Alternative splicing (AS) is a widespread regulatory mechanism in multicellular organisms. Numerous transcriptomic and single-gene studies in plants have investigated AS in response to specific conditions, especially environmental stress, unveiling substantial amounts of intron retention that modulate gene expression. However, a comprehensive study contrasting stress-response and tissue-specific AS patterns and directly comparing them with those of animal models is still missing.Arabidopsis thaliana, PastDB, comprising AS and gene expression quantifications across tissues, development and environmental conditions, including abiotic and biotic stresses. Harmonized analysis of these datasets reveals that A. thaliana shows high levels of AS, similar to fruitflies, and that, compared to animals, disproportionately uses AS for stress responses. We identify core sets of genes regulated specifically by either AS or transcription upon stresses or among tissues, a regulatory specialization that is tightly mirrored by the genomic features of these genes. Unexpectedly, non-intron retention events, including exon skipping, are overrepresented across regulated AS sets in A. thaliana, being also largely involved in modulating gene expression through NMD and uORF inclusion.We generate a massive resource for A. thaliana.Non-intron retention events have likely been functionally underrated in plants. AS constitutes a distinct regulatory layer controlling gene expression upon internal and external stimuli whose target genes and master regulators are hardwired at the genomic level to specifically undergo post-transcriptional regulation. Given the higher relevance of AS in the response to different stresses when compared to animals, this molecular hardwiring is likely required for a proper environmental response in The online version contains supplementary material available at 10.1186/s13059-020-02258-y. Complex multicellular organisms generate a myriad of highly specialized cell and tissue types during their lifetime. Furthermore, they need to efficiently and rapidly react to a wide diversity of external stresses, both abiotic  and biotic , all requiring unique and highly coordinated molecular responses. Remarkably, all this is achieved by differentially regulating a single genome sequence to generate specific transcriptomes and proteomes for each environmental condition and cell or tissue type. In addition to the transcriptional control of gene expression, other less studied regulatory mechanisms are essential. Among them, alternative splicing (AS), the differential processing of introns and exons in pre-mRNAs to produce multiple transcript isoforms per gene, is the most important contributor to transcriptome diversification in both plants and animals –3. The fAS can exert mainly two major molecular functions. On the one hand, it can expand proteome complexity by generating two or more distinct protein isoforms, which in some cases have been shown to present radically different functional properties . On the Arabidopsis thaliana, in particular, detecting transcriptomic variation in up to 60–83% of multi-exonic genes  ), CameliA. thaliana and the three animal species, we used ExOrthist v0.0.1.beta (https://github.com/biocorecrg/ExOrthist). This software is designed to identify orthologous exons based on intron position-aware pairwise protein alignments  for each species using the default settings for “long evolutionary distance” for all pairwise species comparisons . Finally, to generate the four-way Venn diagrams in Additional file https://github.com/vastdb-pastdb/pastdb). First, we checked if any orthologous gene (defined as being in the same gene cluster) in the target species also hosted an AS event (of any type) belonging to the same core set . Second, we assessed whether the exact splice site(s) affected by the AS event were also affected by an AS event (of any type) of the same core set in the target species. In the case of 5′ UTR AS events, these were conservatively considered to fall in the same splice site if orthologous genes in the two species had a core set AS event in the 5′ UTR. It should be kept in mind that these overlaps do not imply evolutionary conservation; rather, for such distantly related species, the overlaps are likely the result of convergent evolution.To assess the overlap among the AS core sets from ribed in , 32). ExBroccoli  with thePastDB, while MediaWiki works as Content Management System platform. MediaWiki BioDB extension, a custom modification of a pre-existing extension named External Data, was installed for reusing data directly from MySQL tables. To allow faster custom searches , selected search fields were mapped into document structures, which were imported into an IBM CouchDB 3 (a NoSQL document DBMS) server instance. Predefined indexes were created for gene descriptions (taking advantage of IBM CouchDB 3 Lucene capabilities) and for coordinates . After that, a custom MediaWiki extension previously created for VastDB [The website was built over a LAMP environment, where MariaDB is used as DataBase Management System for r VastDB  and somePastDB uses a custom UCSC browser instance (http://ucsc.crg.eu). This browser includes a general transcript track , a gene track  [PastDB can be downloaded from http://pastdb.crg.eu/wiki/Downloads.Gene expression and PSI data were represented using Rickshaw, a D3.js-based JavaScript library [n 2.3.4)  and Dison 2.3.4) , respectAdditional file 1: Table S1.A. thaliana RNA-seq datasets compiled for PastDB.Additional file 2. Supplementary Figure S1-S32.Additional file 3: Table S2.A. thaliana RNA-seq datasets used to define the tissue-specific and stress-responsive GE and AS core sets.Additional file 4: Table S3. List of AS events differentially regulated in response to abiotic and biotic stimuli or in different tissues of A. thaliana.Additional file 5: Table S4. List of genes differentially regulated in response to abiotic and biotic stimuli or in different tissues of A. thaliana.Additional file 6: Table S5. Enriched GO terms for genes belonging to the abiotic, biotic and tissue AS and GE core sets in A. thaliana.Additional file 7: Table S6. List of samples related to A. thaliana RNA-processing factor mutants.Additional file 8: Table S7. RNA-seq analyses of RNA-processing factor mutant experiments.Additional file 9: Table S8. RNA-seq datasets used to define the tissue-specific and stress-responsive AS event core sets in C. elegans, D. melanogaster and H. sapiens.Additional file 10: Table S9. List of AS events belonging to the stress and tissue AS core sets in C. elegans, D. melanogaster and H. sapiens.Additional file 11: Table S10. Enriched GO terms for genes belonging to the abiotic, biotic and tissue AS core sets in C. elegans, D. melanogaster and H. sapiens.Additional file 12: Table S11. Representative transcript per gene used for GE quantification in vast-tools and other analyses in this study.Additional file 13: Review history.


Escherichia coli from river-water and river-sediment in pre-, during- and post-mass bathing events in river Kshipra, Central India.Antibiotic resistance is one of the major global health emergencies. One potential source of dissemination of resistant bacteria is mass gatherings, e.g. mass bathing events. We evaluated the physicochemical parameters of water quality and the antibiotic resistance pattern in commensal E. coli to 17 antibiotics were tested.Water and sediment samples were collected from three selected points during eight mass bathing events during 2014–2016. Water quality parameters  were analyzed using standard methods. In river water and sediment samples, antibiotic susceptibility patterns of isolated E.coli with multi drug resistance (MDR) or extended spectrum beta-lactamase (ESBL) production were between 9–44% and 6–24%, respectively in river-water as well as river-sediment. Total coliform count/ml and E. coli count were higher during-and post-bathing in river water than in pre-bathing period. Thus, the percentage of resistance was significantly higher during and post-bathing period (p<.05) than in pre-bathing. Colony forming unit (CFU)/ml in river-sediment was much higher than in river-water. Percentage of resistance was significantly higher in river-water (p<.05) than in river-sediment.pH, turbidity and dissolved oxygen were significantly lower and total dissolved solid, free carbon dioxide were higher during mass bathing, whilst TSS, BOD and COD were lowest in pre-bathing and highest in post-bathing period. E.coli isolated from the Kshipra River showed significant variation during mass bathing events. Guidelines and regulatory standards are needed to control environmental dissemination of resistant bacteria.Antibiotic resistance in  Clean, pure and safe water exists briefly in nature, as it is immediately polluted by prevailing environmental factors and anthropogenic activities. Industrialization and urbanization has polluted rivers mostly via agricultural runoffs and industrial effluents containing many used or unused antibiotics . AntibioEscherichia coli (E. coli), usually a commensal coliform of humans and animals, enters into the river mainly from livestock operations, their waste products and human septate  images which may be copyrighted. 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PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  23 Jan 2020PONE-D-19-25307Mass-bathing events in River Kshipra, Central India- influence on the water quality and the antibiotic susceptibility pattern of commensal E.coliWe are thankful to the Editor and reviewer for the comments and suggestions required for the improvement of the manuscript. Following are the reply to the various queries and concerns.Editors Comments1. When submitting your revision, we need you to address these additional requirements.Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttp://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfAuthor's Reply: Formatting done as suggested.2. 