Integrase inhibitor (INSTI)-based antiretroviral therapies (ART) are first-line for HIV infection. Recent studies have identified metabolic complications of INSTI regimens, especially when TAF is included. The objective of this study was to assess lipid changes associated with INSTI-based ART.This was a retrospective, observational, single-center study. Patients age ≥ 18 with HIV infection, receiving care Jan 2004 to Jul 2019, and prescribed the same ART for ≥ 6 consecutive months were randomly selected from a computer-generated list. Unique ART regimens prescribed per patient were considered “exposures”. Patients were followed up to 2 years per exposure. INSTI-based exposures included an INSTI plus one or more NRTI. Non-INSTI-based exposures included a PI or NNRTI plus one or more NRTI. Lipid panel results were recorded before and during exposures. Lipid changes with INSTI and non-INSTI exposures were compared. Other metabolic outcomes were recorded.2, respectively. 72 (37.5%) were ART-naïve. 53.5% INSTI recipients received BIC or DTG. Hypertriglyceridemia (HTG) occurred more often during INSTI exposures . Mean change in TG concentration was +10.1 and -15.0 mg/dL for INSTI and non-INSTI regimens, respectively (p = 0.105). Patients receiving INSTIs were more commonly prescribed a new lipid-lowering therapy (LLT) or an increase in dose of preexisting LLT . Of those that received an INSTI-regimen and either TAF, TDF, or no tenofovir, 17.4%, 10.3%, and 8.3% developed HTG, respectively (p = 0.49). There was no difference between groups in HDL or LDL changes. Among INSTI and non-INSTI groups, weight increased by +3.8 and +2.1 kg, respectively (p = 0.129). Mean weight change was less in INSTI recipients whose regimen included TDF .192 exposures were identified  among 132 patients. The majority were Black (69.3%) and male (61.9%). Mean age and BMI were 42.0 ± 11.3 and 26.2 ± 5.5 kg/mTreatment with INSTI-based ART was associated with HTG. LLTs were utilized more often among INSTI recipients, and this limited our ability to fully characterize lipid changes in this real-world study. Weight gain among INSTI recipients may be attenuated if TDF is included in the regimen.James Johnson, PharmD, FLGT (Shareholder) Caryn Morse, MD, Chimerix (Scientific Research Study Investigator)Covis Pharma (Scientific Research Study Investigator)Gilead Sciences Inc. (Scientific Research Study Investigator)Ridgeback Biotherapeutics (Scientific Research Study Investigator)Roche (Scientific Research Study Investigator)SCYNEXIS, Inc. (Scientific Research Study Investigator)Theratechnologies (Advisor or Review Panel member)Viiv (Advisor or Review Panel member)


Staphylococcus lugdunensis and determine its association with sequence types (STs) determined by multilocus sequence typing (MLST) and oxacillin susceptibility. Primers were designed to detect and sequence types IIIA and IIC CRISPR-Cas in 199 S. lugdunensis isolates. MLST and oxacillin susceptibility tests were also performed on the isolates. We found that 84 S. lugdunensis isolates had type IIIA CRISPR-Cas, while 46 had type IIC. The results showed a strong association between STs and CRISPR-Cas types. The ST1, ST6, ST12, and ST15 isolates had type IIIA CRISPR-Cas systems, and the ST4, ST27, and ST29 isolates had type IIC CRISPR-Cas. Interestingly, of 83 isolates containing type IIIA CRISPR-Cas, 17 (20.5%) were oxacillin-resistant S. lugdunensis (ORSL), and all of these ORSL isolates belonged to ST6 cluster 1. Moreover, spacers 23 and 21 were found in 16 and 17 ORSL isolates, respectively. In contrast, all 46 isolates with type IIC CRISPR-Cas were susceptible to oxacillin. Our results showed that 41.3% of CRISPR-Cas IIIA spacers were homologous to plasmids and 20.2% were homologous to phages. However, in type IIC CRISPR-Cas, 11.8% and 39.9% of spacers showed sequence homology with plasmids and phages, respectively. In conclusion, we found that the distribution and composition of the CRISPR-Cas system in S. lugdunensis was associated with STs and oxacillin susceptibility.Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) genes (CRISPR-Cas) are present in many bacterial genomes with functions beyond adaptive immunity. We aimed to characterize the CRISPR-Cas system in the pathogenic Gram-positive bacterium IMPORTANCE CRISPR-Cas systems have been characterized as playing several biological roles in many bacterial genomes. Moreover, CRISPR-Cas systems are useful for epidemiological, diagnostic, and evolutionary studies of pathogenic bacteria. However, the characteristics of CRISPR-Cas systems in Staphylococcus lugdunensis have been rarely reported. In this study, we revealed that type IIIA CRISPR-Cas was dominant in S. lugdunensis isolates, followed by type IIC CRISPR-Cas. Moreover, the composition of CRISPR-Cas spacers was strongly associated with multilocus sequence typing and oxacillin susceptibility of S. lugdunensis. These results advance our understanding of the evolution of CRISPR-Cas systems; however, the biological functions of CRISPR-Cas systems in S. lugdunensis remain to be further characterized. Staphylococcus lugdunensis is a Gram-positive, catalase-positive, and coagulase-negative Staphylococcus (CoNS) bacterium. Staphylococcus lugdunensis has emerged as an important pathogen, implicated in clinically invasive infections such as endocarditis and bacteremia in recent years  has been frequently reported over the last decade (Staphylococcus lugdunensis expressing penicillin-binding protein 2a (PBP2a), encoded by the mecA gene, shows a lower affinity for β-lactams and is responsible for β-lactam resistance   , 5.S. lugdunensis isolates from different clinical and geographic sources, using DNA sequence analysis of seven housekeeping genes: aroE, dat, ddl, gmk, ldh, recA, and yqiL (S. lugdunensis isolates could be defined into 20 sequence types (STs) and 5 clonal complexes  and CRISPR-associated (Cas) genes (CRISPR-Cas) are present in many bacterial and archaeal genomes . CRISPR-lococcus –13. Rossococcus –, 15. HowS. lugdunensis  isolates and determined the distribution of CRISPR-Cas in the isolates. The PCR results showed that 130/199 (65.3%) of the S. lugdunensis isolates had CRISPR-Cas systems, including 84 (42.2%) with type IIIA and 46 (23.1%) with type IIC . Most ST1 and ST6 clusters (clusters 1 to 5) had spacers 1 and 2, which showed homology with Campylobacter phage CP220 and Clostridium botulinum strain CDC_67071 plasmid pNPD7, respectively. All ST15 cluster 7 isolates had spacers 94, 95, 96, and 97, of which 94 and 96 showed homology with phages in the S. lugdunensis genome . We were unable to determine the homologs of spacers 12 and 81.To further validate the association between CRISPR-Cas types and STs, we performed a phylogenetic analysis based on the sequences of seven housekeeping genes for MLST. The results revealed a strong association between ST1, ST6, ST12, and ST15 , which had type IIIA CRISPR-Cas systems . Among tisolates . Spacer s genome . Spacer S. lugdunensis isolates containing type IIIA CRISPR-Cas systems was 10.2 (818 spacers/80 isolates) (S. lugdunensis chromosome (The mean number of spacers in solates) . We founsolates) . Moreoveromosome . Overallromosome .S. lugdunensis isolates with type IIC CRISPR-Cas comprised 8 clusters and 33 CTs (S. lugdunensis genome (Table S5). All ST29 isolates had spacers 55 and 56 (Secundilactobacillus paracollinoides strain TMW 1.1979 plasmid pL11979-2 (Table S5). We were unable to determine the homologs of spacer 56. Spacers 20, 21, 29, 31, 32 to 36, 44 to 46, and 53 were highly conserved in ST4 isolates, but ST4 cluster 1 lacked spacer 53 (Staphylococcus genomes (Table S5).Forty-three d 33 CTs . Three id 33 CTs  and 3B. d 33 CTs . Howeverd 33 CTs . Spacers5 and 56 . Spacer pacer 53 . Among tS. lugdunensis containing type IIC CRISPR-Cas systems was 13.7 (591 spacers/43 isolates) (S. lugdunensis containing type IIIA CRISPR-Cas systems (mean 10.2 spacers/strain) (S. lugdunensis chromosome (All isolates with type IIC CRISPR-Cas were susceptible to oxacillin. The mean number of spacers in solates) , higher /strain) . Our res/strain) . Moreoveromosome . Overallromosome . Of spacromosome .S. lugdunensis. We found 84 S. lugdunensis isolates with type IIIA CRISPR-Cas and 46 isolates with type IIC  and related IncF epidemic plasmids . Surprisingly, spacer 23 showed homology with a S. aureus strain 16405 plasmid that expressed PBP2A encoded by mecA. Therefore, the activity of CRISPR-Cas systems in plasmid acquisition and their association with oxacillin susceptibility of S. lugdunensis merits further investigation.Our results demonstrated that CRISPR-Cas spacer sequences were strongly associated with MLST and oxacillin susceptibility. Previous studies have shown that pandemics caused by multidrug-resistant plasmids . The absplasmids , 16. In isolates . In addiisolates . The homS. lugdunensis typing, including a classical length-based multiple loci VNTR analysis (MLVA) method and a sequence-based MLVA method known as the tandem repeat sequence typing (TRST) method (fbl-typing) has also been used for S. lugdunensis typing  method . Sequenc) method . DNA seqs typing . Among tdunensis , 9. CompAcinetobacter baumannii global clone 1 (GC1) isolates  were included in the study. All information associated with the 199 isolates  was collected. Staphylococcus lugdunensis isolates were initially identified by Gram staining, biochemical methods , and rapid PCR detection . The isolates were stored in tryptic soy broth containing 20% glycerol at −80°C until use.A total of 199 etection . All thecas1 gene for CRISPR-Cas types IIIA and IIC, respectively  database, including sequences for type IIIA-positive strain VISLISI_33 (accession no. CP020769.1) and type IIC-positive strain C_33 (accession no. CP020768.1). The PCRs were carried out in a total volume of 20 μl containing 2× master mix , 10 pmol of each primer, and 1 μl DNA template. The PCR cycling conditions for CRISPR-Cas system detection were 95°C for 3 min; 30 cycles of 30 s at 95°C, 30 s of annealing at 50°C, and 1 min of extension at 72°C; and a final extension for 3 min at 72°C.The Crispr-IIIA-F/Crispr-IIIA-R and Crispr-IIC-F/Crispr-IIC-R primers were designed to specifically detect the The primers Crispr-IIIA-F/Crispr-IIIA-R and Crispr-IIC-F/Crispr-IIC-R were designed for also determining type IIIA and type IIC CRISPR-Cas spacer sequences, respectively  for ST type determination. Sequence types were identified based on the allele profiles. Further analysis was performed using eBURST (http://eburst.mlst.net) to identify clonal complexes (CCs) and founders, as well as to determine the overall population structures. A minimum-evolution (ME) tree of the concatenated sequences (aroE-dat-ddl-gmk-ldh-recA-yqiL) for each ST shared by the S. lugdunensis isolates was generated using Mega X and the Kimura two-parameter model to estimate genetic distances. Statistical support of the nodes in the ME tree was assessed by performing 1,000 bootstrap resamplings.MLST was performed for all us study . Seven hS. lugdunensis isolates to oxacillin was determined using an agar dilution assay and interpreted according to the Clinical and Laboratory Standards Institute guidelines (Staphylococcus lugdunensis isolates with a MIC ≥ 4 μg/mL were defined as resistant. Staphylococcus aureus ATCC 29213 was used as a control strain. Antimicrobial susceptibility testing was performed in duplicate to ensure reproducibility.Susceptibility of the idelines . StaphylS. lugdunensis isolates were subjected to SCCmec typing and mecA detection using a multiplex PCR assay to amplify the ccr and mec complexes according to a previous study (All us study .


