Overfeeding a high-grain diet is known to reduce ruminal pH and microbial activity, disrupt carbohydrate metabolism, and consequently lead to subacute ruminal acidosis. Thiamine, as the co-enzyme of pyruvate formate-lyase, plays a critical role in carbohydrate metabolism in dairy cows. Therefore, we investigated the impacts of thiamine supplementation on ruminal carbohydrate-active enzymes using the metagenomic sequencing technique in lactating dairy cows fed high-concentrate diets. The results indicated that feeding high concentrate diets reduced fiber-degrading enzymes and the total carbohydrate-active enzymes. However, dietary thiamine supplementation increased fiber-degrading enzymes, starch-degrading enzymes, and total carbohydrate-active enzymes. These findings demonstrated that thiamine supplementation could enhance rumen carbohydrate metabolism through increasing the abundance of ruminal carbohydrate-active enzymes, providing effective strategies to improve dairy cows’ health and wellbeing under a high-concentrate feeding regime.As the co-enzyme of pyruvate formate-lyase under ruminal anaerobic condition, thiamine plays a critical role in carbohydrate metabolism in dairy cows. The objective of this study was to investigate the impacts of thiamine supplementation on ruminal carbohydrate-active enzymes. Twelve Holstein dairy cows were randomly assigned into three dietary treatments: control diet  basis), high-concentrate diet  and a high-concentrate diet supplemented with 180 mg thiamine/kg DM . Dry matter intake and milk production were recorded for 21 days. Rumen fluid samples were collected, and ruminal pH and volatile fatty acids (VFAs) were measured. The metagenome sequencing technique was used to detect the genes in ruminal microorganisms and identify putative carbohydrate-active enzymes. The total abundances of carbohydrate-active enzymes and fiber-degrading enzymes were both reduced by HC with no effect on starch-degrading enzymes compared with CON. However, the fiber-degrading enzymes and starch-degrading enzymes were both increased after thiamine supplementation. These results indicated that 180 mg thiamine /kg DM might effectively improve rumen carbohydrate metabolism through increasing the abundance of ruminal carbohydrate-active enzymes and consequently balanced the rumen volatile fatty acids and rumen pH, providing a practical strategy in preventing subacute ruminal acidosis in cows offered HC. Carbohydrates, usually making up 70–80% of the diets, are the primary energy sources for the high requirement of lactating dairy cows ,2. The r12H16N4OS), also known as vitamin B1, plays a critical role in carbohydrate metabolism [Our previous studies found that thiamine supplementation could effectively attenuate subacute ruminal acidosis (SARA) induced by a high-grain diet through buffering ruminal pH, shifting rumen fermentation pattern, and balancing the structure of ruminal microorganisms ,13. Thiatabolism  and has http://www.cazy.org/) is the key database that describes the families of structurally-related catalytic and carbohydrate-binding modules  of enzymes that degrade, modify, or create glycosidic bonds [The CAZymes database . Throughout the experimental period, the cows were housed in a single dairy barn area (18 m long × 4.25 m wide × 4.5 m high) with free access to fresh water.The experimental design has been stated in our previous study . In brieg for 15 min at 4 °C. Five mL of clear supernatant samples was transferred into tubes and stored at −20 °C for analysis of thiamine. Another 10 mL of clear supernatant samples was mixed with 2 mL of 250 g/L of metaphosphoric acid and stored at −20 °C for VFA analysis. The concentrations of VFA and thiamine were determined, as described by Pan et al. [During the experimental period, automatic feeding equipment  was used to record dry matter intake (DMI). Milking facilities of Afimilk  were applied to record milk production of each cow. Two hundred and fifty milliliters of rumen semi-solid contents of each cow were sampled at 3 h after the morning feeding on day 21. Rumen contents were strained through 4 layers of cheesecloth with a mesh size of 250 μm. Rumen fluid was then divided into two portions. One was used to analyze the pH value, thiamine, and rumen volatile fatty acid (VFA) concentrations. Ruminal pH was measured immediately using a portable type pH meter . Then the rumen fluid was centrifuged at 10,000× n et al. . The othwww.illumina.com, Illumina Inc., San Diego, CA, USA).Metagenomic sequencing analysis of DNA extraction, library construction, sequence quality control, genome assembly, gene prediction, and taxonomy was performed as described in our previous study . In genehttps://github.com/jstjohn/SeqPrep). The low-quality reads, or reads associated with the cow genome, were removed [http://soap.genomics.org.cn, Version 1.06) based on De Brujin graph construction [The quality control methods of reads were stripped using SeqPrep ( removed . The filtruction . Scaffolhttp://metagene.cb.k.u-tokyo.ac.jp/) [−5 and Score > 60. Based on the NCBI microbial taxonomy information database, species annotation information of genes was obtained, and the relative abundance of species was calculated.Open reading frames (ORFs) from each sample were predicted using MetaGene (.ac.jp/) . DIAMOND.ac.jp/)  was emplhttps://www.kegg.jp/ghostkoala/) [http://www.genome.jp/kegg/) with an e-value cutoff of 1 × 10−5. Annotate sequences in the KEGG Mapper were specially interfaced with the BlastKOALA server and executed in an interactive mode. CAZymes annotations were then conducted using hmmscan (http://hmmer.janelia.org/search/hmmscan) to match the CAZymes database V5.0 (http://www.cazy.org/). The results were considered significantly enriched in CAZymes with an e-value cutoff of 1 × 10−5.GhostKOALA   was emplNucleotide Sequence Accession Number.http://www.ncbi.nlm.nih.gov/Traces/sra/) under accession number SRP144478.All the raw sequences were submitted to the NCBI Sequence Read Archive . Correlation analysis between DMI, milk fat, milk protein, ruminal fermentation parameters  and relative abundances of fiber-degrading enzymes and starch-degrading enzymes, respectively, were assessed using the PROC CORR procedure of SAS 9.22 . A correlation matrix was then created and visualized in a heatmap format using the R package . The relationships were considered significant with the absolute value of correlation coefficients (|r|) > 0.55 and p < 0.05.Effects of high-concentrate diet and thiamine supplementation treatment on the relative abundances of genes encoding carbohydrate-active enzymes, fiber-degrading enzymes, and starch-degrading enzymes were analyzed using the Student–Newman–Keuls (SNK) test of SAS 9.22 . p < 0.05). In contrast, these changes were significantly inversed by thiamine supplementation (p < 0.05). While butyrate and total VFA concentrations were not affected by dietary treatments (p > 0.05).The DMI, milk production, ruminal pH, and ruminal VFAs parameters have been stated in our previous study , which aThe sequencing results are shown in http://metagene.cb.k.utokyo.ac.jp/) [The KEGG database was used to annotate the genes that might participate in ruminal metabolism through MetaGene (.ac.jp/) . More thp < 0.001), while there was no significant difference between CON and HCT. In detail, genes encoding CBMs, CEs, GHs, GTs, and PLs were all less abundant in HC than those in CON, which were then significantly increased after thiamine supplementation in comparison with HC (p < 0.01). The abundances of genes that encoded AAs and GTs in HCT were higher than both of CON and HC (p < 0.001).Differential analysis of all CAZymes categories was then conducted to investigate the effects of HC and thiamine supplementation on CAZymes. The results are shown in The correlations of CAZymes of each sample among the three treatments were investigated using correlation-distance analysis . The reshttp://www.cazy.org/) according to the report of Comtet-Marre et al. [p < 0.001). In contrast, thiamine supplementation significantly increased the total relative abundances of fiber-degrading enzymes compared with both HC and CON (p < 0.001). Specifically, HC significantly reduced the abundances of genes encoding CBM3, CBM30, GH11, and GH48 compared with CON (p < 0.01), and HCT significantly increased the abundances of genes encoding GH10, GH45, and GH5 in comparison with CON and HC (p < 0.01). In which, GH10 and GH45 genes were the main genes that contributed to the significant effect on the higher total abundance of genes encoding fiber-degrading enzymes in HCT than CON and HC.Differential analysis was then used to investigate the effects of the dietary treatments on the relative abundance of genes encoding fiber-degrading enzymes and starch-degrading enzymes based on the functional annotation of CAZymes database , while no difference was detected between the latter two. For individual enzymes, HC had significantly fewer abundances of genes encoding CBM20, GH13, GH4, GH63, and greater GH97 than CON (p < 0.05). Meanwhile, CBM25, GH15, and GH119 genes were all most abundant in HCT in comparison with CON and HC (p < 0.01), which contributed to the significant effect on the higher total abundance of genes encoding starch-degrading enzymes in HCT than CON and HC.On the other hand, HCT significantly increased the total relative abundances of starch-degrading enzymes compared with CON and HC  was applied. The results of the correlation analysis between fiber-degrading enzymes and animal performance are shown in Similarly, the results of correlation analysis between starch-degrading enzymes and animal performance are shown in Ruminants have a special capacity to digest fibers in the feeds with the assistance of ruminal microorganisms . ApproxiIn addition, fiber mainly contains cellulose and hemicellulose. Where cellulose is composed of glucose and hemicellulose is composed of linear and branched heteropolymers of o-xylose, L-arabinose, D-mannose, D-glucose, D-galactose, and D-glucuronic acid . In rumiAcetate is produced from acetyl-coA , while tSimilar to cellulose and hemicellulose, more than 60% of starch is digested by the rumen microbes, varying according to the feed intake and its passage rate through the rumen . In the Streptococcus bovis [Clostridium acetobutylicum [Dietary starch is hydrolyzed into glucose and then digested into pyruvate and acetyl-CoA by microorganisms through the glycolytic pathway and pentose phosphate pathway ,40. Howeus bovis  and Closutylicum . This miTo effectively degrade ruminal starch, many microbial GHs are appended with CBMs . The CBMMegasphaera elsdenii and reduced the lactate producing bacteria Strepococcus bovis and Lactobacillus [Usually, a high proportion of dietary starch results in the accumulation of propionate in the rumen ,48. Howebacillus . Furtherbacillus . TherefoPrevious studies of our team proved that 180 mg thiamine/kg DM in HC could help attenuate SARA by increasing ruminal pH, acetate, and thiamine contents and reducing propionate concentration in rumen fluid compared with HC ,22. ThisIn the present study, we observed that 180 mg/kg DM of thiamine supplementation in HC significantly increased the relative abundances of the genes encoding CAZymes in dairy cow rumen microorganisms by 33.6% compared with HC, including both fiber (52.8% increase) and starch (46.7% increase) degrading enzymes. These results provided genetic evidences to explain how thiamine supplementation promoted the carbohydrate metabolism in the rumen through metagenomic sequencing of the rumen microorganisms and consequently balanced the rumen VFA and rumen pH, which could thus be a practical strategy in preventing SARA in cows offered HC. The findings added some novel information on thiamine’s function in dairy cows under a high-concentrate feeding regime. However, further research is still needed to investigate the relationships between CAZymes and rumen microbes and the mechanism of correlations between CAZymes and rumen fermentation variables.


