage geographic data structures43, such that:mining_polygons layer has five attributes:ISO3_CODE: A string with the country’s ISO 3166-1 alpha-3 codeCOUNTRY_NAME: A string with the country name in EnglishAREA: A number with the area of the feature in square kilometresgeom: A polygon geometry in geographical coordinates WGS84fid: An integer with feature IDthe validation_points layer has four attributes:MAPPED: A string with the class derived from the mining polygons (“mine” or “no-mine”)REFERENCE: A string with the validation class (“mine” or “no-mine”)geom: A point geometry in geographical coordinates WGS84fid: An integer with feature IDthe The mining polygons and validation points are encoded in 44. Each grid cell over land has a float number (data type Float32) greater than or equal to zero representing the mining area in square kilometres; grid cells over water have no-data values. The grid is available in three spatial resolutions, 30 arcsecond, 5 arcminute, and 30 arcminute, extending from the longitude −180 to 180 degrees and from the latitude −90 to 90 degrees in the geographical reference system WGS84.The mining grids include a single layer each (one band raster) encoded in Geographic Tagged Image File Format (GeoTIFF)45 format, including four attributes:COUNTRY_NAME: A string with the country name in EnglishISO3_CODE: A string with the country ISO3 codeAREA: A number with the area of the feature in square kilometresN_FEATURES: An integer with the number of features per countryThe summary of the mapped mining area per country derived from the mining polygons is available in Comma-separated values (CSV)The global mining dataset described here is available from 10.1594/PANGAEA.942325 under the 46, R47, and Python48). Besides, we also provide a tool for visual analysis of the geographical data records at www.fineprint.global/viewer and a Web Map Service (WMS)49 accessible from www.fineprint.global/geoserver/wms.The datasets can easily be overlaid with other geospatial variables for further spatial analysis using software with support Geographic Information System (GIS) (e.g. including QGIS


Background: Burns are considered a critical care problem in emergency medicine, resulting in physical, psychological, and chronic disabilities. Silver sulfadiazine is the gold standard in topical burn treatment but was associated with toxicity to keratinocytes and fibroblasts, which may delay wound healing. In discovering potential alternative treatments for burn wound healing, this study was performed to determine the effect of Labisia Pumila (Blume) Fern.-Vill. Var. Alata (LPVa) extract on thermal-burn wounds in rats.Methods: A total of 50 Sprague-Dawley male rats were categorized into five groups. There were three control groups; normal control (left untreated), negative control (given ointment base) and positive control (given silver sulfadiazine). Meanwhile, the two intervention groups were given with 2% LPVa leaf and root extracts, respectively. Burn wounds were inflicted on the loin region of the rat by applying a heated steel rod at 80°C for 10 s. On days 3, 7, 14, and 21, wounds were measured macroscopically using a digital calliper and one animals of each group were sacrificed, and the wounded skin were excised for histomorphological assessments. The wounds were excised for hydroxyproline content on Day 14 of treatment.Result: For wound contraction percentage, both the leaf and root extracts of LPVa showed a significant reduction in burn wound size on Day 7 onwards, when compared to other groups. For hydroxyproline content, only the leaf extract of LPVa produced significantly higher content compared to both negative and normal control groups. In terms of histological examination, the leaf extract group demonstrated a superior healing effect than the root extract group.Conclusion: Both leaf and root extracts of LPVa could promote wound healing in the thermal-burn wound rat model, with leaf extract being superior to root extract. Burns are a critical care problem requiring specialised care focusing on stabilising the patient, preventing infection, and optimizing functional recovery . Burns cvia localized vasodilation and fluid extravasation  Fern.-Vill. (Primulaceae) (LP) or locally known as ‘Kacip Fatimah’ is one of the herbal plants in the Myrsinaceae family, characterized by lanceolate leaves with a creeping stem and long roots , which is a type of paraffin used as a vehicle in these topically-applied preparations to the rats. This emulsifying ointment was chosen as a vehicle as it is chemically inert and inactive to the skin . A 2.0% ad libitum. All animal experiments were approved by Universiti Kebangsaan Malaysia Animal Ethical Committee (FAR/PP/2018/NAZRUN/25-JULY/935-AUG-2018-MAR-2019).Fifty male Sprague-Dawley rats aged 4–5 months and weighing 250 ± 50 g were obtained from the Universiti Kebangsaan Malaysia Laboratory Animal Research Unit. All rats were housed in individually ventilated cages at temperature-controlled (25 ± 1°C) environment under natural day/night cycle. They were fed with standard laboratory food pellets and given water The rats were acclimatized to laboratory conditions for 1 week prior to the experiment. Initially, the rats were anesthetized with intraperitoneal injection of Ketamine and Xylazil at 1:1 ratio before inducing burn wound. The dorsal regions of the rats were shaven with an electric shaver and sterilized with 70% alcohol. Prior to burn-wound infliction, the rats were injected intramuscularly with tramadol (12.5 mg/kg body weight) for pain control. Burn infliction techniques were performed accordingly to the methods by Following the burn infliction, macroscopic changes of burn wounds were evaluated at day 0 post burn and subsequently at days 3, 7, 14, and 21 post burn. Photographs were taken with digital camera  until complete closure of wound. The images were scored blindly by anatomical expert using a macroscopic scoring tool for burns as described by Wounds were observed and measured at day 0, 3, 7, 14, and 21 by using a digital caliper . Woud contraction will be expressed as reduction in percentage of the original wound size .On the 14th day, one rat from each group was euthanized using ketamine and xylazil (overdose) to excise the wound tissue for determination of hydroxyproline content. The protein content of the wound tissue was estimated using the techniques described by 2O2 in methanol for 30 min. Subsequently, the sections were incubated with normal serum for 20 min and were incubated with primary antibody diluted in buffer at 4°C. The sections were then incubated for 1 hour with diluted biotinylated secondary antibody and incubated for 30 min with Vectastain ABC reagent. In between of each incubations, the sections were washed in buffer for 5 min. Following the incubation with Vectastain, the antibody binding sites were visualized by incubation with DAB signal stain kit for 15 min and counterstained with Haematoxylin for 2 min.Three types of staining methods were used for histomorphological analysis; 1) Hematoxylin & eosin (H&E), 2) Masson’s Trichrome (TRI), and 3) Immunohistochemistry (IHC). Wound bed biopsies were excised at days 3, 7, 14 and 21 post-wounding. The tissue samples were fixed in 10% buffered formalin, processed and embedded in paraffin to prepare tissue blocks. Then, the tissue blocks were sectioned for 5 µm  and 3 µm (IHC). All sections were deparafinized and rehydrated conventionally prior to staining. For H&E and Masson’s trichome, the sections were stained with their respective staining kits. For further investigation, immunohistochemistry was performed to visualize distribution and localization of specific antigen or cellular components responsible for wound healing. In this study, three antibodies were used: Collagen I, Collagen III, and fibroblast. Following deparaffinization and dehydration processes, the sections undergone antigen retrieval process using citrate-based solution in microwave for 10 min and blocking process in 0.3% HAll the tissue sections were then subjected to clearing and mounting processes. The slides were observed under microscope  and photomicrographs were taken at ×20 magnification for two parts; dermis and hypodermis. The stained tissue sections were scored blindly by histological experts using the modified 0 to three numerical scale as described by p values < 0.05 were considered significant. GraphPad Prism software version 9.0  was used to estimate the area under the curve (AUC) with a 95% confidence interval. The AUC was calculated from the wound contraction percentage versus time profiles, which was determined by the area normalized to the baseline for the 21-day period . Normality test was performed using Kolmogorov-Smirnov test. One-way ANOVA was conducted followed by Post Hoc Tukey’s test to determine statistical significance. y period .Macroscopic changes over time for Day 0, 3, 7, 14, and 21 for superficial partial thickness of second-degree burn wounds were shown in On Day 3, all the groups did not show any significant value and demonstrated mildly negative percentage of wound contraction . At Day The LF group demonstrated the highest AUC among all the groups and showed significant difference when compared to normal (NM), negative (NG) and positive (PS) control groups. The RT group showed significant difference when compared to the NM group .On day 14, the hydroxyproline content of LF group demonstrated significant difference when compared to NM and NG groups. For the PS and RT groups, the hydroxyproline content were increased but did not reach significant value .Histopathological view of the burn wounds for all the five groups stained with haematoxylin and Eosin (H&E) were shown in Histopathological views of the burn wounds for all the five groups stained with Masson’s trichome were shown in Oxidative stress plays an important role in burn wound conversion, in which the zone of stasis cannot be rescued and progresses to necrosis. Reduction in oxidative stress may halt or arrest burn injury progression into deeper tissue . Burn woThe LPVa root extract was as effective as the leaf extract in terms of wound contraction percentage and area under the curve. According to via direct interactions with bacterial cell membranes, DNA, enzymes and proteins  of up to 1,000 mg/kg, indicating that the LPVa extract is safe for skin application with appropriate concentration . The silver component is effective in eliminating pathogens proteins . This anolecules . Howeverolecules , which lolecules . Additioolecules . Moreoveolecules . Contradntration . Additiontration . TherefoHydroxyproline which is a basic constituent of collagen is a good marker for wound healing assessment. It is one of the most abundant amino acids in collagen and its concentration indicates the concentration of collagen. High concentration of hydroxyproline symbolises faster rate of wound healing . In thisHistological analyses of all staining used in this study revealed complete destruction of epidermis and partial destruction of dermis for all the groups, observed at Day 3 after wound induction. These characteristics confirmed second-degree or superficial partial thickness of burn wound . InvolveAs for the scoring of Masson’s trichome staining, all the groups received the highest score of 3, indicating the presence of complete and mature collagen in the wounds of all the groups. Most collagens found in the skin are type I and III, that plays an important role of attracting fibroblast and keratinocytes to the wound . The derCollagen III is the type of collagen synthesized during the early stages of wound healing and is progressively replaced by the dominant skin collagen (collagen I) . This coFibroblast is the cell responsible for the production of extracellular matrix and collagen, and therefore plays an important role during tissue repair. The migration and proliferation of fibroblast to the wounded area may initiate proliferative phase of repair which subsequently promote effective wound healing and wound contraction. Based on the H & E staining of this study, the LPVa leaf group demonstrated complete and mature fibroblasts at Day 21 compared to the LPVa root group. The superior effect of leaf extract for fibroblast proliferation was in parallel to the hydroxyproline concentration measurements. In fact, fibroblasts in wound healing areas may proliferate and are encircled by collagen fibrils. The new growth-initiating factor for fibroblasts are present and may attach to collagen fibrils at the wound healing site, generating peptides of collagen including prolyl-hydroxyproline . The proBoth leaf and root extracts of LPVa could promote the healing of thermal-burn wounds, with leaf extract being more superior in terms of the hydroxyproline content and histological analysis (Haematoxylin & Eosin and immunohistochemistry). Further studies related with molecular aspects should be performed to determine the mechanism of wound healing by LPVa leaf.