Arabidopsis thaliana, Gossypium herbaceum (A1), Gossypium arboreum (A2), Gossypium raimondii (D5), and Gossypium hirsutum (AD1) genomes demonstrated that four HSF genes were inherited from a common ancestor, A0, of all existing cotton A genomes. Members of the HSF gene family in G. herbaceum (A1) genome indicated a significant loss compared with those in G. arboretum (A2) and G. hirsutum (AD1) A genomes. However, HSF genes in G. raimondii (D5) showed relative loss compared with those in G. hirsutum (AD1) D genome. Analysis of tandem duplication (TD) events of HSF genes revealed that protein-coding genes among different cotton genomes have experienced TD events, but only the two-gene tandem array was detected in Gossypium thurberi (D1) genome. The expression analysis of HSF genes in G. hirsutum (AD1) and Gossypium barbadense (AD2) genomes indicated that the expressed HSF genes were divided into two different groups, respectively, and the expressed HSF orthologous genes between the two genomes showed totally different expression patterns despite the implementation of the same abiotic stresses. This work will provide novel insights for the study of evolutionary history and expression characterization of HSF genes in different cotton genomes and a widespread application model for the study of HSF gene families in plants.Heat shock transcription factors (HSFs) are involved in environmental stress response and plant development, such as heat stress and flowering development. According to the structural characteristics of the HSF gene family, HSF genes were classified into three major types  in plants. Using conserved domains of HSF genes, we identified 621 HSF genes among 13 cotton genomes, consisting of eight diploid and five tetraploid genomes. Phylogenetic analysis indicated that HSF genes among 13 cotton genomes were grouped into two different clusters: one cluster contained all HSF genes of HSFA and HSFC, and the other cluster contained all HSF genes of HSFB. Comparative analysis of HSF genes in  Gossypium) is one of the most important economic crops worldwide, which provides the major resource of natural fiber for human beings over the past decades. The genus Gossypium contains over 45 diploid (designated as A to G and K) and 7 tetraploid (designated as AD1 to AD7) species (Gossypium herbaceum (A1) (Gossypium arboreum (A2) (Gossypium thurberi (D1) (Gossypium raimondii (D5) (Gossypium turneri (D10)   (Gossypioides kirkii (K) (Gossypium hirsutum (AD1) (Gossypium barbadense (AD2) (Gossypium tomentosum (AD3) (Gossypium mustelinum (AD4) (Gossypium darwinii (AD5) (Bombax ceiba at approximately 24 million years ago (Mya). Subsequently, cotton A0 and D5 genome species diverged from their ancestor at approximately 4.8 Mya. After the split of their ancestor, cotton A0 and D5 genome species crossed and generated cotton allotetraploid AD genome species at approximately 1.6 Mya (G. hirsutum (AD1) and G. barbadense (AD2) (G. herbaceum (A1) and G. arboreum (A2) (Cotton (genus  species . To facieum (A1) , Gossypieum (A2) , Gossypieri (D1) , Gossypidii (D5) , Gossypiri (D10) , Gossypilyx (F1) , Gossypiale (G2) , and Gosrkii (K) , and fivum (AD1) , Gossypise (AD2) , Gossypium (AD3) , Gossypium (AD4) , and Gosii (AD5) . Previou 1.6 Mya . Later, se (AD2) . Less theum (A2) . A cleareum (A2) .Solanum lycopersicum (24)  (Oryza sativa (25) (Zea mays (25) (Glycine max (26) (Brassica oleracea (35) (Brassica rapa (36) (Brassica napus (64) (Sesamum indicum (30) (Prunus mume (18) (Fagopyrum tataricum (29) (Cicer arietinum (20) (Vitis vinifera (19) (Capsicum annuum (25) (Brassica juncea (60) (G. hirsutum (AD1) D genome using EST assembly and genome-wide analyses (Heat shock transcription factors (HSFs) serve as the major activators of heat shock proteins (HSPs) with the help of binding to the promoter regions of HSP genes in plants. This enables HSP genes to regulate transcription in response to heat stress for adapting to various environmental dynamic changes . HSF procum (24) , Arabidoana (21) , Oryza siva (25) , Zea mayays (25) , Glycinemax (26) , Brassiccea (35) , Brassicapa (36) , Brassicpus (64) , Sesamumcum (30) , Prunus ume (18) , Fagopyrcum (29) , Cicer anum (20) , Vitis vera (19) , Capsicuuum (25) , and Bracea (60) . For theanalyses .G. herbaceum (A1), G. arboreum (A2), G. raimondii (D5), and G. hirsutum (AD1), we detected the formation of the HSF gene family and traced the evolution of HSF genes in G. hirsutum (AD1) genome. According to the genome-wide analysis of TD events, we investigated the influence of TD events on the generation of HSF genes among 13 cotton genomes. However, we used the expression analysis to reveal the functional differences of HSF genes under the same abiotic stresses in G. hirsutum (AD1) acc. TM-1 and G. barbadense (AD2) acc. Hai7124. This project will provide novel insights for understanding the evolutionary history and expression characterization of the HSF gene family, paving the way for genetic breeding and molecular improvement of cotton diploid and tetraploid species.In this project, we performed genome-wide comparative genomics analysis for the HSF gene family among 13 cotton genomes. Based on the evolutionary relationship among G. herbaceum , G. arboreum , G. thurberi , G. raimondii , G. turneri , G. longicalyx , G. australe , and G. kirkii , and tetraploid genomes, G. hirsutum , G. barbadense , G. tomentosum , G. mustelinum , and G. darwinii , are downloaded from CottonGen1  acc. TM-1 and G. barbadense (AD2) acc. Hai7124 are retrieved from the Sequence Read Archive (SRA) database with accession number PRJNA490626  program with “trusted cutoff” as threshold . All proA. thaliana and Gossypium species followed the same procedures.ClustalW2 was employed to perform MSA with protein sequences of target HSFs from 13 different cotton species. Then, the MSA file with “meg” format were used to construct the phylogenetic tree through MEGA 7 with maximum likelihood (ML) statistical method and 1,000 bootstrap replications . The phyFollowing the identification of tandemly duplicated genes in PTGBase, all-against-all BLAST of protein sequences in 13 cotton species was employed to identify the paralogous gene pairs within single cotton species with E-value cut-off ≤1e-20 . After cG. herbaceum (A1), G. arboreum (A2), and G. hirsutum (AD1) A genomes, as well as between G. raimondii (D5) and G. hirsutum (AD1) D genomes, were detected by the MCscanX software and validated by phylogenetic analysis (G. herbaceum (A1), G. arboreum (A2), and G. hirsutum (AD1) A genomes and between G. raimondii (D5) and G. hirsutum (AD1) D genomes. So, phylogenetic analysis of these four cottons and A. thaliana genomes was used to validate collinear relationships among cotton genomes. The collinear analysis of G. hirsutum (AD1) and G. barbadense (AD2) A genomes, as well as G. hirsutum (AD1) and G. barbadense (AD2) D genomes, follows the above procedures.Orthologous gene pairs between analysis . First, G. hirsutum (AD1) and G. barbadense (AD2) reference genome sequences through HISAT (version 2.2.1)  , and alln 2.2.1) . The Strn 2.2.1) . The hien 2.2.1) .G. herbaceum , with 17 in G. arboreum , 29 in G. thurberi , 33 in G. raimondii , 36 in G. turneri , 38 in G. longicalyx , 36 in G. australe , 29 in G. kirkii , 78 in G. hirsutum , 78 in G. barbadense , 84 in G. tomentosum , 79 in G. mustelinum , and 80 in G. darwinii  genomes (G. herbaceum (A1) has the least number of HSF genes and G. australe (G2) has the greatest number of HSF genes in cotton diploid genomes. Out of five cotton tetraploid genomes, G. hirsutum (AD1) and G. barbadense (AD2) have the least number of HSF genes, and G. tomentosum (AD3) has the greatest number of HSF genes.With the development of genome sequencing technology, more and more cotton genome sequences were released for the community, which facilitate the analysis of key gene families within plant whole genomes. Totally, 13 latest cotton genomics data, consisting of eight diploid and five tetraploid genomes, were downloaded from CottonGen , G. tomentosum (AD3), G. mustelinum (AD4), and G. darwinii (AD5) genomes, respectively. Finally, we obtained 78 representative HSF genes in G. barbadense (AD2) genome, with 84, 79, and 80 HSF genes in G. tomentosum (AD3), G. mustelinum (AD4), and G. darwinii (AD5) genomes, respectively.To identify accurately the members of the HSF gene family, we used the entire available protein sequences in different cotton genomes. After curation, we found that several cotton tetraploid genomes released multiple protein sequences for one gene due to an alternative splice in genomes. Using the profile HMMs of HSF_DNA-bind, we identified 120, 135, 122, and 106 HSF protein sequences in Based on the characteristics of conserved domains of HSF genes, we obtained 621 target HSF genes among 13 cotton genomes. With protein sequences of the identified HSF genes, we constructed a phylogenetic tree to investigate the evolutionary relationship of all HSF genes among 13 cotton genomes. Following the phylogenetic tree, all HSF genes were grouped into two different clusters, including I and II clusters, among 13 cotton genomes . A previG. hirsutum (AD1), was generated from hybridization between G. raimondii (D5) and the common progenitor, A0 genome, of G. herbaceum (A1) and G. arboreum (A2). However, out of four HSF genes in G. herbaceum (A1) genome, two HSF genes belonged to HSFA, with one HSF gene belonging to HSFB and one HSFC gene belonging to HSFC. For HSF genes in G. arboreum (A2) genome, six HSF genes were classified into HSFA, with eight HSF genes belonging to HSFB and three HSF genes belonging to HSFC. These HSF genes in