The main goal of this collaborative effort is to provide genome-wide data for the previously underrepresented population in Eastern Europe, and to provide cross-validation of the data from genome sequences and genotypes of the same individuals acquired by different technologies. We collected 97 genome-grade DNA samples from consented individuals representing major regions of Ukraine that were consented for public data release. BGISEQ-500 sequence data and genotypes by an Illumina GWAS chip were cross-validated on multiple samples and additionally referenced to 1 sample that has been resequenced by Illumina NovaSeq6000 S4 at high coverage.The genome data have been searched for genomic variation represented in this population, and a number of variants have been reported: large structural variants, indels, copy number variations, single-nucletide polymorphisms, and microsatellites. To our knowledge, this study provides the largest to-date survey of genetic variation in Ukraine, creating a public reference resource aiming to provide data for medical research in a large understudied population.Our results indicate that the genetic diversity of the Ukrainian population is uniquely shaped by evolutionary and demographic forces and cannot be ignored in future genetic and biomedical studies. These data will contribute a wealth of new information bringing forth a wealth of novel, endemic and medically related alleles. Ukraine is the largest country located fully in Europe, with a population that was formed as a result of several millennia of migration and admixture. It occupies the intersection between the westernmost reach of the great steppe and the easternmost extent of the great forests that spread across Europe, at the crossroads of the great trade routes from “Variangians to the Greeks” along the river Dnipro, which the ancient Greeks referred to as Borysthenes, and the Silk Road linking civilizations of Europe and Asia . This laThe rich history shaped genetic diversity in the population living in the country of Ukraine today. As people have moved and settled across this land, they have contributed unique genetic variation that varies across the country. While the ethnic Ukrainians constitute approximately more than three-quarters of the total population, this majority is not uniform. A large Russian minority compose approximately one-fifth of the total population, with higher concentration in the southeast of the country. Smaller minority groups are historically present in different parts of the country: Belarusians, Bulgarians, Crimean Tatars, Greeks, Gagauz, Hungarians, Jews, Moldovans, Poles, Romanians, Roma (Gypsies), and others .This study offers genome data from 97 individuals from Ukraine (Ukrainians from Ukraine [UAU]) to the scientific community to help fill the gaps in the current knowledge about genomic variation in Eastern Europe, a part of the world that has been largely and consistently overlooked in global genomic surveys . To our The new dataset includes 97 whole genomes of self-reported UAU at 30× coverage sequenced using BGISEQ-500  and annotated for genomic variants: single-nucleotide polymorphisms (SNPs), indels, structural variants, and mobile elements. The samples were collected across the entire territory of Ukraine, after obtaining institutional review board (IRB) approval  were additionally genotyped using Illumina Global Screening Array  to confirm the accuracy of base calling between the 2 platforms. In addition, 1 sample (EG600036) was also sequenced on the Illumina NovaSeq 6000 S4  and used for validation of the variant calls  are not included in the study, increasing the aforementioned sample size from 100 to 1,000 individuals is not likely to greatly contribute to discovery of novel mutations . The proFor each sample in the database, we estimated the number of passing bi-allelic SNP calls  Table . Then ∼12r= 0.985, Fig. We report a good correspondence between the SNP calls made using BGISEQ-500 and NovaSeq 6000 S4 data. A comparison of the variants detected using these 3 platforms for sample EG600036 is summarized in Fig. The transition/transversion (TITV) ratio for the novel SNPs ,  large structural variants , and mobile element insertions (MEI) . A number of structural elements were reported, including common and novel ones. While among the small variants most were common (6–9%), a large proportion of large variants and MEIs (38–52%) have not been reported previously in the 1GP Database : 87.9% of the variants called by the BGISEQ-500 were also detected by the Illumina platform. At the same time, there were 822 deletions, or 33.8% of all the indels called by the Illumina that were not detected by the BGISEQ-500 Fig. . A similThe database was compared to the existing global resources of population variation such as gnomAD  and the A particular interest in this study is the distribution of functional variation, not in the least due to the potential impact on phenotypes, especially to those with medical relevance . As muchIn addition to the gene-coding mutations, we report a number of regulatory variants. For example, the database contains a total of 2,229 transcription factor binding site ablation (TFBS) mutations   or ClinVAs expected, our study shared a lot more variants with the GWAS  than witSLC6A19), efficacy of bisphosphonate response , autism , Leber congenital amaurosis , and breast cancer susceptibility in BRCA1 and BRCA2 carriers   and that certain diseases and heritable traits display marked differences in frequency between populations . With thse rs229780,FDPS, Of course, not all the medically related variants are currently known, and many remain to be discovered and verified in local populations. This is, to some extent, a consequence of underreporting of allelic endemism within understudied populations, particularly in Eastern Europe  but alsoWe performed several population analyses, but only to demonstrate the uniqueness and usefulness of this new dataset. Our results indicate that genetic diversity of the Ukrainian population is uniquely shaped by evolutionary and demographic forces and cannot be ignored in future genetic studies. However, we do not evaluate any historical hypotheses on the timing of origins, founding, migration, and admixture of this population and use only the naive approaches, based on the statistical models.To demonstrate the extent to which our dataset contributes to the genetic map of Europe, we explored genetic relationships between Ukrainian individuals within our sample and evaluated genetic differences between this population and its immediate neighbors on the European continent for which population data of full genome sequences were publicly available. A principal component analysis (PCA) of the merged dataset of 654 samples included European populations from the 1KG  andUkrainian genomes from this as well as other studies , 44 formAddition of the new genomic data will most likely add to the resolution of the genetic map of this region and further reveal differences between the populations of Eastern and Central Europe. Our dataset showed a limited amount of inbreeding  and contTo provide a more extended view of the genetic components contributing to the Ukrainian population, we used the population structure plots using the ADMIXTURE package . This alDespite the fact that all of the samples were collected from self-identified ethnic Ukrainians, there were 2 notable outliers: sample EG600048 clustered with the Southern Europeans  Fig. People of Ukraine carry many previously known and several novel genetic variants with clinical and functional importance that in many cases show allele frequencies different from neighboring populations in the rest of Europe, including Poland to the west, Romania to the south, the Baltics to the north, and Russia to the northeast. While several large genome projects already exist contributing to the understanding of global genetic variation, many rare and endemic alleles have not yet been identified by international databases such as 1KG and are currently not available in standard genotyping panels for association testing for human diseases, and glaring white spots still exist on the genetic maps in local populations of Eastern Europe . We fullThe collection and consent procedure was approved as part of the “Genome Diversity in Ukraine” project by the IRB of Uzhhorod National University in Uzhhorod, Ukraine , for DNA extraction immediately on arrival. The excess of the blood and DNA from samples remaining after the genetic analysis is stored frozen at the biobank of the Biology Department, Uzhhorod National University, Ukraine. As a result, blood samples were collected from a total 113 individuals.Immediately upon arrival to the laboratory, DNA isolation from 200 μL of blood was carried out with the innuPREP DNA Blood Minikit . High molecular weight genomic DNA was lightly fragmented by vortexing. The initial DNA concentration was measured with the Implen C40 Nanophotometer , and quality was verified visually on a 2% agarose gel. The 97 successfully extracted DNA samples were normalized to 20–30 ng/μL concentration for downstream application. After extraction the samples were recoded and sent to NIH for the genotyping procedure, whence the aliquots were further shipped to a BGI facility  or to Psomagen Inc.  for the whole-genome sequencing (WGS). The remaining ∼2 mL was frozen for future use.All 97 individuals in this study were sequenced with BGISEQ-500 and 88 individuals were cross-validated by genotyping using Illumina Global Screening Array. The record of which individual samples have been cross-validated by both technologies is presented in All 97 DNA samples were sequenced on BGISEQ-500 . Upon receipt at the BGI facility, and prior to sequencing, samples were checked again for quality. Concentration was once more detected by fluorometer or Microplate Reader . Sample integrity and purity were detected by agarose gel electrophoresis . Aliquots of 1 μg genomic DNA were fragmented by Covaris. The fragmented genomic DNA was selected by Agencourt AMPure XP-Medium kit to a mean size of 200–400 bp. Fragments were end-repaired and then 3′-adenylated. Adaptors were ligated to the ends of these 3′-adenylated fragments. PCR products were purified by the Agencourt AMPure XP-Medium kit. The double-stranded PCR products were heat denatured and circularized by the splint oligo sequence. The single-strand circle DNA was formatted as the final library. The qualified libraries were sequenced by BGISEQ-500: the single-strand circle DNA molecule formed a DNA nanoball (DNB) containing >300 copies through a rolling-cycle replication. The DNBs were loaded into the patterned nanoarray by using high-density DNA nanochip technology. Finally, pair-end 100-bp reads were obtained by combinatorial probe-anchor synthesis. Raw reads were filtered to remove adaptor sequences, contamination, and low-quality reads. Sequencing of all 97 full genome samples submitted for sequencing at BGI was successful.One individual was resequenced by Illumina NovaSeq6000 S4 at Psomagen Inc. . The library was prepared using TruSeq DNA PCR Free 350 bp protocol by Illumina. The library was sequenced at ∼64× depth, producing 150-bp–long reads, resulting in 241.7 Gb of data.We attempted to genotype all 97 of the collected samples using the Illumina Infinium Global Screening BeadChip Array-24 v1.0 (GSAMD-24v1–0) for 700,078 loci at the National Cancer Institute's Division of Cancer Epidemiology and