Staphylococcus aureus (S. aureus) cultures at earlier times of DOX release. Despite the well-known use of PLGA as a drug delivery vehicle, when synthesized with DOX, it presents both characteristics of the desired treatment to prevent healthy tissue damage while avoiding bacterial growth in a microenvironment with anatomical features, such as grooves, projections, and other tough conditions that favor the development of oral diseases.The state-of-the-art sustained drug delivery systems are related to features to improve pharmacological transport through a controlled ratio between drug release and the desired therapeutic effect. Microspheres of biodegradable polymers, such as poly(lactic-co-glycolic acid) (PLGA), play an important role in these approaches, directing the release in a specific region of interest. In this way, the encapsulation of doxycycline (DOX) as a microbial agent turns the PLGA microspheres into a potential device for the treatment of topic oral diseases. Thus, this work aimed to produce DOX-loaded PLGA microspheres and see how they interfered with mesenchymal stem cell viability and in the sustained release in antimicrobial assays. Scanning electron microscopy showed the spherical microstructured pattern, revealing assorted sized distribution, with major diameters ranging 1–3 µm. The encapsulation efficiency presented a mean of 80% in both methods to obtain the microspheres (sonication and magnetic rotation). The DOX release test revealed a gradual and continuous profile of 30–40% between 120 and 168 h. Mesenchymal stem cells cultured in PLGA with or without DOX at several concentrations revealed no effect on the cell metabolic activity. Striking morphology changes were observed by confocal microscopy after 1 to 3 days under culture. The live/dead assay indicated that when microsphere densities were increased (from 10 to 100 µg/mL) cultured cells presented an internalized pattern of microspheres in both groups of PLGA containing DOX or not, while slight cell death signals were identified nearby microsphere clusters. Microbiological assays performed by the agar diffusion test pointed out that an inhibition zone was identified in  Sustained release systems are designed to allow the introduction of therapeutic substances into the body and improve their safety and efficacy in a controlled and targeted pathway ,2. MicroDoxycycline (DOX) is a broad spectrum member of the tetracycline antibiotics, known for its fast bacterial action in Gram-negative and -positive protein synthesis. Oral administration of DOX is usually repeated several times per day to keep within the therapeutic range. Hence, local delivery of DOX using polymeric carriers is a reliable method of drug delivery for abroad applications, using assorted bioresorbable polymer-based carriers, such as chitosan, poly , alginate, and gelatin ,13.S. aureus, this drug loaded in the polymeric system developed and achieved an early antimicrobial effect, while no toxicity level was identified in mesenchymal stem cells.One important issue on the topic of drug administration is the matter that it can generate high drug concentration levels in a microenvironment of inflamed tissues, due to the burst drug effect that commonly occurs in the first hours, which can induce local toxicity . In thisw/v) poly (PVA) solution and then homogenized for 3 min at 10,000 rpm in an Ultra Turrex mixer . Before the solvent evaporation step, half of the obtained emulsion was sonicated for 10 min at 85% potency, setup at 180 W . The other half was directly stirred to evaporate the solvent for 24 h at room temperature. Finally, both emulsions were centrifuged at 5000 rpm for 5 min and washed with distilled water . The microspheres (precipitate) were frozen at −20 °C for 24 h and lyophilized for an additional 24 h at −100 °C . The samples obtained were submitted to a selective mesh of 45 µm to obtain more uniform size microsphere. The same protocol was performed for the PLGA microspheres without the addition of DOX. All reagents were purchased from Sigma-Aldrich .The microspheres were prepared using the single emulsion/solvent evaporation method, adapted from Terukina et al. (2016) . For the−1.FT-IR spectral data were obtained on a spectrometer  with attenuated total reflection (ATR) to confirm Doxycycline encapsulation in the PLGA microspheres. The spectral scan was obtained in the range between 4000 and 500 cmThe morphology of the microspheres was observed by scanning electron microscopy (SEM) (EVO MA15-ZEISS). Both PLGA and PLGA + DOX samples were submitted to a gold-sputter of 15–30 nm (Emitech K550) and visualized by secondary electrons at 15 kV. Randomly selected electron micrographs at 3000× magnification were used to obtain the microspheres diameter distribution by Image J software, counted up to reach the total number of 1000 counts.The EE and DL were determined according to Equations (1) and (2), respectively . The sup® tubes (1.5 mL) under continuous shaking at 300 rpm . At specific time intervals , the tubes were centrifuged at 13,800 rpm at 4 °C for 10 min , and the supernatant was collected. The same amount of fresh PBS was added to each sample for the next readings in a UV–VIS spectrophotometer  at 347 nm. The cumulative release profile was calculated by the calibration curve obtained previously. No external stimuli  were conducted.The in vitro release of DOX from the microspheres was determined as proposed by Misra et al. (2009) . A totalS. aureus by the agar diffusion test, according to the Reinbold et al. (2016) protocol . The particle size distribution was analyzed by Gaussian curves and indicates mean size. The data of the MTT assay were submitted to the one-way ANOVA parametric Tukey statistical test. The results are represented by the mean ± standard deviation.During microsphere preparation, the emulsion was obtained by two different mixing mechanisms: sonication (S) and magnetic rotation (MR). Both methods tested revealed minimal and insignificant yield differences, as shown in −1, which are assigned to the stretching vibration of the carbonyl groups (C=O) present in both monomers, and the 1089 cm−1 peak attributed to C–O stretching [−1, specific for the –COOH group present in doxycycline was identified [The FT-IR spectra of PLGA and PLGA + DOX are depicted in retching . When PLentified . The resAll microspheres synthesized in this work were reproduced independently in sextuplicates, and the samples that reached higher encapsulation efficiency were selected for SEM. Both emulsion mixing methods presented as sonication (Sample 1) and magnetic rotation (Sample 3) are depicted in The size measurement revealed that the average microsphere size ranges from 1 to 3 µm (±50%), while the size range from 1 to 10 µm represents 90% of all particle diameters, as seen in This result also demonstrated that different mixing mechanisms did not show a variation in the size of the microspheres produced.Samples submitted to MR presented an encapsulation efficacy mean of 82.50 ± 2.85% while the S-prepared samples were 83.07 ± 1.54% . The resA proportional release profile between samples was identified and the drug delivery increased with time . The majS. aureus culture  containing PLGA and PLGA + DOX at several concentrations. All concentrations between both groups presented no statistical significance, while an increased absorbance signal was observed by time course due to cell growth .The live/dead identification kit revealed that all groups presented high cell survival rates after 3 days in culture . Few deaSeveral methods have been proposed to obtain PLGA microspheres. In the simple emulsion/solvent evaporation method, the emulsion can be prepared by assorted physical methods, including sonication and magnetic rotation . It is kThe FT-IR spectra identified the DOX presence  that canE. coli cultures after 24 h and indicated that a sustained release formulation is required to control bacterial growth for longer periods. Zhong et al. (2022) [Despite the cumulative release rate identified in . (2022)  showed t−1 in PLGA and PLGA + DOX. However, despite there being no evidence of changes in cell growth, the LSCM showed differences in cell morphology. Interestingly, when aggregated microspheres were identified, the cellular organization also followed a pattern with a high amount of cells surrounding such clusters. This pattern was already reported as an early sign of spheroid formation elsewhere in gelatin microspheres [The evaluation of PLGA or PLGA + DOX in hMSC cell culture was identified by metabolic activity measurement during MTT assay  and corrospheres , while tospheres .A similar work recently presented a sustained release pattern of DOX from PLGA microspheres obtained by a double-emulsion method, indicating that microspheres were not toxic, kept the sustained release for up to 21 days and were indicated as a potential periodontitis treatment . In thisIt is important to emphasize here that the use of PLGA + DOX, or even DOX with any other polymeric model of sustained delivery, has constraints due to the limited DOX activity time identified in microbiological assays. Although the release rate has prolonged features, for some treatments such as periodontitis, or even other oral diseases that form bacterial pockets or abscesses, a new DOX administration should be repeated every 24 h. Thus, in future in vivo works, it is necessary to evaluate the issue of a cumulative effect of the physical presence of the carrier biomaterial to be successfully recurrently added at the infection site until the infectious process is solved.S. aureus growth. The cell viability assays demonstrated that after several concentrations tested of PLGA and PLGA + DOX, the metabolic activity remained similar to controls, while the live/dead survival assay showed differences in cell morphology due to adhesion surrounding microspheres aggregates, with no indication of cell death.Both emulsion preparation methods used generated the same particle size of PLGA + DOX, reaching around 85% of encapsulation efficacy. The FT-IR revealed a physical association of DOX and PLGA, while no burst was identified in release tests. Assorted particle sizes identified by SEM revealed average sizes of 1–3 µm (50%) and 3–5 µm (10%). Microbiological tests were moderately effective up to 24 h to inhibit