G. herbaceum (A1) and G. arboreum (A2) originated from a common progenitor of all existing A genomes, including A genome in G. hirsutum (AD1). So, different types of HSF genes in G. herbaceum (A1) genome indicated a significant loss compared with G. arboreum (A2) and G. hirsutum (AD1) A genomes. However, different types of HSF genes in G. raimondii (D5) experienced relative loss compared with those in G. hirsutum (AD1) D genome.Upland cotton, Arabidopsis thaliana is an important model plant, and its genomic data were released more than 20 years, which brings opportunity for studying HSF genes in A. thaliana , G. arboreum (A2), G. raimondii (D5), and G. hirsutum (AD1) genomes to perform comparative genomics analysis of HSF genes in A. thaliana and cotton genomes. Through phylogenetics analysis of HSF genes in A. thaliana and cotton genomes, all HSF genes among five species were clustered into two different groups, namely the I and II groups  has two orthologous genes (Ghe05G03320 and Ghe08G31530) in G. herbaceum (A1) genome, two orthologous genes (Gar05G03090 and Gar08G31350) in G. arboreum (A2) genome, two orthologous genes (D5.v1.pred_00000361-RA and D5.v1.pred_00033482-RA) in G. raimondii (D5) genome, and three  and two (Ghi_D05G01481 and Ghi_D07G00601) orthologous genes in G. hirsutum (AD1) A and D genomes, respectively (G. arboreum (A2) genome, three orthologous genes  in G. raimondii (D5) genome, and three  and three  orthologous genes in G. hirsutum (AD1) A and D genomes, respectively , two genes (Gar02G18780 and Gar10G10520) in G. raimondii (D5) genome, three genes  in G. raimondii (D5) genome, and three  and three  genes in G. hirsutum (AD1) A and D genomes were clustered together and divided into two groups following the divergence from the common ancestor of A. thaliana and Gossypium lineage  was identified to belong to HSFC. Through the analysis of phylogeny, AT3G24520.1 has one orthologous gene (Ghe06G07640) in G. herbaceum (A1) genome, three orthologous genes  in G. arboreum (A2) genome, three orthologous genes  in G. raimondii (D5) genome, and three  and three  in G. hirsutum (AD1) A and D genomes, respectively (In ectively . AT3G029ectively . This re lineage . In A. tectively .G. herbaceum (A1), G. arboreum (A2), and G. hirsutum (AD1) A genomes and between G. raimondii (D5) and G. hirsutum (AD1) D genomes. This result will help to trace the evolution of HSF genes in G. hirsutum genome clearly. In G. hirsutum (AD1) genome, 78 HSF genes were identified, namely 40 and 38 HSF genes distributed into A and D genomes, respectively. From the comparisons between G. herbaceum (A1), G. arboreum (A2), and G. hirsutum (AD1) A genomes, we got 11 and 64 all-against-all HSF gene pairs, respectively , G. arboreum (A2), G. raimondii (D5), and G. hirsutum (AD1), four HSF genes in G. herbaceum (A1) genome were investigated to get four HSF orthologous genes in G. hirsutum (AD1) A genome, and 17 HSF genes in G. arboreum (A2) genome and 17 HSF orthologous genes in G. hirsutum (AD1) A genome (G. hirsutum (AD1) A genome were detected to have orthologous genes in G. herbaceum (A1) and G. arboreum (A2) genomes, which represented 42.5% of the total HSF genes in G. hirsutum (AD1) A genome. A previous study indicated that all existing A genomes in Gossypium lineage originated from a common progenitor, A0 genome, and G. herbaceum (A1) and G. arboreum (A2) genome experienced independent evolution after the split of the A0 genome (G. herbaceum (A1), G. arboreum (A2), and G. hirsutum (AD1) A genome existed in ancestral genome A0 and remained in G. herbaceum (A1), G. arboreum (A2), and G. hirsutum (AD1) A genomes although they experienced independently long-term evolutionary history, but HSF genes in G. herbaceum (A1) genome missed more according to orthologous analysis between G. herbaceum (A1), G. arboreum (A2), and G. hirsutum (AD1) A genomes.To trace the evolutionary history of HSF genes in the Upland cotton genome, we combined the MCscanX software and validation of phylogeny to confirm HSF orthologous gene pairs between ectively . CombineA genome  were obt0 genome . FollowiG. hirsutum (AD1) D genome, 33 of 38 HSF genes were detected to have orthologous genes in G. raimondii (D5) genome, representing 86.8% of the total HSF genes in G. hirsutum (AD1) D genome. For G. raimondii (D5) genome, all HSF genes were detected to have orthologous genes in G. hirsutum (AD1) D genome. These results meant that members of the HSF gene family in G. raimondii (D5) genome and G. hirsutum (AD1) D genome kept excellent collinear relationships after the formation of G. hirsutum (AD1), but HSF genes in G. hirsutum (AD1) D genome showed significant expansion compared with those in G. raimondii (D5) genome.In G. herbaceum, G. arboreum, G. thurberi, G. raimondii, G. turneri, G. longicalyx, G. australe, G. kirkii, G. hirsutum, G. barbadense, G. tomentosum, G. mustelinum, and G. darwinii, representing 12.9, 11.83, 19.79, 14.76, 12.49, 21.03, 14.71, 13.44, 10.27, 34.66, 33.94, 35.23, and 27.75% of total protein-coding genes in G. herbaceum, G. arboreum, G. thurberi, G. raimondii, G. turneri, G. longicalyx, G. australe, G. kirkii, G. hirsutum, G. barbadense, G. tomentosum, G. mustelinum, and G. darwinii genomes (G. thurberi (D1) genome were identified following the identification of tandemly duplicated genes in plants. However, for the remaining 12 cotton genomes, we did not get any tandemly duplicated genes of the HSF gene family in these cotton genomes. So, the TD events brought an increase of the members of the HSF gene family in G. thurberi (D1) genome, but the HSF gene family in 12 cotton species did not experience TD event, which meant that members of the HSF gene family were discretely distributed into 12 cotton genomes.In this part, we investigated this important mechanism that occurred in different cotton genomes to get more evidence for the expansion of the HSF gene family. With the method implemented in PTGBase, we obtained 5,672, 5,118, 6,237, 6,013, 4,956, 8,070, 5,630, 4,930, 7,635, 25,877, 26,570, 26,306, and 21,726 tandemly duplicated genes in  genomes . ThroughG. hirsutum (AD1) acc. TM-1 and G. barbadense (AD2) acc. Hai7124. These treatments of different periods (hours) include control , heat , and cold  in G. hirsutum (AD1) and G. barbadense (AD2), respectively. After curation, we got 32 treatments in total. Using all available RNA-seq short-reads data of different samples of G. hirsutum (AD1) and G. barbadense (AD2), we implemented an expression analysis of total protein-coding genes in G. hirsutum (AD1) and G. barbadense (AD2) genome and further retrieved expression values of 78 and 78 HSF genes from different G. hirsutum (AD1) and G. barbadense (AD2) samples, respectively (G. hirsutum (AD1) genome, 76 HSF genes were detected to have expression in 16 samples, with 2 HSF genes (Ghi_A01G06131 and Ghi_D01G05506) having no expression in any of the samples. In G. barbadense (AD2) genome, 76 HSF genes have expression among 16 samples and two HSF genes (Gobar.A01G125400.1 and Gobar.D01G132500.1) were not detected to have expression in any of the samples. In G. hirsutum (AD1) genome, the 76 expressed HSF genes were divided into two different groups, I and II, and the I group contained 35 HSF genes, with the II group containing 41 HSF genes (G. barbadense (AD2) genome, 76 expressed HSF genes also were divided into two different groups, I and II, but the I group contained 46 HSF genes and the II group contained 30 HSF genes (G. hirsutum (AD1) and G. barbadense (AD2) indicated different expression patterns despite the implementation of identical experimental materials with identical heat and cold stresses.To investigate the expression differences of cotton HSF genes for heat stress tolerance, we analyzed the expression profiling of HSF genes in leaves under different treatments  of ectively . Out of SF genes . In G. bSF genes . These rG. hirsutum (AD1) and G. barbadense (AD2), we obtained 61 HSF orthologous gene pairs between the two genomes. In G. hirsutum (AD1) genome, 31 and 30 HSF genes from 61 orthologous gene pairs were located on G. hirsutum (AD1) A and D genomes, respectively, with one HSF gene locating on unknown pseudomolecular in G. hirsutum (AD1) D genome. In G. barbadense (AD2) genome, there were 61 HSF genes from 61 orthologous gene pairs and one HSF gene was not detected on any chromosome, with 30 HSF genes locating on A genome and 30 HSF genes locating on D genome. The no expression HSF genes, Ghi_A01G06131 versus Gobar.A01G125400.1 and Ghi_D01G05506 versus Gobar.D01G132500.1, were orthologous gene pairs between G. hirsutum (AD1) and G. barbadense (AD2) genomes. After removing the no expression HSF genes, 59 HSF orthologous gene pairs between G. hirsutum (AD1) and G. barbadense (AD2) genomes were selected to investigate the difference of expression patterns (G. herbaceum (A1), G. hirsutum (AD1), and G. barbadense (AD2), four HSF genes in G. herbaceum (A1) were detected to have four and four orthologous HSF genes in G. hirsutum (AD1) and G. barbadense (AD2), respectively. Only three of four HSF orthologous gene pairs between G. hirsutum (AD1)  and G. barbadense (AD2)  were expressed in different abiotic stress treatments and demonstrated different downregulated expression patterns regardless of control, cold, or heat stresses (According to the collinear relationship between patterns . Throughstresses .G. hirsutum (AD1) genome, we collected five genome-sequenced plant species, including the model plant A. thaliana, three cotton diploid species  and one tetraploid species [G. hirsutum (AD1)]. The allotetraploid cotton, G. hirsutum (AD1), named Upland cotton, is one of