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Dysbiosis of the intestinal microbiota is prevalent among patients with colorectal cancer (CRC). This study aims to explore the anticancer roles of the fecal microbiota in inhibiting the progression of colorectal cancer and possible mechanisms. The intestinal microbial dysbiosis in CRC mice was significantly ameliorated by fecal microbiota transplantation (FMT), as indicated by the restored ACE index and Shannon index. The diameter and number of cancerous foci were significantly decreased in CRC mice treated with FMT, along with the restoration of the intestinal mucosal structure and the lessening of the gland arrangement disorder. Key factors in oxidative stress (TXN1, TXNRD1, and HIF-1α); cell cycle regulators (IGF-1, BIRC5, CDK8, HDAC2, EGFR, and CTSL); and a critical transcription factor of the innate immune signal pathway (IRF5) were among the repressed oncogenic targets engaged in the FMT treatment of CRC. Correlation analysis revealed that their expressions were positively correlated with uncultured_bacterium_o_Mollicutes_RF39, Rikenellaceae_RC9_gut_group, and negatively correlated with Bacillus, Marvinbryantia, Roseburia, Angelakisella, Enterorhabdus, Bacteroides, Muribaculum, and genera of uncultured_bacterium_f_Eggerthellaceae, uncultured_bacterium_f_Xanthobacteraceae, Prevotellaceae_UCG-001, uncultured_bacterium_f_Erysipelotrichaceae, uncul-tured_bacterium_f_Lachnospiraceae, uncultured_bacterium_f_Ruminococcaceae, Eubacterium_coprostanoligenes_group, Ruminococcaceae_UCG-005, and uncultured_bacterium_f_Peptococcaceae. This study provides more evidence for the application of FMT in the clinical treatment of CRC.

IntroductionEmerging evidence suggests that the vaginal microbiota is closely associated with cervical cancer. However, little is known about the relationships among the vaginal microbiota, vaginal metabolites, and cervical lesion progression in women undergoing cervical dysplasia.MethodsIn this study, to understand vaginal microbiota signatures and vaginal metabolite changes in women with cervical lesions of different grades and cancer, individuals with normal or cervical dysplasia were recruited and divided into healthy controls (HC) group, low-grade squamous intraepithelial lesions (LSIL) group, high-grade squamous intraepithelial lesions (HSIL) group, and cervical cancer (CC) group. Vaginal secretion samples were collected for 16S rRNA gene sequencing, liquid chromatography coupled with mass spectrometry (LC–MS)-based metabolomics, and integrated analysis.ResultsThe results demonstrated that bacterial richness and diversity were greater in the CC group than the other three groups. Additionally, Lactobacillus was found to be negatively associated with bacterial diversity and bacterial metabolic functions, which increased with the degree of cervical lesions and cancer. Metabolomic analysis revealed that distinct metabolites were enriched in these metabolite pathways, including tryptophan metabolism, retinol metabolism, glutathione metabolism, alanine, aspartate, and glutamate metabolism, as well as citrate cycle (TCA cycle). Correlation analysis revealed positive associations between CC group-decreased Lactobacillus abundance and CC group-decreased metabolites. Lactobacillus iners was both negative to nadB and kynU genes, the predicted abundance of which was significantly higher in the CC group. The linear regression model showed that the combination of the vaginal microbiota and vaginal metabolites has good diagnostic performance for cervical cancer.DiscussionOur results indicated a clear difference in the vaginal microbiota and vaginal metabolites of women with cervical dysplasia. Specifically altered bacteria and metabolites were closely associated with the degree of cervical lesions and cancer, indicating the potential of the vaginal microbiota and vaginal metabolites as modifiable factors and therapeutic targets for preventing cervical cancer.

