Escherichia coli is a clinically important bacterial species implicated in human- and livestock-associated infections worldwide. The bacterium is known to reside in the guts of humans, livestock, and wild animals. Escherichia coli is a common bacterial species in the gastrointestinal tracts of warm-blooded animals and humans. Pathogenicity and antimicrobial resistance in E. coli may emerge via host switching from animal reservoirs. Despite its potential clinical importance, knowledge of the population structure of commensal E. coli within wild hosts and the epidemiological links between E. coli in nonhuman hosts and E. coli in humans is still scarce. In this study, we analyzed the whole-genome sequencing data of a collection of 119 commensal E. coli strains recovered from the guts of 55 mammal and bird species in Mexico and Venezuela in the 1990s. We observed low concordance between the population structures of E. coli isolates colonizing wild animals and the phylogeny, taxonomy, and ecological and physiological attributes of the host species, with distantly related E. coli strains often colonizing the same or similar host species and distantly related host species often hosting closely related E. coli strains. We found no evidence for recent transmission of E. coli genomes from wild animals to either domesticated animals or humans. However, multiple livestock- and human-related virulence factor genes were present in E. coli of wild animals, including virulence factors characteristic of Shiga toxin-producing E. coli (STEC) and atypical enteropathogenic E. coli (aEPEC), where several isolates from wild hosts harbored the locus of enterocyte effacement (LEE) pathogenicity island. Moreover, E. coli isolates from wild animal hosts often harbored known antibiotic resistance determinants, including those against ciprofloxacin, aminoglycosides, tetracyclines, and beta-lactams, with some determinants present in multiple, distantly related E. coli lineages colonizing very different host animals. We conclude that genome pools of E. coli colonizing the guts of wild animals and humans share virulence and antibiotic resistance genes, underscoring the idea that wild animals could serve as reservoirs for E. coli pathogenicity in human and livestock infections.IMPORTANCEEscherichia coli is a clinically important bacterial species implicated in human- and livestock-associated infections worldwide. The bacterium is known to reside in the guts of humans, livestock, and wild animals. Although wild animals are recognized as potential reservoirs for pathogenic E. coli strains, the knowledge of the population structure of E. coli in wild hosts is still scarce. In this study, we used fine resolution of whole-genome sequencing to provide novel insights into the evolution of E. coli genomes from a small yet diverse collection of strains recovered within a broad range of wild animal species , the coevolution of E. coli strains with their hosts, and the genetics of pathogenicity of E. coli strains in wild hosts in Mexico. Our results provide evidence for the clinical importance of wild animals as reservoirs for pathogenic strains and highlight the need to include nonhuman hosts in the surveillance programs for E. coli infections. Escherichia coli is the most prevalent aerobic bacterial species that resides in the intestines and feces of warm-blooded animals and dominates the corresponding microbiomes  being most prevalent, followed by B2 , A , D , and E   across both domesticated/livestock and wild animals in North America, which motivated us to examine the prevalence of transmissions and recent divergences at a fine resolution.We sequenced 119 strains isolated from 55 wild animal host species predominantly in Mexico and found them to capture much of the known global ins; 6%) . The hig E. coli . Analyzi10.1128/mSphere.00738-20.1FIG S1E. coli from wild hosts with strains from other sources. (A) Alignment-free phylogenetic tree for the E. coli strains in the context of previously sequenced genomes in the Enterobase database and the associated metadata of phylogroup and isolation sites. (B) The distributions of continent of isolation and host in the curated dataset, compared with those in the Enterobase collection. Accession number and metadata for strains are provided in FIG S1, TIF file, 0.9 MB.Phylogeny of Copyright © 2021 Murphy et al.2021Murphy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli strains from wild animals and their hosts to understand the long‐term concordance between the evolutionary histories of E. coli and their host species and the extent to which the genetic distance between the host species agrees with the regional population structure of E. coli. Both comparisons of host and E. coli distance matrices    .We compared the phylogenetic trees for our collection of el test)  and comp0 tests)  rejectednd hosts , with cld family . This wac groups , as 0.95y genes) . Togethe10.1128/mSphere.00738-20.2FIG S2FIG S2, TIF file, 0.7 MB.Comparison between core genome and accessory genome trees. The core genome and accessory genome trees were reconstructed from the SNPs in the core genome alignment and from the pattern of gene presence and absence, respectively. Download Copyright © 2021 Murphy et al.2021Murphy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli collection from wild-animal hosts, we compared the rates of nonsynonymous and synonymous single-nucleotide evolution (aK/sK) since their last shared common ancestor. Of 3,529 genes in the core genome, 242 had a aK/sK value above 1 in at least one strain, with an average of 11.7 genes, i.e., 0.3% of total genes, per strain falling in this category  The distribution of functional classifications for 256 genes that were under positive selection, compared with the baseline distribution in the pan genome. We excluded genes without assigned COG group. The letters S, G, P, C, H, and T represent unknown functions, carbohydrate metabolism and transport, inorganic ion transport and metabolism, energy production and conversion, coenzyme metabolism, and signal transduction, respectively. The full interpretation for other classes is found in reference Copyright © 2021 Murphy et al.2021Murphy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli isolates colonizing wild animals in relation to those of the external collection of E. coli isolates coming from other hosts. Our analysis did not identify E. coli isolates with zero, or close to zero, SNP distance within wild hosts or food animal sources, which would suggest direct, recent transmissions. However, the analysis of SNP clusters revealed two links between wild hosts and clinical strains. One link weakly (38 SNPs) connected a sample (SAMEA4607586) from the avian species Aratinga canicularis to two clinical strains from Mexico (SNP cluster ID PDS000073768.1), and one moderately strongly (17 SNPs) connected an E. coli isolate from the avian species of Gallus gallus and 7 clinical strains (SNP cluster PDS000066827.4). One and nine of the clinical strains were from France and the United States, respectively. Interestingly, of 22 external strains isolated from wild hosts in Mexico, 17 formed parts of SNP clusters with our strains, showing extensive circulation of E. coli across wild hosts in Mexico , food (n = 12), and environmental sources (n = 13) . We reconstructed the Bayesian tree of these 158 strains and found their last common ancestor to have lived about 1,000 years ago, with a substantial expansion of the clade over the past 100 years , habitat breadth , diet breadth , and social group size  of hosts correlated significantly with E. coli virulence gene counts. More diverse habitats and diets of the hosts were associated with fewer virulence genes, and the formation of larger social groups was associated with more virulence genes in E. coli isolates colonizing these hosts  Correlation between social group size and total number of virulence genes for the wild-host E. coli sample set. The blue line is the fitted linear regression model. The grey area corresponds to the 95% confidence interval. The coefficient is 0.22419, with a standard deviation of 0.09071. (C) Social group size values across host orders. Download FIG S4, TIF file, 0.6 MB.Correlation between virulence factor counts and physiological and ecological attributes of wild hosts. (A) Correlation coefficient between the number of virulence genes and ecological features in the panTHERIA dataset. The size and color of the circles correspond to the absolute value and direction of Spearman’s rank correlation coefficient, respectively. Entries with insignificant correlation correlations, i.e., Copyright © 2021 Murphy et al.2021Murphy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli serotypes, which reflect O, H, and K antigen variation, are recognized to cause virulence in human- and livestock-associated infection. We found 53 and 14 serotypes to be shared between our E. coli strains colonizing wild animal hosts and those in domestic animal and human infections, respectively  (n = 40), enterotoxigenic E. coli (ETEC) strains (n = 12), enteropathogenic (EPEC) strains (n = 11), and enteroaggregative E. coli (EAEC) strains (n = 11), across hosts (E. coli (EHEC) serotype, and contained both the enterocyte effacement (LEE) pathogenicity island (PAI) and the toxin stx2 gene. This strain was recovered from a wild sheep close to a city. The other strain belonged to O78:H34 and was isolated from a parakeet carrying EAEC virulence genes, including the gene for the plasmid-encoded, heat-stable enterotoxin toxin (EAST-1) and aatA and aggR, encoding a transporter of a virulence protein and a virulence regulator, respectively  led us to classify these isolates as atypical EPEC (aEPEC), an E. coli class that is widely spread across food animals and humans  pathovars and that affect pathogenicity  , iron uptake (iroN and iha), and serum resistance (iss) proteins  . We founne hosts . Our finne hosts . We alsoproteins . Whetherproteins .E. coli isolates from wild animal hosts to antibiotics in common use against human E. coli infections and found them to be mostly sensitive, except that 65% of the strains were resistant to ampicillin  and chromosomal (n = 3) regions. The genomic contexts varied across host species; for example, while one strain from a member of the Pilosa, a placental mammal, harbored a distinct AMR gene cassette consisting of tet, str, and sul genes, we found the AMR genes of four other strains, from