the most economically important crops which dominates over 98% of cotton production worldwide. Previous reports illustrated that G. hirsutum (AD1) was generated from hybridization between cotton A-genome progenitor and D-genome ancestor by chromosome doubling. Upland cotton D genome originated from G. raimondii (D5), and the A genome originated from the common progenitor, A0 genome, of all existing A genomes in Gossypium lineage. Furthermore, the formation of G. hirsutum (AD1) has preceded the split of G. herbaceum (A1) and G. arboreum (A2). These two cotton A genomes were shown to evolve independently from their common ancestor (G. hirsutum (AD1) genome, we got 40 and 38 HSF genes in G. hirsutum (AD1) A and D genomes, which meant that the D genome in G. hirsutum (AD1) missed two HSF genes compared with G. hirsutum (AD1) A genome. Through comparative genomic analysis between G. raimondii (D5) and G. hirsutum (AD1) D genome, we got 33 HSF orthologous gene pairs between the two genomes, which meant that G. raimondii (D5) missed five HSF genes after independent evolution. For the comparisons of G. herbaceum (A1), G. arboreum (A2), and G. hirsutum (AD1) A genomes, we got 17 HSF orthologous gene pairs between G. arboreum (A2) and G. hirsutum (AD1) A genomes, containing four HSF orthologous gene pairs, which meant that G. herbaceum (A1) and G. arboreum (A2) missed more HSF genes after the split of their common ancestor, but they kept four HSF orthologous genes inherited from their common ancestor, A0 genome. These four common conserved HSF genes in G. herbaceum (A1), G. arboreum (A2), and G. hirsutum (AD1) A genome might experience stronger natural selection pressures and performed a key biological function for resisting heat stress tolerance for adapting to the changing environment. From the evolutionary history of cotton diploid and tetraploid genomes, we knew that ancestral cotton genomes experienced loss of the members of the HSF gene family, but the current cotton tetraploid genomes kept more members of the HSF gene family.To trace the evolutionary history of HSF genes in ancestor . ThroughHeat shock transcription factor gene family analysis among different cotton diploid and tetraploid genomes showed significant differences in number variation of the members of the HSF gene family. Apparently, members of the HSF gene family in cotton tetraploid genomes indicated a larger family size of the HSF gene family than those in cotton diploid genomes. Members of the HSF gene family in subgenomes of cotton tetraploid genomes showed expansion compared with those in cotton diploid genomes. These results demonstrated that HSF genes in cotton diploid genomes experienced weaker selection pressures leading to fast loss of HSF genes during the evolutionary history, but the HSF genes in subgenomes of cotton tetraploid genomes experienced stronger selection pressures after hybridization. The analysis of number variation of the members of the HSF gene family illustrated that the cotton tetraploid species may indicate stronger ability for the resistance of heat stress tolerance than diploid species.G. hirsutum (AD1) and G. barbadense (AD2) diverged after the formation of cotton allotetraploid AD genome at approximately 0.96 Mya, suggesting that these two genomes kept many orthologous gene pairs (G. hirsutum (AD1) and G. barbadense (AD2) genomes, respectively. From collinear analysis, 122 HSF genes from 61 orthologous gene pairs in G. hirsutum (AD1) and G. barbadense (AD2) were used to detect the expression pattern of HSF orthologous genes in cotton tetraploid species. After the analysis of difference expression, 61 and 61 HSF genes in G. hirsutum (AD1) and G. barbadense (AD2), respectively, indicated totally different expression pattern, which meant the functional divergence of HSF orthologous genes between the two genomes. These results provided novel insights for functional studies and molecular breeding for the cotton community.A previous study illustrated that ne pairs . Based oG. thurberi (D1), G. raimondii (D5), G. turneri (D10), G. longicalyx (F1), G. australe (G2), and G. kirkii (K) compared with those in G. herbaceum (A1) and G. arboreum (A2). As expected, cotton tetraploid genomes have more members of the HSF gene family than diploid genomes. Phylogenetic analysis revealed that 621 HSF genes in 13 cotton genomes were clustered into two different groups, and the I group contained all HSF genes of HSFA and HSFC, with the II group containing all HSF genes of HSFB according to the conserved domains and phylogeny of HSF genes. Comparing A. thaliana, G. herbaceum (A1), G. arboreum (A2), G. raimondii (D5), G. herbaceum (A1), and G. arboreum (A2), four HSF genes were inherited from the common progenitor, A0, of all existing cotton A genomes, and members of the HSF gene family indicated a significant expansion in G. arboreum (A2) and G. hirsutum (AD1) A genome compared with G. herbaceum (A1). However, HSF genes in G. hirsutum (AD1) D genome experienced a significant expansion compared with those in G. raimondii (D5). Analysis of TD events of HSF genes revealed that the HSF gene family in G. thurberi (D1) genome has experienced TD event, but not in the remaining 12 cotton genomes. Expression analysis revealed that HSF orthologous gene pairs between G. thurberi (D1) and G. barbadense (AD2) showed a totally different expression trend under identical heat and cold stresses, which is beneficial for cotton molecular breeding and directed screening. This study is the first to trace the evolutionary history and expression features of the HSF gene family in 13 cotton genomes and provides more biological evidence to study the HSF gene family in response to abiotic stress tolerance in cotton genomes, particularly expression diversity of HSF genes in different treatments of leaves among different G. hirsutum (AD1) and G. barbadense (AD2) accessions. We hope this work will provide novel insights for the study of HSF genes under environmental stress in different cotton genomes and a widespread application model for the study of HSF gene families in plants.According to the conserved domains of HSF gene family in plants, we identified 621 HSF genes among 13 genome-sequenced cotton species distributed into eight diploid and five tetraploid species. Comparative genomics analysis demonstrated that members of the HSF gene family in cotton diploid genome showed a significant expansion in The original contributions presented in the study are included in the article/YL and QC conceived and designed the experiments. YL analyzed the data and prepared the manuscript. JW and JZ performed the experiments and analyzed the data. ZG performed phylogenetic analysis and revised the manuscript draft. ZL collected genomic data, performed comparative analysis of HSF genes, and revised the manuscript draft. XA and XL performed the tandem duplication analysis of HSF genes and revised the manuscript draft. QC revised the manuscript. All authors read and approved the final manuscript.ZL was employed by company Adsen Biotechnology Corporation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rapid antigen tests  are cheaper and faster than nucleic acid amplification tests  for SARS-CoV-2 infection, with variable reported sensitivity. A horse racetrack in California experienced a COVID-19 outbreak among staff and used BinaxNOW to supplement RT-PCR. Utility of BinaxNOW in detecting SARS-CoV-2 infection in a workplace outbreak was assessed.t) < 30.Between November 25–December 22, 2020, anterior nasal swabs were collected from racetrack staff for six rounds of paired BinaxNOW and RT-PCR tests. BinaxNOW tests were interpreted according to manufacturer instructions. RT-PCR was performed at the state public health lab using the ThermoFisher TaqPath COVID-19 Combo Kit. Staff with positive results on either test were isolated and removed from subsequent testing. Viral cultures were attempted on specimens with cycle threshold  (17.8 vs. 28.5) (p < 0.001). In dual positive pairs, median time from specimen collected to RT-PCR result reported was 4 days (range 1-6), compared to the 15-minute BinaxNOW reporting time. Of 100 Ct < 30 specimens, 51 resulted in positive virus isolation, 45 (88.2%) of which were BinaxNOW-positive.Overall, 769 paired results from 342 staff were analyzed. Most were of Hispanic ethnicity (62.0%) and ages ranged from 18 to 92 years (median 52). BinaxNOW performance compared to RT-PCR (95% CI) was as follows: positive percent agreement (PPA) 43.3% (34.6%–52.4%); negative percent agreement (NPA) 100% (99.4%–100%); positive predictive value (PPV) 100% (93.5%–100%); negative predictive value 89.9% (87.5%–92.0%). Among 127 RT-PCR-positive specimens, those with paired BinaxNOW-positive results (n = 55) had a lower mean Ct value and positive viral cultures, which could suggest that among RT-PCR-positive specimens, those that are BinaxNOW-negative may be less likely to contain infectious virus than those that are BinaxNOW-positive.High NPA and PPV support immediate isolation of BinaxNOW-positive individuals, while low PPA supports confirmatory testing following BinaxNOW-negative results. BinaxNOW performed better in paired specimens with lower CDavid Seftel, M.D., M.D., M.B.A., Enable Biosciences, Inc