To conclude, seropositivity to BKPyV and JCPyV is common among marital couples in Finland, with only slight differences between genders. In men, the sexual behavior might be associated with JCPyV seroprevalence.BK (BKPyV) and JC (JCPyV) polyomavirus infections are commonly subclinical and known infrequently to cause serious clinical diseases. Longitudinal follow-up studies regarding JCPyV and BKPyV serological outcomes are scanty. We analyzed JCPyV and BKPyV IgG-antibodies in 327 pregnant women and their 132 spouses, enrolled in the longitudinal Finnish Family HPV cohort at Turku University Hospital, Finland. Blood samples taken at baseline, and at 12-, 24-, and 36-month follow-up visits were analyzed for capsid protein VP1-antibodies using multiplex serology. Seroprevalence was constant for both BKPyV and JCPyV across the follow-up, varying between 95–99% and 59–68%, respectively, in women and between 96–97% and 66–72%, respectively, in their spouses. Seroconversion to BKPyV and JCPyV was detected in 15% and 18% of the women and in 13% and 19% of the men, respectively. Waning of BKPyV and JCPyV antibodies was infrequent, present in only 5% of the women (both viruses) and in 1.5% of the male spouses (only BKPyV). The number of lifetime sexual partners ( First two HPyVs were identified in the 1970’s; BK polyomavirus (BKPyV)2 and John Cunningham polyomavirus (JCPyV)3. BKPyV and JCPyV infections are generally subclinical but can cause also serious clinical diseases especially among immunocompromised individuals. BKPyV infection can lead to polyomavirus-associated nephropathy in renal transplant patients, while JCPyV has a causative role in progressive multifocal leukoencephalopathy . Both BKPyV and JCPyV are capable of transforming cells in vitro7. In malignant transformation, both the viral large- and small T antigens play a key role. BKPyV and JCPyV are considered as possible human carcinogens (Group 2B) by IARC (https://monographs.iarc.who.int/list-of-classifications), although their causative role in human carcinogenesis still remains controversial.Human polyomavirus (HPyV) family comprises 13 members of non-enveloped DNA viruses with circular double-stranded genome varying from 5100 to 5400 base pairs9. Both BKPyV and JCPyV can persist in the urinary tract allowing their asymptomatic urinary shedding even in healthy subjects12.BKPyV and JCPyV have been intensively studied, but the natural history of these infections is still incompletely understood. Furthermore, no longitudinal studies on spouses exist. Horizontal transmission via mucosal surfaces or virus-containing fluids has been suggested. BKPyV transmission is believed to take place primarily via upper respiratory and oral routes16. Seroepidemiological studies suggest that primary BKPyV infection might be acquired earlier in life than JCPyV, implicating a different mode of transmission18. Pregnancy has also shown to be associated with impaired immunocompetence, and viral replication and shedding of BKPyV increase during pregnancy22. However, there is only one previous study on pregnancy so far, with a single focus on BKPyV and JCPyV serology during the first and second pregnancy but not after pregnancy14.In healthy adult population, BKPyV seroprevalence is around 90% while the JCPyV is lower, around 50–7023. Subsequently, multiplex detection with glutathione S-transferase (GST)-tagged proteins utilizing the Luminex platform become more popular25, allowing simultaneous analyses of multiple antibodies and more importantly also the comparison of the results provided by different studies.The seroprevalence for BKPyV and JCPyV has varied widely among different studies, possibly explained by the different sensitivity of the assays used. Early studies until late 1990’s, were mostly based on the haemagglutination-inhibition (HI) method. The methods based on virus-like particle (VLP) ELISA became more common since the early 2000’As longitudinal studies are nearly lacking, the goal of our study was to determine the seroprevalence, seroconversion, and waning of antibodies to capsid protein VP1 of JCPyV and BKPyV among spouses during their 3-year prospective follow-up and to determine co-factors that could be associated with seropositivity.rd trimester when enrolled at the Maternity Unit of Turku University Hospital (Finland) as described earlier by Rintala et al. 200526. All women filled in a standardized questionnaire providing comprehensive information on their demographics26. During their first post-partum visit 1.5–4.9 months after delivery, also the spouses of the enrolled mothers completed a standardized questionnaire. These comprehensive questionnaires collected demographic data e.g. on education, employment, marital status, number of sexual partners, alcohol consumption, smoking history and snuff usage, allergy, and atopy. Mean age of the mothers and their male spouses at enrollment was 25.5 years  and 29.0 years (range 19–64 years), respectively. The study was approved by the Research Ethics Committee of Turku University Hospital . All methods were carried out in accordance with relevant guidelines and regulations. Informed consent was obtained from all participants.In total, 327 of the 329 women and 132 of their 133 male spouses enrolled originally in the Finnish Family HPV Study (FFHPV), were eligible for the present study. All women were pregnant at their 327. The number of blood samples of women and their male spouses available for the present study at 12-, 24- and 36-month visits were 278, 220, 261 and 112, 87, 94, respectively. After collection, the samples were centrifuged at 2400 r.p.m. for 10 min . Each serum sample was immediately divided into three 1 ml aliquots and stored first at −20 C for no longer than 1 week and then at −70 C until shipped on dry ice to DKFZ, Heidelberg, Germany, for serological analysis.Blood samples were taken at baseline and at 12-, 24-, and 36 months of follow-up as described earlier16. The method is a GST-capture ELISA28 combined with fluorescent bead technology29. In the present study, sera were scored as positive when the antigen-specific mean fluorescence intensity (MFI) values were greater than the cut-off level of 400 or 1000 MFI (stringent) for VP1 antigen of BKPyV and JCPyV.The samples were analysed for IgG-antibodies to VP1 of BKPyV and JCPyV. Antonsson et al. and Gossai et al. have described the method for BKPyV and JCPyV serology used here in more detailed, earlierSeroconversion was defined by two conditions, both of which must be fulfilled: (1) at least a twofold increase of the previous serum MFI value and (2) MFI value exceeding over the cut-off of 400 MFI. Similarly, antibody waning was defined by two conditions: (1) at least a twofold decrease of the previous serum value, and (2) fall of the MFI value below the cut-off of 400 MFI.rd trimester. Thus, our study design allows us to study BKPyV and JCPyV seropositivity, antibody titers, seroconversion and antibody decay both during and after pregnancy. Demographic data recorded by the questionnaire at enrollment (women pregnant at 3rd trimester) was used to evaluate to the association between different co-factors and BKPyV/JCPyV seropositivity. The same data was analyzed also in male spouses.We first evaluated the seroprevalence, seroconversion, and waning of antibodies to capsid protein VP1 of JCPyV and BKPyV among the couples during their 3-year prospective follow-up using the criteria given above. All women were enrolled in the cohort while pregnant at 3p < 0.05.All statistical analyses were run by using the SPSS for Windows  software package. Conventional 2 × 2 tables were used to analyze categorical variables, tested using the likelihood-ratio test or