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Escherichia coli (E. coli) among poultry-workers. Here we determined the prevalence and genetic relatedness among MDR E. coli isolated from poultry-workers, chickens, and poultry environments in Abuja, Nigeria. This study was conducted to address the gaps identified by the Nigerian AMR situation analysis.Inappropriate use of antimicrobial agents in animal production has led to the development of antimicrobial resistance (AMR) in foodborne pathogens. Transmission of AMR foodborne pathogens from reservoirs, particularly chickens to the human population does occur. Recently, we reported that occupational exposure was a risk factor for multidrug-resistant (MDR) We conducted a cross-sectional study among poultry-workers, chickens, and poultry farm/live bird market (LBM) environments. The isolates were tested phenotypically for their antimicrobial susceptibility profiles, genotypically characterized using whole-genome sequencing (WGS) and in silico multilocus sequence types (MLST). We conducted a phylogenetic single nucleotide polymorphism (SNPs) analysis to determine relatedness and clonality among the isolates.E. coli. Of these, 110 isolates were viable for phenotypic and genotypic characterization. The selection comprised 47 (42.7%) isolates from poultry-workers, 36 (32.7%) from chickens, and 27 (24.5%) from poultry-farm or LBM environments. Overall, 101 (91.8%) of the isolates were MDR conferring resistance to at least three drug classes. High frequency of resistance was observed for tetracycline , trimethoprim/sulfamethoxazole , streptomycin  and ampicillin . Two plasmid-mediated colistin genes—mcr-1.1 harboured on IncX4 plasmids were detected in environmental isolates. The most prevalent sequence types (ST) were ST-155 (n = 8), ST-48 (n = 8) and ST-10 (n = 6). Two isolates of human and environmental sources with a SNPs difference of 6161 originating from the same farm shared a novel ST. The isolates had similar AMR genes and plasmid replicons.A total of 115 (26.8%) out of 429 samples were positive for E.coli isolates were prevalent amongst poultry-workers, poultry, and the poultry farm/LBM environment. The emergence of MDR E. coli with novel ST in two isolates may be plasmid-mediated. Competent authorities should enforce AMR regulations to ensure prudent use of antimicrobials to limit the risk of transmission along the food chain.MDR The online version contains supplementary material available at 10.1186/s13756-021-00930-x. E. coli among chickens, humans, and the poultry environment . It is interesting to note that although the plasmid structures of the two isolates were so similar, there was no clonal relationship between them.All isolates assigned a phylogenetic group and ST were used to construct phylogenetic trees to determine if the isolates were genetically related or very diverse. Three phylogenetic trees were constructed: one for all the isolates Fig. , one focOverall, 110 isolates used to construct a maximum likelihood phylogenetic tree showed that the isolates were genetically diverse. The isolates were grouped based on similarities among them. Whole-genome (wg) SNPs-based phylogenetic analysis showed that some isolates sharing the same ST and phylogroups were not clonally related. The strains were in completely different clades in the SNP tree, separated by strains belonging to other STs. Three isolates with ST-1638 recovered from human, chicken and poultry environment were clustered together on the same clade. Pairwise SNP differences between the genomes of the isolates showed that they were not clonally related ; as well as plasmid replicons , IncHI2A, IncHI2, Col(pHAD28).Two isolates of human and environmental origin with SNPs difference of 6161 were not clonally related although the isolates shared a novel ST and belonged to phylogroup B1  and chicken farmers (22%) when compared with the present study where a prevalence of 34.7% and 38.6% was detected in chickens and poultry workers respectively [E. coli (82%) from chicken faecal samples when compared with the findings from this study, with a much lower prevalence of 32.2% [E. coli prevalence of 67.7% [E. coli from freshly dropped chicken faecal samples as opposed to cloacal swabs. A study conducted in Pakistan, reported a slightly higher E. coli prevalence of 36% from the poultry farm environment when compared to 26.1% obtained from the poultry environment in the present study [E. coli prevalence of 26.8% was obtained from the poultry environment [The first objective of this study was to characterize ectively . Access of 67.7%  and 69.8nt study . Our stuE. coli isolates from poultry farm workers and chicken sellers and compared them to resistance rates of E. coli isolates from chickens and poultry farm/market environment. The patterns of resistance were similar for human and chicken isolates. High resistance rates were observed in isolates recovered from humans, chickens, and poultry farm/market environments for tetracycline, trimethoprim/ sulfamethoxazole, ampicillin, and streptomycin. This is in agreement with the findings of a study conducted in southwest Nigeria, where high resistance rates of E. coli isolates to beta-lactams, tetracyclines, macrolides, and sulfonamides were reported [Our study examined AMR in reported . This fireported .E. coli isolates from all the sources. Interestingly, 83% of human, 97% of chicken, and 100% of poultry environment isolates were MDR E. coli. A possible explanation for this very high level of resistance observed could be because of the lack of prudent use of antimicrobials and the required regulation to support it resulting in over-the-counter availability often without prescription as reported in many studies [E. coli is a known zoonotic bacteria [Our study revealed a very high proportion (91.8%) of MDR  studies , 38–40. bacteria .blaTEM-1 gene, which confers ampicillin resistance in E. coli isolates and is in agreement with a previous study that reported ampicillin-resistant E. coli isolates in food, humans, and healthy animals [isolates although phenotypic characterization showed that 10.9% of the isolates were carbapenem-resistant. This may possibly be as a result of borderline interpretation of breakpoint settings between resistance and susceptibility. The present study identified one of the most important AMR genes [mcr 1.1 harboured on IncX4 plasmids in two isolates recovered from the poultry environment. Evidence shows that the IncX4 plasmids harbouring mcr-1 genes have been detected in human and animal E. coli isolates however, our study recovered these plasmids from the poultry environment [mcr 1 in E. coli isolates sourced from the aquatic environment [mcr 3.1 gene was detected in a human Salmonella case in the US [mcr gene has spread across multiple pathogens.The most common beta-lactamase gene observed in this study was the  animals . Our stuMR genes , being tironment . Anotherironment  however,n the US . This fublaTEM-1, mdf(A) and qnrS13 in the present study and these are consistent with the literature [sul2 genes that confer sulphonamide resistance and this is consistent with reports of other studies [Our study highlights that poultry workers, chickens, and the poultry environments share identical plasmid replicons and this is consistent with the literature , 47. Theterature . The Inc studies , 48. TheE. coli recovered from humans, chickens, and poultry environments. Our results revealed that these isolates showed very diverse genetic profiles. Common STs were assigned based on MLST including ST155, ST48, ST10, ST1638, and ST398 in isolates from humans, chickens, and poultry environments, although ST155 was mostly detected in isolates of poultry origin at the LBM. The most common ST detected among isolates recovered from the poultry farm environment was ST48. Previous studies have reported that E. coli with ST48 in phylogroup A has been detected in healthy volunteers, seafood, and water [E. coli recovered from healthy people, chickens, and the poultry environment. E. coli strains with ST10 have previously been reported as being emerging and pathogenic as implicated in human infections although MDR strains with ST10 have also been detected in poultry and other animal sources [E. coli strains with ST10 in healthy individuals, poultry manure, and water. A possible explanation could be that this is becoming an emerging public health issue arising due to possible mutations in the bacteria.To determine the genetic relatedness of the isolates, we analyzed by WGS, nd water –51. Our  sources . Our stuE. coli isolates in this present study belonged to phylogroup A (55.5%) and phylogroup B1 (32.7%). Most human and poultry environment isolates belonged to phylogroup A while majority of the chicken isolates belonged to phylogroup B1. Our study findings are in agreement with the results of a similar study conducted in Pakistan that reported that phylogroups B1 and A were the most prevalent detected among human and animal E. coli isolates [E. coli isolates were evenly distributed into phylogroups A and B1 while phylogroup B1 was the most prevalent among human isolates [E. coli isolates belonging to phylogroup B2 are usually the most virulent, hence MDR [E. coli belong to phylogenetic groups A and B1 [The majority of isolates . Interesisolates . Previouence MDR –56. Howeence MDR . Our stuA and B1 , 58. HowqnrB19 genes that confer quinolone resistance and consistent with the literature [E. coli strains, are frequently detected in humans and animals serving as reservoirs for the spread of AMR genes and have been associated with MDR E. coli [E. coli at the human-animal-environment interface; however, our findings suggest that mobile genetic elements may have facilitated the horizontal transfer of MDR genes between the plasmids among commensal E. coli which could potentially mutate into real pathogens with serious public health implications [The phylogenetic SNP tree rooted using NCTC11129 reference strain revealed that the isolates were genetically diverse among the identified STs. Two unrelated isolates of human and environmental origin belonging to phylogroup B1 and sharing a novel ST, had Col440I replicons harbouring the terature . In sili E. coli , 59. Thiications .E.coli isolates were found to be prevalent amongst poultry-workers, chickens, and poultry farm/market environments in Abuja, Nigeria. The highest resistance rates among MDR E. coli isolates were observed to tetracycline, sulphonamides, penicillins, aminoglycosides, and quinolones which are classes of antimicrobials commonly used in poultry production for treating avian diseases in Abuja. ST-155, ST-48, and ST-1638 were the only STs detected in humans, chickens, and poultry farm/LBM environments in our study. Our findings showed the emergence and spread of MDR E. coli with novel-ST from a poultry farm environment to a poultry farmer, which may have resulted from horizontal transfer of AMR genes harboured in plasmids. Consequent upon these, healthcare and poultry-workers should be educated on the fact that people in proximity with poultry are a high-risk group for faecal carriage of MDR E. coli. Competent authorities should enforce AMR regulation to ensure prudent use of antimicrobials to limit the risk of transmission along the food chain and to poultry workers. Farmers should be discouraged from indiscriminate use of antimicrobials in poultry production and encouraged to adopt preventive measures by observing biosecurity as well as good management practices on their farms.MDR Additional file1: Supplementary Data.