different mammalian species, to be sporadically distributed across the genomes. Besides plasmid-borne resistance determinants, we identified a set of ciprofloxacin resistance mutations in the parE, parC, and gyrA genes which emerged independently across lineages , through mobile genetic elements, in the recent spread of beneficial E. coli genes across niches in Australia , aminoglycosides (gentamicin and tobramycin), ciprofloxacin, and trimethoprim, as described in reference We acquired a systematic collection of commensal us study . The colwww.github.com/dmoradigaravand/WildHostEcoliMexico). Roary identified 24,060 genes, composed of 2,855 genes in the core genome (present in at least 99% of strains), 357 genes in the soft core genome (present in 95% to 99% of strains), 2,141 genes in the shell genome (present in 15% to 95% of strains), and 18,707 genes in the cloud genome (present in up to 15% of strains). Multilocus sequence typing was performed on assemblies using a publicly accessible typing tool and database (www.github.com/sanger-pathogens/mlst_check) with default parameter values to identify sequence type (ST) clones. Multilocus sequence typing (MLST) results are provided in DNA was extracted with the QIAxtractor (Qiagen) kit according to the manufacturer’s instructions. We prepared Illumina sequencing libraries with a 450-bp insert size and performed sequencing on an Illumina HiSeq2000 sequencing machine with paired-end read lengths of 100 bp. Ninety-six samples were multiplexed to yield an average depth of coverage of ∼85-fold. Reads were then assembled and improved with an automated pipeline, based on Velvet with default parameters. Assemblies were annotated with an improvement assembly and Prokka-based annotation pipeline, respectively –39. Deta–E. coli strains from the environment, livestock/domesticated animals, and humans in the publicly available Enterobase data set (https://enterobase.warwick.ac.uk/). Since we were primarily interested in recent evolution and transmissions between E. coli in wild hosts and other hosts, we retrieved genomic data and metadata for all strains with an identical ST with at least one strain in our collection on 26 April 2020. We included only strains for which prior consent was obtained from the strain’s owners. In total, genomic data for 1,868 strains were retrieved. The accession numbers and associated metadata are provided in We contextualized our collection with 10.1128/mSphere.00738-20.7TABLE S2Table S2, CSV file, 0.1 MB.Sample specification and associated metadata for external isolates from Enterobase, shown in Copyright © 2021 Murphy et al.2021Murphy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00738-20.8TABLE S3aK/sK values and functional annotations for genes present in >70% of isolates. Download Table S3, CSV file, 0.2 MB.Copyright © 2021 Murphy et al.2021Murphy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli strains, available on the NCBI Pathogen Detection database (www.ncbi.nlm.nih.gov/pathogens) on 4 December 2020. We determined whether our strains fell in any SNP cluster, i.e., isolates that differ by <50 SNPs, with external strains from wild-animal, food animal, and human hosts. Assuming a substitution rate of two SNPs/year , with SMALT v 0.7.4 , with a minimum score of 30 for mapping. SAMtools and BCFtools were then employed to annotate SNPs .We mapped short-read sequences to the ate SNPs . SNPs atE. coli genomes are too divergent to map to a single genome, we adopted an alignment-free approach to analyze the phylogenetic tree for the collection composed of wild-type and external strains. To this end, we first enumerated k-mers with a size of 50 from assemblies with the frequency-based substring mining (fsm-lite) package . We subsequently counted the number of identical k-mers for pairs of isolates to produce a similarity matrix, which was then converted into a distance matrix. The distance matrix was used as input for the ape (http://tree.bio.ed.ac.uk/software/figtree/).Because  the ape  and phan the ape  packages the ape  and Figthttps://cge.cbs.dtu.dk/services/) using the Virulence Factor Data Base (VFDB)  online servers (e (VFDB)  and ResFe (VFDB) , respecte (VFDB) . We usedwzx (encoding an O-antigen flippase) and wzy (encoding an O-antigen polymerase) were examined (wzm (encoding an O-antigen ABC transporter permease gene) and wzt (encoding an ABC transporter ATP-binding gene), involved in O-antigen synthesis, were studied. All the databases for typing are available in the srst2 package, and results of typing are provided in The genomic context of the AMR genes was explored in two ways. First, we searched the nucleotide database to find similar annotated genomic regions with the contig that contains the resistance gene with BLASTN. Second, to further examine whether genes are located on the plasmid or the chromosome, we also utilized PlasmidSPAdes (version 3.9.0)  to firstexamined . For theE. coli strains with Dendroscope  categories of genes were extracted from the annotation by Prokka and assigned to functional classes. We used COG categories  for Escherichia coli K-12 substrain MG1655 on https://www.ncbi.nlm.nih.gov/research/cog/. We repeated the analysis on genes that were present in 3,529 strains, which corresponds to >70% of strains. The results of positive selection analysis for these genes are detailed in We analyzed positive selection by reconstructing the ancestral sequence for each gene in the core genome, identified by Roary, with FastML using the general time-reversible (GTR) model as the evolutionary model for nucleotide substitution . SubsequE. coli from wild hosts and other strains in a clone in the B1 phylogroup. The clone was identified with the clustering tool in the adegenet package .The multiple alignment encompassed 2,176 variant sites and included 128 strains from the global collection. We ran Gubbins  with 5 iThe multiple alignment was then used as input for BEAST. We examined a range of prior models, including a strict molecular clock and a log-normal model of a relaxed molecular clock with constant population size. Markov chain Monte Carlo (MCMC) simulations were performed three times for 50 million generations with sampling every 10 generations. A cutoff 100 was chosen for the effective sample size (ESS) of key parameters, i.e., the substitution rate, the tree root height, and the population size, for the convergence of simulations. We used TreeAnnotator v1.10.4 to aggregate trees after removing 0.2 of the tree as the burn-in phase. The 95% highest posterior interval (HPI) was used to report the certainty on ages of ancestral nodes.PRJEB23294.Short-read data were submitted to the European Nucleotide Archive under the BioProject accession number


Pediatric Neurology Briefs (PNB) remainsthe same: “PNB is a continuing education service designed to expedite andfacilitate the review of current scientific research and advances in childneurology and related subjects.”In 2020, the mission of  Pediatric Neurology Briefs (PNB) remains thesame: “PNB is a continuing education service designed to expedite and facilitatethe review of current scientific research and advances in child neurology and relatedsubjects.” PNB has been published since 1987 and consists of 34 volumes withgreater than 3700 articles and includes over 10,000 citations referencing the works ofapproximately 28,000 scholarly authors. PNB was relaunched as an open access,peer-reviewed journal with an expanded editorial board in January 2015. In 2018, thepublishing model of PNB evolved even further. The canonical version of the article waspublished online as soon as it was accepted and typeset. The published articles arecollected into online volumes, and an annual collection for print will be available atthe end of every year.In 2021, the mission of The Editors of PNB select source articles using criteria that include recent publicationin a peer-reviewed journal and a topic of clinical value to practicing pediatricneurologists. Contributing Editors provide detailed summaries of published articles,followed by commentaries based on their experience and corroborated by appropriatesupplementary citations. In 2015, PNB launched a new website and content managementsystem capable of organizing peer-review and providing improved indexing, DOIassignment, and online full-text article view. Before the internet, the Editorperiodically compiled and published the PNB articles in book form with index, accordingto subject heading and chronological order .In 2016, digitization of the 30 years of back issues was completed. There are now over3700 full-text open access articles available on the journal website. Also, in 2016, PNBwas selected for inclusion in PubMed Central® (PMC). PMC is a free archive ofbiomedical and life sciences journal literature at the U.S. National Institutes ofHealth's National Library of Medicine (NIH/NLM). The most highly accessed articlespublished in 2020 covered topics related to new epilepsy treatment , mechaniPediatricNeurology Briefs - Vol. 34 - 2020. The Editors would like to take thisopportunity to sincerely thank the 2020 Contributing Editors for their effort andexpertise (listed below in publication order):Despite the immense personal and professional demands of the global COVID-19 pandemic,PNB published an increased number of manuscripts this year. The annual collectionorganizes the articles under 17 different sections and is available for download in PDFformat here: Matheus G. FerreiraEthan J. RosenbergAbigail SchwaedeVamshi K. RaoJun T. ParkIga A. FudymaJoanna Garcia PierceRifali PatelSameh S. MorkousPeter PenzesLalitha SivaswamyNancy L. KuntzMark S. WainwrightDarius LoghmaneeTracy S. GertlerLalitha SivaswamyGurpreet KhairaDaniel A. FreedmanHesham T. GhonimJena KruegerSafiullah ShareefSue J HongHélio A. G. TeiveJonathan E. KurzAmber N. BuehnerAndrea C. PardoGuadalupe Fernandez-Baca VacaNitin R. WadhwaniDivakar S. MithalJamie K. CapalMarc P. ForrestMitchell T. WilliamsAbigail N. SchwaedeMichael CroninInnessa DonskoyBridget R. McGowanTuhina Govil-DalelaJason CoryellJonathan E. KurzJorge VidaurreArayamparambil C. AnilkumarAbhinaya GaneshElizabeth Maynej-millichap@northwestern.edu.In 2021, the Editor, Dr. John J. Millichap, is thankful for the assistance from theAssociate Editors, Dr. Andrea Pardo and Dr. Stephen Nelson. Topic experts interested incontributing to PNB as authors or reviewers may contact the Editor at The author has declared that no competing interests exist.