Authors would like to correct the errors in their publication.The original article has been corrected.Incorrect Supplementary Information (ESM2) deleted now.The line “A color version of this figure is available online” to be deleted from all figure captions since the color figures in print version is not charged now.Missing references for the text citations were updated hereAlongi, 2002Alongi DM (2002) Present State and Future of the World’s Mangrove Forests. Environ Conserv 29:331–349. 10.1017/S0376892902000231Alongi and Dixon, 2000Alongi DM, Dixon P (2000) Mangrove primary production and above-and below-ground biomass in Sawi Bay, southern Thailand. Phuket Mar Biol Cent Spec Publ 22:31-38.Arfi et al. 2012Avicennia marina and Rhizophora stylosa. FEMS Microbiol Ecol 79:433–444. 10.1111/j.1574-6941.2011.01236.xArfi Y, Buée M, Marchand C, et al (2012) Multiple markers pyrosequencing reveals highly diverse and host-specific fungal communities on the mangrove trees Krishna and Mohan, 2017Krishna MP, Mohan M (2017) Litter decomposition in forest ecosystems: a review. Energy, Ecol Environ 2:236–249. https://doi.org/10.1007/s40974-017-0064-9Murdiyarso et al. 2015Murdiyarso D, Purbopuspito J, Kauffman JB, et al (2015) The potential of Indonesian mangrove forests for global climate change mitigation. Nat Clim Chang 5:1089–1092. 10.1038/nclimate2734Quadros et al. 2019Quadros AF, Nordhaus I, Reuter H, Zimmer M (2019) Modelling of mangrove annual leaf litterfall with emphasis on the role of vegetation structure. Estuar Coast Shelf Sci 218:292–299. 10.1016/j.ecss.2018.12.012


We observed altered gene expression in several DNA repair pathways including homologous recombination repair, non-homologous end-joining and mismatch repair, with hypoxia primarily resulting in downregulation of gene expression. The extent of gene expression changes was dependent on hypoxic severity. Some, but not all, of these downregulations were directly under the control of HIF activity. For example, the downregulation of LIG4, a key component of non-homologous end-joining, was reversed upon inhibition of the hypoxia-inducible factor (HIF). In contrast, the downregulation of the mismatch repair gene, PMS2, was not affected by HIF inhibition. This suggests that numerous molecular mechanisms lead to hypoxia-induced reprogramming of the transcriptional landscape of DNA repair. Whilst the global impact of hypoxia on DNA repair gene expression is likely to lead to genomic instability, tumorigenesis and reduced sensitivity to anti-cancer treatment, treatment re-sensitising might require additional approaches to a simple HIF inhibition.Glioblastoma, a grade IV astrocytoma, has a poor survival rate in part due to ineffective treatment options available. These tumours are heterogeneous with areas of low oxygen levels, termed hypoxic regions. Many intra-cellular signalling pathways, including DNA repair, can be altered by hypoxia. Since DNA damage induction and subsequent activation of DNA repair mechanisms is the cornerstone of glioblastoma treatment, alterations to DNA repair mechanisms could have a direct influence on treatment success. Our aim was to elucidate the impact of chronic hypoxia on DNA repair gene expression in a range of glioblastoma cell lines. We adopted a NanoString transcriptomic approach to examine the expression of 180 DNA repair-related genes in four classical glioblastoma cell lines  exposed to 5 days of normoxia (21% O Surviva options . Alterna options . Despite options .2  components leading to reduced HRR capacity . Also, cWe adopted a NanoString transcriptomic approach to assess the impact of chronic hypoxia on DNA repair genes in glioblastoma cell lines. We found that hypoxia specifically affects mismatch repair (MMR), non-homologous end-joining (NHEJ) and homologous recombination repair (HRR). Additionally, we provide evidence that the hypoxia inducible factor (HIF) play a role in downregulation of some, but not all, key DNA repair genes. Downregulation of DNA repair genes by hypoxia will have significant clinical impact for cancer management and should be considered when designing new treatment methods and protocols.Cell culture reagents were from Gibco Life Technologies and Foetal Calf Serum from Harlam Seralab (UK). Acriflavin was purchased from Sigma Aldrich. β-Actin (Ab8226), PMS2 (Ab110638) and anti-mouse HRP (Ab6808) antibodies were from abcam. HIF-1α (20960-1-AP) and HIF-2α (A700-003) were from Bethyl. Anti-rabbit HRP (7074S) antibody was from Cell Signalling.2. For hypoxic experiments, cells were incubated in a Don Whitley H35 Hypoxystation for 1% O2 or a New Brunswick Galaxy 48R incubator for 0.1% O2. Cells were routinely tested for mycoplasma infection.U87-MG  and T98G  were purchased from ATCC. U251-MG  were purchased from CLS. D566-MG cells  were a kind gift from Prof. DD Bigner . U87-MG and T98G were cultured in modified Eagle’s Medium (EMEM) with 10% FCS, 1% Sodium pyruvate. D566-MG and U251-MG were cultured using EMEM, 10% FCS, 1% sodium pyruvate and 1% non-essential amino acids. Cells were maintained at 37 °C in 5% CO2. RNA was extracted using High Pure RNA Extraction kit . A total of 100 ng of total RNA was used in the NanoString assay. The expression of 180 DNA repair genes, plus 12 housekeeping genes, were assessed using the NanoString nCounter Vantage™ RNA Panel for DNA Damage and Repair (LBL-10250-03) following the manufacturer’s protocol. Analysis of the NanoString data was conducted using nSolver™ Analysis Software (3.0) with nCounter® Advanced Analysis plug-in (2.0.115). Data are available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE139250 and inCells were cultured in 6 cm dishes for 5 days at 21%, 1% or 0.1% O® VILO  following the manufacturer’s guidelines. RT-PCR experiments were performed and analysed as described in LIG4 Forward: TCCCGTTTTTGACTCCCTGG Reverse: GGCAAGCTCCGTTACCTCTG, ABL Forward: TGGGGCATGTCCTTTCCATC Reverse: GATGTCGGCAGTGACAGTGA, ERCC4 Forward: CTCCCTCGCCGTGTAACAAA Reverse: ACACCAAGATGCCAGTAATTAAATC, FEN1 Forward: GTTCCTGATTGCTGTTCGCC Reverse: ATGCGAATGGTGCGGTAGAA, MSH5 Forward: GTTTGCGAAGGTGTTGCGAA Reverse: GTCTGAGACCTCCTTGCCAC, PARP1 Forward: GCCCTAAAGGCTCAGAACGA Reverse: CTACTCGGTCCAAGATCGCC, UBE2T Forward: ATGTTAGCCACAGAGCCACC Reverse: ACCTAATATTTGAGCTCGCAGGT, WRN Forward: TCACGCTCATTGCTGTGGAT Reverse: CAACGATTGGAACCATTGGCA, PMS2 Forward: AGCACTGCGGTAAAGGAGTT Reverse: CAACCTGAGTTAGGTCGGCA, CYCLOA Forward: GCTTTGGGTCCAGGAATGG Reverse: GTTGTCCACAGTCAGCAATGGT. Cyclophillin A was used as a housekeeping gene.RNA was extracted using High Pure RNA Extraction kit . Reverse transcription was conducted using SuperScriptLIG4 (NM_001352604.1) and PMS2 (NM_000535.7) was obtained through the UCSC Genome Browser using human genome assembly h38 , and GLUT1 or VEGFA levels assessed by RT-PCR to confirm hypoxia had indeed resulted in hypoxia-induced gene expression changes , D566-MG (0.1% = 2.6-fold) and U87-MG (0.1% = 1.6-fold)  at 0.1% O2  and MutLα  actively translocates along the DNA in search of discontinuity . EXO1 is 0.1% O2 , yet litsistance , was als.6-fold)  and 3B, .6-fold) . However studies . Other c 0.1% O2 . MSH5 is 0.1% O2 , therefo 0.1% O2 , potentiXRCC6, Ku80-XRCC5, DNA-PK-PRKDC) remain largely unaffected by hypoxia. However, in all cell lines tested, LIG4 (DNA Ligase IV) was downregulated at least 1.5-fold in hypoxia (1% and 0.1% O2.), although, this was only statistically significant in U87-MG  complex recognises the DSB . The endn U87-MG  and 3D.  hypoxia  and 3D. in XRCC4 .PMS2 and LIG4, essential components of their respective repair pathways, are involved in chemoresistance   , resulti2 (PHD2) . To testactivity . Treatmeget gene . In bothnificant . In contreatment , suggestPMS2 (MMR) and LIG4 (NHEJ), essential components of their respective pathways, were downregulated across multiple cell lines after chronic and acute hypoxic exposure, providing potential targets for re-sensitisation of hypoxic tumour cells to DNA damaging therapies.Tumour hypoxia plays an essential role in tumour progression, metastasis and drug resistance, leading to poor patient outcome, particularly for GBM patients. Hypoxia has been shown to impact numerous signalling pathways including the cell cycle, metabolism and apoptosis, yet additionally, hypoxia can have a significant impact on DNA repair mechanisms. We have shown here that several key DNA repair pathways were downregulated by hypoxia, including MMR and NHEJ, both essential pathways for the repair of modified/mismatch bases and double strand breaks respectively. Further analysis determined that PMS2 , and siRNA of LIG4 enhanced cell lethality of both chemotherapeutics U87-MG, (B) U251-MG and (C) D566-MG was utilised in RT-PCR experiments to assess the expression levels of selected genes. Data are expressed as logClick here for additional data file.10.7717/peerj.11275/supp-3Supplemental Information 32 and treated with 0–10 µM Acriflavin for 24 h. Control cells remained in 21% O2. RT-PCR was performed to assess GLUT1 expression levels. Data represent the fold change in GLUT1 expression relative to 21% O2. Data are from a single experiment. (B) HIF inhibition does not restore PMS2 mRNA levels in hypoxia. D566‑MG and U87-MG cells were incubated in 21% and 1% O2 for 24 h with and without 5 µM Acriflavin, the plots show the log2 fold change in mRNA levels with respect to levels for the 21% O2 samples. Data are the mean of at least two independent experiment.(A) D566-MG cells were incubated in 0.1% OClick here for additional data file.10.7717/peerj.11275/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.11275/supp-5Supplemental Information 5The data for each figures are on separate tabs.Click here for additional data file.10.7717/peerj.11275/supp-6Supplemental Information 62 for 5 days before examination of gene expression using the NanoString nCounter Vantage™ RNA Panel for DNA Damage and Repair. Differential gene expression analysis was performed using nSolver™.U87-MG, D566-MG, U251-MG and T98G were exposed to 21%, 1% or 0.1% OClick here for additional data file.