Genetics  . Data weThe sequencing data produced using the BGISEQ-500 platform for 97 samples were analyzed using the Sentieon tools  high-performance implementation of the BWA/GATK best practices pipeline on servers hosted by the Cornell University Biotechnology Resource Center. Reads were aligned to the GRCh38 human reference genome using BWA-MEM (Version: 0.7.16a-r1181), and mapped reads were prepared for variant calling using GATK (v3.8-1-0-gf15c1c3ef by Broad), including marking duplicates , indel realignment , and base quality score recalibration . SNP and indel discovery were performed for each individual using GATK HaplotypeCaller and merged into a single pVCF using bcftools. Sample EG600036 was also run without joint calling, which was used when calculating concordance between the Illumina and BGISeq variant callsets, estimated with TiTvtools and visualized by plotTiTv .Mobile element discovery was performed using MELT (Version 2.2.0)  and struVariant files were compared for consistency across the 3 different platforms: BGISEQ-500 sequencing, Illumina genotyping, and Illumina NovaSeq6000 S4 sequencing. Illumina genotyping was performed on 86 of the 97 samples previously sequenced with BGISEQ-500. Additionally, 1 sample (EG600036) was also sequenced with Illumina NovaSeq6000 S4. The variant detection programs were rerun without joint calling for the BGISEQ-500 sequencing for sample EG600036 for comparison with the single Illumina-sequenced sample. In this sample, the SNPs derived from the WGS platforms were compared to those identified using the Illumina SNP array for both matching position and matching genotype. Structural variants and MEIs were compared between the WGS platforms in EG600036. Variants were considered the same if they had 95% reciprocal overlap. Overall, we found that Illumina identified a higher number of larger variants than BGISEQ-500. This could potentially be due to its higher coverage (∼60×) compared to BGISEQ-500 (∼30×). However, because both have high coverage, we may see diminishing returns for coverage >30×. An alternative explanation is that the variant identification tools have been built to detect variation from Illumina sequencing data and therefore may not be able detect variants in BGISEQ-500 data as accurately.RRID:SCR_012821) [RRID:SCR_005191) [RRID:SCR_006169) [RRID:SCR_015624) [Sequence variant files were annotated using ANNOVAR   and SNPE_005191)  software_005191) , InterVa_005191) . For SNP_005191)  was comp_006169)  and GWAS_006169)  database_015624) .RRID:SCR_004965) [For PCA, we used WGS variants of our samples and merged them with samples from neighboring countries available from the European samples from 1KG  and_004965) .To produce a meaningful number of alleles to analyze, the resulting dataset was filtered by genotyping rate (1) and pruned for variants in linkage disequilibrium by excluding those with high pairwise correlation within a moving window (–indep-pairwise 50 10 0.5). This resulted in 677 samples with 208,945 variants. We used EIGENSOFT  to calcuRRID:SCR_001263) [For the naive (model-based) structure analysis, we used the same dataset described in the PCA (above). The analysis was performed using ADMIXTURE software  . For ide_001263) , 60 to c_001263) , 39 and _001263) , as well_001263) , and Sim_001263) . The resWe estimated inbreeding coefficients for all the genotype samples in the same dataset. For this analysis the samples were pruned for genotyping rate (>0.9) and linkage disequilibrium by excluding those with high pairwise correlation within a moving window (PLINK parameter –indep-pairwise 50 10 0.1). Using the resulting dataset containing the remaining 117,641 loci from 84 samples, we performed several inbreeding estimates: (i) method-of-moments F-coefficient estimates, (ii) variance-standardized relationship minus 1 estimates, and (iii) F-estimates based on correlation between uniting gametes . All theSince the publication of the first human genome , 63 and The genome diversity of Ukraine is an important clue to advance modern genome studies of the population history of Europe. The country is positioned in the crossroads of the early migration of modern humans and the westward expansion of the Indo-Europeans, and represents an aftermath of centuries of migration, admixture, and demographic and selective processes. As wave after wave of great human migrations moved across this land for millennia, they were followed by the exchange of cultural knowledge and technology along the great trade routes that continue to transect this territory until the present day.The justifications for collecting, sequencing, and analyzing populations from this part of Europe have been outlined previously , 66, andThe present project is built upon the open release/access philosophy. The data have been released and can be used to search for population ancestry markers, as well as medically related variants, in subsequent studies. The public nature of the data deposited on the specially created web resource located at Uzhhorod National University will ensure that the nation's biomedical researchers will receive access to a useful information resource for future projects in genomics, bioinformatics, and personalized medicine. Engaging local Ukrainian scientists in this collaborative international project lays the foundation for future studies and ensures their participation in the worldwide research community.GigaScience GigaDB [The raw reads are available at the SRA . All other datasets mentioned in this project are available in e GigaDB .Supplementary Table S1: Sequencing summaries of output from BGISEQ-500 and Illumina NovaSeq6000 S4. Full sequencing statistics for individual samples in Table S1.2.Supplementary Table S2: Filtering summary of the data obtained from 97 whole genomes sequenced with BGISEQ-500.Supplementary Table S3: The full list of high-impact functional variants  that had an allele count of ≥2 with their predicted function, number of gene transcripts of the gene affected, and frequencies.Supplementary Table S4: List of the medically relevant functional markers found in the Ukrainian population and reported in (A) GWAS catalog [ catalog  and (B)  catalog  databaseSupplementary Table S5: Complete list of the highly differentiating markers, reported in ClinVar [ ClinVar , with hi ClinVar , 39, 40 Supplementary Table S6: A list of markers with the highest non-reference allele frequency (NAF) differences in the Ukrainian population evaluated by the Fisher exact test (FET) compared with the frequencies in neighboring populations: the combined population from Europe (EUR) [pe (EUR)  and Russpe (EUR) . This dape (EUR)  that canpe (EUR) , 47.Supplementary Table S7: Inbreeding estimates in a dataset of 117,641 loci from 84 samples: (a) method-of-moments F-coefficient estimates, (b) variance-standardized relationship minus 1 estimates, and (c) F-estimates based on correlation between uniting gametes [ gametes .Supplementary File S1: IRB approval of the study “Genomic Diversity of Ukraine's Population” (in Ukrainian).Supplementary File S2: Genomic Diversity of Ukraine's Population Project: Protocol description, questionnaire, and informed consent to participate and publish (in Ukrainian with English translation).Supplementary File S3: The list of the samples in this study, their characteristics and geographical locations, and sources of genomic data for each .Supplementary File S4: List of the samples from different studies used in the present population analysis.Supplementary File S5: Sample sources.Supplementary Figure S1: Transition/transversion (TITV) ratio for the novel SNPs , doubleton, rare , and common (>5%) to more closely approximate the 1KGP definitions. B. Percent novel mutations in various classes of SNPs.Supplementary Figure S3: Genetic structure of Ukrainian population in comparison with other European populations. For identification of the optimal K parameter, we used the 10-fold cross-validation function of ADMIXTURE in the range 2–8, with K = 3 resulting in the lowest error [st error . This anst error , 39 and st error , as wellst error .Supplementary Figure S4: Distribution of inbreeding coefficients in the Ukrainian sample. The individual values corresponding to the samples are presented in Table S7.1KG: 1000 Genomes Project; AIM: ancestry informative marker; ALU: Alu elements; bp: base pairs; BWA: Burrows-Wheeler Aligner; DNB: DNA nanoball; DUP: duplications; EGDP: Estonian Biocentre Human Genome Diversity Panel; GATK: Genome Analysis Toolkit; Gb: gigabase pairs; gnomAD: Genome Aggregation Database; GWAS: genome-wide association studies; HGDP: Human Genome Diversity Project; INV: inversions; IRB: institutional review board; MEI: mobile element insertions; NCBI: National Center for Biotechnolgy Information; NIH: National Institutes of Health; NUMT: nuclear mitochondrial DNA; PC: principal component; PCA: principal component analysis; QC: quality control; SRA: Sequence Read Archive; SVA: non-autonomous retroelements; TFBS: transcription factor binding site ablation; TITV: transition/transversion; UAU: Ukrainians from Ukraine; WGS: whole-genome sequencing.The collection procedure was approved as part of the “Genome Diversity in Ukraine” project by the IRB of Uzhhorod National University, Uzhhorod, Ukraine (Supplementary File S1). Each participant had an opportunity to review the informed consent materials (Supplementary File S2) and received an explanation of the issues around the sharing of genome data before making any decisions about making them public in this manner.Y.L. and H.Y. are employed by BGI, which owns DNBSEQ™ technology; Olga Levchuk, Alla Patrus, and Nelya Lazar represent AstraDIA (Ukraine), which collected and extracted DNA samples. All other authors declare that they have no competing interests.This research was funded in part by internal funding from BGI (China), Uzhhorod National University (Ukraine), Division of Cancer Epidemiology and Genetics, National Cancer Institute (USA), and the startup fund of Oakland University, Rochester, Michigan.Conceptualization: T.K.O., K.S., W.W.W., S.C., H.Y., Y.L., J.L.R.F., F.B, O.T.O., M.D., M.Y., R.E.M., V. SmolankaData curation: W.W.W., K.S., A.W., A.U., P.B., V. Stakhovska, K.M., Y.H., N.L., O.T.O., M.N., S.C., O.P., N.V.Formal analysis: W.W.W., A.W.Funding acquisition: T.K.O.Investigation: K.S., W.W.W., A.W., S.O.C.M.Methodology: T.K.O., M.D., R.E.M.Project administration: T.K.O., M.D., V. SmolankaResources: T.K.O., H.Y., Y.L., R.E.M., M.Y., M.D., O.T.O., V. SmolankaSoftware: W.W.W., A.W.Supervision: T.K.O., V. Smolanka, M.D., R.E.M.Visualization: W.W.W., K.S., A.W.Writing: T.K.O., K.S., W.W.W., A.W. , and T.K.O. and R.E.M. (review and editing).giaa159_GIGA-D-20-00230_Original_SubmissionClick here for additional data file.giaa159_GIGA-D-20-00230_Revision_1Click here for additional data file.giaa159_GIGA-D-20-00230_Revision_2Click here for additional data file.giaa159_Response_to_Reviewer_Comments_Original_SubmissionClick here for additional data file.giaa159_Response_to_Reviewer_Comments_Revision_1Click here for additional data file.giaa159_Reviewer_1_Report_Revision_1Jong Hwa Bhak, Ph.D. -- 8/25/2020 ReviewedClick here for additional data file.giaa159_Reviewer_2_Report_Original_SubmissionChaochun Wei -- 9/14/2020 ReviewedClick here for additional data file.giaa159_Reviewer_2_Report_Revision_1Chaochun Wei -- 11/10/2020 ReviewedClick here for additional data file.giaa159_Supplemental_FilesClick here for additional data file.