Fisher’s exact test. Differences in the means of continuous variables were analyzed by ANOVA or Mann–Whitney/Kruskal–Wallis test for two and multiple independent samples, respectively. All statistical tests performed were two-sided and declared significant when The mean and median levels of BKPyV and of JCPyV IgG-antibody levels of the pregnant women and their male spouses during their 3-year followed-up are summarized in Table Figure Similarly as with BKPyV, the seroprevalence of JCPyV (MFI 400) was lowest in mothers to come at baseline visit (59.0%) and at the 2-year visit (58.6%). Nearly 70% of the women were seropositive for JCPyV at the last 3-year visit. Among the male spouses, seroprevalence of JCPyV (MFI 400) was lowest at the baseline (65.9%) and highest after two years (73.6%). JCPyV seropositivity was slightly less common in women than in their male spouses. As compared with BKPyV, seroprevalence of JCPyV was more sensitive to the cut-off change resulting in 14% and 6–12% decline in women and their spouses, respectively.p = 0.038) between JCPyV seroconverted and non-converted men. However, JCPyV seropositivity did not associate with the increase of lifetime sexual partners. Education, employment, smoking, alcohol consumption, using of snuff, marital status, allergy, and atopy did not show any association with BKPyV or JCPyV seropositivity.The association between demographic data and BKPyV and JCPyV seropositivity in women and their spouses are shown in Supplement Table The proportion of women and their male spouses who seroconverted for BKPyV or JCPyV during the follow up are presented in Fig. rd trimester pregnancy with their seropositivity one year later  and higher sample dilution (1:1000)16. They found no gender differences in BKPyV seropositivity (88%), while seroprevalence of JCPyV was lower in women (49%) than in men (60%), exactly as shown in our study. Several studies have confirmed that seropositivity for JCPyV increases with age especially among men30. In the present cohort, these couples were healthy adults with the mean age of 25 years (women) and 29 years (men), thus precluding calculations of age-specific data. In general, JCPyV seroprevalence was higher in the present study than reported by earlier studies17. The JCPyV seroprevalence found here is similar as reported earlier in age groups older than 50 years16. However, Viscidi et al. reported findings closely matching with our data, the seroprevalence of JCPyV being 60–68% in the age group of 20–49 years31.To our knowledge, this is the first study evaluating BKPyV and JCPyV serology among spouses in a longitudinal setting. We showed that over 94% of the couples were seropositive for BKPyV, whereas JCPyV seroprevalence was lower, varying between 59–68% and 66–72% among women and their male spouses, respectively. Our results are in perfect alignment with those by Antonsson and coworkers who reported 96% seroprevalence for BKPyV and 60% and for JCPyV in an Australian population14. As expected, this seroprevalence was very close to that in our cohort, because the study subjects in this study represented also mothers to be. Similarly as reported by Antonsson et al.15, we also found that overall BKPyV seropositivities were relative robust to changes in the MFI cut-offs used, i.e., 1000 MFI or 400 MFI. JCPyV seroprevalence was more sensitive to cut-off changes than BKPyV as described also by Antonsson et al.15. Interestingly, seroprevalence for JCPyV declined more in women than in men. We could also confirm that among the seropositive individuals, there was a marked fluctuation in the MFI values as also reported by Antonsson et al.15.Stolt and coworkers described that BKPyV seropositivity increased rapidly with increasing age of the children, reaching 99% seroprevalence at 7–9 years of age. This was followed by approximately 10–20% decline, attributed to the antigenic variants of BKPyV. In contrast, seroprevalence of JCPyV increased more slowly with increasing age being highest among the mothers over 25 years of age (72%)14. This discrepancy can be explained partly by the use of different assays for BKPyV and JCPyV serology. We used GST-capture ELISA in combination with fluorescent bead technology, while Stolt et al. used VLP-based ELISA14. Another source of discordant data can be due to the different criteria used to define seroconversion. Importantly, we did not look only seroconversion from seronegative to seropositive exceeding the 400 MFI cut-off but seroconversion was recorded when there was at least a twofold increase of the previous serum MFI value (e.g. from 1000 to 2000MFI or 4000 MFI to 8000MFI). Antonsson and co-workers found in their longitudinal study that none of the subjects seroconverted to BKPyV while serorevision was found in 1% and fluctuation in 2% of the participants. According to their data, seroconversion for JCPyV was more prevalent and found in 4%. They reported that seroconversion and fluctuation were more prevalent, detected in 4% and 3%, respectively15. Our study confirmed their findings implicating that JCPyV antibody status over time was less stable than that of BKPyV.In our study, we also found seroconversion for BKPyV and JCPyV in both women and their male spouses during their 3-year follow-up. Seroconversion for BKPyV was of similar magnitude in both genders (13% in men and 15% in women). It was shown also for JCPyV, albeit seroconversion of JCPyV was slightly more prevalent (19% men and 18% women) than that of BKPyV. We also detected BKPyV antibody waning in 5% of these couples while JCPyV antibody waning was found only among women (2%). Stolt and coworkers have studied seroconversion or antibody waning of BKPyV and JCPyV among Finnish pregnant women previously. They did not find any seroconversion or loss of seropositivity during 5-year follow up9. On the contrary, JCPyV seroprevalence increases slowly and markedly later than for BKPyV, reaching 50–80% among healthy adults. The previous evidence supports the view that exposure to JCPyV occurs mostly in adult life.All previous studies support the view that the exposure to BKPyV occurs in early life because seroprevalence studies indicate that nearly 50% of children at the age of 2 years are seropositive, and more than 90% of children have acquired seropositivity by the age of 10 years. This pattern of seropositivity support the view that BKPyV transmission primarily takes place by upper respiratory and oral routes16. Only few studies have analyzed other risk factors associated with JCPyV seropositivity or antibody stability16. As far as the authors are aware, the present study seems to be the first to find evidence that sexual behavior, determined by the number of sexual partners when graded into four different categories, was different between JCPyV seroconverted and non-converted men. JCPyV is known to be asymptomatically and intermittently shedding in the urinary tract even with high viral load32.However in the current study, the number of lifetime sexual partner was lower among JCPyV seropositive men. In the present analysis, education, employment, smoking, alcohol consumption, using of snuff, allergy, atopy, or glucocorticoid use did not have any significant association in predicting seropositivity either for BKPyV or JCPyV. In line with our results, two previous studies have confirmed that smoking had no association with BKPyV or JCPyV serostability or seropositivity16.This different age profile between JCPyV and BKPyV exposure might indicate that different risk factors are being involved in developing seropositivity for these two viruses. The only covariates recognized so far are age and gender, the latter being still controversial22. We found that the mean antibody levels of BKPyV were lower in pregnant mothers at their 3rd trimester (and their spouses as well) as comparted with the antibody levels one year after the pregnancy. One could speculate that reactivation or reinfection of BKPyV among spouses during the pregnancy could result in elevated antibody levels. Stolt and coworkers also studied BKPyV and JCPyV serology in pregnant mothers (at their 1st trimester) and later when they were pregnant for the second time. However, they could not estimate the effect of pregnancy because all samples were derived from pregnant women14. In addition, McClure et al. have showed that asymptomatic urinary shedding of BKPyV was more common in pregnant women than in non-pregnant, although there was no difference in the level of JCPyV excretion33.We also demonstrated that the index pregnancy (FFHPV cohort) had no impact on the profile of either the BKPyV or JCPyV serology among the mothers. One would expect a rise in BKPyV seropositivity as BKPyV reactivation has been documented in up to 53% of pregnant women through PCR amplification of viral sequencesTaken together, the present longitudinal cohort study demonstrates that both BKPyV and JCPyV are ubiquitous among young healthy couples, with only minor gender-related differences.Supplementary Information.