Withania is a small genus belonging to the Solanoideae subfamily. Here, we report the de novo assembled chloroplast genome sequences of W. coagulans, W. adpressa, and W. riebeckii. The length of these genomes ranged from 154,162 to 154,364 base pairs (bp). These genomes contained a pair of inverted repeats (IRa and IRb) ranging from 25,029 to 25,071 bp that were separated by a large single-copy (LSC) region of 85,635–85,765 bp and a small single-copy (SSC) region of 18,457–18,469 bp. We analyzed the structural organization, gene content and order, guanine-cytosine content, codon usage, RNA-editing sites, microsatellites, oligonucleotide and tandem repeats, and substitutions of Withania plastomes, which revealed high similarities among the species. Comparative analysis among the Withania species also highlighted 10 divergent hotspots that could potentially be used for molecular marker development, phylogenetic analysis, and species identification. Furthermore, our analyses showed that even three mutational hotspots  were sufficient to discriminate the Withania species included in current study.Within the family Solanaceae,  Withania Pauq., belonging to the subfamily Solanoideae, contains 10–20 species [Withania species, ashwagandha or winter cherry (W. somnifera (L.) Dunal) and paneer booti or ashutosh booti (W. coagulans (Stocks) Dunal) are considered highly important due to their therapeutic potential. Withania species are pivotal in the Ayurvedic medicine system in Southeast Asia, and W. somnifera has been used for medicinal purposes for around 3000 years [Withania have described various pharmacological properties of these species,  [Withania species) products show irregularities in quality through the herbal medicinal value chain affecting their impact and safety [The globally distributed megadiverse Solanaceae family includes 93 genera and 2700 species ,2,3. The00 years ,5. Many tective) ,7,8,9. Td safety . The quad safety .The structure and composition of the chloroplast genome (or plastome) can be utilized to generate molecular markers that can be used in DNA barcoding . ChloropW. coagulans, W. adpressa Coss., and W. riebeckii Schweinf. ex Balf.f. in addition to the previously reported W. somnifera genome [Withania plastomes such as repeats, indels, and substitutions, and to identify mutational hotspots for future DNA barcoding.Here, we aimed to assemble and compare the complete chloroplast genome sequences of a genome . We alsoW. coagulans were obtained from Mianwali, Pakistan (32.5839° N 71.5370° E). The leaf segments were washed in 70% ethanol and total genomic DNA extraction was carried out according to the CTAB (cetyltrimethylammonium bromide) method of Lu et al. [W. adpressa (5 Gb) and W. riebeckii (5 Gb) were acquired from the sequence read archive (SRA) deposited under accession numbers SRR8718119 and SRR8718120. The raw sequencing read quality was verified with the FastQC tool [Withania species plastomes were determined by mapping the reads to the de novo assembled plastome through the Burrows–Wheeler aligner (BWA) [W. coagulans (MN216390), W. adpressa (BK010847), and W. riebeckii (BK010849). The plastome of W. coagulans was also deposited in the GWH database of the National Genomics Data Center [Fresh green leaves of u et al. . DNA quatQC tool  with k-mtQC tool . The juntQC tool  and CPGAtQC tool . The restQC tool . CPGAVAStQC tool  and Clicer (BWA)  and visuer (BWA) . The nova Center   to create a Circos output.All de novo chloroplast genomes were aligned with multiple alignment using fast Fourier transform (MAFFT) 7.309 , using d species , using trcoletto  was usedWithania, using W. somnifera as reference. The data were interpreted as: Ka/Ks > 1, Ka/Ks = 1, Ka/Ks < 1, representing positive, neutral, and purifying selection, respectively. Microsatellites in Withania plastomes were detected with the microsatellite-web (MISA) [The predictive RNA editor for plants-chloroplast genes (PREP-cp) was used to predict putative RNA editing sites using default settings , while cb (MISA) , using ab (MISA)  was alsob (MISA)  using deWithania plastome sequences in our analysis and added further Solanaceae plastomes  from closely related groups of Physaleae and additional taxonomic groups from the so-called ‘x = 12 clade’. This group encompasses species of the traditional subfamily Solanoideae, Nicotiana L. and the Australian endemic tribe Anthocercideae belonging to Nicotianoideae. This strongly supported group is united with the cytological synapomorphy of chromosome numbers based on 12 pairs [Petunia × atkinsiana (Sweet) D. Don ex W.H. Baxter (Syn.: Petunia × hybrida Vilm.) as an outgroup to root our tree, since this was the only available complete chloroplast genome sequence outside the x = 12 clade. For phylogenetic analysis, we removed one of the IR regions (IRa), and subsequently excised all protein-encoding genes from the plastomes. The reading frames were manually verified during extraction by checking the start and stop codons. We discarded accD, ycf1, and ycf15 from our final alignment, because these genes were highly variable in size. The trans-spliced rps12 was also not included in the phylogenetic alignment together with sequence of the infA pseudogene. The nucleotide sequences of 74 protein-coding genes were aligned with MAFFT (default setting) via the Geneious shell. IQ-TREE [We included all available  IQ-TREE  was used IQ-TREE . The max IQ-TREE ) with 10 IQ-TREE . Branch  IQ-TREE ,55.6 generations sampling every 100th tree per generation, with unlinked parameters across partitions. Branch length and topology parameters were set unlinked. Convergence was checked using the average standard deviation of split frequencies  was used to measure convergence of the runs. A majority-rule consensus tree was constructed from the runs with a 25% burn-in removal.For further analyses we divided the dataset into protein-coding gene subsets according to the heuristic searches carried out with Partition Finder v1.1.1  and defaWithania species have similar plastome structures  content of the de novo assembled Withania plastomes was 37.7%, as was the previously sequenced species.Our comparative analysis revealed that ructures . The lenWithania species had 132 genes from which 18 were represented in duplication in the inverted repeats  and rRNAs (55.3%). Hydrophobic amino acids were abundant, while the acidic amino acids were present in the least amount in plastomes of the genus Withania. These amino acids were adenine-thymine (AT)-rich sequences in all species (Withania plastomes (The IRs showed a higher GC content (43.2%) compared to the large- (35.7%) and small-single copy regions (31.8%), which could have been due to the occurrence of rRNA genes containing GC-rich regions ,59,60,61 repeats . All Wit species A. The anlastomes B. The colastomes .Withania genomes had similar nucleotide compositions in all structural  and coding regions, which extended even to IGSs  to 0.0119 . We assessed the efficacy of these regions to discriminate among the four species of Withania and found that three regions  provided enough information for successful barcoding.The indels and SNP mutational events in the plastome showed uneven distributions and clustered as “hotspots” ,65. ThesaccD, ycf2, ycf1, and ndhF) had Ka/Ks ratios > 1. ycf1 and psbC showed Ka/Ks ratios > 0–1 for W. riebeckii, ndhF for W. coagulans, and rps15 for W. adpressa and accD, ycf2 showed Ka/Ks > 0 for W. adpressa and W. riebeckii, and rpoC2 for W. coagulans and W. riebeckii. The low divergence of most chloroplast genes showed signs of purifying selection to conserve the sequence and function of proteins related to photosynthesis.We also investigated the Ks and Ka substitutions and their ratio Ka/Ks; . We seleW. coagulans) to 379 (W. adpressa). Poly-A and T SSR motifs were frequent in Withania chloroplast genome sequences, while AT/TA dinucleotide stretches were also highly abundant. The mononucleotide motifs occurred in 7–17-unit repeats, while dinucleotide repeats had a frequent 4–5-units, whereas other types of SSRs were present mainly in 3–5-unit repeats. Most SSRs occurred in the LSC, followed by IR and SSC  and a large fraction of the repeats was located in the LSC and existed in IGS regions, followed by gene, intron, and coding DNA sequence (CDS) regions , and a portion of this gene (8–59 bp) was copied in the IRa in all Withania genomes. The ndhF gene was entirely present in the SSC region in W. somnifera and W. adpressa, but in W. coagulans (5 bp) and W. riebeckii (3 bp) it was located in the IRb region.The plastome of land plants has a conserved quadripartite structure while variation frequently occurs in a form of expansion and contraction of the junction sites that could rarely even lead to the loss of the entire IR regions ,71. The lastomes . The resWithania species, we predicted putative sites in the plastomes using PREP-cp. This revealed 37 putative sites in 15 genes of W. somnifera, while 35, 39, and 37 editing sites were found in 13 genes of W. coagulans and in 14 genes of W. adpressa and W. riebeckii, respectively. The gene clpP has editing sites only in W. somnifera and ccsA only in W. adpressa. Among Withania species ndhB (9), ndhD (7), and rpoB (5) had the highest number of RNA-editing sites. All species had high levels of conversion for serine (Ser) to leucine , followed by proline (Pro) to Leu , and Ser to phenylalanine . Of the putative RNA-editing sites detected, 33 (94.2%), 34 (87.1%), and 33 (89.1%) codons were substituted on the second nucleotide and two (5.71%), five (12.8%), and four (10.81%) codons were substituted in the first nucleotide in W. coagulans, W. adpressa, and W. riebeckii, respectively. Many amino acids were converted from Ser to Leu helping to form hydrophobic amino acids, e.g., valine , Leu, and Phe  and Bayesian analysis for phylogenetic reconstruction for 19 Solanaceae species, based on selected protein-coding gene sequences extracted from whole-plastome sequences. Based on a 69,582-bp alignment, our tree was reconstructed and resolved identical topologies for both methods and a phylogenetic tree were supported by high bootstrap values and posterior probabilities . The genWithania . Our resL spacer , ndhF annd trnLF .Withania species, which was further used in phylogenetic inference. Withania species belong to a rather diverse and widely distributed Withaninae clade within the so-called physaloid group. Species of the genus Withania are morphologically similar: the flowers are found in lateral clusters (fascicles) lacking a supporting inflorescence stem (peduncle). The flower petals (corollas) are bell or elongated cup-shaped, sometimes