ObjectivesOropouche virus (OROV) causes systemic infections including the nervous and blood systems, posing a significant and growing public health challenge. However, a comprehensive review of the bibliometric analysis of OROV is still lacking. Therefore, the objective of this study was to provide insight into the research dynamics and current hotspots of OROV.MethodsThis study used bibliometric analysis to explore the current status of research related to OROV. 148 publications from 1961 to 2024 were retrieved from the Scopus database. Countries, authors, institutions, journals, references, and keywords were visualized using VOSviewer, CiteSpace, R studio, and Bibliometrix. Microsoft Excel was used for statistical analysis.ResultsBrazil is the country with the highest number of publications, total cited frequency, and the most extensive international collaboration. The most popular journal in this field is the American Journal of Tropical Medicine and Hygiene. Instituto Evandro Chagas is the institution with the highest number of publications, and Eurico Arruda is involved in the highest number of publications. Keyword co-occurrence analysis showed that Oropouche bunyavirus, virology, bunyavirus, priority journal, and nucleotide sequence are the main research hotspots in this field.ConclusionOur study provides a comprehensive overview of the research trends and key areas of focus in OROV. The field is currently experiencing rapid growth, as evidenced by the rising number of annual publications, which not only highlights increased research activity but also lays a solid foundation for further in-depth investigations. This trend offers valuable insights for developing effective strategies for outbreak prevention and control in public health. Presently, researchers are concentrating on the detailed study of Bunyavirus infections, employing both virological and genetic approaches to elucidate their complex pathogenic mechanisms.

Clostridioides difficile is an emerging pathogen of One Health significance. Its highly variable genome contains mobile genetic elements (MGEs) such as transposons and prophages that influence its biology. Systematic deletion of each genetic element is required to determine their precise role in C. difficile biology and contribution to the wider mobilome. Here, Tn5397 (21 kb) and ϕ027 (56 kb) were deleted from C. difficile 630 and R20291, respectively, using allele replacement facilitated by CRISPR-Cas9. The 630 Tn5397 deletant transferred PaLoc at the same frequency (1 × 10−7) as 630 harboring Tn5397, indicating that Tn5397 alone did not mediate conjugative transfer of PaLoc. The R20291 ϕ027 deletant was sensitive to ϕ027 infection, and contained two unexpected features, a 2.7 kb remnant of the mutagenesis plasmid, and a putative catalase gene adjacent to the deleted prophage was also deleted. Growth kinetics of R20291 ϕ027 deletant was similar to wild type (WT) in rich medium but marginally reduced compared with WT in minimal medium. This work indicates the commonly used pMTL8000 plasmid series works well for CRISPR-Cas9-mediated gene deletion, resulting in the largest deleted locus (56.8 kb) described in C. difficile. Removal of MGEs was achieved by targeting conjugative/integrative regions to promote excision and permanent loss. The deletants created will be useful strains for investigating Tn5397 or ϕ027 prophage contribution to host virulence, fitness, and physiology, and a platform for other mutagenesis studies aimed at functional gene analysis without native transposon or phage interference in C. difficile 630 and R20291.

BackgroundEngaging in anal sexual intercourse markedly increases the risk of developing HIV among men who have sex with men (MSM); oral sexual activities tend to uniquely introduce gut-derived microbes to salivary microbiota, which, combined with an individual’s positive HIV status, may greatly perturb oral microecology. However, till date, only a few published studies have addressed this aspect.MethodsBased on 16S rRNA sequencing data of bacterial taxa, MicroPITA picks representative samples for metagenomic analysis, effectively revealing how the development and progression of the HIV disease influences oral microbiota in MSM. Therefore, we collected samples from 11 HIV-negative and 44 HIV-positive MSM subjects (stage 0 was defined by HIV RNA positivity, but negative or indeterminate antibody status; stages 1, 2, and 3 were defined by CD4+ T lymphocyte counts ≥ 500, 200–499, and ≤ 200 or opportunistic infection) and selected 25 representative saliva samples (5 cases/stage) using MicroPITA. Metagenomic sequencing analysis were performed to explore whether positive HIV status changes salivary bacterial KEGG function and metabolic pathway in MSM.ResultsThe core functions of oral microbiota were maintained across each of the five groups, including metabolism, genetic and environmental information processing. All HIV-positive groups displayed KEGG functions of abnormal proliferation, most prominently at stage 0, and others related to metabolism. Clustering relationship analysis tentatively identified functional relationships between groups, with bacterial function being more similar between stage 0-control groups and stage 1-2 groups, whereas the stage 3 group exhibited large functional changes. Although we identified most metabolic pathways as being common to all five groups, several unique pathways formed clusters for certain groups; the stage 0 group had several, while the stage 2 and 3 groups had few, such clusters. The abundance of K03046 was positively correlated with CD4 counts.ConclusionAs HIV progresses, salivary bacterial function and metabolic pathways in MSM progressively changes, which may be related to HIV promoting abnormal energy metabolism and exacerbate pathogen virulence. Further, infection and drug resistance of acute stage and immune cell destruction of AIDS stage were abnormally increased, predicting an increased risk for MSM individuals to develop systemic and oral diseases.