In this research, different clays such as laponite and montmorillonite (NaMMT) are used as fillers in the preparation of thermoplastic starch/clay nanocomposites. Thin films are produced by casting and evaporation in a wide composition range, using glycerol as the plasticizer at two different concentrations. The surface energy of clay fillers is measured by inverse gas chromatography (IGC); X-ray diffraction (XRD) and light transmission measurements (UV-VIS) are carried out to characterize the structure of nanocomposites; and mechanical properties and water vapor permeability are also studied. While all the starch/montmorillonite nanocomposites possess intercalated structures, significant exfoliation can be noted in the starch/laponite nanocomposites, mainly at low clay contents. Due to the larger surface energy of montmorillonite, stronger polymer/clay interactions and better mechanical properties can be assumed in starch/NaMMT composites. The smaller surface energy of laponite, however, can facilitate the delamination of laponite layers. Thus, the specific surface area of laponite can be further increased by exfoliation. Based on the results, the better exfoliation and the much larger specific surface area of laponite lead to higher reinforcement in starch/laponite nanocomposites. Polysaccharides are promising biopolymers that are widely used in the packaging, agricultural, healthcare, and medical areas as an alternative to petroleum-based polymers. Films, nanoparticles, gels, and scaffolds are usually developed from these biopolymers ,3,4,5,6.Polymer/clay nanocomposites are assumed to exhibit improved barrier, thermal, and mechanical properties compared with traditional composites. Recently, researchers have reported in the literature several attempts at preparing TPS nanocomposites. In most cases, TPS/montmorillonite (NaMMT) nanocomposite films were prepared by melt blending or by film casting. The results demonstrated that the incorporation of NaMMT results in an intercalated/exfoliated structure of TPS nanocomposites ,19,20,21Since NaMMT is a natural mineral produced by mining, it usually contains contaminants and irregularities in its crystalline structure, making it necessary to explore and find other silicate fillers that do not have these drawbacks. A synthetic layered silicate, laponite, is an obvious alternative because its chemical and crystalline structures are well known. Laponite is a synthetic silicate (Laponite XLG composition: Na+0.7[(Si8Mg5.5 Li0.3)O20(OH)4]-0.7) with the same structure as the clay mineral hectorite, another smectite similar to NaMMT. Compared to natural NaMMT, synthetic laponite presents more well-defined compositions and is free of impurities and accessory minerals. The individual laponite particles have a more regular disk shape, and their lamellae are smaller than natural NaMMT lamellae, with an average diameter of 25 nm and thickness of 1 nm . Our forSince the properties of polymer composites strongly depend on the specific surface area of the filler and the polymer/filler interfacial interaction ,11,15,20Although laponite is also a popular clay nowadays, there are few publications on TPS/laponite nanocomposites ,33,34,35Unfortunately, there is also no publication in the scientific literature that compares the effects of NaMMT and laponite on TPS nanocomposite properties, though the effects of the two fillers have been compared for several other polymers ,38,39,40High-quality corn starch produced in Hungary  was used in the experiments. Its water content was 12%. Glycerol with a 0.5 wt% water content was obtained from Sigma-Aldrich Kft. Hungary and was used without further purification or drying.Two kinds of layered silicate were studied during the experiments: sodium montmorillonite (Nanofill 116) supplied by Southern Clay Products  and Laponite XLG obtained from Byk Additives and Instruments . The most important characteristics of the silicates are summarized in TPS nanocomposite films were prepared by the solution casting method ,20. SoluThe films were obtained by casting the hot suspension in Petri dishes covered by a Teflon sheet and dried in a climate chamber at 35 °C for 24 h at 50% RH. The clay content changed between 0 and 20 wt % based on the amount of dry starch. The thickness of the films was 0.10 ± 0.02 mm. Before testing, the films were stored at 23 °C and 50% RH (in a climate chamber) until a constant weight was reached.The crystalline structure of the TPS and its composites was studied by X-ray diffraction (XRD) using Philips PW1830/PW1050 equipment with Cu Kα radiation at 40 kV and 35 mA. Samples were scanned in the diffraction angle range of 2–15° in 0.04° steps. Diffractograms were recorded on the powders (starch) or films using a multipurpose sample stage. The basal spacing of the silicate layers was calculated using Bragg’s equation. The light transmission of films at 700 nm was determined using a UV-VIS spectrometer (UnicamW500). Tensile properties  were measured by Instron 5566 apparatus according to ISO 527/4A. The Young’s modulus was determined at 0.5 mm/min while ultimate properties were determined at a cross-head speed of 5 mm/min. All characteristics were derived from five parallel measurements.−1·s−1·Pa−1) was calculated using Equation (1), where m is the weight loss (g) of the test cell, x is the film thickness (m), and A is the exposed area (m2) during Δt duration (s) under Δp partial water vapor pressure (Pa).The water vapor permeability (WVP) of films prepared by casting was investigated at a 100–50% relative humidity difference. The film was placed between two metal rings on top of a glass cell containing distilled water, and then the glass cell was introduced to a climate-controlled chamber regulated at 50% RH and 23 °C. The test cell was periodically weighted to a constant variation rate of weight. WVP (g·msd) of the clay samples was determined by inverse gas chromatography (IGC) at an infinite dilution using n-alkanes with different chain lengths. IGC measurements were carried out using Perkin Elmer Autosystem XL apparatus at 160 °C without conditioning the samples. A new column was prepared for each set of experiments. Vapor samples of 5–20 μL were injected into the column, and an FID detector recorded retention peaks. Each reported value is the result of three parallel measurements. High-purity nitrogen was used as the carrier gas, the flow rate of which changed between 40 and 60 mL/min depending on the types of adsorbent and adsorbate. The γsd values were calculated by the method put forward by Dorris and Gray [The dispersion component of the surface tension . Eventually, n is a parameter taking into account strain hardening, and φ is the volume fraction of the filler. Equation (2) can easily be transformed into a linear form by taking the logarithm of both sides:At large deformations, mechanical properties such as the tensile strength are determined by the composition, matrix properties, structure, and interactions. Interfacial interactions considerably influence and occasionally determine the properties of all heterogeneous materials. They also play an important role in the studied TPS/clay composites. Reinforcement, or the strength of interaction, can be estimated from the composition dependence of the tensile strength of composites. The use of a simple semi-empirical model developed earlier  allows uThe slope of this linear equation provides parameter B, which is proportional to the extent of reinforcement and determined by the specific surface area of the filler, the strength of interfacial interaction, and the matrix strength ,45.In From In addition to the importance of appearance and mechanical properties, water permeability is also essential for packaging materials. It is well-known that the strong hydrophilic character of TPS results in high water permeability. During the investigation, the effects of laponite and montmorillonite on WVP were studied. The results determined for 100–50 RH % are presented in Only a limited number of publications in the literature provide data on the water vapor permeability of TPS/NaMMT ,46,47 an2) can facilitate the delamination of laponite layers. Thus, exfoliation further increases the already significant specific surface area of Laponite XLG clay. Although due to the larger surface energy of NaMMT (229 mJ/m2), stronger polymer/clay interaction and better mechanical properties could be assumed of TPS/NaMMT composites, the results proved that the better exfoliation and the much larger specific surface area of laponite result in higher reinforcement in TPS nanocomposites . This was proven by the model of composition dependence of tensile strength. The TPS/laponite nanocomposites with better mechanical properties can act as excellent biodegradable packaging materials in addition to or instead of TPS/montmorillonite nanocomposites.We investigated the effect of particle characteristics and surface properties of laponite and sodium montmorillonite on the structure and properties of TPS nanocomposites. TPS/clay nanocomposites in a wide composition range and using two different concentrations of glycerol content were prepared by film casting. The XRD spectra and light transmission measurements showed that all TPS/NaMMT nanocomposites possess an intercalated structure. In TPS/laponite nanocomposites, effective exfoliation can also be supposed mainly at low clay contents. The small surface energy of laponite (153 mJ/m