Circusaeruginosus) breeding in Belgium and the Netherlands. The Western marsh harrier is included as a threatened bird species in Annex I of the European Bird Directive due to the steep decline in population densities. In order to collect data of habitat use and migration behaviour, Western marsh harriers were equipped with light-weight solar powered GPS trackers developed by the Institute for Biodiversity and Ecosystem Dynamics (IBED) at the University of Amsterdam . These trackers automatically collect and store data on the bird’s activity and 3D position in time and transmit these data to ground stations.In this data paper three datasets are described containing GPS tracking and acceleration data of Western marsh harriers (INBO) and the Dutch Montagu’s Harrier Foundation. Tracked Western marsh harriers were breeding in the northeast of the Dutch province of Groningen and on the opposite side of the river Ems in Germany (H_GRONINGEN), in the region of Waterland-Oudeman near the Belgian-Dutch border (MH_WATERLAND), and at the left bank of the Scheldt estuary, close to the Belgian-Dutch border and north of the city of Antwerp (MH_ANTWERPEN). Most individuals remained within 10 km from their nesting sites during the breeding season and wintered in West Africa. H_GRONINGEN contains 987,493 GPS fixes and 3,853,859 acceleration records of four individuals since 2012. MH_WATERLAND contains 377,910 GPS fixes of seven individuals. Sampling in this region began in 2013. Three more Western marsh harriers were tagged in the Scheldt estuary near Antwerp more recently in 2018  and 2019  for the MH_ANTWERPEN study, which contains 47,917 GPS fixes and 227,746 acceleration records.The datasets were collected by the Research Institute for Nature and Forest (https://www.movebank.org) and archived as data packages in Zenodo (https://www.zenodo.org) to ensure long-term preservation and versioning of the data.The three Western marsh harrier datasets were published as separate studies in Movebank ( H_GRONINGEN – Western marsh harriers  breeding in Groningen (the Netherlands). Dataset. https://doi.org/10.5281/zenodo.3552507Koks B, Schlaich A, Schaub T, Klaassen R, Anselin A, Desmet P, Milotic T, Janssens K, Bouten W (2019) MH_WATERLAND – Western marsh harriers  breeding near the Belgium-Netherlands border. Dataset. https://doi.org/10.5281/zenodo.3532940Anselin A, Desmet P, Milotic T, Janssens K, T’Jollyn F, De Bruyn L, Bouten W (2019) MH_ANTWERPEN – Western marsh harriers  breeding near Antwerp (Belgium). Dataset. https://doi.org/10.5281/zenodo.3550093Spanoghe G, Desmet P, Milotic T, Janssens K, De Regge N, Vanoverbeke J, Bouten W (2019) Circusaeruginosus Linnaeus, 1758) is a large harrier species native to temperate and subtropical Eurasia and Africa. Due to the steep population decline observed in Europe since the 1970s, the species has been included as a threatened species in annex I of the European Birds Directive in 1979. In Flanders, the Western marsh harrier appears as an endangered species on the red list of breeding bird species  initiated a GPS tracking study in the northeastern part of the Netherlands (Groningen) using lightweight, solar powered GPS tags. The research objectives for this monitoring study were to determine habitat use of Western marsh harriers in agricultural landscapes, to reveal their migration behaviour, and to study flying behaviour in the vicinity of wind turbines for estimating collision risks.In 2012, the Dutch Montagu’s Harrier Foundation (INBO) started studying the ecology of the Western marsh harrier in Belgium in 2011. One of the aims was to study detailed habitat use and migration patterns. In 2013, the INBO started a GPS sensor network for birds as part of the Belgian contribution to the LifeWatch observatory, using the same technology as GKA. In this network, individuals of a breeding population of Western marsh harriers in the northern part of Flanders (Waterland-Oudeman region) were equipped with GPS trackers in collaboration with GKA. In 2018, a third population was tagged with GPS trackers in the Scheldt estuary north of the city of Antwerp. The research objectives of these projects were to study the trade-off between migratory behaviour, reproductive performance and survival, and to study the home-range area, habitat preference, and foraging behaviour of Western marsh harriers in agricultural areas. To allow greater use of the data beyond our research questions, all data are now published as open data.The Research Institute for Nature and Forest (H_GRONINGEN), seven individuals breeding near the Belgian-Dutch border (MH_WATERLAND), and three individuals breeding near Antwerp (Belgium) (MH_ANTWERPEN)   Figure .Kingdom: AnimaliaPhylum: ChordataClass: AvesOrder: AccipitriformesFamily: AccipitridaeGenus: CircusSpecies: Circusaeruginosus H_GRONINGEN), in the region of Waterland-Oudeman near the Belgian-Dutch border (MH_WATERLAND), and at the left bank of the Scheldt estuary close to the Belgian-Dutch border and north of the city of Antwerp (MH_ANTWERPEN). All individuals from which data from the non-breeding period were available wintered in West Africa , the polder area in the north-western part of Belgium , and in the polder area at the left bank of the Scheldt estuary in the northern part of Belgium . The tracked birds nested on the ground in small reed beds and cereal fields.The studied Western marsh harrier populations breed in agricultural landscapes in the northeast of the Dutch province of Groningen (H_GRONINGEN and MH_WATERLAND) or a bal-chatri (MH_ANTWERPEN). As trapping proved very difficult, only a few individuals could be tagged per breeding season (Table H_GRONINGEN and MH_WATERLAND) and MH_ANTWERPEN). Sex was determined on sight. UvA-BiTS GPS-trackers  came back to his breeding grounds, but the tracker got broken and he has not been spotted again in 2019 (status unknown). In the MH_ANTWERP study, two individuals were tagged in 2019, while one animal (Suzanna) was tagged in 2018 but did not come back after the migration in 2019 (status unknown). One of the individuals (Raymond) in MH_WATERLAND was found dead in the Italian Alps in spring 2016. His tracker was reused for another male (Ben). Another individual in the MH_WATERLAND dataset (Jozef) was tagged in 2013 in his breeding area in the Waterland-Oudeman region but moved to another breeding area at the Moeren close to Veurne at 70 km from his previous breeding ground in 2016, 2017 and 2018. The other individuals in this dataset have not been seen in their original breeding grounds in the past few years and are assumed dead.In total, 14 Western marsh harriers were tracked (Table UvA-BiTS) developed by the Institute for Biodiversity and Ecosystem Dynamics (IBED) at the University of Amsterdam. These lightweight, solar powered GPS trackers automatically record 3D position and air temperature. The built-in tri-axial accelerometer can be configured to collect body movements and bird behaviour and was deployed in the H_GRONINGEN and MH_ANTWERPEN studies. Each individual tri-axial accelerometer measurement consists of x (acceleration-raw-x), y (acceleration-raw-y) and z data points (acceleration-raw-z). Tilt values  are derived from the raw acceleration measurements (M), the calibration factors offset (O) and sensitivity (S).Thus acceleration for heave (tilt-z), surge (tilt-x) and sway (tilt-y) is calculated as: Az = (Mz-Oz)/Sz ; Ax = (Mx-Ox)/Sx; Ay = (My-Oy)/Sy (Harriers in the three studies were equipped with the University of Amsterdam Bird Tracking System (y-Oy)/Sy . Tilt daData are stored in the tracker’s 4 MB built-in flash drive. Depending on the settings, up to 60,000 GPS records can be stored in the internal memory . TrackerH_GRONINGEN study, “high-resolution” GPS data with an interval of 3 s were collected during parts of the day using hourly blocks or virtual geographic fences in order to increase the positional accuracy of the GPS fixes , which is accessible to the involved researchers only. In order to make our data available to the whole scientific community, all tracking data are eventually published as open data. We decided to upload the data to Movebank (https://www.movebank.org) as it is a specific repository for this type of data and it is well adopted by the scientific community . This allows us to repeat the process when new data become available for active studies. These data were then uploaded to the Movebank database, with one study for each dataset  currently does not support versioning and version-agnostic DOIs, we opted to archive our studies in the Zenodo data repository (https://www.zenodo.org). For each Movebank study, one Zenodo data package has been created  and automatically before uploading to Movebank for GPS-fixes with speeds above 30 m/s (indicated as TRUE in import-marked-outlier). Using this approach, 376, 97 and 16 observations were marked as outliers in H_GRONINGEN, MH_WATERLAND and MH_ANTWERPEN respectively. The workflow and scripts for querying data from the UvA-BiTS database and transforming these into the Movebank data format are publicly documented on GitHub (https://github.com/inbo/bird-tracking).GPS fixes that are likely incorrect  are marked in two ways: manually by the researcher in the Data recording1. Researcher captures bird, takes biometrics, attaches GPS tracker, and releases bird.2. Researcher records or updates metadata about bird, GPS tracker and deployment.3. Researcher sets a measurement scheme, which can be updated anytime.4. GPS tracker records data.5. GPS tracker automatically receives new measurement settings and transmits recorded data when a connection can be established with the mobile base station.UvA-BiTS.6. Recorded data are automatically harvested, post-processed, and stored in a central PostgreSQL database at 7. Data stream stops when birds no longer return to the nesting site or if GPS trackers no longer function.Data publicationUvA-BiTS in the Movebank data format.1. Data  are periodically exported from 2. GPS outliers are marked.3. Data are uploaded to the appropriate study on Movebank and made publicly available.4. Data are exported from Movebank and archived on Zenodo, where each update is a version with a DOI.H_GRONINGEN is the largest dataset containing 987,493 GPS fixes in the period 2012–2018, while the MH_WATERLAND study started in 2013 with 377,910 GPS fixes until 2018, and MH_ANTWERPEN started in 2018 and contains 47,917 GPS fixes in the Movebank study for the period 2018–2019 . H_GRONINGEN datasetObject name: H_GRONINGEN – Western marsh harriers  breeding in Groningen (the Netherlands)• Format name: Movebank data format• Format version: 2 (http://vocab.nerc.ac.uk/collection/MVB/2/)• Language: English• License: http://creativecommons.org/publicdomain/zero/1.0/• Usage norms: http://www.inbo.be/en/norms-for-data-use• Publication date: 2019-11-26• Derived from: https://www.movebank.org/cms/webapp?gwt_fragment=page=studies,path=study922263102• DOI of version described in this paper: https://doi.org/10.5281/zenodo.3828298• DOI for all versions: https://doi.org/10.5281/zenodo.3552507• MH_WATERLAND datasetObject name: MH_WATERLAND – Western marsh harriers  breeding near the Belgium-Netherlands border• Format name: Movebank data format• Format version: 2 (http://vocab.nerc.ac.uk/collection/MVB/2/)• Language: English• License: http://creativecommons.org/publicdomain/zero/1.0/• Usage norms: http://www.inbo.be/en/norms-for-data-use• Publication date: 2019-11-12• Derived from: https://www.movebank.org/cms/webapp?gwt_fragment=page=studies,path=study604806671• Source of: https://doi.org/10.15468/rbguhj • DOI of version described in this paper: https://doi.org/10.5281/zenodo.3826591• DOI for all versions: https://doi.org/10.5281/zenodo.3532940• MH_ANTWERPEN datasetObject name: MH_ANTWERPEN – Western marsh harriers  breeding near Antwerp (Belgium)• Format name: Movebank data format• Format version: 2 (http://vocab.nerc.ac.uk/collection/MVB/2/)• Language: English• License: http://creativecommons.org/publicdomain/zero/1.0/• Usage norms: http://www.inbo.be/en/norms-for-data-use• Publication date: 2019-11-21• Derived from: https://www.movebank.org/cms/webapp?gwt_fragment=page=studies,path=study938783961• DOI of version described in this paper: https://doi.org/10.5281/zenodo.3827918• DOI for all versions: https://doi.org/10.5281/zenodo.3550093• http://creativecommons.org/publicdomain/zero/1.0/). We would appreciate however, if you read and follow these norms for data use (http://www.inbo.be/en/norms-for-data-use) and provide a link to the original dataset using the DOI whenever possible. If you use these data for a scientific paper, please cite the dataset(s) following the applicable citation norms and/or consider us for co-authorship. We are always interested to know how you have used or visualized the data, or to provide more information, so please contact us via the contact information provided in the metadata or opendata@inbo.be.To allow anyone to use these datasets, we have released the data to the public domain under a Creative Commons Zero waiver (