Immune-related genes (IRGs) play considerable roles in tumor immune microenvironment (IME). This research aimed to discover the differentially expressed immune-related genes (DEIRGs) based on the Cox predictive model to predict survival for lung squamous cell carcinoma (LUSC) through bioinformatics analysis. First of all, the differentially expressed genes (DEGs) were acquired based on The Cancer Genome Atlas (TCGA) using the limma R package, the DEIRGs were obtained from the ImmPort database, whereas the differentially expressed transcription factors (DETFs) were acquired from the Cistrome database. Thereafter, a TFs-mediated IRGs network was constructed to identify the candidate mechanisms for those DEIRGs in LUSC at molecular level. Moreover, Gene Ontology (GO), together with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, was conducted for exploring those functional enrichments for DEIRGs. Besides, univariate as well as multivariate Cox regression analysis was conducted for establishing a prediction model for DEIRGs biomarkers. In addition, the relationship between the prognostic model and immunocytes was further explored through immunocyte correlation analysis. In total, 3,599 DEGs, 223 DEIRGs, and 46 DETFs were obtained from LUSC tissues and adjacent non-carcinoma tissues. According to multivariate Cox regression analysis, 10 DEIRGs  were retrieved to establish a prognostic model for LUSC. Immunocyte infiltration analysis showed that dendritic cells and neutrophils were positively correlated with IRGs, which possibly exerted an important part within the IME of LUSC. Our study identifies a prognostic model based on IRGs, which is then used to predict LUSC prognosis and analyze immunocyte infiltration. This may provide a novel insight for exploring the potential IRGs in the IME of LUSC. Lung cancer remains a leading factor leading to cancer-related deaths worldwide . Lung caRecently, immunotherapy has been widely recognized to be the efficient therapy for many cancer types –11. ReceThis study aimed to obtain the differentially expressed genes (DEGs), differentially expressed IRGs (DEIRGs), and differentially expressed TFs (DETFs) in LUSC, so as to establish a Cox prediction model based on the DEIRGs to predict the prognosis for LUSC. The regulatory network between DEIRGs and DETFs possibly exerts an important part in exploring the underlying mechanisms at molecular level. Meanwhile, correlation analysis of immunocytes and risk score also sheds new light on the tumor immune microenvironment (IME) status.https://portal.gdc.cancer.gov/), including 502 LUSC as well as 49 non-LUSC tissue specimens. Additionally, FPKM data were downloaded for differential analysis. Meanwhile, IRGs were obtained using the Immunology Database and Analysis Portal (ImmPort) (http://www.immport.org/) (http://www.cistrome.org/)  . Besidesme.org/) .http://www.bioconductor.org/packages/release/bioc/html/limma.html)  of >2. DEIRGs were obtained from DEGs based on the ImmPort database, whereas DETFs were extracted from DEGs using the Cistrome database.All transcriptome RNA-Seq data, IRGs, and cancer TF targets were differentially analyzed using the “limma R” package (ma.html) , accordihttps://david.ncifcrf.gov/)  (http://www.bioconductor.org/packages/release/bioc/html/clusterProfiler.html) , together with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, was conducted on DEIRGs using the Database for Annotation, Visualization, and Integrated Discovery (DAVID: rf.gov/) . Upon GOon.html) . In addier.html) , and a dP < 0.05). TFs stand for the significant molecules that can regulate gene expression level directly. Therefore, exploring the mechanism of TFs in the regulation of prognosis-related DEIRGs is of great necessity. The Cistrome Cancer database provides the regulatory interactions between TFs and transcriptomes from TCGA profiles (http://cistrome.org/CistromeCancer/CancerTarget/)  . To furtP < 0.05). Then, LUSC cases were classified into low or high-risk group according to the median risk score value. The receiver operating characteristics (ROC) curve is based on the specificity and sensitivity of various critical values of continuous diagnostic tests judged according to the binary gold standard  and the expression levels of 10 DEIRGs in the prediction model.https://zenodo.org/record/57669#.Xeezu9V5uMo).Tumor Immune Estimation Resource (TIMER), an online database, is able to analyze and visualize the tumor-infiltrating immunocyte levels . It reant-test was performed to validate the heterogeneities in clinical characteristics. A difference of P < 0.05 indicated statistical significance.The “limma R” package in R software was used for differential analysis, whereas the “GOplot R” and “cluster profile R” packages were adopted for functional enrichment analysis. The prediction model was constructed and applied in univariate as well as multivariate Cox regression analysis. Besides, the “survival ROC R,” “survival R,” “risk Plot R,” and “beeswarm R” packages were adopted to validate the prognostic model in LUSC. The independent P < 0.01 and fold change of >2), 3,599 DEGs, 223 DEIRGs, and 46 DETFs were screened in LUSC as well as non-LUSC tissue specimens. In line with these criteria, we screened 2,598 up-regulated DEGs and 995 down-regulated ones, 110 up-regulated DEIRGs and 113 down-regulated ones, 31 up-regulated DETFs and 15 down-regulated ones. Among them, the top 50 DEGs, DEIRGs, and DETFs are shown in In the present study, a total of 551 tissues were analyzed, which included 502 LUSC as well as 49 normal tissue specimens. According to the set thresholds (P < 0.05), among which, “GO: 0005576 extracellular region” was the most significant GO term . Then, 37 OS-related DEIRGs were identified, incorporating 31 high-risk DEIRGs and 6 low-risk DEIRGs  (n = 215) or low- (n = 216) risk group. According to survival analysis based on the prediction model with the 10 DEIRGs, LUSC cases of high-risk group were associated with notably poor prognosis compared with those of low-risk group (P = 0)  . Then, t < 0.05) . Besides (P = 0) . To eval (P = 0) . The risP < 0.05) (P < 0.001). Moreover, the multivariate independent prognostic analysis showed that, pathological M stage and the riskscore might serve as the independent prognostic factors to predict the survival for LUSC  ( < 0.05) . Meanwhifor LUSC .P < 0.05) (P < 0.05) (P < 0.05) (P = 0.005)  . The exp < 0.05) . The exp < 0.05) . The exp= 0.005) .P > 0.05) (P < 0.05)  . Dendrit < 0.05) .In recent years, IRGs show increasing importance to cancer development and immunotherapies –27. HoweIn recent years, the prognostic or predictive biomarkers associated with the tumor IME are promising to identify new molecular targets and to enhance the treatment for patients during immunotherapy development –34. SeveA study demonstrates that the NSCLC IME may be adopted to be the potential prognostic biomarkers to predict patient prognosis after receiving immune checkpoint inhibitor treatments . Howeverex-vivo NSCLC models exerts an important part in predicting patient sensitivity to such therapies  can be used to be the tumor suppressor gene to suppress the Akt signal transduction pathway, which is achieved via the neuropilin-1 receptor in lung cancer cells  can be found in the article/supplementary material.Y-QQ and RL designed the research. J-PL, Y-HY, and XL analyzed the data. X-JZ and XC collected the literature. RL drafted the manuscript. Y-QQ revised the manuscript. All authors read and approved the final manuscript.The 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.


Ficus altissima plays an important role on biodiversity in tropical forests. In this study, the complete chloroplast genome sequence and the genome features of F. altissima were analyzed using the Illumina NovaSeq platform. The whole chloroplast genome sequence of F. altissima is 160,251 including a large single-copy region , a small single-copy region , and a pair of repeat regions . Further gene annotation revealed the chloroplast genome contains 124 genes, including 79 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. A total of 82 simple sequence repeats (SSRs) were identified in the chloroplast genome. Phylogenetic development was analyzed based on F. altissima with other species of Moraceae. This information will be useful for study on the evolution and genetic diversity of F. altissima in the future. Ficus L. constitutes the most distinctive of the widespread genera in tropical area since figs (syconium) have a complex obligatory mutualism with their pollinating agaonid fig wasps  is a monoecious fig tree species distributed across Asia . A voucher specimen  was deposited at Yunnan Academy of Biodiversity, Southwest Forestry University, Yunnan, China. Then we sequenced, assembled and annotated the accurate chloroplast genome with the next-generation sequencing method to reveal the phylogenetic relationship of F. altissima.F. altissima was extracted from fresh leaves according to the modified CTAB methods described by Doyle and Doyle (F. religiosa chloroplast genome (GenBank accession number: NC_033979) as a reference, and assisted with manual correction. The raw sequencing reads used in this study has been deposited in SRA with the accession number SRR12665077 and the annotated chloroplast genome sequence has been deposited into the GenBank with the accession number MW013819.For this study, the total genomic DNA of nd Doyle . A genomnd Doyle , and thend Doyle  with F. F. altissima was 160,251 bp in length, consisting of a large single copy region , a small single copy region , and two inverted repeat regions . The total GC content was 36.92%, with IR regions (42.60%) higher than that in LSC (33.56%) and SSC regions (28.9%). The chloroplast genome of F. altissima contains a total of 124 genes, including 79 protein-coding genes, 8 rRNA genes, and 37 tRNA genes. Furthermore, a total of 82 SSR markers ranging from mononucleotide to pentanucleotide repeat motif were identified in F. altissima chloroplast genome.The complete chloroplast genome of F. altissima, based on complete chloroplast genomes of 25 species within the family Moraceae, with Debregeasia orientalis and Boehmeria nivea var. nipononivea as outgroup (F. altissima closely clustered with F. religiosa. The complete chloroplast genome of F. altissima will provide essential data for future research on the phylogenetic and evolutionary relationship in genus of Ficus.To determine the phylogenetic relationship of outgroup , chloropoutgroup , and phyoutgroup  with 100outgroup . The phy