Congenital generalized lipodystrophy type 1 (CGL1) is a very rare autosomal recessive genetic mutation with generalized lipoatrophy and metabolic complications. We report CGL1 in two Saudi female siblings with lipoatrophy, diabetes mellitus, hypertriglyceridemia, steatohepatitis, and acanthosis due to very rare homozygous 1‐acylglycerol‐3‐phosphate O‐acyltransferase β (AGPAT2) genetic variant. Genetic lipodystrophies are inherited in autosomal recessive or autosomal dominant pattern.O‐acyltransferase 2 (AGPAT2) in CGL type 1 and seipin in CGL type 2 are responsible for 95 percent of reported cases.CGLs, or Berardinelli–Seip congenital lipodystrophy (BSCL), are autosomal recessive disorders frequently recognized at birth or shortly thereafter.People with CGL usually have generalized lipoatrophy and metabolic complications related to insulin resistance and abnormal deposition of fat in other organs such as the liver, skeletal muscles, and the heart. Complications may include diabetes mellitus, hypertriglyceridemia, steatohepatitis, acanthosis nigricans, and hypertension.The prevalence of CGL in Saudi Arabia is unknown; only four cases were reported in the literature, although the true prevalence may be higher due to the high prevalence of consanguinity in this country.2This study was reviewed and approved by the institutional ethical committee, Jazan, Saudi Arabia, and was conducted in agreement with the Declaration of Helsinki Principles. The two sisters are offspring of a consanguineous Saudi family living in Jazan. The written informed consent was obtained from patients and their father.Two sisters, 22 and 14 years old, were referred to diabetes and endocrinology center in Jazan, Saudi Arabia, with history of uncontrolled diabetes mellitus, HbA1c , hypertriglyceridemia, and hepatomegaly. Both were on high‐dose insulin, total doses of 200 iu/day in the older and 290 iu/day in the younger girl. Diabetes had been diagnosed at the age of 13 and 8 years, respectively. They had never achieved good glycemic control despite high‐dose insulin and oral antidiabetic medications. Their history did not reveal ketoacidosis despite continuous high blood sugar. The older sister had a history of admission to a regional hospital due to an episode of acute pancreatitis. Family history revealed a consanguineous relationship between their parents who did not complain of any chronic medical disease. The older sister menarche started at the age of 12, and then, she had irregular periods (oligomenorrhea) with scanty menstruation; the younger sister had not yet started her menstrual periods. Ovarian ultrasound reveals appearance of multiple small cysts, so these in addition to oligomenorrhea and androgenic features signify that she has polycystic ovary. Hepatic fibroscan and DEXA scanning were not performed as they are not available in our center.2 and the younger height was 154 cm, weight 41 Kg, and BMI at the 24th percentile for age and sex. Systolic and diastolic arterial blood pressures were in the normal range for their age, and they are not on antihypertensive medications.On physical examination, the older sister height was 160 cm, weight 46.8 Kg, and body mass index (BMI) 18 kg/mBoth had clear patterns of generalized fat loss on cheeks, trunks, upper and lower extremities, acromegaloid features, hypertrophy of the skeletal muscles, prominent subcutaneous veins, acanthosis nigricans, and hirsutism. Their livers were palpable more than 3 cm below the right costal margins.Laboratory tests presented in Table A clinical diagnosis of CGL was suspected, and genetic analysis was performed, which confirmed autosomal recessive GCL type 1 with a homozygous variant identified at AGPAT2 gene, c.158del/p.(Gly53Alafs*8) in both sisters. This genetic variant can predict the change of the amino acid glycine residue at position 53 with alanine creating a shift in the reading frame starting at codon 53 and ending in a stop codon 7 positions downstream.Current management includes nutritional and exercise program in addition to metformin, multiple daily insulin injections, statin, and fenofibrates. They are scheduled for close follow‐up aiming to control metabolic comorbidities and prevent micro‐ and macrovascular complications and pancreatitis.3AGPATs are key enzymes required for triglyceride and phospholipid biosynthesis, and for the growth and development of adipocytes.Patients with CGL type 1 are either homozygous or compound heterozygous for AGPAT2 gene mutations and inherited in autosomal recessive pattern. Heterozygous carriers are asymptomatic and are at increased risk of diabetes.Our two cases have pathogenic homozygous variants recognized at AGPAT2 gene, c.158del/p.(Gly53Alafs*8). This genetic variant was reported in one Emirati female presented with diabetes firstly diagnosed at 14 years of age with hypertriglyceridemia and acanthosis.Adipose tissue can be divided into mechanical and metabolic types.The clinical presentation of CGLs could be very heterogeneous with variation in the correlation between genotype and phenotype of these syndromes.The gradual progress of ectopic fat deposition, hyperinsulinemia, and insulin resistance increased in the second decade of life making the diabetes difficult to control with conventional therapy. Our patients were diagnosed with diabetes at 8 and 13 years of age and were uncontrolled despite oral therapy and high insulin doses. The very high triglyceride level could be explained by insulin resistance and defective AGPAT2 function.Hepatic involvement in these two siblings manifested with mild elevations of liver enzymes and hepatomegaly attributed to the accumulation of triglycerides in the livers. Another mechanism of steatohepatitis hypothesized that functional insufficiency of AGPAT2 could induce upregulation of liver activity of AGPAT1 enzyme isoform leading to overproduction of triglycerides with subsequent steatosis and hepatomegaly.Adipocytes secrete several bioactive substances including leptin, adiponectin, and tumor necrosis factor‐α, which play an important role in the pathophysiology of insulin resistance and dyslipidemia.Management in these patients includes lifestyle modifications, diet regulation, control of metabolic complications, and early screening for other comorbidities. Insulin is used combined with metformin, and continuous glucose monitoring is performed aiming to control metabolic comorbidities. Although not prescribed for our patients due to high cost and unavailability in the health system, recombinant leptin is the most promising treatment option for patients with CGL. Metreleptin decreases hyperphagia with marked improvement of glucose levels, triglycerides, and steatohepatitis4In summary, we report two sisters with CGL type 1 due to AGPAT2 variant c.158del/p.(Gly53Alafs*8) frameshift mutation. The description of the clinical, biochemical, and molecular features of these cases is of great importance for the clinicians to build up data regarding this disease and for early diagnosis and appropriate screening and prevention of complications.The authors declare that they have no competing interests.AH planned the study. AAN supervised the molecular testing. AGH contributed to data interpretation and clinical investigations. RAT provided the clinical care and counseled the family. IZ critically reviewed the manuscript. SY wrote the draft of the manuscript and coordinated the research plan. All authors agreed on the final manuscript.The written informed consent was obtained from the patient and their father for publication of this case report.


P = 0.036), cervical microbiota (P = 0.010) and microbiota from uterine lavage fluid (P = 0.001). In addition, dramatic decreases in gamma interferon and interleukin-6 cytokine levels were observed in the RSA group. In conclusion, our data suggested altered microbial biodiversity in the vagina, cervix and uterine lavage fluid in the RSA group. Alterations in the microbiota in the uterine cavity could be associated with altered cytokine levels, which might be a risk factor for RSA pathogenesis. Moreover, the microbiota composition differed markedly from the lower genital tract to the uterine cavity, and the microbiota in the uterine cavity also distinctly varied between endometrial tissue and uterine lavage fluid in the RSA group. Hence, sampling with these two methods simultaneously allowed a more comprehensive perspective of microbial colonization in the uterine cavity.Recurrent spontaneous abortion (RSA) is a complex multifactorial disease. Recently, the microbiota of the female reproductive tract, as an emerging factor in RSA, has gradually attracted the attention of many clinical researchers. Here, we reported that the microbiota of the lower and upper female reproductive tracts from patients with RSA showed no significant differences in alpha diversity compared to that of controls. Beta diversity was significantly higher in the RSA group than in the control group in the vaginal microbiota  can be caused by a variety of factors, and a current understanding of the etiology of RSA is still lacking; half of cases have an unknown cause. A substantial fraction of patients show no improvement after treatment. Since the microbiota of the female reproductive tract has been proposed as an emerging factor in RSA patients, further investigation is needed to provide guidance for clinical therapy. In general, this is the first report describing the distinct alterations of the vaginal, cervical, and uterine microbiota in RSA, not just that in the vagina. Furthermore, another major strength of this study derived from the further in-depth investigation and analysis of the characteristics of the microbiota colonizing the upper female genital tract in RSA, which provided a more comprehensive view for investigating the uterine microbiota. Lactobacillus can prevent infection, and a decrease in its abundance might be associated with infertility and adverse pregnancy outcomes in in vitro fertilization (IVF), with undefined mechanisms (Atopobium and Prevotella) exhibited significantly greater abundance, along with higher expression of CCL2/CCL8/CCL3/CCL4/CCL5, in RSA patients -positive recurrent miscarriage patients have been observed (The definition of recurrent spontaneous abortion (RSA) mostly refers to two or more consecutive miscarriages before 20 weeks, and 1–3% of childbearing women suffer from this situation . Therefochanisms , 8. The chanisms , 9. The patients –13. Howeoversial , 13. A roversial . Howeverobserved . Hence, To obtain a comprehensive view of microbiota alterations in RSA patients, samples of 25 RSA and 25 control patients were included for analysis. The basic clinical information of the included patients in this study was analyzed and is shown in Six uterine cavity samples were excluded due to DNA extraction issues. In total, 194 samples were analyzed by 16S rRNA gene sequencing . A totalP = 0.036) was demonstrated  was the dominant phylum in both groups. Actinobacteria , the second dominant phylum in the vagina, was slightly decreased in abundance in the RSA group  was the dominant genus in the vagina in both groups. Compared with those in the RSA group at the genus level, two bacterial taxa, Bifidobacterium and Gardnerella spp., showed a higher relative abundance in the control group, but the difference was not significant  was used to determine the trends of variation in the microbiota between the two groups via dimensionality reduction, and each dot distributed within the figure represents a single sample. Slight separation of these two groups in the VM was observed, and PCo1 and PCo2 accounted for 43.4% and 10.1% of the variation, respectively . To detenstrated . The taxP = 0.010). Similar to the VM, Firmicutes and Actinobacteria were also the top two most important phyla in the CaM in both groups. Both Firmicutes  and Actinobacteria  showed higher relative abundances in the control group, while Proteobacteria  showed a higher relative abundance in the RSA group. Bacteroidetes  and Crenarchaeota  showed distinctly higher abundances in the RSA group , Atopobium , and Staphylococcus spp. , showed a significant