Thalassioisira weissflogii, coastal Thalassiosira pseudonana and oceanic Thalassiosira oceanica) during transient shifts from a moderate to high growth irradiance (85 to 1200 μmol photons m-2 s-1). Transient high light exposure caused T. weissflogii to rapidly downregulate PSII with substantial nonphotochemical quenching, protecting PSII from inactivation or damage, and obviating the need for induction of O2 consuming  pathways. In contrast, T. oceanica retained high excitation pressure on PSII, but with little change in RCII photochemical turnover, thereby requiring moderate repair activity and greater reliance on LDR. T. pseudonana exhibited an intermediate response compared to the other two diatom species, exhibiting some downregulation and inactivation of PSII, but high repair of PSII and induction of reversible PSII nonphotochemical quenching, with some LDR. Together, these data demonstrate a range of strategies for balancing light harvesting and utilization across diatom species, which reflect their adaptation to sustain photosynthesis under environments with inherently different light regimes.Marine phytoplankton, and in particular diatoms, are responsible for almost half of all primary production on Earth. Diatom species thrive from polar to tropical waters and across light environments that are highly complex to relatively benign, and so have evolved highly divergent strategies for regulating light capture and utilization. It is increasingly well established that diatoms have achieved such successful ecosystem dominance by regulating excitation energy available for generating photosynthetic energy via highly flexible light harvesting strategies. However, how different light harvesting strategies and downstream pathways for oxygen production and consumption interact to balance excitation pressure remains unknown. We therefore examined the responses of three diatom taxa adapted to inherently different light climates (estuarine  Diatoms account for the majority of marine primary production , 2 and asensu  to plot vs. dynamic non-photochemical quenching [1-Q] under each actinic light intensity following equations from Suggett et al.   that span highly divergent photophysiological responses.Measurements were performed for each species using at least three independent samples collected during acclimated exponential growth. Methods described from this point onwards apply to only the three selected diatoms (-1. Specific growth rates (divisions [day]-1) were calculated as μ = ln(N2/N1)/(t2-t1), where μ is the specific growth rate (day-1), N1 and N2 are the cell concentrations (mL-1) at time 1 (t1) and time 2 (t2), respectively. Cell volume was calculated from the same sample used for cell count determinations using shape-specific geometric formulas from Sun & Liu  and [1-Q]  in T. oceanica and T. pseudonana were not statistically distinguishable from NPP in T. weissflogii  were generally higher  for the larger T. weissflogii compared to the smaller T. oceanica and T. pseudonana [Cellular growth rates increased with decreasing cell size from eudonana . Chl a:Ceudonana , T. weisa and μ, , 99), anoceanica . Net prissflogii . Consisteudonana .a and were largely similar for samples incubated for 10 min under HL (1200 μmol photons m-2 s-1) compared to those at the growth irradiance  , were different among species (p < 0.05) with fucoxanthin:Chl a highest in T. oceanica compared to T. pseudonana and T. weissflogii , differed between light treatments where all species exhibited an increase in DPS,  from Ig to HL. T. pseudonana exhibited the lowest DPS under Ig but also the greatest difference in DPS between Ig and HL   within essflogii . Most nog and HL .T. weissflogii exhibited much higher nonphotochemical quenching than T. oceanica (p < 0.001) despite similar DPS. Whilst T. pseudonana had lower total DPS, the corresponding increases of DPS and YNPQ from Ig to HL were more similar to those of T. weissflogii  for 120 min, followed by a recovery period at 15 μmol photons m-2 s-1  measured immediately after exposure to treatment or recovery light. Systematic photophysiological assessment of samples removed over the 120 min HL exposure and subsequently subjected to a 10 min dark period before fluorometric assessment highlighted the influence of nonphotochemical quenching on PSII function. This is particularly evident in T. oceanica and T. pseudonana where initial Fv′/Fm′ largely relaxed to higher Fv/Fm , tracked with the black dots connecting the orange dashed and green solid lines, respectively (see T. weissflogii showed a progressive decrease in the relaxation amplitude over 120 min HL indicative of sustained (longer lived) nonphotochemical quenching, consistent with the failure of T. weissflogii to fully counter photo-inactivation through induction of repair [In parallel with the effects of photo-inactivation and repair, vely see , over thf repair . It is tPI. These kPI values determined for each species in the absence of PSII repair were then inputted into fit models [REC (PI) decreased with increasing diatom cell size from the smaller T. oceanica and T. pseudonana to the larger T. weissflogii (REC) also decreased with increasing cell size from T. pseudonana to T. weissflogii, but was also low in the offshore T. oceanica, consistent with patterns of higher capacity for PSII repair observed previously for onshore versus offshore diatoms [Replicate time courses for PSII photo-inactivation in the presence of lincomycin were fit for each species to extract the rate constant for photoinactivation, kt models , 76 usinels [REC . As expessflogii . The rat diatoms .T. oceanica compared to T. weissflogii from shorter incubations  via MIMS were consistent across light treatments where there was higher production under HL than Ig for all three species . Measurements of energy losses are shown in O2 allocated to respiration in the dark (RDARK) and in the light (LDR), with the remainder being net O2 production (NetO2). Extent of LDR (% of GPO2) was comparable for all species under Ig but not HL; specifically, %LDR for T. weissflogii decreased from 11.8% at Ig to 8.1% at HL but increased from an average of 10.5% at Ig to 16.6% at HL for the other two species, with T. oceanica exhibiting the highest LDR increase from Ig to HL. Patterns of %RDARK were more variable across species and treatment, whereby %RDARK in T. weissflogii and T. pseudonana was 16.6–21.7% regardless of irradiance and %RDARK in T. oceanica was always <10% (T. oceanica differed from both T. weissflogii (p = 0.017) and T. pseudonana  resulted in an increase and decrease of NetO2 from Ig to HL for T. weissflogii and T. oceanica, respectively . Remarkably, for all species under HL only 0.05 of the fluorescence-derived yields for absorbed light energy went to YII (p = 0.236), which contradicts [T. weissflogii (~0.7) under HL while, instead, YNO represents ~0.7 of excitation flux in T. oceanica. T. pseudonana has balanced YNO and YNPQ at ~0.48 each, once again revealing the divergence of photoprotective strategies employed by these diatoms. Since FRRf-based values of YII appear to, generally, be directly proportional to GPO2 in microalgae [e.g. DARK and %NetO2 allocations of GPO2 to be comparable to YII (2 recycling’ (loss of electrons in the membrane electron transport chain) through %LDR across these diatom species; specifically, 4–6% under Ig and <1% under HL.The collective energy loss yields via YII, YNPQ and YNO as per  revealedt to YII  , exhibit a high capacity to rapidly initiate nonphotochemical quenching whereas oceanic diatoms (e.g. T. oceanica) have slower initiation of nonphotochemical quenching as light intensifies, with coastal diatoms (e.g. T. pseudonana), exhibiting an intermediate response [T. pseudonana was accompanied by greatest change in DPS capacity from Ig to HL. For T. oceanica, slower nonphotochemical quenching induction, and greatest susceptibility to PSII inactivation, was in turn accompanied by greater proportion of O2 evolved from PSII (GPO2) flowing to LDR, and less to RDARK. Thus, under transient high light exposure T. weissflogii adopts a strategy of rapid and sustained PSII downregulation, thereby requiring relatively little RCII inactivation/repair, or the need to induce O2 and electron consuming (LDR) pathways. In contrast, T. oceanica appears to not initiate protective mechanisms to alleviate excess excitation pressure on PSII, as evident by relatively little downregulation, inactivation and only moderate repair, but, instead, places greater reliance on LDR to dissipate excess excited energy downstream of PSII. Although these experiments were conducted under nutrient repletion, T. oceanica is evolved for low nutrient growth. Limiting reliance upon PSII repair thereby lowers the requirement for mineral nutrient investment into metabolically expensive systems for protein turnover [T. pseudonana is intermediate, with moderate downregulation and inactivation of PSII, but high repair of PSII and relaxation of nonphotochemical quenching, and some LDR. These trends show inherent trade-offs in how these different species deploy downregulation and repair of PSII, versus modulating subsequent re-consumption of oxygen and electrons  gradient across the thylakoid membrane (ΔpH) and xanthophyll cycling (XC), involving the de-epoxidation state (DPS) of diadinoxanthin  to diatoxanthin  [T. pseudonana was shown to be inhibited at HL due to the presence of a proton gradient, which maintains high concentrations of this photo-protective pigment. Dt epoxidation is also inhibited by complete darkness after HL exposure [T. pseudonana showed rapidly reversible nonphotochemical quenching, T. weissflogii appeared to sustain nonphotochemical quenching upon transition to low light, in parallel with its low capacity for PSII repair , therefore the trends observed appear robust. Interestingly, T. oceanica had slow initiation of nonphotochemical quenching under HL  . Dt epoxexposure . Such inexposure . Diatomsexposure , 110. Suexposure , howeverir