urn-shaped, circular and flattened (rotate), or trumpet-shaped , while the filaments often form nectar grooves with lateral attachments. The Withaninae clade consists of approximately seven small often monotypic genera mostly found in the Old World, e.g., Tubocapsicum (Wettst.) Makino, Mellissia, Aureliana, or Discopodium Hochst. D’Arcy [Withania to be one of the truly Old-World genera, while Symon [Withania has a center of distribution around Spain, NW Africa extending to the Canary Islands, while another is located in India, the southern region of the Arabian Peninsula, and the Horn of Africa. The phylogenetic relationships within the genus are poorly known, and the biology, chromosome numbers, and the exact number of species are also lacking. Chromosome counts showed that most species of Withania are polyploids with 2n = 2x = 48 [Withania taxa, there are 35 unresolved botanical names that need further investigation to clarify the taxonomy of the genus.Chloroplast DNA (cpDNA) is frequently used in plant phylogenetics at various levels  ,66,78. I. D’Arcy  considerle Symon  regarded 2x = 48 , derivedWithania consisted of 10 species, which were extended by Hunziker [Mellissia Hook. and Physaliastrum Makino. These additions extended the geographical range of the genus from the Canary Islands in the west, through Asia to China and Japan in the east. Symon [Mellissia  to Withania but retained them as distinct genera. In contrast, Hunziker [Mellissia within Withania and molecular results support this placement [Withania, while its closest relatives are also debated. In our analysis, Withania formed a clade together with Physalis, similarly to the findings of Deanna et al. [In the Hepper’s treatment , WithaniHunziker  with thet. Symon  also empHunziker  includeda et al. , althougMelissia and other monotypic genera. For such barcoding studies our results could provide valuable reference genomes for assemblies. The hotspot regions described in our study could be useful in such phylogenetic or even population genetic investigations. It was previously demonstrated that identifying highly variable regions by comparative plastomics could provide reveal loci that could be used in DNA barcoding [Withania in our study could be applied for DNA barcoding. Moreover, similar to the aforementioned studies, our identified mutational hotspots showed high discrimination properties and from the 10 mutational hotspots, three regions were found to be sufficient for the identification of the four Withania species included in our study.Plastome sequences could be used as tools to further elucidate species boundaries and investigate the phylogenetic relationships among the small clades of Withaninae and resolve the taxonomic debate over the placement of arcoding ,86,87. Sarcoding ,88,89,90atpB, ndhD, ndhF, rpoA, rpoC1, rps2, and rps12 showed greater Ka/Ks values (>1), possibly indicating selective pressure acting over these genes that was previously proposed in other groups [Withania clade was limited to explore the biological causes of the elevated Ka/Ks ratios observed in cases involving these genes. Here, we suggest that the following set of genes could be the principal candidates in investigations of these environmental interactions and their effects on plastid genes. Such investigations should include a nearly complete phylogenetic sampling of Withania and consider the effects of arbitrary variations in Ka and Ks values leading to false positive inferences and values higher than 1. These shortcoming can be bypassed by complete sampling and additional tests of selective pressure also stressed here for future analyses.We analyzed Ks substitutions and Ka substitutions of protein-coding genes and recorded greater Ks substitutions relative to the Ka substations. Such observations are essential markers in evolution for defining slow- and fast-evolving genes . The Ka/r groups ,93,94,95Solanum brevicaule complex [S. melongena complex [Petunia [Physalis [trnH-psbA, matK) could lack enough polymorphism and thus could fail to provide species-specific information necessary for differentiation [S. melongena complex to trace the ancestors of cultivated eggplant and differentiate closely related wild species [Withania species and investigated if plastid genomes based “super barcoding” could be applied among closely related ashwagandha species. The structure of these genomes showed synteny with a previously reported organization of Solanaceae species. We identified sequence divergence hotspots and located repeat sequences and indels in the plastomes of Withania species. These regions may constitute a useful means to develop suitable molecular markers for species identification and DNA barcoding of ashwagandha medicinal products. It is hoped that our study will aid the development of DNA barcoding markers to clarify the taxonomic identity of Withania species in medicinal plant production. Such plastome-based “super barcoding” could be repeatable, reliable, and sensitive enough to distinguish look-alike species of ashwagandha.It has been shown that DNA barcoding can fail in complicated groups . Solanac complex , or the  complex ) but in [Petunia  or closePhysalis . In thesntiation . It has  species . Here, w


A. baumannii bacteremia and identified possible independent risk factors for 14-day mortality. There were 379 patients with A. baumannii bacteremia admitted to a tertiary care center in northern Taiwan between August 2008 and July 2015 enrolled for data analysis. Among them, 89 patients (23.5%) had polymicrobial bacteremia and 290 patients (76.5%) had monomicrobial bacteremia. No significant difference in 14-day mortality was observed between patients with monomicrobial and polymicrobial A. baumannii bacteremia . Logistic regression controlled for confounders demonstrated that polymicrobial bacteremia was not an independent predictor of mortality, whereas appropriate antimicrobial therapy was independently associated with reduced mortality. Higher 14-day mortality rates were observed in the polymicrobial bacteremic patients with concomitant isolation of Escherichia coli, Pseudomonas aeruginosa, and Enterobacter spp. from the bloodstream. Compared with patients with monomicrobial multidrug-resistant A. baumannii (MDRAb) bacteremia, those with MDRAb concomitant with Gram-negative bacilli bacteremia had a worse outcome. Polymicrobial A. baumannii bacteremia was not associated with a higher 14-day mortality rate than that of monomicrobial A. baumannii bacteremia, although more deaths were observed when certain Gram-negative bacteria were concomitantly isolated. Appropriate antimicrobial therapy remains an important life-saving measure for A. baumannii bacteremic patients.This retrospective observational study assessed the differences between monomicrobial and polymicrobial  Polymicrobial bacteremia occurs in 6%–27.4% of patients with bloodstream infections and was first identified as an important problem in the 1960s for its higher attributable mortality rate compared with that for monomicrobial bacteremia ,4. The mAcinetobacter baumannii is an emergent nosocomial pathogen with high morbidity and mortality rates [A. baumannii occurs as polymicrobial infections in 19%–35% of cases [A. baumannii (CRAb) provided a sheltering effect for carbapenem-susceptible pathogens and enhanced the pathogenesis of polymicrobial infection [A. baumannii bacteremia on clinical outcomes. Therefore, this retrospective observational study assessed whether polymicrobial infection was an independent risk factor for 14-day mortality in patients with A. baumannii bacteremia. ty rates ,9,10. Baof cases ,9,11,12.nfection . HoweverAcinetobacter calcoaceticus–Acinetobacter baumannii (Acb) complex accompanied by two or more of the following symptoms and signs of infection: fever, hypothermia, tachypnea, tachycardia, leukocytosis, or leukopenia. We included only the first blood culture for patients with two or more positive blood cultures. Patients under 20 years of age and those with incomplete medical records were excluded. The protocol was approved by the institutional review boards (IRBs) of T-VGH (IRB No. 2016-05-019CC). This retrospective study was conducted at Taipei Veterans General Hospital (T-VGH), a 2900-bed tertiary-care teaching hospital located in Taipei, Taiwan, during a seven-year period from August 2008 to July 2015. Charts were reviewed for all patients with at least one blood culture positive for A. baumannii was drawn. Polymicrobial bacteremia was defined as the simultaneous isolation of A. baumannii and one or more microorganisms from the blood during the same bacteremic episode. However, Corynebacterium spp., Bacillus spp., Propionibacterium acnes, and skin flora  were considered contaminants and not included in the analyses unless they were related to device infection and grown in two or more blood cultures [2. Neutropenia was defined as an absolute neutrophil count <0.5 × 109 neutrophils/L. Immunosuppressive therapy was defined as the receipt of cytotoxic agents within six weeks [A. baumannii bacteremia. The primary outcome measure was all-cause 14-day mortality following bacteremia onset. Fourteen-day mortality was chosen as the endpoint to allow adequate ascertainment of treatment response. We reasoned that 30 days is too long for critically ill patients, as there are many competing causes of death, and seven days is too short a time to witness a response to treatment.We reviewed the medical records to retrieve clinical information, including demographic characteristics, comorbidities, duration of intensive care unit (ICU) and hospital stays, time of receipt, dose and route of therapy with individual antimicrobial drugs, receipt of invasive procedures, time of bacteremia onset, and infectious foci. The onset of bacteremia was defined as the day when the blood culture that eventually yielded cultures ,14. Episix weeks , corticoix weeks ,16. A hiix weeks  within 2ix weeks . ApproprA. baumannii to the genomic species level [A. baumannii and other concomitant isolated bacterial pathogens were determined by the agar dilution method [Bacteria were phenotypically identified to Acb complex using a Vitek 2 system . Other bacterial species were also identified using this system. All isolates were regrown from –80 °C storage, identified to the species level, and tested for their susceptibility to various antimicrobials. A multiplex polymerase chain reaction (PCR) method was used to identify es level . Antimicn method  and inten method .Multidrug resistance was defined as resistance to three or more of the following classes of antimicrobials: anti-pseudomonal cephalosporins, anti-pseudomonal