tendency toward increasing abundance in the RSA group , but no statistically significant changes in the EnM were observed (P = 0.133)  became the predominant phylum in the RSA patients, while Firmicutes  still played a dominant role in the control group  belonging to the Proteobacteria became the predominant genus in RSA group  in the EnM showed a tendency toward lower abundance in the RSA group  and Proteobacteria  showed higher abundances in the RSA group  in the UfM also showed a tendency toward lower abundance in the RSA group . Four genera, Anaerobacillus , Erysipelothrix , Bacillus , and Hydrogenophilus spp. , showed significantly different abundances in the UfM, while nonsignificant differences were observed in the EnM between the RSA and control groups  highly overlapped, significant separation of the PCoA results for the microbiota of uterine lavage fluid (UfM) was found between the RSA and control groups  and k. T= 0.133)  and l. FEscherichia coli infection and Wnt signaling pathway) with significant differences.As shown in Lactobacillus, Bifidobacterium, Atopobium and Gardnerella spp. showed a sharply decreasing trend, while other bacterial taxa, such as Bacillus, Schlegelella, Anaerobacillus, Erysipelothrix, Halomonas, etc., showed an opposite trend from the lower genital tract to the upper genital tract in both groups. A total of 215 common OTUs in the control group and 208 common OTUs in the RSA group were also found in different samples along the female genital tract . Network analysis was performed to find the inherent patterns of co-currence or co-exclusion of specific microbial communities driven by spatiotemporal changes and environmental processes. The microbiota in the control group displayed a more complex network than the RSA group but most nodes showed positive correlations along the female reproductive tract in both groups .The alpha diversity was significantly increased along the lower to upper female reproductive tract, as the richness and diversity increased in both groups . PCoA and beta diversity analysis also showed distinct differences between the microbiota in the lower and upper female reproductive tracts in both groups . Based on the results in Figure S2i and l, the relative abundances of P = 0.281) . However, a different comparison result was shown in the case of the RSA group. PCoA of the RSA group suggested that the UfM and EnM samples were scattered in a relatively greater fashion . As shown in Figure S3f, a significant difference between the EnM and UfM in the RSA group was found (P = 0.001). The overlapping area shown in the Venn diagram indicates the common OTUs between the corresponding groups. There were 3767 and 3446 common OTUs between the 2 groups of samples in the control and RSA groups, respectively .To investigate the differences in microbial signatures between the two types of uterine samples, further analysis of the uterine microbiota in the RSA and control groups was performed. The Chao1 and Shannon indices of the EnM and UfM showed no statistically significant differences in the control and RSA groups, respectively . Regarding beta diversity, PCoA showed that in the control group, the EnM and UfM samples highly overlapped, and PERMANOVA based on Bray–Curtis dissimilarity showed no distinct difference in the comparison of the EnM and UfM (Lactobacillus spp. could be found in the uterine cavity with low relative abundance (<10%). In the control group, the relative abundances of Lactobacillus , Acinetobacter , and Vibrio spp.  in the EnM were significantly lower than those in the UfM . In the RSA group, the relative abundances of Lactobacillus , Acinetobacter , Cutibacterium , Bacteroides , Halomonas , and Muribaculaceae spp.  in the EnM were significantly higher than those in the UfM .Nesterenkonia, Lactobacillus, Acinetobacter, Vibrio spp. and so on) and 2 genera (Sphingobacterium and Magnetovibrio spp.) in the EnM with an LDA score >2 were found in the control group . Highly abundant bacterial taxa with an LDA score >2 in the RSA group included 13 genera  in the UfM and 3 genera  in the EnM.As shown in Figure S3m and n in the supplemental material, the relative abundances of microbiota constituents from the kingdom to genus level with significant differences between groups were described by the logarithmic scores of the linear discriminant analysis (LDA). Different uterine cavity samples displayed with different expression of bacterial taxa. At the genus level, 8 types of differentially abundant genera in the UfM . Spearman’s correlation analysis between the top 15 most abundant bacterial taxa in the UfM and human Th1/Th2/Th17 cytokines in uterine lavage fluid were performed. Statistically significant correlations between Aliihoeflea and IL-17A, Acinetobacter and IFN-γ, Serratia and TNF, and Staphylococcus and Serratia and IL-6 were found . Human Th1/Th2/Th17 cytokines, including IL-17A, IFN-γ, TNF, IL-10, IL-6, IL-4, and IL-2, in uterine lavage fluid were quantified and analyzed. As shown in re found .This study was the first attempt to systematically characterize microbial dysbiosis in both the lower and upper reproductive tracts in RSA patients. Alterations in microbial biodiversity in the vagina, cervix and uterine lavage fluid were found in RSA patients. The microbiota composition differed markedly from the lower genital tract to the uterine cavity, and the microbiota in the uterine cavity also distinctly varied between endometrial tissue and uterine lavage fluid in the RSA group. Furthermore, alterations in the microbiota in the uterine cavity could be associated with altered cytokine levels, which might be a risk factor for RSA pathogenesis. However, this is only a preliminary study; validation of the findings with a larger sample size of patients and investigation of the underlying mechanism are in need of further research.RSA, as a clinical challenge with largely unknown factors, has imposed heavy psychiatric and financial burdens on families and society . Despite16–11–Bifidobacterium spp. showed a tendency toward decreasing abundance in the RSA group, while the abundance of Atopobium spp. increased in the RSA group. Bifidobacterium strains with a long history as probiotics have been demonstrated to have markedly higher abundance in healthy women  and the control group (n = 25), different samples, including vaginal secretions, cervical secretions, intrauterine lavage fluid, and endometrial tissue, were collected from patients on their receptive days . To more comprehensively understand the characteristics of intrauterine microorganisms, endometrial tissue samples and uterine lavage fluid were collected simultaneously. The ovulation date was determined by transvaginal ultrasound and/or urine LH test paper and further confirmed by serum progesterone quantification on the day of sampling. Chronic endometritis was diagnosed according to the pathological results with immunohistochemical staining of CD138 positive plasma cells . All patients in this study were enrolled according to the inclusion and exclusion criteria..1Patients whose informed consent was obtained after having been duly informed of the nature of the study and who voluntarily agreed to participate after being fully aware of the potential risks, benefits and any discomfort involved..2Maternal age: 18–42 years..32 (both inclusive).BMI: 18.5–30.0 kg/m.4Nonpregnant, with a regular menstrual cycle..5Negative serological tests for hepatitis B surface antigen (HBsAg), hepatitis C virus (HCV), and human immunodeficiency virus (HIV)..6No antibiotic use, vaginal drug use or cervical treatment in the past 30 days and no sexual activity in the past 2 weeks..7Two or more consecutive spontaneous abortions occurred in the individuals in the RSA group, and infertile patients due to male factors were included in the control group.Inclusion criteria:.1Patients with chromosome abnormalities according to the analysis of the chromosome karyotypes in peripheral blood..2Any pathological finding affecting the endometrial cavity preliminarily detected by transvaginal ultrasound, such as adenomyosis, submucosal myomas or intramural myomas > 4 cm, or hydrosalpinx, must have been previously confirmed and operated on at least 3 months before the endometrial samples were obtained ..3Polycystic ovary syndrome and hypothyroidism, hyperthyroidism, diabetes, antithrombotic syndrome or Sjogren's syndrome..4Patients with acute genital tract inflammation, such as bacterial vaginosis and vulvovaginal candidiasis, or HPV infection..5Illness or unstable medical conditions that may have put the patient at risk of safety and compliance in the study.Exclusion criteria:Before sampling, patients were placed in the lithotomy position, and the cervix and vagina were fully exposed with a sterile speculum. The operator wore sterile gloves for sampling and performed the whole sampling process in a sterile situation, and the sampling process is shown in Vaginal secretions from the upper one-third of the vaginal sidewalls were obtained with a sterile cotton swab, and the lower rod of the swab was separated into a tube containing sterile transfer medium .After wiping the secretion from the external opening of the cervix with sterile dry cotton, a flocked cotton swab was placed into the cervix canal, and the vaginal wall was carefully not touched.According to the standard operating procedure of hysteroscopy examination, the vagina and the external opening of the cervix were disinfected by iodophor after vaginal and cervical canal secretions were collected, and a sterile dry cotton ball was used to clean the disinfectant the surface of the cervix to prevent the disinfectant flowing into the uterine cavity. The inner sheath of an artificial insemination tube  was then gently introduced trans-cervically into the uterine cavity under the protection of the outer sheath and connected with a 5 mL sterile syringe. Then, 2 mL of sterile normal saline was injected, quickly withdrawn and transferred to an empty sterile tube.Under the protection of the outer sheath, the endometrial negative pressure catheter was placed into the uterine cavity for suction and curettage. The catheter was removed, and then the endometrial tissue was transferred into a sterile tube.In this study, negative controls including normal saline and transfer medium were used to exclude potential bacterial contaminations from DNA processing and library preparation. All samples were sealed, put into liquid nitrogen immediately and stored at −80°C before DNA extraction. Then, total bacterial genomic DNA samples were extracted using an OMEGA Soil DNA Kit  according to the manufacturer’s instructions and stored at −20°C prior to further analysis. Samples were labeled by abbreviations as follows: VM is the abbreviation for the vaginal microbiota, CaM