P. aeruginosa is a common infection among hospitalized patients, with increased levels of morbidity and mortality. This pathogen exhibits multiple resistance mechanisms to antibiotics. We analyzed the molecular epidemiology and activity of the main therapeutic options against P. aeruginosa isolated from RTI in Latin America (LATAM).Respiratory Tract Infection (RTI) caused by Isolates were collected from 36 sites in 10 countries during 2017-2019. Non-duplicate samples were consecutively collected. MICs were determined by broth microdilution and interpreted by CLSI criteria. A subset of imipenem non-susceptible isolates was selected for characterization of carbapenemase encoding genes via multiplex PCR and DNA sequencing. β-lactamase genes encoding ESBLs, carbapenemases, and plasmid-mediated AmpCs were investigated.P. aeruginosa were collected from RTI. Overall C/T [87.8% susceptible (S)] was the most active antimicrobial tested against P. aeruginosa isolates followed by amikacin (85.8% S) and imipenem/relebactam . Other antimicrobials had less than 80% susceptibility. C/T remained the most active agent including activity against imipenem and piperacillin/tazobactam non-susceptible isolates . 583 imipenem non-susceptible P. aeruginosa were selected for molecular analysis (Table 1). Thirty  isolates were confirmed to be producers of serine-carbapenemases , while 83 (14.2%) were MBL producers. KPC-2 was found in Colombia (9), Chile (6), Puerto Rico (4), Guatemala (3), and Brazil (2). GES-5 was identified in Mexico (3), Argentina (2) and Brazil (1). VIM-2 was the most common MBL encoding gene identified. IMP variants were observed in Brazil , Ecuador (IMP-13), Mexico (IMP-18), Panama (IMP-18) and Puerto Rico (IMP-18). SPM-1 was only encountered in Brazil. The production of ESBLs was low in most LATAM countries, except for Guatemala (80%) . A total of 2,044 Figure 1. Activities of selected antimicrobial agents against 2,044 P. aeruginosa isolated from respiratory tract infections in Latin America (2017 to 2019).TABLE 1. Molecular analysis of imipenem non-susceptible P. aeruginosa isolates in Latin America (LATAM) from Respiratory Tract Infection (N=583).Figure 2. Carbapenemases identified in 583 imipenem non-susceptible P. aeruginosa isolated from patients with respiratory tract Infections in Latin America (LATAM).P. aeruginosa infections. The prevalence of carbapenemases-encoding genes varied geographically in LATAM.CT, amikacin and IMI/REL showed good activity against RTI isolates and could represent effective treatment options for Leandro Cardinal, PharmD, PhD, MSD (Employee) Cicera P. Marcelino, n/a, MSD (Employee) Aline Okuma, n/a, MSD (Employee)MSD Brazil (Employee) Gustavo Mizuno, PharmD, Merck Sharp Dohme (Employee) Felipe Tuon, PhD, Merck Sharp Dohme Brazil (Scientific Research Study Investigator) Ana C. Gales, MD, MSD Pfizer  Marina Della Negra, Medical Doctor, MSD Brazil (Employee) Thales Polis, Medical Doctor, MSD Brazil (Employee)


OBJECTIVES/GOALS: We compared the validity of an International Classification of Diseases, Clinical Modification (ICD) algorithm for identifying high-grade cervical intraepithelial neoplasia and adenocarcinoma in situ (together referred to as CIN2+) from ICD 9th revision (ICD-9) and 10th revision (ICD-10) codes. METHODS/STUDY POPULATION: Using Tennessee Medicaid data, we identified cervical diagnostic procedures in 2008-2017 among females aged 18-39 years in Davidson County, TN. Gold-standard cases were pathology-confirmed CIN2+ diagnoses validated by HPV-IMPACT, a population-based surveillance project in catchment areas of five US states. Procedures in the ICD transition year (2015) were excluded to account for implementation lag. We pre-grouped diagnosis and procedure codes by theme. We performed feature selection using least absolute shrinkage and selection operator (LASSO) logistic regression with 10-fold cross validation and validated models by ICD-9 era  and ICD-10 era . RESULTS/ANTICIPATED RESULTS: Of 7864 cervical diagnostic procedures, 880 (11%) were true CIN2+ cases. LASSO logistic regression selected the strongest features of case status: Having codes for a CIN2+ tissue diagnosis, non-specific CIN tissue diagnosis, high-grade squamous intraepithelial lesion, receiving a cervical treatment procedure, and receiving a cervical/vaginal biopsy. Features of non-case status were codes for a CIN1 tissue diagnosis, Pap test, and HPV DNA test. The ICD-9 vs ICD-10 algorithms predicted case status with 68% vs 63% sensitivity, 95% vs 94% specificity, 63% vs 64% positive predictive value, 96% vs 94% negative predictive value, 92% vs 89% accuracy, and C-indices of 0.95 vs 0.92, respectively. DISCUSSION/SIGNIFICANCE OF IMPACT: Overall, the algorithm’s validity for identifying CIN2+ case status was similar between coding versions. ICD-9 had slightly better discriminative ability. Results support a prior study concluding that ICD-10 implementation has not substantially improved the quality of administrative data from ICD-9.