kREC; . Sustain gravida . However gravida , support gravida . While ounder HL  suggestiT. oceanica does not rapidly initiate nonphotochemical quenching to dissipate excess incident light energy in the antennae bed and, subsequently, suffers high excitation pressure on the RCIIs that split water and, potentially, higher excitation pressure through the subsequent electron carrier network. Increased ‘traffic’ of excitation energy was clear from the higher photo-inactivation rates (kPI) for T. oceanica compared to the other two species. Previous studies on diatoms have established a link between diatom cell size and susceptibility to photo-inactivation, whereby cell size is inversely proportional to photo-inactivation [REC) for the smaller T. pseudonana than the larger T. weissflogii. Importantly, cell size may explain some photo-inactivation trends, but protein synthesis and regeneration, that alters in accordance with photosynthetic architecture also needs to be considered [tivation  and thustivation . This considered , 73. It nsidered  a sustaiT. oceanica with the highest kPI synthesis of photosynthetic machinery comes at the expense of photosynthetic production . Chlorophest kPI , alterna species , Fig 5. piration . A recenDd to Dt . Thus, iT. oceanica) have been found to have up to 10 PSII:PSI while coastal diatoms generally have 2 PSII:PSI [6f, and PSI are 3, 6, and 12 Fe atoms, respectively, but also provide insight into potential evolutionarily conserved species-specific photo-protective strategies amongst diatoms. Fe availability greatly influences growth rates of diatoms from various habitats whereby T. pseudonana and T. weissflogii growth rates were lowered by approximately 75% under Fe limitation while T. oceanica showed no significant change in growth rate [T. oceanica upon recycling electrons back to O2 under excess light. This cyclic flux of electrons trades biosynthetic reductant for ATP generation. If inorganic nutrients are limiting, the requirement for ATP for maintenance and nutrient uptake increases relative to the requirement for actual reductive biosynthesis. Conversely, for coastal/estuarine waters, where light is dynamic and nutrients plentiful, diatoms can afford to invest more energy in biosynthesis of macromolecules and division as well as energetically expensive photosynthetic machinery, such as PSI, that are more efficient trapping excitation energy than PSII [Synechococcus [T. oceanica cannot “afford” to synthesize new photosynthetic machinery and instead evolved strategies to allocate harvested light energy towards chemical energy for maintenance and growth while the slowly induced nonphotochemical quenching provides a fail-safe in the event of prolonged light stress.Diatoms exhibit distinct alterations in photosynthetic architecture based on ecological niche, where oceanic diatoms  at HL and lower kPI. T. oceanica, on the other hand, does not initiate nonphotochemical quenching as a rapid primary response mechanism to dissipate excess light energy and therefore had an accumulation of photochemical energy resulting in higher rates of LDR but also higher kPI. This supports the idea that photo-protective strategies are evolutionarily conserved based on ecological niche for diatoms. These diatoms possess similar core machinery to dissipate excess light energy but have balanced the mechanistic dissipation strategies employed to best suit their respective niche.In summary, we have built on previous studies demonstrating differences in nonphotochemical quenching amongst diatom species, and strategies in dealing with transient high light exposure (e.g. ) to elucS1 FigPhaeodactylum tricornutum (orange inverted triangles), Chaetoceros muelleri (yellow circles), Ditylum brightwellii (green diamonds), Thalassiosira rotula (black Xs), Thalassiosira pseudonana (black squares), Thalassiosira weissflogii (blue triangles), and Thalassisosira oceanica (red circles) with increasing light intensity. Error bars represent the standard error of the mean of at least n = 3 for independent biological replicates.Conventional non-photochemical quenching NPQ; see  capaciti(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Biscogniauxia mediterranea and Diplodia corticola has been reported in the last decade. Due to the high socio-economic and ecologic importance of this plant species in the Mediterranean Basin, the search for preventive or treatment measures to control these diseases is an urgent need. Fungal endophytes were recovered from cork oak trees with different disease severity levels, using culture-dependent methods. The results showed a higher number of potential pathogens than beneficial fungi such as cork oak endophytes, even in healthy plants. The antagonist potential of a selection of eight cork oak fungal endophytes was tested against B. mediterranea and D. corticola by dual-plate assays. The tested endophytes were more efficient in inhibiting D. corticola than B. mediterranea growth, but Simplicillium aogashimaense, Fimetariella rabenhorstii, Chaetomium sp. and Alternaria alternata revealed a high potential to inhibit the growth of both. Simplicillium aogashimaense caused macroscopic and microscopic mycelial/hyphal deformations and presented promising results in controlling both phytopathogens’ growth in vitro. The evaluation of the antagonistic potential of non-volatile and volatile compounds also revealed that A. alternata compounds could be further explored for inhibiting both pathogens. These findings provide valuable knowledge that can be further explored in in vivo assays to find a suitable biocontrol agent for these cork oak diseases.An increase in cork oak diseases caused by  Quercus suber L.) is an evergreen tree species that covers between 1.7 and 2.7 million ha in the western Mediterranean, distributed among Portugal, Spain, France, Italy, Morocco, Tunisia and Algeria  and poured into 9 cm Petri dishes. A mycelial plug of an actively growing pathogen (B. mediterranea or D. corticola) was placed in the center of the plate. Control plates were similarly prepared but containing PDA without a fungal filtrate. Mycelial growth areas were determined as previously referred and mycelial growth inhibition was determined according to the formula: (Ac−Af)/Ac × 100, where Ac is the area of fungal growth in the control plate and Af is the area of fungal growth in the presence of the filtrate. Three independent experiments were performed (with at least 3 replicates each) for all endophyte/pathogen combinations. Statistical analysis was performed using ANOVA in GraphPad Prism 7.00 software  to understand the impact of endophytic compounds on pathogen growth.The antagonistic effect of the metabolites produced by the eight selected fungal endophytes was tested using a method adapted from Campanile et al. . Three m2/h; The effect of volatile compounds produced by the eight selected fungal endophytes in inhibiting pathogen growth was evaluated by an inverted plate method . For eacFimetariella rabenhorstii, Discosia sp.), including 18 that have never been described as plant endophytes   or the lowest  precipitation levels.Endophytic fungi of cork oak twigs were recovered from eight different forests in Portugal. From a total of 1117 fungal isolates, 440 were molecularly identified and grouped into 128 OTUs comprising 18 orders, 38 families, 45 genera and 39 species . Only OTcus sp.) . Among pB. mediterranea was the only fungus identified in all cork oak stands and Fusarium sp. was present in all forests, with the exception of LI and GR. Further, Penicillium sp., Sarocladium kiliense and Neocucurbitaria sp. were recovered from almost all cork oak stands. In contrast, there were fungi  that were only recovered from one sampling location. Since many fungi are difficult to be cultured, culture-dependent methods are known to underestimate fungal communities [Some OTUs were more widespread throughout Portuguese cork oak forests than others. munities . The usemunities . Accordimunities ,34. Cultmunities . DespiteB. mediterranea [Coryneum sp. [Cryphonectria naterciae [D. corticola [D. quercivora [Discula quercina [Neofusicoccum parvum [B. mediterranea, Coryneum sp. and C. naterciae were isolated from trees in all disease severity levels, while D. corticola, D. quercivora and Discula quercina were only isolated from trees with declining symptoms (B.mediterranea and Coryneum sp.) were isolated from healthy cork oak trees in Italy [D. corticola, D. quercivora and Discula quercina in declining cork oak trees agrees with their role as emerging pathogens to Quercus spp. in different regions of the world [Trichoderma harzianum T22 strain to direct growth toward the plant host [The ecological role of each identified fungal species was determined based on the literature. As the functional role of certain endophytes could change according to their plant host genotype, during different stages of the plant life cycle or in extreme conditions , many enterranea , Coryneuneum sp. , Cryphonaterciae , D. cortorticola , D. querercivora , Disculaquercina  and Neofm parvum . Interessymptoms . In agrein Italy ,40. The he world ,41, whiche world ,42. Conche world ,44. For ant host . HoweverB. mediterranea and D. corticola growth, based on their potential beneficial ecological role, availability and culture readiness. D. corticola mycelial growth was persistently inhibited by the presence of these cork oak endophytes, while B. mediterranea was differentially inhibited by endophytes , A. alternata (19.3%), Chaetomium sp. (13.6%) and S. aogashimaense (12.6%), whereas the other fungal endophytes promoted or did not affect pathogen growth  . Both endophytes displayed a similar interaction with the D. corticola pathogen, resulting in antagonism/co-antagonism with pathogen inhibition . In contrast, A. alternata and Chaetomium sp. increased their growth while inhibiting B. mediterranea, thus displaying agonist interactions (+/-) with this pathogen. Both endophytes displayed a distinct interaction with D. corticola. A. alternata was not affected  and Chaetomium sp. was inhibited by this pathogen . Except for F. oxysporum , all the other tested endophytes also revealed a co-antagonism interaction with D. corticola.Eight endophyte isolates were selected for testing their ability to inhibit dophytes . Dual-pln growth . Regardie growth , S. aogaon (0/-) . AlthougS. aogashimaense revealed a deadlock at distance (B) interaction type , as well as a deadlock with mycelial contact (A)  .B. mediterranea and D. corticola pathogens. Endophytic soluble compounds seem to have more impact on inhibiting mycelial growth than volatile compounds, mainly against B. mediterranea  produced a soluble extract with high anti-fungal activity against both pathogens. These endophytes inhibited from 28% to 51% of B. mediterranea growth  and from 26% to 72% of D. corticola growth . The volatile emissions from the same endophytes also revealed inhibitory activities against B. mediterranea and D. corticola . Multiple studies have already revealed the antifungal activity of genera from tested endophytes, in particular of Chaetomium spp., e.g., [Coniothyrium spp., e.g., [Diaporthe spp., e.g., [Penicillium spp., e.g., [Simplicillium spp., e.g., [Fimetariella spp. or the antifungal activities of A. alternaria and F. oxysporum species, which have been mainly recognized as important plant pathogens. This work provides new information related to the production of antimicrobial compounds, mainly from F. rabenhorstii and A. alternata, against cork oak pathogens. For