carbapenems, ampicillin/sulbactam, fluoroquinolones, and aminoglycosides. Carbapenem resistance was defined as resistance to imipenem, meropenem, or doripenem. p <0.20 in the univariate analysis were considered for inclusion in the logistic regression model in the multivariable analysis. A backward selection process was utilized. Interactions between APACHE II score and the covariates were also examined in the logistic regression model. Time to mortality, defined as the interval between bacteremia onset and death, was analyzed by Kaplan–Meier survival analysis with log-rank tests. p < 0.05 was considered statistically significant. SPSS Statistics for Windows, version 22.0  was used for all data analyses.Chi-square tests with Yates correction or Fisher’s exact tests were used to compare categorical variables; Student’s t or Mann–Whitney rank-sum tests were used to analyze continuous variables, as appropriate. Logistic regression models were used to explore independent risk factors of 14-day mortality. We performed univariate analyses separately for each risk factor to ascertain the odds ratios (ORs) and 95% confidence intervals (CIs). All biologically plausible variables with a A. baumannii bacteremia. The demographic and clinical characteristics of patients with polymicrobial and monomicrobial A. baumannii bacteremia are summarized in A. baumannii bacteremia were older, more likely to have coronary artery disease, less frequently experienced shock within three days of bacteremia onset, and more frequently received inappropriate antimicrobial therapy , compared with those in patients with monomicrobial bacteremia. No significant difference in 14-day mortality  and 30-day mortality  was observed between these two groups. To analyze the duration of in-hospital stay after the onset of bacteremia, we separated the population into two groups, survivors and non-survivors, at hospital discharge. In the patients who survived to discharge, those with polymicrobial A. baumanii bacteremia and monomicrobial A. baumanii bacteremia had a comparable hospital stay after the onset of bacteremia . In terms of the patients who died in hospital, those with polymicrobial A. baumanii bacteremia and monomicrobial A. baumanii bacteremia also had a comparable hospital stay after the onset of bacteremia . The 30-day re-admission rates of survived patients with polymicrobial and monomicrobial A. baumannii bacteremia were not significantly different .During the seven-year study period, we enrolled 732 patients with at least one set of blood cultures positive for Acb complex. A total of 379 patients met the inclusion criteria, of which 89 (23.5%) patients had polymicrobial A. baumannii bacteremia, as shown in p = 0.001). The predictors for 30-day mortality among patients with A. baumannii bacteremia were analyzed in the same fashion as the above analyses. The results were similar to those for prediction of 14-day mortality , followed by Pseudomonas aeruginosa (50.0%) and Enterobacter spp. bacteremia (37.5%). The 14-day mortality rate was higher in patients who had polymicrobial A. baumannii bacteremia with concomitant Gram-negative bacilli (GNB) than in patients with concomitant Gram-positive cocci (GPC) (p = 0.053) . The rates of methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae, ESBL-producing E. coli, and AmpC-producing E. coli were 80.0%, 16.7%, 14.3%, and 14.3%, respectively  or carbapenem-resistant A. baumannii (CRAb) infections for further analysis. Patients with MDRAb and concomitant GNB bacteremia had a higher 14-day mortality rate than patients with monomicrobial MDRAb bacteremia . Patients with CRAb and concomitant GNB bacteremia had a higher 14-day mortality rate than patients with monomicrobial CRAb bacteremia . The difference was not statistically significant in this small cohort (n = 82). To investigate the influence of The survival analysis at 30 days was analyzed in the same fashion as the above analyses . The resA. baumannii bacteremia is not uncommon, its clinical significance has not previously been elucidated. This retrospective study assessed the differences between monomicrobial and polymicrobial A. baumannii bacteremia and identified possible independent risk factors for 14-day mortality for these infections. We found that polymicrobial bacteremia was not an independent predictor of mortality, whereas appropriate antimicrobial therapy was independently associated with reduced mortality. While polymicrobial A. baumannii bloodstream infections presented with polymicrobial bacteremia. Owing to its high prevalence, investigation of the clinical significance of polymicrobial A. baumannii bacteremia is warranted. These patients were older and more likely to have coronary artery disease and experienced shock at the time of bacteremia. Moreover, as few as one out of seven patients received appropriate antimicrobial therapy directed against every microbial species isolated from the blood. As the clinical characteristics differed between monomicrobial and polymicrobial A. baumannii bacteremia, studies on the clinical outcomes of patients with monomicrobial and polymicrobial A. baumannii bacteremia should evaluate the infections separately. Furthermore, when evaluating the efficacy of various regimens on the clinical outcome of A. baumannii bacteremia, polymicrobial A. baumannii bacteremia should be excluded, unless the antibiotic efficacy against the concomitant isolated microorganisms was also evaluated.This study, in agreement with previous studies ,9,11,12,A. baumannii bacteremia. This finding is consistent with those of previous studies and suggests that polymicrobial infection was not an independent risk factor for mortality of A. baumannii bacteremia [Staphylococcus aureus [Enterococcus spp. [P. aeruginosa [Klebsiella pneumoniae bacteremia did not result in a worse prognosis [Our results showed that the 14-day mortality rate was not significantly higher in patients with polymicrobial than in patients with monomicrobial cteremia ,11,22,23cteremia ,6,24, thcteremia ,26. Comps aureus , Enteroccus spp. , and P. ruginosa  have higrognosis . These fA. baumannii bacteremia, the higher mortality rate observed in patients with concomitant GNB bacteremia and lower mortality rate in those with concomitant GPC bacteremia suggested that the interactions between A. baumannii and GNB had negative effects, while those between A. baumannii and GPC positively influenced the disease severity. The interbacterial interactions in polymicrobial infection, which vary among co-existing species, include metabolite exchange, co-aggregation, interspecies signaling, antibiotic resistance, and biofilm formation . While mA. baumannii would protect the concomitant GNB and result in a worse clinical outcome. Our findings were consistent with those of previous studies reporting higher 14-day mortality in patients with polymicrobial bacteremia with concomitant MDRAb and GNB than in those with monomicrobial MDRAb bacteremia. Although patients with polymicrobial bacteremia with concomitant CRAb and GNB had a higher 14-day mortality rate than those with monomicrobial CRAb bacteremia, the difference was not statistically significant in this small population. On the basis of previous studies showing that beta-lactamase-producing pathogens can provide indirect pathogenesis by protecting the other pathogens in polymicrobial infection environments , we asseA. baumannii bacteremia also reported inappropriate antimicrobial therapy to be an independent risk factor for mortality [A. baumannii bacteremia. Ours and previous studies consistently found that inappropriate antimicrobial therapy was significantly associated with survival in patients with acquired polymicrobial bacteremia [A. baumannii bacteremia, appropriate therapy should be defined as a single or a combination of antimicrobial agents covering all isolated microbes.Previous studies of monomicrobial ortality ,12,22,32A. baumannii bacteremia, consistency of patient care in a single center, stringent definition of the appropriateness of antimicrobial therapy, and a well-defined primary endpoint of 14-day mortality. We also conducted multivariate analysis to assess clinical variables that were significantly associated with mortality owing to A. baumannii bacteremia. The weakness of this study was the retrospective design required to enroll sufficient patients for detailed analysis at a single tertiary care medical center. The major strength of our study was its inclusion of a number of patients with genomically-defined A. baumannii bacteremia was not an independent risk factor for 14-day mortality. Regardless of monomicrobial or polymicrobial infection, appropriate antimicrobial therapy remains an important life-saving measure for A. baumannii bacteremia patients.Polymicrobial


We report a complete genome sequence of a pepper yellow leaf curl Indonesia virus (PepYLCIV) isolated in Bali, Indonesia. This virus shares around 90% identity with other PepYLCIV DNA-As and 86% identity with DNA-Bs, suggesting that it is a novel isolate of PepYLCIV. We report a complete genome sequence of a pepper yellow leaf curl Indonesia virus (PepYLCIV) isolated in Bali, Indonesia. This virus shares around 90% identity with other PepYLCIV DNA-As and 86% identity with DNA-Bs, suggesting that it is a novel isolate of PepYLCIV. Geminiviridae, genus Begomovirus) possess bipartite circular, single-stranded DNA genomes. Begomoviruses are transmitted by whitefly and cause typical mosaic, yellowing, curling symptoms on leaves and stunted growth of plants, resulting in serious damage to crop production in tropical and subtropical regions.Most begomoviruses . Total DNA was extracted from collected tomato leaves using a DNeasy plant minikit , followed by PCR amplification  of a part of the DNA-A components of the begomovirus genome, using universal primers  , and at https://www.ncbi.nlm.nih.gov/orffinder/). A BLASTn search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) indicated that this virus shared high similarity with PepYLCIV isolates  was predicted to have six open reading frames (ORFs) and DNA-B  two ORFs, typical for bipartite Old World begomoviruses, using ORFfinder (isolates . From a isolates .LC381258 through LC381280). These sequences and this isolate shared 80.8 to 89.2% sequence identity, suggesting that our isolate is different from those previously reported.A total of 23 partial DNA-A sequences of PepYLCIV were reported  and LC542630 (DNA-B).The genome sequences of PepYLCIV-[IN:Bali:Tom:19] were deposited in the DDBJ/ENA/GenBank database under accession numbers