IntroductionPakistan is an agricultural country; most of its income is based on livestock rearing. The increasing prevalence of tick-borne pathogens among animals may affect the animal production and livelihood of owners, which eventually derange the economy of a country.MethodologyTo further comprehend TBPs, 213 ticks were collected from different animals, including ruminants, pets, and poultry. After molecular and phylogenetic analysis identification, ticks were managed into different pools based on their species level (Hyalomma anatolicum = 80, Rhipicephalus microplus = 35, Hyalomma scupense = 23, Rhipicephalus turanicus = 70, and Rhipicephalus sanguineus = 5).Results and discussionAfter tick species identification, further molecular PCR amplification was carried out to screen out the pathogens for the presence of Theileria, Rickettsia, Anaplasma, and enzootic nasal tumor virus (ENTV). The following pathogens were detected: 11 (5.16%) for Anaplasma, 1 (0.47%) for Rickettsia, and 9 (4.23%) for Theileria. Nevertheless, other TBPs that had not been reported so far in Pakistan 3 (1.41%), were positive for enzootic nasal tumor virus (ENTV). Besides, phylogenetic analysis of the enzootic nasal tumor virus (ENTV) strain confirmed its resemblance to the Chinese strain, while Anaplasma has comparability with Pakistan and China, Rickettsia with Pakistan, China, and Iran, and Theileria with India, South Africa, United States, Japan, and Spain.ConclusionThis study reveals that there is a considerably wider range of TBPs held in Pakistan that take in various contagious zoonotic pathogens than was previously thought. This information advances TBP epidemiology and will contribute to upgrade future control measure.

ObjectiveThis study aimed to investigate sudapyridine (WX-081) antibacterial activity against Mycobacterium abscessus in vitro and its effect on in vivo bacterial growth and host survival using a zebrafish model of M. abscessus infection.MethodsWX-081 in vitro antibacterial activity was assessed based on growth inhibition of M. abscessus standard strain ATCC19977 and 36 clinical isolates. Maximum tolerated concentrations (MTCs) of WX-081, bedaquiline, and azithromycin and inhibition of M. abscessus growth were assessed in vivo after fluorescently labelled bacilli and drugs were injected into zebrafish. Bacterial counts were analysed using one-way ANOVA and fluorescence intensities of zebrafish tissues were analysed and expressed as the mean ± SE. Moreover, Kaplan-Meier survival analysis was conducted to assess intergroup differences in survival of M. abscessus-infected zebrafish treated with different drug concentrations using a log-rank test, with a p value

Many lines of evidence demonstrate the associations of colorectal cancer (CRC) with intestinal microbial dysbiosis. Recent reports have suggested that maintaining the homeostasis of microbiota and host might be beneficial to CRC patients, but the underlying mechanisms remain unclear. In this study, we established a CRC mouse model of microbial dysbiosis and evaluated the effects of fecal microbiota transplantation (FMT) on CRC progression. Azomethane and dextran sodium sulfate were used to induce CRC and microbial dysbiosis in mice. Intestinal microbes from healthy mice were transferred to CRC mice by enema. The vastly disordered gut microbiota of CRC mice was largely reversed by FMT. Intestinal microbiota from normal mice effectively suppressed cancer progression as assessed by measuring the diameter and number of cancerous foci and significantly prolonged survival of the CRC mice. In the intestine of mice that had received FMT, there were massive infiltration of immune cells, including CD8+ T and CD49b+ NK, which is able to directly kill cancer cells. Moreover, the accumulation of immunosuppressive cells, Foxp3+ Treg cells, seen in the CRC mice was much reduced after FMT. Additionally, FMT regulated the expressions of inflammatory cytokines in CRC mice, including down-regulation of IL1a, IL6, IL12a, IL12b, IL17a, and elevation of IL10. These cytokines were positively correlated with Azospirillum_sp._47_25, Clostridium_sensu_stricto_1, the E. coli complex, Akkermansia, Turicibacter, and negatively correlated with Muribaculum, Anaeroplasma, Candidatus_Arthromitus, and Candidatus Saccharimonas. Furthermore, the repressed expressions of TGFb, STAT3 and elevated expressions of TNFa, IFNg, CXCR4 together promoted the anti-cancer efficacy. Their expressions were positively correlated with Odoribacter, Lachnospiraceae-UCG-006, Desulfovibrio, and negatively correlated with Alloprevotella, Ruminococcaceae UCG-014, Ruminiclostridium, Prevotellaceae UCG-001 and Oscillibacter. Our studies indicate that FMT inhibits the development of CRC by reversing gut microbial disorder, ameliorating excessive intestinal inflammation and cooperating with anti-cancer immune responses.