The article presents the results of studying the biodiversity and biotechnological potential of halophilicmicroorganisms from the thermal highly mineralized Berikey Lake, the salty Lake Tarumovskoye and saline soils ofthe Peri-Caspian Lowland (Republic of Daghestan). Denitrifying halophilic bacteria of the genus Halomonas andVirgibacillus were identified using microbiological methods and 16S rRNA gene analysis. A new species Halomonas sp. G2 (MW386470) with a similarity of the nucleotide sequences of the 16S rRNA genes is 95 %. Strain G2 isan extreme halophile capable of growing in the range of 5–25 % NaCl (optimum 25 %) and forming a carotenoidpigment. Mesophil, 30–37 °С (optimum 30 °С); neutrophil, pH 6–8 (optimum 7.2–7.4). Strain G2 chemolithotroph;reduces nitrate or nitrite as electron donors; catalase-, amylase-, protease- and β-galactosidase-positive; lipase-,oxidase- and urease-negative. Not able to hydrolyze inositol, indole; produces lysine, gelatin, ectoine; uses citrateand sodium malate as a source of carbon and energy; does not produce ornitin, H2S or acid from d-mannose, sucrose, glycerol, cellobiose, except for lactose and d-glucose. Susceptible to trimethoprim, ciprofloxacin, ofloxacin,kanamycin, vancomycin, rifampicin, cefuroxime, ampicillin, ceftazidime, fosfomycin, clarithromycin, cefepime, cefaclor. The G+C content in DNA is 67.3 %. A distinctive characteristic of the isolate was the production of industrially significant hydrolytic enzymes such as amylase, protease, β-galactosidase, and oxidoreductase  at aNaCl concentration of 25 % in the medium. Habitat: saline soils on the territory of the Tersko-Kumskaya lowland. The rest of the halophilic isolates of H. ventosae G1 (MW386469), H. elongata G3(MW386471), V. salinarius B2 (MW386472), and V. salinarius B3 (MW386473) had a high degree of similarity (100 %)with the type strains of H. elongata DSM 2581Тand V. salarius DSM 18441Т; the content of G+C in DNA was 65.8,66.5, 42.8 and 37.3 %, respectively. The strains had a high biotechnological potential at NaCl concentrations of 5 and25 % in the medium. The data obtained expanded the understanding of the diversity and ecological significanceof denitrifying bacteria in the functioning of arid ecosystems and make it possible to identify strains producingenzymes of industrial importance. The interest in extremophilic microorganisms is relativelyhigh due to their biological uniqueness. Agreat contributionto the study of natural microbial communities was made bythe school of Russian scientists . Archaeaand highly-specialized bacteria of the genera Alcaligenes,Bacillus, Halobacillus, Virgibacillus, Micrococcus, andPseudomonas  occupy a dominant place in ecologicalniches with a high salt content  and anthropogenic ecosystems with an increasedlevel of mineralizationThe largest ecosystems on the planet are saline and hypersaline environments . Daghestan isa unique natural province of Russia that has a variety ofnatural landscapes due to the influence of tectonic processes,the erosive activity of flowing waters, transgressive andregressive dynamics of the Caspian Sea, and arid climate.There are a number of works on the study of microbial communities of various ecological niches of the region: lithotrophic sulfur-oxidizing representatives of sulfide sources,hydrocarbon-oxidizing bacteria of the geothermal sourceof the Kizlyar field .The highly mineralized lakes of the Tersko-Kumskayalowland with high salinity form conditions for the existence of halophilic bacteria. Microorganisms from extremehabitats are the producers of valuable industrially importantenzymes, antibiotics; they can participate in soil biodegradation, and are highly resistant to contamination by foreignmicroflora .The study considers the spatial distribution of halophilicmicrobial communities of halophyte plants, saline soils, and highly mineralized lakes in arid regions of the Peri-CaspianLowland . Chemoorganoheterotrophic bacteria of the genera Virgibacillus, Bacillus,Halomonas and Salimicrobium from the phylum Firmicutesand Proteobacteria have proved to be the main componentsof the microbial flora of the Tersko-Kumskaya and TerskoSulakskaya provinces. A major correlation was revealedbetween isolated microbial communities and concentrationsof chemicals Na, K, Ca, Mg, Cl, Cu, Sr, SO4, Cl, and HCO3,as one of the main regulators of microbiological activity insoils and lakes.The aim of the paper is a molecular taxonomic studyof isolated halophilic bacteria and their biotechnologicalpotential.The objects of research are natural microbial communitiesof saline reservoirs and soils in the territory of the PeriCaspian Lowland of the Republic of Daghestan  (Table 1). Samples were taken in July–September 2014.Cultivation. For the cultivation of halophilic bacteria,a modified medium of the following composition (g/l) wasused: bacto yeast extract – 10.00, Na3C6H5O7 ∙5.5H2O –3.0, NaCl – 50, 100, 250, KCl – 2.0, MgSO4 ∙ 7H2O –20.0, glycerin – 4.0 . Bacto-peptone  – 5 g/lwas used as a substrate; the pH of the medium was adjustedto 7.2–7.4 by 1N HCl or 4M KOH (Russia) using a HannaInstrumentals pH 211 pH meter (Germany). The cultureswere incubated in a Binder-115 microbiological incubator(USA) at an operating temperature of (30–37)±1 °C for3–20 days.Morphology of bacterial cells  was studied using a light microscope CX21 FS1  and thePowerShot A640 digital camera  at a working magnification of ×600.Ecological and physiological characteristics of growth. Effect of NaCl concentration in an amount of 2 % ofthe medium volume for cell growth in liquid and solid mediawas determined at 30–37 °C in the Binder-115 incubator(USA). The growth was monitored at 24-hour intervals for7 days by measuring turbidity using a Genesys-20 spectrophotometer . The effect of temperature (30 and 37 °C) on the growth rate was determinedby cultivation under the same conditions.The use of growth substrates  was studied using standard methods.The use of electron acceptors. The ability to use nitrateas an electron acceptor was determined using the BD BBLTaxo Differentiation Discs Nitrate , in compliance with the manufacturer’sinstructions. The discs were impregnated with a solutioncontaining 40 % potassium nitrate and 0.1 % sodium molybdate. The reduction of nitrate to nitrite was detected by theaddition of sulfanilic acid and N,N-dimethyl-α-naphthylamine, which reacts with nitrite to form a red-colored substance-n-sulfobenzenazo-α-naphthylamine (positive result).In the absence of a change in color after the addition ofreagents (negative result), zinc dust was added to determinethe presence of unrecoverable nitrate or products other thannitriteDetermination of enzymatic activity. Hydrolase-producing bacteria were screened for amylase, proteinase,β-galactosidase, lactase, lipase, urease, and oxidoreductases on dishes of starch, tributyrin, andgelatin agar, depending on the concentrations of NaCl.Amylase activity – on the elective medium . The isolateswere incubated at 45 °C for 24–36 hours, tested with Lugolsolution . Potential amylase producers were selected based onthe ratio of the clearance zone diameter to the colony diameter. Protease was determined on media with agar and 10 %skimmed milk; β-galactosidase (lactase) – using indicatordisks impregnated with a special reagent-ortho-nitrophenylβ-d-galactopyranoside ; urease – using CLOtest ; lipase – tested on dishes with1 % tributyrin. Isolates showing clear zones of tributyrinhydrolysis were identified as lipase-producing bacteria.Determination of oxidoreductases: catalase – using 3 %H2O2 as a substrate in the medium for 24–48 hours, oxidase – by Kovac’s method . All screening testsfor enzymatic activity were performed in three repetitions.The bacteria were incubated at 37 °C for seven days.Antibiotic resistance  was determined by the intensity of bacterialgrowth on the basic agarized medium “B” in Petri dishesusing standard disks “Indicator paper systems for identifyingmicroorganisms” of NPO Microgen of Natsimbio Holding(Russia) with 10–30 μg of antimicrobial agent .G+C content and phylogenetic analysis. Genomic DNAwas isolated according to Marmur (1961) and Thomas  methods. The DNA nucleotide compositionwas determined by thermal denaturation (0.5 °C∙min–1)using a Cary-100 Bio UV-VIS spectrophotometer . The GC content in the composition of DNA –according to the method . Escherichiacoli K-12 DNA (51.7 %) was used as the standard.For phylogenetic analysis, the DNA was isolated fromsamples using the modified Birnboim–Doly alkaline DNA method  and the Wizard technologyof Promega (USA) . The concentration of the resulting DNA sample when using this methodwas 30–50 μg /ml. RNA in the resulting preparation is present in trace amounts .For polymerase chain reaction (PCR) and further sequencing of PCR fragments of the 16S rRNA gene for each ofthe studied samples, universal primer systems were usedto detect both eubacteria (11f-1492r)  and archaea (8fa-A915R) . The volumeof the amplification mixture was 50 μl with the followingcomposition: 1× buffer of BioTaq DNA polymerase (17 mM(NH4)2SO4, 67 mM Tris-HCl, pH 8.8, 2 mM MgCl2);12.5 nmol of each dNTP, 50 ng of the DNA matrix; 5 pmolof the corresponding primers and 3 units of BioTaq DNApolymerase . The temperature-timeprofile of the PCR was as follows: the first cycle – 94 °C×9 min, 55 °C×1 min, 72 °C×2 min; the next 30 cycles –94 °C×1 min, 55 °C×1 min, 72 °C×2 min; the final cycle –72 °C×7 min. The PCR products were analyzed by electrophoresis in 2 % agarose gel at an electric field strengthof 6 V/cm. Isolation and purification of PCR products werecarried out from low-melting agarose using a set of reagentsby WizardPCRPreps , according to themanufacturer’s recommendations.Sequencing of PCR products was carried out at the Centerof “Bioengineering” of the Russian Academy of Sciences,Moscow, by the method of  using a setof BigDyeTerminatorv.3.1 reagents on the ABIPRIZM 3730genetic analyzer . Standardprimers were used for sequencing .Analysis of 16S rRNA sequences. The primary analysisof the similarity of the nucleotide sequences of the 16S rRNAgenes of the studied strains was performed using the BLASTprogram on the following web-site: https://blast.ncbi.nlm.nih.gov .The 16S rRNA gene sequences of all studied strains aredeposited in GenBank: G1 – MW386469, G2 – MW386470,G3 – MW386471, B2 – MW386472, B3 – MW386473.Strains of halophilic bacteria G1, G2, G3, B2 and B3,isolated from salt lakes and salt marshes of the TerskoKumskaya and Tersko-Sulakskaya lowlands, grew at atemperature of 30–37 °C and pH 6.4–7.4. The culturesshowed steady growth in the agarized elective mediumin the presence of 5–25 % NaCl with an optimum of 5,10, 25 %, which indicated that they belonged to moderateand extreme halophiles following the known classification.The analysis of the 16S rRNA gene sequence allowed usto determine their phylogenetic position. The 16S rRNAgene sequences of the new halophilic strains were analyzedand compared