Granular and lattice corneal dystrophies (GCDs & LCDs) are autosomal dominant inherited disorders of the cornea. Due to genetic heterogeneity and large genes, unraveling the mutation is challenging.in silico analysis was accomplished.Patients underwent comprehensive clinical examination, and targeted next-generation sequencing (NGS) was used for mutation detection. Co-segregation and TGFBI. The variant co-segregated with the phenotype in the family. Homozygous patients manifested with more severe phenotypes. Variable expressivity was observed among heterozygous patients. Patients suffered from GCD. NGS disclosed a known pathogenic variant, c.371G>A (p.R124H), in exon 4 of The results, in accordance with previous studies, indicate that the c.371G>A in TGFBI is associated with GCD. Some phenotypic variations are related to factors such as modifier genes, reduced penetrance and environmental effects.  ARSC1, CHST6, COL8A2, GLA, GSN, KRT3,KRT12, M1S1 and UBIAD1 have been reported in various types of CDs (TGFBI)-linked corneal dystrophies and heterozygous mutations in TGFBI , on human chromosome 5q31 is responsible for the disease  containing 683 amino acid protein called Keratoepithelin  are heterogeneous autosomal dominant disorders. Gradual accumulation of hyaline, amyloid and non-amyloid deposits within anterior stromal layer of the cornea lead to decrement of sight acuity and visual impairment in the first or second decades of life . Mutatio disease . TGFBI mes of CD . Two hotn, Italy . ACD is n, Italy . The TGFpithelin . The propithelin , 15. It Subjects Four large isolated pedigrees from a village in South Khorasan province of Iran with several affected members suffering from visual problems were recruited. Precise clinical examinations including slit-lamp examination for available normal and affected members were performed by an ophthalmologist. A complete family history was obtained and the pedigrees were drawn by a medical geneticist. Subsequently, full investigation revealed familial relationship between these four selected pedigrees. After taking informed written consent, peripheral blood samples were obtained in EDTA-containing tubes.Molecular analysis®, Dusseldorf, Germany).Genomic DNA was extracted from peripheral blood lymphocytes using Prime Prep Genomic DNA Extraction kit  according to the manufacturer’s instruction. Qualitative and quantitative assessment of genomic DNA was checked using 1.2% agarose gel and Nanospec cube biophotometer , Picard for removal of duplicate reads and GATK for variant calling. Annotation was performed by ANNOVAR. Heterozygous missense, start codon change, splice site, stop gain, stop loss and nidel variants with MAF < 1%, were filtered in dbSNP version 137, 1000 genomes database, NHLBI GO exome sequencing project (ESP) and exome aggregation consortium (ExAC). We applied online software tools including MutationTaster2, FATHMM, SIFT and PolyPhen-2 to investigate in silico pathogenicity prediction of the missense variant. Candidate variant was investigated in the Human Gene Mutation Database (HGMD) and in the literature to seek the variant novelty and its association with a phenotype. A custom designed Nimblegen chip was used to capture exons and exon-intron boundaries of the TGFBI gene was amplified. PCR products were sequenced bi-directionally on an automated Genetic Analyzer ABI 3130XL  using Big Dye Terminator Cycle Sequencing Kit  and analyzed with Sequencer 5.4.5 (Gene Codes Corporation). Sequences were compared with reference sequence NM_000358. Variant nomenclature was based on HGVD and variant interpretation was according to the ACMG guideline.  Using forward: 5’ TCCCTCCTTCTGTCTTCTGC 3’ and reverse: 5’ CTCGGGGAAGTAAGGCAGTT 3’ primers, exon 4 of the Clinical and molecular findingsTGFBI geneand it was confirmed by Sanger sequencing. In silico prediction tools revealed the disruptive effect of the variant , within exon 4 of the  variant . Pedigree AProband was a 27-year-old female with light corneal dystrophy. She belonged to a large family with three generation history of visual impairment. The p.R124H variant was detected in the proposita, her mother and her affected siblings . Pedigree BProbands were two offsprings of a first cousin consanguineous marriage. A 5-year-old girl within a pedigree was diagnosed with mild, slowly progressive CD . This coPedigree CTwo mild form affected Probands referred to know about their offspring risk evaluation and genetic counseling. Several affected members were found in their maternal family with a wide spectrum of the disease phenotype. The heterozygous variant co-segregated with the phenotype in the pedigree .Pedigree DA large association with individuals affected with CD in three generations was identified with mild form of the disease (1D). Sequencing results revealed heterozygous pathogenic variant, p.R124H, in all of these patients. Healthy individuals were carrying wild type alleles in homozygous status. TGFBI gene, in four large Iranian pedigrees. This position is considered as a hotspot codon in TGFBI , at exon 4 of the in TGFBI . The varin TGFBI . Althougin TGFBI . Indeed,in TGFBI . It seemin TGFBI . R124H hin TGFBI . Alavi ain TGFBI , 21. Mor studies . Becauseanations -27. Despanations . We did anations .TGFBI mutation, p.R124H, in Iranian population can result from a founder mutation or genetic drift. The results are useful for genetic counseling, cascade screening and prenatal diagnosis to reduce disease burden as there is not any treatment for the disease right now. High frequency of


Physical function impairment can cause great stress to older adults.The purpose of the study is to investigate the association betweenself-reported and directly-observed physical function on perceived stressamong U.S. Chinese older adults.Data were from the Population Study of Chinese Elderly in Chicago(PINE) of 3,157 Chinese older adults who were 60 and above in the GreaterChicago Area. Self-reported and directly-observed physical functionmeasures, and Perceived Stress Scale were used.p<0.01), Rosow–Breslauindex of mobility scale , and Nagiindex of basic physical activities scale  were associated with higher levels ofperceived stress. In addition, higher scores of directly-observed physicalfunction measurements, including chair stand (OR=0.93), tandem stand, timed walk , and the overall measurement  were associated with lower level ofperceived stress.Participants had a mean age of 72.8 ± 8.3 years old (range60–105). Higher scores of Katz activities of daily living impairments(odds ratio [OR]=1.77), Lawton instrumental activities of daily livingimpartments (OR=1.10, Findings suggested that poor physical function was associated withperceived stress among U.S. Chinese older adults. Longitudinal studies areneeded to obtain a more comprehensive understanding of the pathways betweenphysical function and perceived stress.Health care professionals could provide personalized physicalactivity interventions to encourage older adults to engage in regularexercise in order to maintain and promote older adults’ physicalfunction and psychological well-being. Chinese Americans are the oldest and largest Asian population in the UnitedStates. Approximately 16% of the 4.4 million U.S. Chinese population were over age65. More than 80% of Chinese older adults residing in the U.S. were foreign-born,and about 30% of them immigrated to the U.S. at the age of 60 or over . AccordiPhysical function is critical to maintaining independence and socialinteractions in later life . IndividThere is growing evidence that older Asian Americans have a higher prevalenceof physical functional impairments and disabilities in comparison with theirU.S.-born counterparts . BecauseThe Population Study of Chinese Elderly in Chicago (PINE) is acommunity-engaged, population-based epidemiological study. The PINE study wasconducted to examine key cultural determinants of health and well-being incommunity-dwelling Chinese older adults in the Greater Chicago Area. The projectwas initiated by a synergistic community-academic collaboration among RushInstitute for Healthy Aging, Northwestern University, and over 20community-based social services agencies and organizations throughout theGreater Chicago Area. The PINE study implemented extensive culturally andlinguistically appropriate recruitment strategies through a community-basedparticipatory research approach. The inclusion criteria of participantsincluded: 1) older adults over the age of 60 years old, (2) self-identified asChinese; (3) resided in the Greater Chicago Area. Details of the PINE studydesign were published elsewhere  older ad.Self-reported physical function was assessed with fourwell-established measures. The first was Katz activities of daily living(ADL), which measured physical function impairment in participants’ability to perform basic self-care tasks, such as dressing, bathing,toileting, etc. . The secAs for the directly-observed physical function testing, the ShortPhysical Performance Battery (SPPB) was used. The first test was the chairstand, measuring participants’ ability to rise to a standing positionfrom a chair. The second test was the tandem stand, which asked participantsto perform semi-tandem, then full tandem or side-by-side (depending on theperformance on semi-tandem). The third test was an 8-foot timed walk. Higherscores of physical performance testing indicated better functionalperformance. The SPPB in the PINE sample had good validity and inter-raterreliability  [Perceived stress was assessed with the 10-item Perceived StressScale (PSS-10) . ParticiSocial-demographic characteristics included age, gender, years ofeducation completed , annual personalincome , living arrangement , years in the community , years in the United States , and medical comorbidities. The number of medicalcomorbidities was calculated by summing the number of “yes”responses to the nine groups of the following medical conditions: (1) heartdisease; (2) stroke or brain hemorrhage; (3) cancer, malignancy or tumor ofany type; (4) high cholesterol; (5) diabetes; (6) high blood pressure; (7) abroken or fractured hip; (8) thyroid disease; and (9) osteoarthritis,inflammation, or problems with joints Confounding factors were chosen andcategorized based on the previous studies [Chi-square and t-test were used to compare the mean differences insocio-demographics and physical function between participants with and withoutperceived stress. Spearman correlations were used to measure the correlationbetween perceived stress and physical function. Multivariate logistic regressionmodels were used to examine the association between physical function andperceived stress. Potential covariates were controlled in the models. Model Awas adjusted for basic socio-demographic characteristics, including age, gender,education. Income and living arrangement were added to Model B. In Model C,years in the community and years living in the United States were included inaddition to Model B. We then added medical comorbidities to build Model D. Oddsratios (ORs), 95% confidence intervals (CIs), and significance levels werereported. All statistical analyses were conducted using SAS, Version 9.2 .p<.001), female(p<0.05), have lower income(p<0.01), fewer household members(p<0.01), fewer years in the community(p<0.05), and more medical comorbidities(p<0.05). In terms of physical function, older adults withperceived stress were more likely to have higher scores in ADL impairment(p<0.05), IADL impairment(p<0.001), index of mobility scale(p<0.001), index of basic physical activities scale(p<0.001), chair stand (p<0.01),tandem stand (p<0.001), timed walk(p<0.001), and SPPB (p<0.001)compared with those without perceived stress, indicating that older adults withperceived stress were more likely to have poorer self-reported and directly-observedphysical function. This study of Chinese older adults used a total of 3,157 participants with amean age of 72.8 years . Of the participants in thestudy, 58.9% were female, while 71.3% were married. 