BackgroundIdentification of transposons without close homologs is still a difficult task. IS630/Tc1/mariner transposons, classified into a superfamily, are probably the most widespread DNA transposons in nature. Tc1/mariner transposons have been discovered in animals, plants, and filamentous fungi, however, not in yeast.ResultsIn the present study, we report the discovery of two intact Tc1 transposons in yeast and filamentous fungi, respectively. The first one, named Tc1-OP1 (DD40E), represents Tc1 transposons in Ogataea parapolymorpha. The second one, named Tc1-MP1 (DD34E), represents Tc1 transposons in the Rhizopodaceae and Mucoraceae families. As a homolog of Tc1-OP1 and Tc1-MP1, IS630-AB1 (DD34E) was discovered as an IS630 transposon in Acinetobacter spp.ConclusionTc1-OP1 is not only the first reported Tc1 transposon in yeast, but also the first reported nonclassical Tc1 transposon. Tc1-OP1 is the largest of IS630/Tc1/mariner transposons reported to date and significantly different from others. Notably, Tc1-OP1 encodes a serine-rich domain and a transposase, extending the current knowledge of Tc1 transposons. The phylogenetic relationships of Tc1-OP1, Tc1-MP1 and IS630-AB1 indicated that these transposons had evolved from a common ancestor. Tc1-OP1, Tc1-MP1 and IS630-AB1 can be used as reference sequences to facilitate the identification of IS630/Tc1/mariner transposons. More Tc1/mariner transposons will be identified in yeast, following our discovery.

ABSTRACT Metagenomic next-generation sequencing (mNGS), mostly carried out in independent clinical laboratories, has been increasingly applied in clinical pathogen diagnosis. We aimed to explore the feasibility of mNGS in clinical laboratories and analyze its potential in the diagnosis of infectious ascites. Two reference panels composed of 12 strains commonly appearing in peritonitis were constructed to evaluate the performance metrics based on in-house mNGS protocols. The mNGS clinical detection value was analyzed in 211 ascitic samples and compared with culture and composite standards. Finally, eight patients with cirrhosis were prospectively enrolled to verify the clinical value of mNGS in peritoneal infection diagnosis. The mNGS analytical performance showed that the assay had great linearity, specificity, stability, interference, and limits of detection of 33 to 828 CFU/mL. The sensitivity and specificity of mNGS for bacterial or fungal detection using culture standards were 84.2% and 82.0%, respectively. After adjustment using digital PCR and clinical judgment, the sensitivity and specificity increased to 87.2% and 90.1%, respectively. Compared with culture, mNGS detected a broad range of pathogens and more polymicrobial infections (49% versus 9%, P < 0.05). The pathogen results were obtained within 24 h using mNGS in eight prospective cases, which effectively guided antibiotics therapy. mNGS testing in clinical laboratories affiliated with a hospital has certain advantages. It has unique superiority in pathogens detection, particularly in patients with polymicrobial infections. However, considering spectrum characteristics and test cost, pertinent pathogen panels should be developed in clinical practice. IMPORTANCE This study established and evaluated a complete metagenomics next-generation sequencing assay to improve the diagnosis of suspected ascitic infection in a clinical laboratory affiliated with a hospital. The assay is superior to traditional culture testing and will aid in the early and accurate identification of pathogens, particularly in patients with polymicrobial infections. This assay is also essential for precision therapy and can reduce the incidence of drug resistance stemming from irrational use of antibiotics.