understanding the production of inhibitors by the tested endophytes, their non-volatile and soluble compounds (produced in liquid culture) and volatile emissions were tested against terranea . However., e.g., , Conioth., e.g., , Diaport., e.g., , Penicil., e.g., , and Sim., e.g., , as wellB. mediterranea in dual-plate methods also revealed the production of inhibitors for this phytopathogen. For example, the soluble compounds of P. olsonii revealed the highest inhibition  of all against B. mediterranea, and volatiles from C. carteri and F. oxysporum also exhibited high inhibitory activity against the same pathogen . These results suggest that the inhibitory effect of a specific fungal isolate against a phytopathogen may be due to the production of multiple compounds that could act in a synergistic or antagonistic way [S. coffeanum volatile compounds against Aspergillus species was reported by Gomes et al. [S. lamellicola was used to produce a fungicide against Botrytis cinerea [Alternaria sp. was already reported for the control of fungal and bacterial growth [F. oxysporum phytopathogen [Interestingly, endophytes with no inhibitory activity against stic way . Accordis et al.  and S. l cinerea . The prol growth , but soml growth , represepathogen ,138. TheAlternaria alternata and Fusarium oxysporum) have revealed a strong in vitro inhibitory effect against cork oak pathogens (B. mediterranea and D. corticola). In particular, an A. alternata isolate revealed a high inhibitory activity against both pathogens, promoting hyphal deformations on B. mediterranea. Although this was not the case when interacting with D. corticola, this pathogen was similarly inhibited by A. alternata and F. oxysporum, displaying a mycelial interaction type based on the partial replacement of mycelia after an initial deadlock with mycelial contact. Non-volatiles and volatiles obtained from these isolates  revealed inhibitory activity and their potential to be used in a biocontrol strategy for restraining cork oak diseases should be further explored. The use of naturally adapted endophytes (for a specific plant host/environment) in a biocontrol strategy has gained increasing interest for restricting plant diseases. In this cork oak endophyte survey, we detected a high number of fungal OTUs from trees displaying different disease severity levels. The number of OTUs belonging to the pathogenic functional group (including described pathogens to other plant species) was high, even when considering healthy trees. Isolates of potential pathogens . A high ecological and economical value has been given to Simplicillium species due to their biocontrol role and production of bioactive compounds. For example, soybean plants when inoculated with S. lanosoniveum before infection with soybean rust pathogen (Phakopsora pachyrhizi) revealed reduced disease severity [S. aogashimaense could similarly be explored to control cork oak diseases, better understanding of its potential role as a biocontrol agent is still required. We thus conclude that cork oak endophytes could be further explored as biocontrol agents against cork oak diseases.The colonization of plants by beneficial endophytes has been a useful biocontrol strategy. This work suggests severity . Althoug


Mycobacterium chelonae. Clarithromycin with thermotherapy was effective. A review of the second biopsy confirmed coinfection of CMV and Gram-positive mycobacteria. Immunostaining using a panel of anti-bacterial antibodies visualized the mycobacteria in the lesion. Positive findings were obtained with antibodies to Bacillus Calmette-Guérin, Bacillus cereus, MPT64 (Mycobacterium tuberculosis-specific 24 kDa secretory antigen), LAM (Mycobacterium tuberculosis-related lipoarabinomannan), and PAB (Propionibacterium acnes-specific lipoteichoic acid).A mildly diabetic 58-year-old male had traumatic ulceration on the left popliteal fossa, and the lesion progressed to a painful 6 cm deep ulcer. After surgical debridement and skin grafting, ulceration recurred. Pyoderma gangrenosum was clinically diagnosed after the first biopsy, indicating a noninfective ulcer. Immunosuppressive therapy (prednisolone and cyclosporine A) induced complete epithelialization in three months. Four months later, subcutaneous nonulcerated nodules appeared on the anterior area of the left lower leg. Subcutaneous induration progressed and ulceration recurred, so that immunosuppressive therapy continued for one year. Cytomegalovirus (CMV) viremia was detected, and the second biopsy demonstrated CMV inclusions of endothelial and perivascular cells in fibrosing septolobular panniculitis. Cyclosporine A was cancelled, prednisolone was tapered, and ganciclovir started. Viremia soon disappeared, but the lesion progressed to large induration with multiple ulcers measuring up to 3 cm. The third biopsy disclosed infection of Gram-positive mycobacteria, accompanying fat droplet-centered suppurative granulomas without CMV infection. Microbial culture identified  Mycobacterium chelonae on the left lower leg. Histopathological and immunohistochemical features of this rare combination of pathogens are detailed.Immunosuppressive conditions may provoke opportunistic skin infection of a variety of bacteria, fungi, and viruses , 2. The A 58-year-old Japanese man slipped and fell on the road to have his left knee contused, resulting in a 1.5 cm sized shallow ulceration. The patient had suffered from hypertension and mild type 2 diabetes mellitus for two years, but no history of autoimmune disorders. Serum antibodies against insulin and glutamic acid decarboxylase were negative, excluding the possibility of type 1 diabetes mellitus. He had smoked 15 cigarettes per day for more than 40 years. Five months later, a painful skin ulcer, 6 cm in size, occurred on the inner side of the left popliteal fossa (at the same site of contusion). No pathogens were identified in the biopsy specimen, and microbial culture was negative. Surgical debridement and full-thickness grafting from the abdominal skin were performed by a plastic surgeon, but half of the skin flap was eventually impaired, leaving a 3 cm sized deep ulcer with severe pain. The patient was then consulted to dermatologists. Clinical findings, as well as the first biopsy features displaying nonspecific and noninfective ulcer, were consistent with pyoderma gangrenosum . The serFour months later, subcutaneous nonulcerated, painful nodules appeared on the anterior area of the left lower leg, 20 cm distal from the primary ulcer caused by pyoderma gangrenosum. Ulceration recurred, and subcutaneous induration soon progressed toward both the distal and proximal directions. Immunosuppressive therapy restarted, but the painful ulcer persisted for one year. At this point of time, CMV viremia was identified, and the second skin biopsy from one of the subcutaneous nodules identified CMV inclusions in the subcutaneous tissue with fibrosing septolobular panniculitis. The CMV antigens were immunohistochemically visualized in the viral inclusions with 1 : 200 diluted cocktail monoclonal antibodies, CCH2+DDG9, available from Agilent Technologies . Both enMycobacterium chelonae was identified by analyzing with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) by using the MALDI Biotyper  (M. chelonae was more than 2.0 (2.208).Microbial culture on 2% Ogawa medium at 37°C identified rapidly growing nontuberculous mycobacteria. The bacteria were acid-fast and Gram-positive. okohama)  4]. The. TheMycoClarithromycin administration (800 mg/day), combined with thermotherapy using disposal pocket body warmers for one hour twice a day, resulted in marked regression of the ulcers. PSL tapering induced subcutaneous nodularity with ulceration, and thus, clarithromycin treatment was kept to date. After chemotherapy for 15 months, epithelialization was almost achieved. Valganciclovir was administered when CMV viremia reappeared.Metformin  and sitagliptin phosphate hydrate (a selective inhibitor of dipeptidyl peptidase-4) continued for controlling type 2 diabetes mellitus. Mitiglinide calcium hydrate (a stimulator of insulin secretion) was added after the appearance of the skin ulceration for one year. During the clinical course, the patient's blood glucose levels were persistently hyperglycemic, ranging from 106 to 186 mg/dL but without glycosuria. Mild proteinuria ranging from 15 to 100 mg/dL was noted, indicating the complication of stage 2 (early) diabetic nephropathy. His HbA1c levels ranged from 6.4 to 7.3% (standard levels: 4.1–6.2%). The patient thus suffered from mild and controlled diabetes mellitus.Bacillus cereus [M. tuberculosis-specific 24 kDa secretory antigen) [M. tuberculosis-related lipoarabinomannan) [Propionibacterium acnes-specific lipoteichoic acid) [Treponema pallidum gave weak immunoreactivity. Escherichia coli antigens were scarcely observed. The background staining was minimal.For characterizing the mycobacteria colonizing the skin lesion in the second and third skin biopsy specimens, immunostaining using selected antibodies was performed, as indicated in s cereus , MPT64 (antigen) , and monomannan)  and PAB ic acid) . AntiserPathology of Skin Infections published in 2013 [M. chelonae, provoked by immunosuppressive therapy against pyoderma gangrenosum. The patient suffered from mild and controlled type 2 diabetes mellitus. Pyoderma gangrenosum is characterized by noninfectious autoimmune-type progressive skin ulceration [Opportunistic infection caused by a variety of bacteria, fungi, or viruses occurs in the skin under the immunosuppressive state such as acquired immunodeficiency syndrome (AIDS) and after the use of immunosuppressive agents , 2. One  in 2013 . We descceration , successceration .Opportunistic skin infection of CMV is rare. CMV mainly affects vascular endothelial cells and perivascular stromal cells, and multifocal anogenital ulcerations are most frequently encountered in AIDS patients . CutaneoM. avium-intracellulare complex, M. marinum, M. ulcerans, M. fortuitum, and M. chelonae-abscessus complex [Varied nontuberculous mycobacteria affect the skin, including  complex , 18. Rep complex .M. chelonae was first isolated from the tortoise by Friedmann in 1902 [M. chelonae belongs to nonchromogenic RGM (Runyon group IV), widely distributed in environmental water [M. chelonae-abscessus complex, causes opportunistic infection in the human skin, as well as infection in aquatic animals such as the reptiles and fish [M. chelonae, provokes abscess-forming granulomas (suppurative granulomas): some lesions are recognized as abscess surrounded by abortive granuloma formation, while the others predominantly reveal epithelioid granulomas [ in 1902 . M. chelal water . This zoand fish . Of notiand fish . It is pand fish , 18, 22.anulomas , 25. Acianulomas .The cell wall of mycobacteria contains voluminous mycolic acids, extremely long fatty acids, as molecular forms of mycolic acid-containing glycolipid such as trehalose dimycolate and trehalose monomycolate, giving lipophilic nature of mycobacteria . RGM areCorynebacterium kroppenstedtii [C. kroppenstedtii lacks mycolic acid but instead contains tuberculostearic acid in the cell wall [Lipid droplet-centered suppurative granulomas are commonly observed in granulomatous mastitis caused by a lipophilic bacterium, nstedtii . The Graell wall . It is uell wall  that admell wall . In the ell wall , 33. Theell wall , based oell wall , 18.B. cereus was observed in the infected lesion. T. pallidum antiserum also showed weak cross-reactivity. Positive signals with low background staining were observed not only on the mycobacteria in the lipid droplets but also in the cytoplasm of macrophages, so that the detection sensitivity was very high [M. chelonae. It has been reported that MPT64 and LAM are specific to M. tuberculosis, and PAB is solely expressed on Propionibacterium (Cutibacterium) acnes [P. acnes antigens were detected in granulomas of sarcoidosis [Immunohistochemical cross-reactivity of rabbit antisera against BCG and ery high . Similarery high . The visery high . Anotherm) acnes –9. The Pcoidosis . The lipcoidosis .M. chelonae, a rapidly growing nontuberculous mycobacterium, after immunosuppressive therapy against pyoderma gangrenosum. It is of note that M. chelonae provoked suppurative granulomas with lipid droplet-centered colonization of Gram-positive and acid-fast rods. Unique immunohistochemical reactivities with a variety of antibacterial antibodies were applicable to visualizing the causative mycobacteria in the lesion.We reported here rare opportunistic skin coinfection of CMV and