Gammarus fossarum is widely distributed throughout Europe and an important species for stream biomonitoring. It is known to consist of several cryptic species. We here report the complete mitochondrial genome of G. fossarum clade 11/type B with a length of 15,989 bp, encoding for 13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes. Protein-coding and ribosomal genes have a similar arrangement as in other gammarid amphipods. A phylogenetic analysis clarifies the placement of G. fossarum within the Gammaridae.The freshwater amphipod  Gammarus is especially speciose and species are essential components of freshwater ecosystems, often forming important trophic links  and belongs to G. fossarum nine closely related species and two outgroups were taken. The mitochondrial genes were obtained from annotated mitochondrial genomes (NCBI GenBank), unannotated genome or transcriptome assemblies (NCBI WGS and TSA) and from raw transcriptome data assembled anew (NCBI SRA)  . The datCBI SRA) . After mCBI SRA)  and MrBaCBI SRA)  based onCBI SRA) .Gammarus fossarum clade 11/B forms a monophylum with other Gammarus species, rendering the genus monophyletic, albeit without a strong branch support ( support . This st support . The Gam support .G. fossarum clade 11/B was deposited in GenBank under the accession number KY197961. Extended methods description and sequence data are available at figshare (DOI:10.6084/m9.figshare.4487912).The mitochondrial genome of


Pseudoperonospora humuli is an obligate biotrophic oomycete that causes downy mildew (DM), one of the most destructive diseases of cultivated hop that can lead to 100% crop loss in susceptible cultivars. We used the published genome of P. humuli to predict the secretome and effectorome and analyze the transcriptome variation among diverse isolates and during infection of hop leaves. Mining the predicted coding genes of the sequenced isolate OR502AA of P. humuli revealed a secretome of 1,250 genes. We identified 296 RXLR and RXLR-like effector-encoding genes in the secretome. Among the predicted RXLRs, there were several WY-motif-containing effectors that lacked canonical RXLR domains. Transcriptome analysis of sporangia from 12 different isolates collected from various hop cultivars revealed 754 secreted proteins and 201 RXLR effectors that showed transcript evidence across all isolates with reads per kilobase million (RPKM) values > 0. RNA-seq analysis of OR502AA-infected hop leaf samples at different time points after infection revealed highly expressed effectors that may play a relevant role in pathogenicity. Quantitative RT-PCR analysis confirmed the differential expression of selected effectors. We identified a set of P. humuli core effectors that showed transcript evidence in all tested isolates and elevated expression during infection. These effectors are ideal candidates for functional analysis and effector-assisted breeding to develop DM resistant hop cultivars. Arabidopsis DM pathogen Hyaloperonospora arabidopsidis due to the wealth of genetic information available on the model plant, Arabidopsis (Pseudoperonospora cubensis (cucurbit DM) (Peronospora tabacina (tobacco DM)  (Plasmopara viticola (grape DM) (Bremia lactucae (lettuce DM) (Pseudoperonospora humuli (hop DM) (Downy mildew (DM) pathogens are a group of obligate biotrophic oomycetes that belong to the Peronosporales lineage oomycetes and have caused epidemics in many agriculturally important plants including grapes , spinachbidopsis . Advancerbit DM) , b, Peroacco DM) , Plasmopower DM) , Plasmoprape DM) , 2019, Btuce DM) , and Pse(hop DM) .R proteins, in which case an effector acts as an avirulence factor that activates effector-triggered immunity (ETI) in host plants   . RXLR efathogens . Severalathogens . A leveldopsidis , QXLRs icubensis , GKLR inlactucae , and RXLalstedii .Phytophthora infestans effector Avr3a . Symptoms including arrest in shoot development, abortion of developing cones, and defoliation, result in reduced yield, decrease in bittering acids, and even plant death in some cultivars  and over wet sterile paper towels. Leaves were drop inoculated using 10 μl of a zoospores solution and incubated at 25°C with a 12 h light/dark cycle in a precision plant growth chamber . Twenty leaf disks were collected with a sterile core borer size 4  at 2, 3, and 4 days post inoculation (DPI). Leaf samples were ground in liquid nitrogen with a mortar and pestle to a fine powder and stored at −80°C for later RNA extraction.PRJNA354153: SRX2363032 (2 DPI), SRX2363027 (3 DPI), SRX2363031 (4 DPI). The predicted coding genes from P. cubensis isolate MSU1 (study number SRP011018) reported by P. humuli OR502AA. The nuclear genome assembly and annotation of P. humuli OR502AA  and submitted to The Genomic Sciences Laboratory at North Carolina State University for library preparation and sequencing in an Illumina NextSeq 500 platform . cDNA libraries of 350 bp insert size were prepared and sequenced as described in  OR502AA  can be fP. humuli OR502AA were used to extract homologs shared among 11 oomycete species The oomycete species included: P. cubensis MSU1  obtained by CEGMA version 2.5  followinsis MSU1 , P. tabaa 968-J2 , P. halsdii BLA4 , H. arabis EMOY2 , P. infens T30-4 , Phytophm PR102a , P. sojaae P6497 , Pythiumum BR144 , Albugo hii NC14 , and SapBS223.65 . All genn 3.8.31  and subsn 3.8.31 , with 10P. humuli from published genome assembly by P. cubensis MSU1 reported by P. cubensis MSU1 were then analyzed for potential secreted proteins as described above for P. humuli. Apoplastic effectors were predicted using ApoplastP  using a PowerUpTM SYBRTM Green Master Mix (Applied Biosystems). Gene-specific primers (P. humuli actin (Phum_OR502AA_v1_g_08820), Glycerol-3-phosphate dehydrogenase (Phum_OR502AA_v1_g_18083), and ubiquitin-conjugating enzyme E2 (Phum_OR502AA_v1_g_00443) were used as reference genes and the values were averaged for calculation. ΔΔCt values were calculated for each time-point using the expression in sporangia as a control by the method published by P-values were calculated by a paired t-test using the GraphPad Prism 8.00 software for Mac.Quantitative RT-PCR was performed using gene-specific oligonucleotides. Tissue was collected and RNA was extracted as described in Sections “Pathogen and Plant Material” and “RNA Extraction, Library Preparation, and Sequencing of  primers  were gen primers . P. humuP. humuli showed a 97.8% complete and 98.8% partial completeness based on the coverage of eukaryotic genes -like proteins (NLPs). However, the NLPs in P. humuli had highly degenerate heptapeptide “GHRHDWE” motifs -like extracellular proteins, and a myriad of proteins of unknown function  of scripts used for analysis. The first 150 residues of the gher FDR .P. humuli, an HMM search was performed using a previously published HMM model (P. humuli secretome had a single candidate that possessed an LFLAK-like (LYLARK) and an HVLVXXP-like motif. On the other hand, an HMM search on the 941 P. cubensis secreted proteins produced no hits. Since the low number of CRN effectors was surprising, an HMM search was performed on all 18,656 predicted P. humuli proteins, which resulted in 62 hits of which nine had no LFLAK or HVLVXXP motifs. Among the remaining 53 CRNs, 20 had both LFLAK-like and HVLVXXP-like motifs  family of effectors in MM model . The P. e motifs . Apart f peptide .P. humuli isolates. RNA-Seq performed on sporangia of the P. humuli isolate OR502AA and eight others used by P. humuli, we found transcript evidence with an RPKM value > 0 for 10,536 genes in all the sequenced isolates  at all time-points post inoculation  did not show upregulation at any time-point. Out of the 296 predicted RXLR effectors, 62 were upregulated  at all time-points. It was interesting to observe that most of the expressed effectors showed consistent levels of expression across the time-points tested  in P. cubensis. Despite similar secretome sizes between P. humuli and P. cubensis, there was an increased number of predicted effector proteins in P. humuli. The apoplastic effector content in P. humuli was much higher than in P. cubensis with a notable increase in glucanase inhibitors and CAZymes. The most astonishing increase in the number of effectors between P. humuli and P. cubensis, however, was observed in the RXLR class of effectors, especially in the number of WY-EERS. Also, despite the close phylogenetic relationship between P. humuli and P. cubensis, the number of orthologous RXLRs were few. This lack of orthology between species of the same genus is not surprising considering the high divergence in the RXLR family that has previously been reported in Phytophthora spp., with P. sojae and P. ramorum having a low number of shared orthologs  proteins in rthologs . Moreovenew host . It is anew host . There w71 RXLRs . However71 RXLRs . Due to athogens .P. humuli and P. cubensis could also be attributed to our initial filtering for contaminants in the P. cubensis proteome. The presence of contaminants is not surprising in next generation sequencing data from biotrophic pathogens like P. cubensis and P. humuli  responses. For example, NLPs belong to a gene family that induces cell death and their role has been indicated in the switch from the biotrophic to necrotrophic phase in P. infestans  showed elevated expression in planta. However, variation in the time-points tested could have contributed to this discrepancy since the highest expression in H. arabidopsidis was observed during the early stages of infection  to a bacterial shikimate kinase. The lack of homology to known RXLRs is not surprising since RXLRs are a class of diverse proteins that show a high level of sequence divergence outside of the conserved N-terminal RXLR and EER motifs (Phum_OR502AA_v1_g_06904 across the P. humuli isolates and its high expression during infection may indicate a role during the host-pathogen interaction making it a good candidate for downstream functional analysis.The R motifs . DespiteP. humuli effectorome contained effectors that exhibited transcript evidence in all P. humuli isolates and showed enhanced expression during infection. We propose that these core effectors are ideal candidates for downstream functional analysis aiming toward the identification of robust sources of resistance to DM in hop.Natural sources of DM resistance in hop are rare and for this reason, identifying new sources of resistance is a priority . HoweverPRJNA354153.The datasets generated for this study can be found in the National Center for Biotechnology Information database under the Bioproject number LQ-O, SP, and LC conceived and designed the experiments and wrote the manuscript. SP, LC, MB, KC, and DG performed the experiments. SP, LC, MB, and KC analyzed the data. LQ-O, DG, and KC contributed reagents, materials, and analysis tools. All authors contributed to the article and approved the submitted version.MB is employed by the company Ball Horticultural Company, West Chicago, IL, United States.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.