78.9% had less the equivalent ofhigh school education, or less. Most of them (85.1%) had an annual income less thanUS$10K. More than half of the participants (55.6%) had three or more children. 21%of the participants lived alone. 73.3% of the participants had resided in the U.S.for more than 10 years. A total of 96.08% of study participants perceived certainlevels of stress. p<0.01) significantly morelikely to report perceived stress, compared to those reported no stress. Chineseolder adults with a higher score of index of mobility scale were 39%  significantly morelikely to report perceived stress, compared to reporting no stress. Chineseolder adults with a higher score of index of basic physical activities scalewere 19% significantly more likely to report perceived stress, compared with those whoreported having no perceived stress.p<0.05; timed walk: OR=0.73, 95%CI=0.63–0.86, p<0.001; and SPPB: OR=0.87, 95%CI=0.80–0.95, p<0.01). Every one-point increasesin the tandem stand, timed walk, and SPPB were associated with 29%, 27%, and 13%decreased odds of reporting perceived stress, respectively.This is the first and largest population-based epidemiological study toinvestigate the association between physical function and perceived stress amongU.S. Chinese older adults in the Greater Chicago Area. Our study found that olderadults who had perceived stress were more likely to be an older age, female, havelower income, fewer household members, and fewer years in the community comparedwith those who reported having no perceived stress. Our study indicates that stresslevels were higher among participants with an older age as older age is associatedwith declined in physical function, and increased risk of losing significant othersand life partners ,21. WithOur study demonstrated that both self-reported and directly-observedphysical function were associated with perceived stress among Chinese older adults.We found that U.S. Chinese older adults with poorer self-reported IADL impairment,index of mobility scale, and index of basic physical activities scale were morelikely to perceive higher levels of stress compared with those with betterself-reported physical function. When older adults cannot take care of their basicneeds  and have difficultygoing out shopping alone, or going to a medical appointment, they may feel incapableand dependent, and thus feel distressed and pessimistic . Our resWith respect to directly-observed measures, our participants with betterfunctional performance on tandem stand, timed walk, and SPPB were less likely toperceive stress compared with those with poorer physical performance. Our resultssupported Bellon et al.  study thSupporting the transactional model of stress , the preChinese culture has been enforcing filial piety as an essential moralstandard . ChineseThe study was presented with several limitations. First, this study wasrepresentative of Chinese older adults in the Greater Chicago Area. Hence, thefinding may not be generalizable to Chinese older adults in other geographic areas.Second, this was a cross-sectional study, so we did not know the causal directionsof physical function and perceived stress. Futures studies with longitudinal designsare needed to obtain a more comprehensive understanding of the pathways betweenphysical function and perceived stress among U.S. Chinese older adults. Third, somefactors  may influence theperceived stress on older Chinese immigrants. Future studies could include potentialrisk or protective factors to examine possible mediating or moderating effectsbetween physical function on perceived stress.Despite the limitations, our findings have important implications. Forresearch implications, our results showed that poor physical function was associatedwith perceived stress among U.S. Chinese older adults. This study allowed us to testwhether the physical function was associated with perceived stress and whetherfindings differed when using a self-reported measure of physical function comparedwith a directly-observed measure of physical function. The results lay thegroundwork for research investigating the longitudinal relationship between physicalfunction and perceived stress in U.S. Chinese older adults. In addition,cross-cultural research is needed to examine the association between physicalfunction and perceived stress in different ethnic/ racial groups. For clinicalimplications, health care professionals should be aware that U.S. Chinese olderadults with poor physical function may experience high levels of perceived stress.Health care professionals should identify older adults who are vulnerable to stressand provide coping strategies for alleviating psychological distress. In addition,health care professionals should provide educational programs to encourage olderadults to engage in regular exercise in order to maintain and/or promote olderadults’ physical function and psychological well-being.


Helicobacter pylori infection does not belong to the spectrum of opportunistic infections in people living with HIV (PLHIV). To evaluate the Helicobacter pylori infection prevalence rate trends in HIV co-infected individuals in comparison to the HIV-negative population, we compared histopathological findings of H. pylori positive gastritis (gastritis topography and histopathology) between 303 PLHIV and 2642 HIV-negative patients who underwent esophagogastroduodenoscopy (EGD) between 1993 and 2014 due to dyspeptic symptoms. The prevalence of H. pylori infection was significantly higher in HIV-negative controls than in PLHIV (50.2% vs. 28.1%). A significantly positive linear trend of H. pylori co-infection in PLHIV was revealed in the observed period , while this trend was significantly negative in HIV-negative patients . Patients with HIV/H. pylori co-infection had significantly higher CD4+ T cell counts and more often had undetectable HIV viremia, due to successful anti-retroviral therapy (ART). Stomach histopathological findings differed between HIV co-infected and H. pylori mono-infected patients. Our findings confirm that the ART has changed the progression of HIV infection, leading to a significant increase in the prevalence of H. pylori infection in dyspeptic PLHIV over time. Our data also suggests that a functional immune system may be needed for H. pylori-induced human gastric mucosa inflammation. Helicobacter pylori (H. pylori) is the most common bacterial infection in the world .The survey protocol was conducted according to the Declaration of Helsinki. All survey participants signed informed consent forms before EGD was performed.All patients who had dyspeptic symptoms lasting up to three months before esophagogastroduodenoscopy (EGD) and who underwent their first EGD between January 1993 and December 2014 were included in the study. Dyspeptic symptoms comprised epigastric discomfort (pain), nausea, vomiting, heartburn, burping, body weight loss, bloating and stomach cramps. The study group were PLHIV, while the controls consisted of HIV negative patients from the general population. Patients with gastrointestinal bleeding , odynophagia, dysphagia (indicating possible opportunistic diseases), chest pain, and those younger than 18 years were excluded from the study.Each patient/control was enrolled in the study only once.+ cell counts, HIV viral load, and the history of antiretroviral therapy (ART) from the hospital database. The CD4+ cell counts and HIV viral loads were considered relevant if the blood sample for their determination was taken within 1 month before or after EGD. Histopathological data were collected from the database of the Institute of Pathology, Faculty of Medicine, University of Belgrade.We collected demographic data , risk factors for HIV acquisition, duration of HIV, CD4The HIV status was determined using commercial immunoassays, in accordance with the manufacturer´s protocol. The CD4 cells were quantified by flow cytometry (Becton Dickinson BD FACS Count). Plasma HIV-1 RNA loads were measured with EDTA collected plasma samples, frozen at -20°C, using commercial RT-PCR tests. The usage of PCR tests and their limits of detection, expressed as copies per milliliter (cps/ml), varies over time according to what was available on the market. From 1997 to 2008 Roche Amplicor HIV-1 Monitor Test was used, with the range of detection of 400 cps/ ml– 750000 cps/ml, while from 2008 to 2016 COBAS AmpliPrep/CobasTaqMan HIV version 1.5, with the range of detection from 48 cps/ml– 10 000 000 cps/ml was used, and recently, in 2016, COBAS HIV-1 4800/6800/8800 with the range of detection from 20 cps/ml– 10 000 000 cps/ml of plasma was introduced.All patients underwent EGD after the informed consent was signed. EGD was performed in the standard manner using Olympus video endoscope. Four gastric biopsy specimens were taken from each patient (two from the corpus and two from the antrum). Endoscopic diagnosis was recorded using standard terminology. Biopsies were taken from macroscopically normal tissue and macroscopically pathological areas. Biopsy samples were fixed in formalin, embedded in paraffin, cut into 4-μm sections, and stained by hematoxylin and eosin (H&E) and modified Giemsa stain. The stage of gastritis was scored in accordance with the Sydney classification .H. pylori infection was conducted by histopathological examination using a modified Giemsa staining protocol outlined by Gray S et al. , while detectable viremia was more common