E. coli and Klebsiella species, are problematic, particularly in hospitalized patients. Poultry meat vendors are at risk of carrying ESBL-producing bacteria when processing and handling meat products in an unhygienic environment. There is limited information on the carriage rate of ESBL-producing pathogens among poultry meat vendors that necessitated the conduction of the study.  Bacteria possessing extended-spectrum beta-lactamase (ESBL), especially p value < 0.05 was considered statistically significant.  A cross-sectional study was conducted among poultry meat vendors in Dar es Salaam, Tanzania. Participants provided rectal swabs in transport media upon instruction. The primary isolation of ESBL-producing bacteria was carried out using MacConkey agar supplemented with ceftazidime. Identification of isolates relied on conventional methods. Double-disk synergy was the method used to confirm ESBL-producing isolates. We performed descriptive statistics using Statistical Package for Social Sciences version 23. A E. coli and Klebsiella spp. The majority of confirmed ESBL-producing isolates was E. coli (78.5%). Participants from Ubungo District had significantly higher carriage of ESBL-producing Escherichia coli and Klebsiella spp.  than Temeke District . Only 28.0% of participants had access to latrines at the workplace, and all working areas lacked access to running water.  A total of 300 participants were recruited from five districts, with a mean age of 27.2 ± 6.7 years. The majority was male (67.3%), and 74.7% worked as poultry meat vendors for more than one year. Out of 300 participants, 107 (35.7%) had confirmed ESBL-producing E. coli and Klebsiella spp. among poultry meat vendors. Poor working environments and hygienic practices are risks for spread of these multidrug-resitant pathogens. The study revealed a relatively high fecal carriage rate of ESBL-producing  Infectisistance , 3. Thersistance , 5. Of csistance , 7.The irrational use of antimicrobial agents in the animal influence the emergency of antibiotic-resistant strains , 9 and sE. coli and K. pneumoniae are reported in these studies as the common ESBL-producing pathogens [E. coli and Klebsiella spp. among the poultry meat vendors in Dar es Salaam, Tanzania. The findings provide baseline information to guide strategies for the control of infection and antimicrobial resistance in community settings.Most studies in Tanzania on the carriage rate of ESBL-producing pathogens in humans were conducted in hospital settings , 23. E. athogens , 23. On This was a cross-sectional study conducted in Dar es Salaam, the largest city in Tanzania. The study involved poultry slaughterhouses and poultry meat-selling centers from selected wards in five districts of the Dar es Salaam region. The study recruited healthy poultry meat vendors involved in either poultry slaughtering, washing, or selling raw meat for more than three months. The study excluded vendors with signs of illness and those who failed to provide written informed consent.We determined the sample size using the Kish Leslie formula , consideThe investigator visited poultry meat vendors at their working station for data collection. The interviewer provided a detailed explanation about the study to every study participant who provided written informed consent before recruitment. The data were collected using a structured questionnaire. The collected information includes sociodemographic characteristics , working environments, and hygienic practice conditions.The research assistant clearly instructed each participant and illustrated the rectal swab's self-collection and how to insert the swab onto Cary-Blair transport media. The samples were transported daily in a cool box with ice packs within six hours after collection to the microbiology and immunology laboratory at Muhimbili University of Health and Allied Sciences (MUHAS) for processing.E. coli and Klebsiella spp. based on colonial characteristics and Gram stain reaction. Colonies with distinctive morphological appearance of dry or mucoid lactose-positive (pink) and Gram-negative rods were selected. Suspected colonies were subcultured into nutrient agar  to get enough pure colonies for further identification. Conventional biochemical tests of Kligler's iron agar, sulfide-indole-motility test, and citrate test were used  and cefotaxime (30 μg) disks were placed at a distance of 20 mm around the amoxicillin-clavulanic acid (30 μg) disk. The incubation environment for inoculated media was 37°C for 18 to 24 hours. Any distortion or increase in the inhibition zone towards the disk of amoxicillin-clavulanic acid indicated positive for ESBL production. Klebsiella pneumoniae ATCC 700603 represented a positive control strain for ESBL production. The laboratory workflow for isolation, identification, and confirmation of ESBL-producing E. coli and Klebsiella spp. is summarized in As previously described, it was confirmed that all isolates were ESBL-producing pathogens by the double-disk synergy method . In brieData were analyzed using Statistical Package for Social Sciences version 23.0. Categorical variables were summarized as proportions, while continuous variables were summarized as mean and standard deviation. A chi-square test was calculated to determine the differences between proportions. A 95% confidence interval of proportion was calculated to provide more information on the upper and lower bound of proportion estimates. The level of significance was specified at 0.05.The study enrolled 300 poultry meat vendors with a mean age of 27.2 ± 6.7 years. The majority, 202 (67.3%), was males, and 138 (46.0%) were below 25 years. More than a quarter, 80 (26.7%), were recruited from the Kinondoni District, followed by Temeke District 70 (23.3%). Of all, 224 (74.7%) worked as poultry meat vendors for more than one year. Around half, 151 (50.3%), reported a preference for self-medication with antibiotics before going to the hospital .Most of the study participants, 216 (72%), had no latrines at their workplace, and all (100%) reported having access to water for washing hands, clearing the environment, cleaning knives, washing poultry, bowel, and plates. However, all working areas lack access to running water. No participant used hand protection when handling poultry meat (data not presented).E. coli and Klebsiella spp. E. coli, 101 (77.7%), were the predominant bacteria. Of 130 ESBL-suspected isolates, 107 (82.3%) were confirmed as ESBL-producing E. coli and Klebsiella spp. Out of 107 ESBL producers, 84 (78.5%) were E. coli isolates, and 23 (21.5%) were Klebsiella spp.  bacterial isolates were suspected to be ESBL-producing lla spp. .E. coli and Klebsiella spp. was 35.7% (95% CI: 30.5–41.2). The frequency of carriage was high (38.4%) among participants aged less than 25 years, but the observed difference in age groups was not significant. There was a significant difference in the proportion of participants with carriage of ESBL-producing E. coli and Klebsiella spp. in the district of business. Ubungo District had higher proportion of fecal carriage  than Temeke District  (p=0.030). The proportion of fecal carriage of ESBL-producing E. coli and Klebsiella spp. observed in sex, duration in business, and preference for self-treatment was not significantly different (p > 0.05)  .E. coli and Klebsiella spp. among poultry meat vendors in Dar es Salaam. Although there is no study done in a similar population in Tanzania, the finding is relatively higher than the fecal carriage of ESBL-producing bacteria in the community among people sharing latrines in Dar es Salaam [E. coli and Klebsiella spp. may be due to direct contact with poultry meat that may be carrying or contaminated with ESBL strains, poor hygiene practices in their working environment, and lack of safe and running water for washing hands and other uses.The present study demonstrates a high carriage rate of ESBL-producing s Salaam . The stuE. coli was the most common ESBL producer isolated among poultry meat vendors than Klebsiella spp.; the finding agrees with the study reported in Spain [E. coli was also reported in another study conducted in Tanzania, where E. coli was the most common cause of community-acquired infection [E. coli at a rate of 21.7% [E. coli [E. coli among poultry meat vendors indicates a community problem as these poultry meat vendors are part of the community.In the current study, in Spain . The prenfection . Furtherof 21.7% . The stu[E. coli . Thus, tE. coli in humans related to contamination during slaughtering and in contact with food animal products, including poultry meat [E. coli exposure of the Dutch population through chicken meat consumption [Our study revealed the lack of running water in the working area for all poultry meat vendors. Instead, they used water kept in the buckets or tins for washing hands, poultry meat, and cleaning tables. Hand washing from the same bucket may increase the contamination rate, thus spreading the ESBL-producing bacteria. Studies in France reported the prevalence of 10.7% of ESBL-producing try meat . A Dutchsumption . Comparesumption . Handlinsumption , 20. In E. coli and Klebsiella spp. among poultry meat vendors. The present study's findings suggest the need for a regular screen for colonization with ESBL-producing bacteria for a specific group of poultry meat vendors and improving the working environment and hygienic practices. The government should have a policy that requires the screening of food handlers before engaging in business to control the spread of ESBL-producing bacteria and antimicrobial resistance in the community. Based on the laboratory methods used to identify E. coli and Klebsiella spp., we might have underestimated these organisms' magnitude. Some strains may have an opposite reaction with the conventional biochemical tests used for identification, which advanced identification technique.To the best of our knowledge, this is the first report from the Dar es Salaam region investigating the prevalence of ESBL-producing bacteria in poultry meat vendors. Therefore, our study presents the baseline information on the prevalence of ESBL-producing E. coli and Klebsiella spp. among poultry meat vendors. For food handlers, the availability of clean running water is essential as washing hands in the same water as where the chickens are washed is a risk for public health as well as for the poultry workers.The study demonstrates a relatively high fecal carriage rate of ESBL-producing