Lung disease is a leading cause of morbidity and mortality. A breach in the lung alveolar-epithelial barrier and impairment in lung function are hallmarks of acute and chronic pulmonary illness. This review is part two of our previous work. In part 1, we demonstrated that CdM is as effective as MSCs in modulating inflammation. Herein, we investigated the effects of mesenchymal stromal cell (MSC)-conditioned media (CdM) on (i) lung architecture/function in animal models mimicking human lung disease, and (ii) performed a head-to-head comparison of CdM to MSCs.Adhering to the animal Systematic Review Centre for Laboratory animal Experimentation protocol, we conducted a search of English articles in five medical databases. Two independent investigators collected information regarding lung: alveolarization, vasculogenesis, permeability, histologic injury, compliance, and measures of right ventricular hypertrophy and right pulmonary pressure. Meta-analysis was performed to generate random effect size using standardized mean difference with 95% confidence interval.A total of 29 studies met inclusion. Lung diseases included bronchopulmonary dysplasia, asthma, pulmonary hypertension, acute respiratory distress syndrome, chronic obstructive pulmonary disease, and pulmonary fibrosis. CdM improved all measures of lung structure and function. Moreover, no statistical difference was observed in any of the lung measures between MSCs and CdM.In this meta-analysis of animal models recapitulating human lung disease, CdM improved lung structure and function and had an effect size comparable to MSCs. Pulmonary illness is a leading cause of morbidity and mortality . In chilA unifying theme in many lung diseases includes inflammation –8. WhileMSCs are promising agents as they are easily harvested, can be rapidly expanded, and can secrete factors  known to reduce inflammation –18. The This review is an extension of our previous work. In this review, we examined the effects of CdM on (i) lung architecture/function in animal models recapitulating lung disease and (ii) compare these findings to MSCs. Given that the therapeutic benefit of MSCs is attributed to a paracrine fashion, we believed CdM would have comparable effects to MSCs.The methods in our review abide to those outlined by the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) . Our proWe conducted a literature search in five databases using the following terms: mesenchymal stem cell-conditioned media, lung disease, and animal. The last search was performed on March 17th, 2020. Three independent investigators evaluated titles and abstracts, followed by full-text review.We included studies administering MSC-CdM to animal models of acute lung injury or acute respiratory distress syndrome (ALI/ARDS), asthma, bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), pneumonia, pulmonary fibrosis (PF), and pulmonary hypertension (PH). Refer to Supplementary File Measures of lung structure and/or function were our primary endpoint. Lung architecture and function were assessed under the following categories: alveolarization, vasculogenesis, right ventricular hypertrophy, fibrosis, permeability, pulmonary pressures, compliance, and lung injury. Although the pathogenesis of the included lung diseases are heterogeneous, we combined all processes irrespective of disease. This was conducted to obtain a scoping overview of the impact of CdM on biologic processes implicated in lung disease. Subsequently, we assessed lung structure/function by disease in our subgroup analysis. Excluded studies were those which did not provide data concerning our primary outcome of inflammation.Three groups of investigators were used  to collect data. Uniformity of data was assessed by the primary author. This data included general study design, animal model characteristics, conditioned media characteristics, and outcomes of interest.I2 metric, and funnel plots were used to examine publication bias. If more than six articles were included per outcome, we conducted a subgroup analysis for disease, animal species, and route and dose of CdM administration. All statistical analyses were performed in R version 3.6.2; packages used included dmetar, metafor, and meta.A random effects model was used to generate forest plots. A minimum of three studies were required for each outcome to proceed with a meta-analysis. The estimated effect size of CdM or MSC on lung architecture/function was determined using standardized mean difference (SMD) with a 95% confidence interval (CI). Statistical heterogeneity between studies was calculated using the Our literature search resulted in 245 articles. After removing duplicates and viewing the titles and abstracts, 55 articles underwent full-text review. Twenty-nine articles met inclusion , followed by ALI/ARDS (n = 5) and asthma (n = 5). All of the studies used rodents to induce their lung model.Table Conditioned media properties are summarized in Supplementary File CdM: improved alveolarization with an SMD of 1.32 .MSC: improved alveolarization with an SMD of 1.80 .CdM vs. MSC: no significant difference ; 6 studies; Fig. I2 = 70%; p < 0.01).MSC: favored over the control with an SMD of − 1.05 .CdM vs. MSC: no significant difference .MSC: favored MSC over the control with an SMD of − 1.99 .CdM vs. MSC: the comparison between CdM and MSCs was similar .MSC: superior to control with an SMD of − 2.29 .CdM vs. MSC: overall effectiveness between CdM and MSCs again showed no significant difference .MSC: in the evaluation of permeability, the MSC was favored over the control with an effect size of − 1.54 .CdM vs. MSC: equal effectiveness .MSC: superior to control with an SMD of − 1.63 .CdM vs. MSC: comparable .MSC: superior to control with an SMD of − 2.01 .CdM vs. MSC: less than 3 studies; comparison not performed.CdM: improvement in lung compliance compared to control with an SMD of 1.75 .MSC: improvement in lung compliance compared to control with an SMD of 2.33 .CdM vs. MSC: not applicable as less than three studies performed a head-to-head comparison.CdM: Supplementary Figure MSC: Supplementary Figure CdM vs. MSC: no difference was appreciated between CdM and MSC when all outcomes were combined  had similar groups at baseline. Risk of bias was large regarding allocation concealment, whether authors mention random housing of animals, and blinding of caregivers or random selection of outcome. All studies were found to sufficiently report complete data and being free from other bias. Refer to Supplementary File No study was judged as low risk across all ten domains. Eight studies stated that the allocation selection was random. Most studies n = 5 had simSupplementary Figures Preclinical studies reiterate the ability MSCs have on dampening lung inflammation. This capacity is largely due to the paracrine secretion of MSC factors  that provide a basis for future cell-free therapies for human disease –31. ThisE. coli pneumonia-induced ALI mouse model, MSC microvesicles  reduced lung permeability and histologic injury score and were equivalent to MSCs [The bioactive factors contained in the CdM of MSCs have been the focus of multiple studies and review articles –34. Cong to MSCs . Togethe to MSCs , 38.This year, Augustine et al. published a network meta-analysis comparing stem cell and cell-free therapies in preclinical measures of BPD. MSC-CdM had a similar effect size to MSCs regarding alveolarization (MSC SMD 1.71 vs. CdM SMD1.68), angiogenesis (SMD 2.24 vs. 1.79), and pulmonary remodeling (1.29 vs. 1.22) . Similarn = 10 intravenous injections). Conversely, the optimal source and dose of CdM is dependent on the variable or the lung disease. This brings to light that it will be incredibly challenging to find a single CdM product that is ideal for all lung diseases. Thus, the idea of “one-size-fits-all” does not hold true for regenerative cells or products. Illustrating this concept, Rathinasabapathy et al. showed greater improvement in measures of RVH compared to other studies measuring right ventricular size. Important differences seen in the study by Rathinasabapathy and colleagues was that they used a different animal model (PH vs. BPD) and age of rodents  [In the study by Hayes et al., they found that MSCs were superior to CdM in a rodent model of ventilator-induced lung injury. However, our review suggests that when you compile the literature, there were no significant benefits of using cells over CdM. We cannot explain why CdM was not comparable in this study; however, an important challenge that remains in the field includes the rigorous testing of key variables  that may impact the quality of CdM –44. For eonatal) .As investigators, we should attempt to tease out these characteristics in order to have the ideal product(s) for our lung disease of interest. In this way, we may have translational success in future clinical studies. Refining these features will take time but will play a vital role in efficacy. Moreover, pinpointing small and large animal models of lung disease that will recapitulate what occurs at the patient bedside is essential if we want to move the needle in the field .The plausibility of using a cell-free product as a therapeutic agent for lung disease is substantiated by newly registered human clinical trials. For instance, NCT04235296 and NCT04234750 are evaluating safety of MSC-CdM in regulating wound inflammation and promoting wound healing in burn injury. Another Phase I trial (NCT04134676) plans to study the therapeutic potential of umbilical cord tissue-derived stem cell CdM on chronic skin ulcers. Trials valuing the safety of stem cell CdM constituents (exosomes) are also underway for ischemic stroke (NCT3384433) and ocular conditions .There are several limitations to our systematic review and meta-analysis, many of which mirror those published in our previous report. We incorporated multiple animal models of lung disease that have diverse pathologic processes resulting in their etiology. Also, most of the studies lacked methodologic details rendering them with an unclear risk of bias. Moreover, although preclinical models of lung disease have been helpful in identifying targetable mechanisms/processes, they oftentimes lack the intricacies of human disease. Thus, meticulous efficacy studies in large animals may be one approach to mitigate translational failure in human trials.This review demonstrates that the administration of CdM in animal models of lung disease improves lung architecture and function. When compared to MSCs, CdM is as efficacious and provides a basis that cell-free products are a viable option for future studies. However, mores studies are needed to identify how specific variables  may impact/strengthen their therapeutic potential.Additional file 1: Figure S1. Flow diagram demonstrating study selection process.Additional file 2: Figure S2. Effect size of CdM vs. MSC on lung alveolarization. . Forest plots demonstrate SMD with 95% confidence interval.Additional file 3: Figure S3. Effect size of CdM on right ventricular hypertrophy. Forest plots demonstrate SMD with 95% confidence interval.Additional file 4: Figure S4. Effect size of MSC on lung fibrosis. Forest plots demonstrate SMD with 95% confidence interval.Additional file 5: Figure S5. Effect size of CdM vs. MSC on pulmonary vasculogenesis. Forest plots demonstrate SMD with 95% confidence interval.Additional file 6: Figure S6. Effect size of CdM vs. MSC on lung permeability. Forest plots demonstrate SMD with 95% confidence interval.Additional file 7: Figure S7. Effect size of CdM vs. MSC on pulmonary pressures. Forest plots demonstrate SMD with 95% confidence interval.Additional file 8: Figure S8. Effect size of CdM (a), MSCs (b), and CdM vs. MSC (c) on all eight outcomes. Forest plots demonstrate SMD with 95% confidence interval.Additional file 9: Figure S9. Effect size of CdM on lung alveolarization by disease (a), source (b), dose (c), and route (d). Forest plots demonstrate SMD with 95% confidence interval.Additional file 10: Figure S10. Effect size of CdM on right ventricular hypertrophy by disease (a), source (b), dose (c), and route (d). Forest plots demonstrate SMD with 95% confidence interval.Additional file 11: Figure S11. Effect size of CdM on lung fibrosis by disease (a), source (b), dose (c), and route (d). Forest plots demonstrate SMD with 95% confidence interval.Additional file 12: Figure S12. Effect size of CdM on pulmonary vascularization by disease (a), source (b), dose (c), and route (d). Forest plots demonstrate SMD with 95% confidence interval.Additional file 13: Figure S13. Funnel plot assessing for publication bias of CdM on lung alveolarization.Additional file 14: Figure S14. Funnel plot assessing for publication bias of CdM on right ventricular hypertrophy.Additional file 15: Figure S15. Funnel plot assessing for publication bias of CdM on lung fibrosis.Additional file 16: Figure S16. Funnel plot assessing for publication bias of CdM on pulmonary vasculogenesis.Additional file 17: Figure S17. Funnel plot assessing for publication bias of CdM on lung permeability.Additional file 18: Figure S18. Funnel plot assessing for publication bias of CdM on pulmonary pressures.Additional file 19: Figure S19. Funnel plot assessing for publication bias of CdM on histologic lung injury.Additional file 20: Figure S20. Funnel plot assessing for publication bias of CdM on lung compliance.Additional file 21: File S1. List of articles included in this review.Additional file 22: File S2. SYRCLE risk of bias.Additional file 23: File S3. CdM characteristics.