is the abbreviation for the cervical microbiota, UfM is the abbreviation for the microbiota of uterine lavage fluid and EnM is the abbreviation for the microbiota of endometrial tissue. C is the abbreviation for the control group, and A is the abbreviation for the RSA group.2O. Thermal cycling consisted of initial denaturation at 98°C for 5 min, followed by 25 cycles consisting of denaturation at 98°C for 30 s, annealing at 53°C for 30 s, and extension at 72°C for 45 s, with a final extension of 5 min at 72°C. PCR amplicons were purified with Vazyme VAHTSTM DNA Clean Beads  and quantified using a Quant-iT PicoGreen dsDNA assay kit . After the individual quantification step, amplicons were pooled in equal amounts, and paired-end 2 × 250 bp sequencing was performed using the Illumina NovaSeq platform with a NovaSeq 6000 SP reagent kit (500 cycles) at Shanghai Personal Biotechnology Co., Ltd. .PCR amplification of the bacterial 16S rRNA gene V3–V4 region was performed using the forward primer 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and the reverse primer 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Sample-specific 7-bp barcodes were incorporated into the primers for multiplex sequencing. The PCR components contained 5 μL of buffer (5×), 0.25 μL of Fast PFU DNA polymerase (5 U/μL), 2 μL (2.5 mM) of dNTPs, 1 μL (10 μM) of each forward and reverse primer, 1 μL of DNA template, and 14.75 μL of ddHhttps://docs.qiime2.org/2019.4/tutorials/)  . Brieflyorials/) . Nonsingorials/) . Alpha dorials/) , 39.https://www.kegg.jp/) database . OTU-level alpha diversity indices, such as the Chao1 richness estimator and Shannon diversity index, were calculated using the OTU table in QIIME2. Beta diversity analysis was performed to investigate the structural variation in microbial communities across samples using Bray–Curtis metrics , 41 and database .n = 9; Control: n = 9) was collected, and inflammatory cytokine levels were measured via a BD CBA Human Th1/Th2/Th17 Cytokine Kit . According to the manufacturer’s instructions, cytokine capture beads, including those for IL-17A, IFN-γ, TNF, IL-10, IL-6, IL-4, and IL-2, were mixed with 50 μL samples and then incubated with PE-conjugated detection antibodies at room temperature for 3 h. The mixture was then analyzed on a BD FACSCelesta , and data were analyzed using FlowJo software.Uterine lavage fluid  and SPSS version 26.0 were used for statistical analysis. One-way analysis of variance followed by Tukey’s PRJNA774109 and PRJNA813906.Sequence reads for the 194 specimens included in this study have been deposited in the National Center for Biotechnology Information (NCBI) under reference number


SOX10 gene. Three of them were associated with reproductive traits, whereas two were associated with plumage color.A total of six novel SNPs were found from 11 identified SNPs in the duck SOX10 gene affect the plumage color of chickens and pigeons. The mutation also causes abnormal pigmentation of the skin and hair color, as well as postnatal growth retardation and reproduction problems in humans and mice. In this study, we investigated the association between the SOX10 gene and plumage color and reproductive traits of ducks using SNPs. We found six novel SNPs from 11 identified SNP sites using direct sequencing for PCR products from three different mixed DNA pools. We found two coding SNPs to be associated with the plumage color of ducks (ZJU1.0 Chr1. g.54065419C>T and g.54070844C>T), and found three coding SNPs associated with the reproductive traits of ducks , which were age at sexual maturity, body weight at sexual maturity, and the Haugh unit for egg quality traits and egg production in different productive periods. These results also indicated that the T alleles of the three SNPs of the coding region of SOX10 contribute to lower reproductive traits.Mutations in the  Pigmentation in ducks is a substantial issue for down feather commodities. Although color changes do not necessarily affect quality, the pigmentation of poultry products can be critical to visual acceptance and consumer preferences. Plumage color has become an important consideration in the feather and down industries in China, the USA, and some other countries ,2. ChinaKaiya and white Liancheng   .Based on individual genotyping, we calculated minor allele frequency (MAF) and heterozygosity for the three SNPs. We performed a statistical test to determine if the Hardy–Weinberg Equilibrium (HWE) existed in our population of ducks using PLINK v1.07 . We then2 individuals, we categorized three plumage color groups, including 282, 399, and 218 individuals with white, grey, and black plumage, respectively. The summary statistics for each reproductive trait can be found in By observing the 899 FSOX10 gene from the initial screening with PCR and pooled sequencing  revealed three LD blocks between g.54065419C>T, g.54070844C>T, and g.54070904C>T in the X10 gene . HaplotyX10 gene . Such efX10 gene  Table A1p > 0.05). According to the genotyping and association analysis, we found significant associations between mutations and duck plumage color. Two of the three SNPs were associated with plumage colors .The genotypic frequencies of the three examined SNPs for different plumage colors are presented in SOX10 mutations lead to melanin pigment reductions [The genotype frequency of the C/T of g.54065419 was higher in ducks with grey plumage, whereas the C/C frequency of g.54070844 was higher in ducks with white plumage. These association results suggested that the relationship of the three examined SNPs with plumage pigment intensity is not independent, and any mutation in this particular site may lead to variations in duck plumage pigment intensity. For the two SNPs, the presence of the C allele, whether in homozygous or heterozygous genotypes, might lighten the pigment intensity of the plumage to grey or white. These results support the idea that ductions .p < 0.003) with at least one of the duck egg production traits, especially when the cumulative egg numbers were recorded for longer time periods (EP390 and EP420). Generally, the T allele of each SNP was associated with lower reproductive traits. This indicated that a delayed start to egg production might not be compensated by the increased duration of egg production.All three SNPs that we examined in this study were associated with duck reproductive traits  Table A1SOX10 transcription factor gene considerably decrease the regulation of the target genes of Tyrp1 in the feathers of recessive red birds [SOX10 mutation. Although mutations in the SOX10 gene of humans and mice (in the heterozygous state) lead to a dominant-negative effect by disrupting the functions of the wild-type SOX10 protein, it has a milder effect on the Waardenburg–Shah (WS4) phenotype than that seen in homozygotes due to the incomplete blocking of SOX10 functions [In this study, the frequency of the heterozygous genotype (C/T) of g.54065419C>T was highest in ducks with gray plumage, which is in agreement with the results of our previous study . This emunctions ,27. WS4 unctions .SOX10 have pleiotropic effects affecting plumage color, cumulative egg production, and age at sexual maturity. The ducks were reared with ad libitum feeding program. Sexual maturity with an ad libitum feeding program should be reached by 16 weeks or 112 days of age [SOX10 reached sexual maturity three weeks later at an average age of 135 days. Our results also suggested that the delayed start in egg production might be unable to be compensated during later stages of egg production. These are consistent with those of Cherry and Morris [In this study, we proposed that the mutations on duck s of age . Therefod Morris  and Wrigd Morris , who sugd Morris . Thus, iSOX10 gene mutations. Similar to other breast cancer problems, BLBC shows symptoms of a lack of expression of estrogen receptors and progesterone receptors [SOX10 gene action can be linked with sexual development and skeletal or bone modeling. In the present study, ASM was associated with the SOX10 genotype.SOX10 has been identified as a driver of melanoma progression, a cancer that develops from melanocytes which are neural crest derivatives. Reproductive disorder diseases, such as basal-like breast carcinoma (BLBC), is also linked with eceptors . Estrogeeceptors . From heαERKO gene (αER knock out) fails to differentiate into the follicle and seemed incapable of ovulation. The mice with βERKO gene also shows a reduction in the oocyte number, ovarian dysregulation, and an increasing number of unruptured follicles [ER1) is negatively correlated with SOX10 [FOXA1 gene, which opposes SOX10 expression, cooperates with ER1 to maintain luminal identity in breast cancer, whereas GATA3, XBP1, and CA12 showing a negative correlation with SOX10, also cooperate in ER1 signaling. Thus, ER1 and FOXA1 activities are essential for the function of SOX10. González-Morán revealed that ER1  is differentially expressed in both chicken ovaries during development. Both the left and right chicken ovaries can respond to estrogen, which directly acts through their interaction with ER-α [Drummond and Fuller demonstrated that mice with the ollicles . Ivanov th SOX10 . The FOXith ER-α . The difp ≤ 0.01) between age at first egg and egg weight (r = 0.35). This r value is larger than that reported by Cankaya et al. [p ≤ 0.01), one possible explanation could be that egg weights at the same age while observing HU values are relatively heavier.We found a significant positive correlation  [Mutation positions of all synonymous SNPs, Arg67Arg, His162His, and Gly182Gly, are on the s region . Becausectively) . Howeverctively) .SOX10 gene of ducks might be responsible for duck plumage pigmentation and reproductive trait variation. An example of third base changes in synonymous SNPs is CGT>CGG (arginine), which was demonstrated by Sauna and Kimchi-Sarfaty [In several occurrences, synonymous SNPs are associated with a particular disease or trait. Codons changing from frequent codons to rare codons in a cluster of infrequently used codons affecting cotranslational folding timing may result in altered function . Silent -Sarfaty , having SOX10 gene. This study shows that mutations in the SOX10 gene affected duck plumage color and reproductive traits, as two out of the three observed SNPs were associated with duck plumage color variations, and all three of these SNPs were associated with reproductive traits. All the genotype variations of the observed effects of the three SNPs were more intense at older production ages, with ducks with the T/T genotype having the lowest reproductive traits. This is the first report stating that synonymous mutations of the SOX10 gene are associated with duck reproductive traits.We found six novel SNP from 11 identified SNP sites in the