with the 16S rRNA sequences of the validlydescribed bacterial species. The analysis has shown that thenew isolates belong to two genera of bacteria containing halophilic microorganisms Halomonas and Virgibacillus. Table 2 and Figure 1 demonstrate that theG2 strain represents a new species in the genus Halomonas.The H. ventosae G1 (MW386469) and H. elongata G3(MW386471) strains seem to belong to the species H. ventosae and H. elongata, respectively, while the V. salinariusB2 (MW386472) and V.salinarius B3 (MW386473) strainsbelong to the group of species related to V. salinarius.Characteristics of the Halomonas sp. G2 strainThe main object of further research is the strain Halomonas sp. G2. The GC content in the G2 strain DNA was67.3 %.Morphology of cells and colonies. Rod-shaped, gramnegative, mobile bacillus of the size of 0.8–1.0×1.5–3.0 µm.The cells were observed singly, in pairs, or in short chains. Cell mobility was ensured by one or two lateralflagella located on one side of the cell, forming endospores.On an elective solid medium, the strain formed an activegrowth of colonies of a round shape with a wavy edge, yellow and pale-yellow color with a gloss. With an increase inthe concentration of NaCl in the culture medium, a brightcarotenoid pigment appeared in the beige-colored colonies.On meat-peptone agar (MPA) – small colonies located closeto each other in a chain, rounded shape with a wavy edge,turning into a solid growth; smooth, shiny, light beige witha pinkish tinge. In all variants, a smearing consistency wasobserved .Physiology of strain growth . When determining the optimal growth parameters, theG2 strain was assigned to mesophiles  and moderate alkalophiles . As a representative of the genus Halomonas, it cangrow in a wide range of NaCl concentrations from 10 to25 % with an optimum of 25 %; an extreme halophileThe substrates and electron acceptors used. Oxygenratio. The G2 strain is capable of denitrification by usingnitrates as an electron acceptor, reducing them to nitrites.The differentiating characteristics of the G2 strain arepresented in Table 3. The strain had a positive reaction tolysine, gelatin, ectoine, lactose, and d-glucose; utilized citrate and Na malonate. Tests for β-galactosidase, amylase,protease, and catalase are positive; for oxidase, lipase, andurease – negative. Growth does not occur under anaerobicconditions.Antibiotic sensitivity. The G2 culture differed in sensitivity to trimethoprim of the sulfanilamide group; fluoroquinolones of the 1st and 2nd generations ; vancomycin of the macrolide group;rifamycins of the rifampin group; cefuroxime and ampicillin of the penicillin group; antibiotics of the 3rd generationcephalosporins of the macrolides ; antibiotics of the 4th generation cephalosporins .Characteristics of isolates G1, G3, B2, B3Rod-shaped, mobile cells of strains G1, G3 have the following sizes: 0.6–0.8 × 1.6–1.9 µm (G1), 0.7–1.0 × 1.5–2.5 µm (G3) . Single cells and chains ofthem were observed. Mobility was provided by flagellalocated on one side of the cell. The cells of the B2 andB3 strains were mobile, in the form of rods, with sizes:0.5–0.7×1.0–2.5 µm (B2) and 0.2–0.7×1.0–5.0 µm (B3);formed endospores. The biomass of isolated strains on theMPA medium is represented by a chain of colonies locatedone after another, differing in shape, color, size, pigment andmorphology . On an elective agar mediumwith 5–25 % NaCl  and 5–10 % NaCl , thecultures formed colonies with lipochromic pigment.The results of phylogenetic analysis of 16S rRNA genesequences indicated that the closest type strains (100 %) forG1 and G3 are the type strain of H. elongata DSM 2581T,and for B2 and B3 – V. salarius DSM 18441T. On thedendrogram, the cultures formed a common cluster withthe typical strains, making it possible to classify isolated cultures as these species. Related cultures for G1 and G3 areH. ventosae GQ903443, H. elongata NR 074782; for B2 andB3 – V. salarius MK785132, B. brevis JF802177, V. olivaeNR 043572, H. arcis MK063873, V. salarius NR 041270,which combined the typical features of moderate and extreme halophiles.The distinctive characteristics differentiating the culturesof H. ventosae G1 and H. elongata G3 were: optimumgrowth at 5–25 % NaCl vs. 32 %; pH 7.2–7.4 vs. 7–9; noutilization of sucrose, glycerol, d-mannose, cellobiose,lactose, and production of urease, oxidase, and protease(except G3) at a concentration of 5–25 % NaCl in themedium (see Table 3). At the same time, such traits forV.salarius B2 and B3 strains in comparison with the typicalV. salarius DSM 18441T were: no need for d-mannose, theability to produce amylase, protease, and β-galactosidaseenzymes at a concentration of 5–10 % NaCl in the medium(Table 4).Characteristics of genera Halomonas and VirgibacillusThus, based on phenotypic and genetic studies, we providea brief description of the strain of the new species Halomonas sp. G2 and halophilic strains of H. ventosa G1, H. elongata G3, V. salinarius B2, V. salinarius B3.Currently, the genus Halomonas includes 91 species,where H. elongata acts as the type species . The Halomonadaceaefamily was first described in 1988 by combining moderatelyhalophilic and marine bacteria of the genera Deleya andHalomonas . Over the past threedecades, many species have been assigned to the genusHalomonas, the domain Bacteria, the phylum Proteobacteria, the class Gammaproteobacteria, the order Oceanospirillales, and the family Halomonadaceae; however, at the timeof writing, 7 species have been reclassified. Representativesof the genus – gram-negative, facultative anaerobes, aerobes,prototrophs, mesophiles, denitrifying, produce exopolysaccharides; they mainly utilize oxygen, nitrate or nitrite as anelectron acceptor; under conditions of saline stress, theysynthesize ectoine, which protects cells from adverse environmental influences .The genus Virgibacillus was created as a result of thereclassification of Bacillus pantothenticus, Virgibacilluspantothenticus and, subsequently, established as a speciesof Virgibacillus . At the moment, the genus consists of 27 species,representatives of which are gram-positive, obligate aerobesor facultative anaerobes, moderate halophiles, chemotaxonomic; the main fatty acid is C15:0 .Description of the strain of the new species Halomonas sp. G2. The G2 strain cells are encapsulated, motile,aerobic, gram-negative bacillus 0.8–1.0 × 1.5–3.0 µm;observed singly or in a chain of 2 to 4 interlocked cells.The G2 strain is an extreme halophile, capable of growingin the range of 10–25 % NaCl (25 % optimum) and forming a carotenoid pigment. On an elective solid mediumwith 25 % NaCl, it forms colonies of a round shape with awavy edge, beige color with a gloss; forms areas of brightcarotenoid pigment. The strain grows on meat-peptonebroth. The species is mesophile, the temperature range is30–37 °C (30 °C optimum). Neutrophil, pH 6–8 (7.2–7.4optimum). Denitrifying strain; chemolithotrophic; reducesnitrate or nitrite as electron donors; catalase-, amylase-,protease-, and β-galactosidase-positive; lipase-, oxidase-,and urease-negative. Unable to hydrolyze inositol, indole;produces lysine, gelatin, ectoine; uses sodium citrate andmalate as a source of carbon and energy; does not produceornithine; H2S and acid from d-mannose, sucrose, glycerol,cellobiose, except lactose and d-glucose. Susceptible to trimethoprim, ciprofloxacin, ofloxacin, kanamycin, vancomycin, rifampicin, cefuroxime, ampicillin, ceftazidime, fosfomycin, clarithromycin, cefepime, cefaclor. The GC contentin DNA is 67.3 %.Based on its physiological, biochemical, and phylogeneticproperties, the G2 strain represents a new species, calledHalomonassp. G2. Adistinctive characteristic of the isolateis the production of hydrolytic enzymes protease, amylase,β-galactosidase, and oxy-reductase-catalase at a concentration of 25 % NaCl in the medium.Habitat: soil  on the territory of theTersko-Kumskaya lowland .Tersko-Kumskaya lowland .Description of the strains Halomonas ventosae G1(MW386469), and Halomonas elongata G3 (MW386471).Halomonas G1 and G3 strains are aerobes, gram-negative,denitrifying; mesophiles, prototrophs, chemolithotrophs,and extreme halophiles (5 to 25 % NaCl). Unable to hydrolyze inositol; produce lysine, ornithine, gelatin, ectoine;reduce nitrate or nitrite as electron donors; utilize citrate(excluding G3) and sodium malonate as a source of carbonand energy; do not produce H2S and acid from d-mannose,sucrose, glycerol, cellobiose, except for d-glucose. TheGC content in the DNA for G1 and G3 are 65.8 and 66.5 %,respectively. On the basis of phenotypic and genotypic characteristics, isolated bacteria are classified as H. ventosae G1(MW386469), and H. elongata G3 (MW386471).Habitat: saline soils  and Lake Tarumovskoe on the territoryof the Tersko-Kumskaya lowland . The type strain of H. elongata DSM 2581Т wasisolated from equipment for extraction of salt from brine.Description of V. salinarius B2 (MW386472) andB3 (MW386473) strains. The strains are gram-positive;mesophiles, neutrophils, chemolithotrophs, moderate halophiles . The cultures are unable tohydrolyze inositol, sodium malonate; they do not producelysine (except for B3), indole, H2S and acid from d-mannose,sucrose, except for d-glucose, reduce nitrate to nitrite andare capable of utilizing the polypeptide substrate gelatin andsodium citrate as a carbon source. The GC content in theDNA of strains B2 andB3 was 42.8 and 37.3 %, respectively.Based on the phenotypic and genotypic characteristics, theisolated cultures have been classified as V. salinarius B2(MW386472) and V. salinarius B3 (MW386473) strains.Habitat: waters of technogenic, highly mineralizedBerikey Lake . The type strain Virgibacillus salarius DSM 18441Тisolated from the salt crust of the Gharsah Salt Lake in Shattal Gharsah (Sahara) in Tunisia .Biotechnological potentialof halophilic microorganismsHalophilic bacteria are increasingly being studied for theirbiotechnological potential for the production of biochemically active and resistant enzymes to alkaline pH, hightemperature and salt concentration . These multifaceted properties are useful fora variety of industries , suchas fermented food, textiles, pharmaceuticals, cosmetics,and leather production .Most producers of extracellular hydrolytic enzymes lipase,amylase, protease, inulinase, xylanase, cellulase, DNase,and pectinase are halophilic bacteria, including strains ofthe genera Halomonas and Virgibacillus .Isolation of natural strains in these studies made it possibleto discover a new species Halomonas sp. G2 (MW386470)and new strains Halomonas G1 (MW386469) and G3(MW386471), Virgibacillus B2 (MW386472) and B3(MW386473), capable of producing hydrolytic enzymesand oxidoreductase .The study confirms the biotechnological and scientific importance of halophilic denitrifying bacteria inhabiting theextremophilic ecological niches of the Peri-Caspian Lowland in the Republic of Daghestan. 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