Staphylococcus aureus (S. aureus), particularly methicillin-resistant Staphylococcus aureus (MRSA), remains the predominant cause of infections in drug users. This cross-sectional study aims to elucidate the prevalence, risk factors, phenotypic and molecular characteristics of S. aureus carriage among community-based drug users.S. aureus. Antimicrobial susceptibility test and polymerase chain reaction assays were used to detect phenotypic and molecular characteristics for identified isolates. Univariate and multivariate logistic regression analyses were used to assess risk factors for S. aureus carriage.All eligible drug users, with both injection and non-injection route of drug administration, were asked to complete questionnaires and collect nasal swabs by trained personal during the period between May and December 2017 in Guangzhou, China. Swabs were processed for identification of S. aureus carriage was 15.01% (53/353). The prevalence of MRSA carriage was 6.80% (24/353). Cohabitation was a risk factor for S. aureus . The proportion of multidrug resistance was 54.72% for S. aureus isolates and most of these isolates were resistant to penicillin, erythromycin and clindamycin. Seventeen MRSA isolates were multidrug resistant. The results of clonal complexes (CCs) and sequence types (STs) for S. aureus were diverse. The three predominant types for CCs were CC5 , CC59 , and CC7 ; and for STs were ST188 , ST5 , and ST59 .Overall, 353 drug users were included in the study and the prevalence of S. aureus nasal carriage was lower while the prevalence of MRSA carriage was moderate compared to previous studies. Phenotypic and molecular characteristics of S. aureus isolates, particularly MRSA isolates, revealed high proportions of antibiotic resistance, indicating the existence of cross-circulation, and implying high opportunity of virulence-related diseases. Decolonization and antibiotic stewardship might be implemented for drug users with MRSA carriage.The prevalence of  Staphylococcus aureus (S. aureus), particularly methicillin-resistant S. aureus (MRSA), continues to be a major pathogen in both hospital- and community-associated infections  and erythromycin-resistant genes . All S. aureus isolates were further tested to confirm the presence of toxin genes including Panton-Valentine leukocidin genes (lukF-PV and lukS-PV), Toxic shock syndrome toxin-1 gene (tst), Exfoliative toxin A gene (eta), Exfoliative toxin B gene (etb) and Staphylococcal enterotoxins (SEs)  genes. Multilocus sequence typing (MLST) was performed to confirm clonal complexes (CCs) and sequence types (STs). Additionally, all MRSA isolates were tested for Staphylococcal cassette chromosome mec (SCCmec) typing. More details were described in previous work [All ous work .S. aureus carriers and relevant characteristics by the following methods. Univariate analyses were conducted using the Pearson’s chi-squared test or the Fisher’s exact test when appropriate. Multivariate logistic regression models were used to determine risk factors associated with S. aureus carriage. Independent risk factors with a P < 0.1 in univariable logistic regression analysis were included in the multivariable models. Potential confounding covariates were adjusted in the models. A two-sided P-value of ≤0.05 was defined as statistical significance.The data were entered using Epidata 3.1  and exported to Stata 14.2  software for further analysis. We assessed the associations between S. aureus nasal carriage in drug users was 15.01% (53/353). The prevalence of MRSA carriage was 6.80% (24/353).A total of 353 drug users were eligible for inclusion in the study. The prevalence of S. aureus carriage among drug users. After adjusting for confounding covariaes, current cohabitation was still a risk factor for S. aureus carriage  in drug users , erythromycin (49.06%), clindamycin (45.28%) and tetracycline (32.08%)   and one (1.89%) was positive for the erm(A) gene. Only one S. aureus isolate was positive for both the erm(C) and erm(A) genes. For tetracycline-resistant genes, four (7.55%) S. aureus isolates were positive for the tet(K) and no isolate was positive for tet(M) gene. Additionally, only one S. aureus isolate was positive for erm(C), erm(A) and tet(K) genes. These gene-positive S. aureus isolates were all MRSA isolates.In terms of macrolide-resistant genes, five (9.43%) S. aureus isolates , sei (34.96%) and sad (32.08%). All S. aureus isolates were negative to the sea and see genes.In terms of virulence genes Table , 5.66% omec types were detected from the 24 MRSA isolates, in which 12 isolates were type IVd, five were type IVa, one was type V, one was type II, and five were non-typeable  is lower than previously reported estimates which ranged from 19.79 to 45.05% [S. aureus carriage, further studies about the impact of snorting drugs on S. aureus carriage need to be conducted in the future The prevalence of MRSA nasal carriage (6.80%) in the study is similar to the previous studies in other countries [S. aureus isolates was higher than the observed studies [To the best of our knowledge, this is a relatively comprehensive study which contributes to the understanding of the prevalence, risk factors, phenotypic and molecular characteristics for o 45.05% –11. Partountries –14,but hhe study .01% is lS. aureus carriage in drug users, which is different from another study [S. aureus carriage [S. aureus carriage in drug users.In this study, we found that current cohabitation might be a risk factor for er study . One of er study . HIV infcarriage , howeverS. aureus isolates are consistent with limited available studies [S. aureus isolates could partially demonstrate the high use of antibiotics in community-based drug users and provide evidence that healthcare workers need to be more careful with selection of antibiotics for drug users. Therefore, the administration of antibiotics for drug users should be strengthened.The patterns of antibiotic resistance on  studies , 17, 18, studies , 20, whiThe proportions of virulence genes were high in MRSA, suggesting the higher risks of MRSA isolates in causing virulence-related diseases, including Staphylococcal scalded skin syndrome, toxic shock syndrome, Staphylococcal food poisoning, etc. –23. The S. aureus isolates could demonstrate the multiple transmissions among human beings, livestock and environment, which are similar to previous studies [mec types, we could know the source of MRSA isolates might be both communities and healthcare settings, which is similar to the observed studies [S. aureus isolates displayed identical molecular characteristics, suggesting the possibility of cross-transmission between the communities and healthcare settings and this might be a potential risk for other populations. Relevant decolonization methods could be taken for drug users with MRSA carriage, which would help prevent further MRSA circulation [We found high proportions of ST5 and ST59 in this study and these STs were also globally reported in communities . We also studies , 24. Accculation .S. aureus carriage, particularly MRSA carriage, among drug users in China. Despite the strengths of this study, there are several limitations. First, it was a cross-sectional study. Thus, we could not determine the persistence of S. aureus carriage. Secondly, we only collected nasal swabs instead of nasopharyngeal swabs, which may lead to underestimation of the prevalence of S. aureus carriage. Thirdly, we did not collect information whether male participants were those who have sex with men due to confidentiality. We will explore it in future research. Finally, the generality of this study is limited owing to the small number of drug users.Our study contributes to the understanding of the prevalence, risk factors, phenotypic and molecular characteristics for S. aureus nasal carriage was lower, while the prevalence of MRSA nasal carriage was moderate among community-based drug users but higher than that of general population in China. Cohabitation is a risk factor for S. aureus carriage. Phenotypic and molecular characteristics of MRSA isolates reveal serious antibiotic resistance, indicate the cross-circulation of MRSA isolates between communities and healthcare settings, and imply high opportunity of virulence-related diseases. Decolonization and antibiotic stewardship might be implemented for drug users with MRSA carriage, especially for those with risk factors.In summary, the prevalence of


Caldicellulosiruptor, including genomic and metagenomic analyses in addition to recent breakthroughs in enzymology and genetic manipulation of the genus. Ten years after completing the first Caldicellulosiruptor genome sequence, the tools required for systems biology of this non-model environmental microorganism are in place.Plant polysaccharides continue to serve as a promising feedstock for bioproduct fermentation. However, the recalcitrant nature of plant biomass requires certain key enzymes, including cellobiohydrolases, for efficient solubilization of polysaccharides. Thermostable carbohydrate-active enzymes are sought for their stability and tolerance to other process parameters. Plant biomass degrading microbes found in biotopes like geothermally heated water sources, compost piles, and thermophilic digesters are a common source of thermostable enzymes. While traditional thermophilic enzyme discovery first focused on microbe isolation followed by functional characterization, metagenomic sequences are negating the initial need for species isolation. Here, we summarize the current state of knowledge about the extremely thermophilic genus  Hungateiclostridium thermocellum (formerly ‘Clostridium thermocellum’), were isolated and described as being capable of rapidly degrading cellulose [H. thermocellum. Reports of cellulolytic microbial isolates enriched from terrestrial hot spring samples grown above 70 °C [Over 50 years have passed since Thomas Brock’s discovery that Yellowstone National Park’s hot springs were teeming with microbial life . In the ellulose , followeellulose . The queve 70 °C ,9,10 raiBacterial lignocellulose deconstruction at elevated temperatures (above 70 °C) evolved to leverage specific carbohydrate active enzyme (CAZyme) families and carbohydrate binding modules (CBM) . EnzymesCaldicellulosiruptor, the original focus was the function of modular, multi-functional enzymes that were cloned and sequenced . Howeve in 2008 . Adding database , consistCaldicellulosiruptor saccharolyticus and Caldicellulosiruptor bescii (formerly ‘Anaerocellum thermophilum’) identified that the glucan