in the subgroup without H. pylori co-infection [0 (0–21000) cp/ml vs. 20 (0−107) cp/ml ]. PLHIV with H. pylori co-infection had significantly lower prevalence of AIDS  (HIV viremia was more frequently undetectable in patients with = 0.008) .H. pylori co-infection, the CD4+ T cell counts were significantly higher at the time of EGD compared to the time when HIV infection was detected (p <0.001) (In PLHIV with  <0.001) .H. pylori co-infection, the CD4+ T cell count was not related to gastritis topography (p = 0.470), H. pylori density (p = 0.807), gastritis activity (p = 0.333), nor the presence of lymphoid follicles (p = 0.365) and intestinal metaplasia (p = 0.331) (In PLHIV with = 0.331) .H. pylori infection was detected in 1174 (50.2%) participants. H. pylori-infected subjects were significantly older than those without this infection . H. pylori infection did not show any gender-related correlation (p = 0.241)  males vs. 1327 (56.7%) females, with a mean age of 46.3±14.7 years (age range 18–86). = 0.241) .H. pylori infection was significantly more prevalent in the HIV-negative group of patients in comparison to PLHIV (50.2% vs. 28.1%). The control group of patients with H. pylori infection was significantly older than the PLHIV group with H. pylori co-infection . The histopathological grading of gastritis activity differed significantly between those two subgroups (p = 0.006). The mildest cases prevailed in PLHIV, while in the control group there were more patients with moderate and severe activity. There was no difference between two H. pylori positive subgroups in gastritis topographical localization (p = 0.452), H. pylori density (p = 0.905), presence of lymphoid follicles (p = 0.074) nor intestinal metaplasia (p = 0.817) (= 0.817) .H. pylori infection among PLHIV, this is the first report in which the joinpoint regression analysis was performed. Also, to the best of our knowledge, this is the first EGD-based morphological comparison of histopathological findings in H. pylori-positive patients with and without HIV co-infection covering 21 years.Considering the already published research data on H. pylori infection is significantly lower among PLHIV in comparison with the HIV-negative population, over 21 years covered by our study. These data are in concordance with those already published addressing this issue [H. pylori infection during more than 20 years of EGD follow-up among patients with dyspeptic symptoms, had opposed linear trends between the two investigated groups. Decreasing prevalence rates of H. pylori infection over time were observed in the HIV-negative population, contrary to the increasing trend in PLHIV. Moreover, the APC increase was 3% in the PLHIV group while in the general population the APC decrease was 2.7%. Even though we did not demonstrate any significant changes in two APCs, which converge towards similar prevalence rates during last two years of the observed period, we may speculate that these changes in the two observed linear trends could be related to the gradual ART introduction in HIV-positive patients, as well as the eradication therapy for H. pylori among controls.Our data suggest that the prevalence of is issue , 19–21. The first patient with AIDS in Serbia was diagnosed in 1985, while ART, including mono and dual therapy, became available in 1987 and was in use up to 1998, when HAART became widely available . We demoH. pylori eradication therapy has been available in Serbia since it had been advocated by most guidelines, and this treatment approach has led to the decreased shedding of the pathogen, along with the decreased prevalence of the infection in the general population [H. pylori infection in the PLHIV population over time could probably be explained by the less effective mono and dual ART in comparison to HAART with respect to immune reconstitution, which we consider to be important for the pathogenesis of H. pylori-induced gastric mucosa inflammation.Standard and highly efficient triple pulation . This epH. pylori infection in PLHIV is the immune restoration, corresponding to the CD4+ T cell count. In this study we showed that there was no difference in the CD4+ T cells counts between PLHIV with and without H. pylori co-infection at the time of HIV diagnosis. Contrary to this, at the time when EGD was performed, PLHIV with H. pylori co-infection had a significantly higher CD4+ T cell counts, mostly over 200/mm3, and less progression to AIDS. However, once a higher CD4+ T cell count is achieved, suggestive of immune reconstitution, it does not affect the histopathological findings of the gastric mucosa. The topographic localization of gastritis, H. pylori density, activity, the presence of lymphoid follicles and intestinal metaplasia in patients with HIV/H. pylori co-infection was not dependent on the level of immune restoration, monitored by the CD4+ T cell counts. Definitely T cell subpopulations affect H. pylori colonization, but there is not yet sufficient evidence that T cell deficiency causes H. pylori removal.It seems that the most important factor related to the occurrence of H. pylori infection, we only found the difference in activity: most of the PLHIV had mild gastritis activity (58%) while HIV-negative patients mostly had moderate/severe gastritis activity (53.6%), which may be attributed to the incomplete immune recovery in co-infected patients. However, in our previous investigation, where we enrolled 212 HIV/H. pylori co-infected patients, we concluded that most of the patients treated in the pre-HAART era had mild gastritis activity while those on HAART mostly had mild/moderate gastritis activity [When we compared histopathological findings between HIV-positive and HIV-negative subjects with H. pylori infection over time. Antibiotics are used for H. pylori eradication in both PLHIV and HIV-negative patients. H. pylori eradication therapy consists of simultaneous usage of two antibiotics from different classes along with proton pump inhibitor [H. pylori eradication [In this retrospective study we did not evaluate if the usage of antibiotics affected the prevalence of nhibitor , and antdication .Mycobacterium tuberculosis infection. Rifampicin has an excellent in vitro and good efficacy in vivo against H. pylori , but does not necessarily result in eradication of H. pylori infection [H. pylori infection in PLHIV treated for tuberculosis. Their findings, however, suggest that progressive HIV disease rather than antibiotic usage may be responsible for the diminution in frequency of H. pylori co-infection [H. pylori infection in PLHIV more significantly than in the HAART era. Still, we cannot exclude immunodeficiency of these patients as an important contributing factor to their lower incidence of H. pylori infection. It is important to note that not all of the patients included in this study had been treated for tuberculosis. Future research is needed to clarify the impact of previous antibiotic therapy, primarily antituberculosis therapy, on the incidence of H. pylori infection in PLHIV. Co-trimoxazole has not been reported to have activity against H. pylori [In addition, some PLHIV in our cohort had been taking antibiotics earlier to treat opportunistic infections, such as nfection . While wnfection . We can . pylori .H. pylori induced gastric inflammation is less severe in PLHIV, it is possible that H. pylori infection in these patients was underdiagnosed due to mild symptoms, especially before HAART introduction. In addition, dyspeptic symptoms are common in PLHIV and are usually not associated with H. pylori as they may be due to opportunistic infections and/or tumors, diet, ART side effects, etc., with peptic ulcer disease and gastroesophageal reflux disease frequently detected by EGD [Since d by EGD , 37.H. pylori gastritis was higher among PLHIV with less advanced HIV infection and hence more preserved immune function. We previously demonstrated that the prevalence of H. pylori gastritis had been increasing in concert with the HAART efficacy evolution and the consequent improved immune reconstitution [H. pylori gastritis among PLHIV.Our previous studies demonstrated that the prevalence of titution . More reH. pylori co-infection in addition to the ART, e.g. exposure to antibiotics, PPI and H2 blocker therapy, body mass index, etc. Also, we did not explore the H. pylori acquisition in the control, HIV-negative group in detail.This is a retrospective case-control study and therefore some potentially relevant information was not available, such as other possible risk factors for HIV/H. pylori positive gastritis in HIV/H. pylori co-infection and annual percentage changes in H. pylori infection rates.Despite these limitations we believe that the study presents new research findings on the histopathology of H. pylori co-infection could be the consequence of slow and steady immune reconstitution ranging from a rather weak mono or dual ART in the beginning to the more potent modern HAART, resulting in a similar level of H. pylori gastritis development in all our patients.In conclusion, according to our data, we believe that in the population of HIV infected individuals the linear trend of the slowly rising prevalence of gastritis associated with H. pylori infection is present only in PLHIV who have preserved CD4 counts, hence, immunity. Although more research is needed in this area, we believe that our conclusions are extremely important for the longer life expectancy of PLHIV as well as the carcinogenicity of H. pylori. We believe that the future outcome and course of HIV infection will be quite different and that diseases not previously studied in the context of HIV will become as common in PLHIV as in HIV-negative patients. No studies conducted after HAART became available have implicated that PLHIV individuals are at a decreased risk of gastric cancer. Therefore, PLHIV with H. pylori co-infection, regardless of their CD4 status, could be at an equal risk for of gastric cancer as HIV negative patients, but less likely to be diagnosed with H. pylori due to reduced gastric inflammation. Testing for and treatment of H. pylori in the HIV positive population should be as aggressive as it is for the HIV negative population.Our research has shown that H. pylori co-infection, future prospective studies are needed to address the epidemiological characteristics of H. pylori infection in HIV positive population.To definitively determine the risk factors for HIV/S1 TextHelicobacter pylori prevalence rate trends in HIV-positive (1) and HIV-negative (2) patients in Serbia in the period of 1993–2014.A joinpoint regression analysis of (TXT)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.