degradation locus (GDL) could vary in complexity between strongly cellulolytic species [C. bescii encoded for a number of cellulases and xylanases [C. saccharolyticus [C. saccharolyticus and C. bescii possess a high number of carbohydrate binding modules and glycoside hydrolases in their respective genomes when compared to other thermophiles, and over 86% of the open reading frames (ORFs) in their respective genomes were orthologs [The initial comparison between  species . Based oylanases , and thaylanases  that shaolyticus . Overallrthologs .Caldicellulosiruptor core genome has been reduced from 1543 open reading frames (ORFs) shared among eight species [Caldicellulosiruptor led to an increase of the pangenome from 3493 ORFs (13 species) [Caldicellulosiruptor pangenome was constructed from the genomes of eight strongly to weakly cellulolytic members of the genus Caldicellulosiruptor [Caldicellulosiruptor, that would assumedly be crucial to their strongly cellulolytic lifestyle [As expected, the  species  to 1367  species . In compspecies)  to 3791 species) . The inisiruptor ,25, and ifestyle . Caldicellulosiruptor genomes encode for enzymes containing at least one GH48 domain, with six of those species encoding for two (C. obsidiansis and C. changbaiensis) or three GH48 domains  [Caldicellulosiruptor species grow at similar rates on microcrystalline cellulose. However, their overall ability to solubilize biomass or crystalline cellulose varies, with C. bescii and C. morganensis outperforming other cellulolytic species [While most species had been isolated and described as being cellulolytic see  when tesnoensis) ,22,23. R species .C. bescii, C. kronotskyensis) and one moderately cellulolytic species (C. saccharolyticus), C. saccharolyticus was observed to more efficiently ferment the limited amount of soluble sugars released by its enzymes in comparison to the highly cellulolytic Caldicellulosiruptor species [C. kronotskyensis and weakly cellulolytic C. hydrothermalis have the highest and second highest number of glycoside hydrolase (GH) domains [C. morganii, C. danielii, and C. naganoensis [This variability in enzymatic capacity may be related to the functional role each species plays during plant biomass degradation in situ. Accordingly, in a physiological comparison between two highly  domains are one such class and are required for the interface between cellulosomes and cells in the Clostridiales [Caldicellulosiruptor, each species sequenced possesses multiple S-layer located enzymes and proteins (between 10 and 19) [Caldicellulosiruptor core-genome (GH5-CBM28-SLH-SLH-SLH) [Caldicellulosiruptor species were also involved in the attachment to cellulose and pectin [Aside from using comparative genomics to determine the cellulolytic capacity of the genus ridiales . However and 19) . Interes and 19) , includi and 19) . One S-lSLH-SLH) ,34 and wSLH-SLH) . Latter d pectin  or xylanC. hydrothermalis and C. kristjanssonii) that share a similar shape to the highly cellulolytic tāpirin proteins [Caldicellulosiruptor use classical tāpirins to adhere to cellulose, as atypical tāpirin proteins similar to those encoded for by C. owensensis and C. acetigenus were identified in the genome of C. changbaiensis [Lastly, high-temperature cellulose adhesins (tāpirins) were identified through comparative genomics and proteomics efforts ,36. Geneproteins . Furtherproteins , and grabaiensis . AdditioCaldicellulosiruptor strains [Caldicellulosiruptor were identified from isolated species, such as C. bescii [C. saccharolyticus [Caldicellulosiruptor  allowedptor see . Furthereratures ,45,46,47Caldicellulosiruptor species identification from hot springs microbial community analyses from the Azores [Caldicellulosiruptor species were isolated from most sites sampled, the only report of Caldicellulosiruptor from the African continent was a single 16S rRNA gene clone identified from a hot spring in Tunisia, which was predominantly colonized by Firmicutes [Caldicellulosiruptor, along with genera Fervidobacterium, Dictyoglomus, and Sulfurihydrogenibium were most abundant in a slightly alkaline, highly reduced solfatara effluent stream in the Azores [Caldicellulosiruptor, Fervidobacterium, and Dictyoglomus were also detected by 16S rRNA gene clones isolated from separate enrichment cultures sampled from springs in the Uzon Caldera, Kamchatka, Russia [Caldicellulosiruptor predominated enrichment cultures between 70 and 75 °C [Caldicellulosiruptor species in extremely thermophilic hydrogen- or methane-producing consortia that were sourced from the Ta Na Ma Rao hot spring in Thailand [Caldicellulosiruptor species present in a hot spring from Yongtai, China, that was identified through 16S rDNA phylogeny [Caldicellulosiruptor, supporting their primary role in cellulose hydrolysis at elevated temperatures. Interestingly, geothermally heated springs are not the only potential sources of isolated Caldicellulosiruptor species, for example, C. owensensis was isolated from solar heated mud flats in Owens Lake, CA [C. sp. F32 was isolated from a compost enrichment in China [Caldicellulosiruptor spp. most similar to C. kristjanssonii based on 16S rDNA analysis were identified from manure-supplemented distillery waste streams [Caldicellulosiruptor cells came from the distillery wastewater or the digested cow manure. Additional reports of Caldicellulosiruptor species identified from animal microbiota included community analyses of a rumen microbial community [Caldicellulosiruptor (“Mesocellulosiruptor”), similar to the discovery of mesophilic members of the phylum Thermotogae  that may also possess robust enzymes of interest.The availability of genome sequences has bolstered e Azores , Icelande Azores , Russia e Azores , Tunisiae Azores , and thee Azores ,52 . Caldicin Japan , and by in Japan , India [in Japan , China [in Japan , and Hunin Japan . The ana Germany , and a h Germany  also ideommunity . Of all  surface . Due to Caldicellulosiruptor isolates present in microbial communities is possible. Interestingly, the initial studies using next generation ‘-omics’ techniques that identified Caldicellulosiruptor species as predominant members of microbial communities started from anaerobic digestion sludge [Caldicellulosiruptor spp. were identified through metaproteomics as one of the predominant bacteria involved in polysaccharide hydrolysis in a cellulolytic microcosm that was initially inoculated with anaerobic digester sludge [Caldicellulosiruptor cellulase, CelA, along with CAZymes, more commonly associated as being intracellular [Caldicellulosiruptor enzymes were identified in this study since neither the supernatant nor substrate were sampled for peptide identification. Other proteomic efforts on the genus Caldicellulosiruptor have highlighted the differences in the peptide profiles of whole cell versus supernatant [With rapid advancements in next generation sequencing and proteomics, an assembly of partial genomes or identification of active metabolic pathways from environmental n sludge ,65. For r sludge . Analysicellular . It is nernatant ,72 or beernatant .Caldicellulosiruptor species was previously isolated from Obsidian Pool (C. obsidiansis), other global sites, New Zealand, for example, host a diverse community of Caldicellulosiruptor species [Caldicellulosiruptor species in other hot spring environments. Initial community analysis of these cellulose-enriched communities determined that members of the Caldicellulosiruptor genus ranged from 89.9% to 38.7% of the total community population, although the relative abundance of Caldicellulosiruptor species only marginally correlated with solubilization of lignocellulose [Caldicellulosiruptor genomes assembled from metagenomes shared a high degree of homology and synteny with the C. obsidiansis genome. Regardless, key differences were noted in their GDL composition, including the presence of an additional modular cellulase (GH10-CBM3-CBM3-GH48) in two of the enrichments that C. obsidiansis lacks [Caldicellulosiruptor strains, considering that half of the communities enriched encoded for the three-key synergistic cellulases, CelA (GH9-CBM3-CBM3-CBM3-GH48), CelC (GH10-CBM3-CBM3-GH48), and CelE (GH9-CBM3-CBM3-CBM3-GH5) [In addition to the identification of hydrolytic enzymes from anaerobic digestor metaproteomics, metagenomes were sequenced from enriched cellulolytic communities harvested from Obsidian Pool, Yellowstone National Park in the United States . While a species . Celluloellulose , emphasiis lacks . These cBM3-GH5) . Caldicellulosiruptor that has been genome sequenced encode for a locus of modular, multifunctional enzymes. This genomic region, named the glucan degradation locus (GDL), contains one or more enzymes that possess the marker for the highly cellulolytic phenotype, GH 48 [Caldicellulosiruptor were under considerable focus, and the first report of large (>180 kDa) glycosylated cellulases was noted from C. naganoensis (formerly ‘Thermoanaerobacter cellulolyticus’) [celB (GH10-CBM3-GH5) from C. saccharolyticus [C. saccharolyticus [Caldicellulosiruptor sp. Tok7B.1 [C. saccharolyticus [Every strongly cellulolytic member of the genus e, GH 48 ,25. Histyticus’) . The modolyticus , which wolyticus ,42. Init Tok7B.1 ,77 or C.olyticus ,78. Caldicellulosiruptor genomes, and one metagenome assembled genome [C. morganii [C. bescii outperforming CelA from C. danielii [C. saccharolyticus [C. bescii [C. danielii have been characterized, it remains to be seen if there is a superior candidate to CbCelA. As individual enzymes from various Caldicellulosiruptor species have been cloned, each research group has used differing gene naming conventions. In this review, the naming convention for the GDL in C. bescii will follow unless noted.Now, with the benefit of comparative genomics including 14 d genome ,22,23, td genome ,79. Fromolyticus , C. besc. bescii ,80,81, aEscherichia coli is commonplace for the initial characterization of recombinant enzymes, the size of the modular cellulases may be a limiting factor for the adoption of these cellulases in other systems. The largest Caldicellulosiruptor modular enzyme, CelA (GH9-CBM3-CBM3-CBM3-GH48), was initially identified from screening a C. saccharolyticus genome library for activity on amorphous cellulose, and was subsequently sequenced [C. bescii [C. bescii was observed to solubilize all components of plant biomass, lignin included, during growth on switchgrass, which could alleviate CelA inhibition by lignin adsorption [C. bescii was unable to solubilize lignin present in poplar [While heterologous expression in equenced . However. bescii . Further. bescii . Both ca. bescii . While C. bescii  and can . bescii . Interessorption . Howevern poplar .C. bescii celA knockout mutant, which was reduced in its ability to grow on cellulose and overall cellulase activity when induced by cellobiose [celA in a genetically stable C. bescii parental strain [C. bescii genome (NCBI accession NC_012034.1) [celA knockout vector will amplify a 1 kb downstream sequence that is present in multiple regions and could result in the deletion of an additional two GDL genes . Expansion and diversity of the Caldicellulosiruptor GDL has been proposed to occur through gene recombination after duplication [C. bescii celA, by insertion of N-terminal aspartate repeats, improved glucose release by the extracellular protein fraction [Contribution of CelA to cellulose hydrolysis was also demonstrated by a llobiose . In contl strain  only resl strain . One exp12034.1)  used to 12034.1) . These a12034.1) . Primerslication ,77 and wfraction  and imprfraction .E. coli or Bacillus megaterium [C. bescii ΔcelA strain [C. bescii strain did not result in a dramatic effect, and the GT deletion strain was still capable of solubilizing microcrystalline cellulose [C. bescii GT knock out colonies were observed by Russell et al., [C. bescii strains are also deficient in their ability to form colonies on solid medium, resulting in a false negative result for growth on microcrystalline cellulose. Recent characterization of full length and truncated CelA has used natively purified enzyme , native gaterium , where tgaterium . GlycosyA strain ,90, onlyA strain  and the A strain ,91. Initellulose . Glycosyellulose . Lastly,E. coli) truncations of CelB (*-CBM3-CBM3-GH44), with CelE (GH9-CBM3-CBM3-CBM3-*), CelD (*-CBM3-CBM3-CBM3-GH5), or the S-layer located cellulase (GH5-CBM28-*) was detected [C. bescii cells are present [C. danielii, C. morganii, and C. naganoensis, including two enzymes that include three catalytic domains (GH12-Gh5-CBM3 × 3-GH48 and GH10-CBM3-GH12-GH48) [C. bescii GDL, hydrolyze microcrystalline cellulose synergistically [C. bescii biocatalyst.In addition to the synergy between catalytic domains in the modular cellulases, it would be expected that the enzymes themselves work in concert to hydrolyze plant biomass. Synergism between heterologously produced  . Homologstically  and are C. acetigenus, C. bescii, C. changbaiensis, C. kronotskyensis, C. owensensis, and C. saccharolyticus encode for a second locus of modular enzymes capable of hemicellulose hydrolysis [C. bescii [Caldicellulosiruptor species may be superior sources of xylanases that will work synergistically with cellulases, since they have evolved to specialize on the other carbohydrate components of plant biomass. One example are the extracellular enzymes from weakly cellulolytic C. owensensis, which significantly increased the amount of sugar released from non-pretreated, high-hemicellulose plant biomass when used sequentially with commercial cellulase mixtures [C. lactoaceticus was noted for five-fold higher activity on oat spelt xylan in comparison to birchwood or sugar case xylan [C. bescii may be the inclusion of additional xylanases and other hemicellulose hydrolyzing enzymes from the more xylanolytic members of the genus, or other species, for example, Acidothermus cellulolyticus. Overexpression of two different xylanases from A. celluloyticus resulted in a significant improvement of C. bescii growth on birchwood xylan and sugar release from oat spelt or birchwood xylan [C. kronotskyensis S-layer located xylanase in C. bescii [In addition to the GDL, six isolated species, drolysis . While n. bescii . Other, mixtures . The exase xylan . It is fod xylan , which w. bescii . H. thermocellum which is notable for its cellulosomes  the native H. thermocellum cellulosome during extended cellulose hydrolysis times at 75 °C [Other thermophilic genera also serve as promising consolidated bioprocessing platforms, like iewed in ) and celiewed in , but is iewed in . Prior siewed in , or withiewed in . Since tse, CelS  where itse, CelS . Howeverse, CelS . This iscosidase . In this strains . More reat 75 °C , demonstCaldicellulosiruptor modular cellulases, CelA (Csac_1076) and CelB , from C. saccharolyticus were introduced and expressed in Thermotoga sp. RQ2 [celB’ from C. saccharolyticus (*-CBM3-GH5) was included in an ionic liquid (IL)-tolerant enzyme cocktail (JTherm) for concurrent saccharification and IL treatment, which released over three times more sugars from IL-pretreated biomass at elevated temperatures [Aspergillus niger, and was again able to retain a high level of activity in comparison to other bacterial EGs expressed [C. naganoensis genomic library [Saccharomyces cerevisiae [E. coli.In addition to heterologous expression in other Firmicutes, elA Csac_6 and Celeratures . The samxpressed . This is library ,111 wererevisiae ,113 and Caldicellulosiruptor, the development of genetic tools afforded the opportunity to develop C. bescii as a potential platform organism for consolidated bioprocessing (CBP) where the same organism would enzymatically attack and ferment plant biomass to usable products . De systems  that wer[E. coli ,117. Genn method ,119 or kn method  togetherngements . Reverse of CelA  and a do of CelA  on the aom pBAS2 , one of plasmids . Aside fmocellum , expandiCaldicellulosiruptor , includ6 sugars . Conversnstrated , and recnotrophs  or chemonotrophs  convertiC. bescii for either increased productivity or redirection of reducing equivalents to other valuable metabolic pathways. Accordingly, the initial genetic manipulations of C. bescii were conducted with the goal of developing C. bescii as a suitable platform organism for biofuel production. Deletion of the lactate dehydrogenase gene (ldh) redirected reducing equivalents from the reduction of pyruvate to lactate to hydrogenases, which increased hydrogen gas production [adhE) from H. thermocellum, C. bescii produced ethanol when grown on switchgrass with no exogenously added CAZymes [C. bescii was capable of second generation consolidated bioprocessing, where no physiochemical pretreatment of the plant biomass was required for fermentation. Further improvements in ethanol titers during the growth on cellulose or xylan were made by introducing a reduced ferredoxin NAD oxidoreductase complex (Rnf) from Thermoanaerobacter sp. X514 [C. bescii, which increased the NADH/NAD+ ratio [Hydrogenesis also represents an attractive pathway to engineer in oduction . Further CAZymes . This wasp. X514  into C. D+ ratio . C. bescii (Topt 78–80 °C) [H. thermocellum (Topt 60 °C) [C. bescii from Thermoanaerobacter pseudethanolicus (Topt 65 °C), which would likely perform better at the elevated growth temperatures used for C. bescii [T. pseudethanolicus encodes for two bi-functional alcohol dehydrogenases, and between the two enzymes, C. bescii produced a higher titer of ethanol when expressing the NADH-dependent T. pseudethanolicus ADH during growth on microcrystalline cellulose or plant biomass [H. thermocellum ADH [T. pseudethanolicus ADH, annotated as a butanol dehydrogenase, increased the tolerance of C. bescii to furfurals, a byproduct of thermal and acid-pretreatment of plant biomass, by reducing furfurals to their cognate alcohols which are less toxic [Since the optimal growth temperature of 8–80 °C)  is highet 60 °C) , a bi-fu. bescii . T. pseu biomass . Howeverllum ADH . Anotherss toxic .C. bescii with additional, synergistic enzymes. Initially, modular Caldicellulosiruptor β-glucosidases were introduced in H. thermocellum to relieve cellobiose inhibition of cellulases [A. cellulolyticus [C. bescii, which increased the efficiency of cellulose hydrolysis. An important first step in increasing cellulolytic activity in C. bescii was demonstrated by chromosomal expression of a β-glucanase and/or endoglucanse from A. cellulolyticus, which resulted in synergistic increases in cellulose hydrolysis, and over a 17-fold improvement of C. bescii growth on cellulose [Thermotoga maritima to relieve cellobiose inhibition improved the performance of the C. bescii endoglucanase knock-in strain [C. bescii [C. bescii had evolved to maximize synergy between the endogenous C. bescii enzymes, there are opportunities to engineer superior cellulolytic strains. Improvements in xylan solubilization were also noted for C. bescii xylanase knock-in strains. As described above, expression of multiple GH10 enzymes from A. cellulolyticus [C. kronotskyensis  in C. bescii increased xylan hydrolysis in both cases and increased xylan attachment by the C. bescii  knock-in strain [Aside from metabolic engineering, there also has been a great deal of interest in supplementing the CAZyme repertoire of llulases ,104,105,olyticus  and cellolyticus , were prellulose . Heteroln strain , and the. bescii . Clearlyolyticus  or an S-n strain .Caldicellulosiruptor genome, immense strides towards Caldicellulosiruptor strain development for consolidated bioprocessing have been made. While Caldicellulosiruptor enzymes are robust and have demonstrated promise when heterologously expressed in other systems [C. bescii to produce value added chemicals from plant biomass is now more promising than ever. Critical developments, like the design of C. bescii genetic tools, now allow for reverse genetics and heterologous production of synergistic enzymes. Coupled with the previously established genomics, transcriptomics, and proteomics data available for the genus, the stage is set for further advancements towards targeted strain development. Initial reports of an inducible promoter [C. bescii are supporting the nascent field of Caldicellulosiruptor synthetic biology. Moving forward, the use of established ‘-omics’ tools to identify additional regulated promoters, optimal ribosomal binding sites, and other 5′ untranslated sequences will be critical for continued advancements in genetic manipulation of the genus Caldicellulosiruptor (see In the twelve years since the release of the first  systems ,141,142,promoter  and idenpromoter  in C. beptor see .Caldicellulosiruptor is found to be increasingly versatile for other difficult biotransformations. Enzymes identified from the genus have been demonstrated to be useful for food and pharmaceutical production, in addition to textile and paper processing. Additionally, their fermentative physiology is now being investigated for the formation of metal nanoparticles [Aside from lignocellulose deconstruction, the genus articles ,146 and articles . More so