It is realized that the first intron plays a key role in regulating gene expression, and the interactions between the first introns and other introns must be related to the regulation of gene expression. In this paper, the sequences of mitochondrial ribosomal protein genes were selected as the samples, based on the Smith-Waterman method, the optimal matched segments between the first intron and the reverse complementary sequences of other introns of each gene were obtained, and the characteristics of the optimal matched segments were analyzed. The results showed that the lengths and the ranges of length distributions of the optimal matched segments are increased along with the evolution of eukaryotes. For the distributions of the optimal matched segments with different GC contents, the peak values are decreased along with the evolution of eukaryotes, but the corresponding GC content of the peak values are increased along with the evolution of eukaryotes, it means most introns of higher organisms interact with each other though weak bonds binding. By comparing the lengths and matching rates of optimal matched segments with those of siRNA and miRNA, it is found that some optimal matched segments may be related to non-coding RNA with special biological functions, just like siRNA and miRNA, they may play an important role in the process of gene expression and regulation. For the relative position of the optimal matched segments, the peaks of relative position distributions of optimal matched segments are increased during the evolution of eukaryotes, and the positions of the first two peaks exhibit significant conservatism.

ObjectiveBacterial DNA (bactDNA) detection can be used to quickly identify pathogenic bacteria and has been studied on ascitic fluid. We aimed to retrospectively analyze the diagnostic value and applicational prospect of the bactDNA load in spontaneous bacterial peritonitis (SBP).MethodWe extracted viable bactDNA from ascitic samples of 250 patients with decompensated cirrhosis collected from October 2019 to April 2021 and detected the bactDNA by droplet digital polymerase chain reaction (ddPCR). We used ascitic samples of a baseline cohort of 191 patients to establish diagnostic thresholds for SBP and analyze the patients’ diagnostic performance based on ascites polymorphonuclear (PMN) and clinical manifestation. We performed bactDNA quantification analysis on 13 patients with a PMN less than 250 cells/mm3 but with clinical symptoms. The dynamic changes of the bactDNA load from eight patients (before, during, and after SBP) were analyzed.ResultsAfter the removal of free DNA, the bactDNA detected by ddPCR was generally decreased (1.75 vs. 1.5 log copies/µl, P < 0.001). Compared with the traditional culture and PMN count in the SBP diagnosis, the bactDNA showed that the ddPCR sensitivity was 80.5%, specificity was 95.3%, positive predictive value was 82.5%, and negative predictive value was 94.7%, based on clinical composite criteria. In patients with a PMN less than 250 cells/mm3, the bactDNA load of 13 patients with symptoms was significantly higher than those without symptoms (2.7 vs. 1.7 log copies/µl, P < 0.001). The bactDNA in eight patients had SBP that decreased by 1.6 log copies/µl after 48 h of antibiotic treatment and by 1.0 log copies/µl after 3 days of continued treatment.ConclusionBactDNA detection can be used to further enhance the diagnostic efficiency of SBP. Therefore, the application of ddPCR assay not only can be used to discriminate and quantify bacteria but also can be used in the clinical assessment for antibiotics treatment.

Francisella tularensis is a dangerous pathogen that causes an extremely contagious zoonosis in humans named tularemia. Given its low-dose morbidity, the potential to be fatal, and aerosol spread, it is regarded as a severe threat to public health. The US Centers for Disease Control and Prevention (CDC) has classified it as a category A potential agent for bioterrorism and a Tier 1 Select Agent. Herein, we combined recombinase polymerase amplification (RPA) with CRISPR/Cas12a system to select the F. tularensis target gene (TUL4), creating a two-pronged rapid and ultrasensitive diagnostic method for detecting F. tularensis. The real-time RPA (RT-RPA) assay detected F. tularensis within 10 min at a sensitivity of 5 copies/reaction, F. tularensis genomic DNA of 5 fg, and F. tularensis of 2 × 102 CFU/ml; the RPA-CRISPR/Cas12a assay detects F. tularensis within 40 min at a sensitivity of 0.5 copies/reaction, F. tularensis genomic DNA of 1 fg, and F. tularensis of 2 CFU/ml. Furthermore, the evaluation of specificity showed that both assays were highly specific to F. tularensis. More importantly, in a test of prepared simulated blood and sewage samples, the RT-RPA assay results were consistent with RT-PCR assay results, and the RPA-CRISPR/Cas12a assay could detect a minute amount of F. tularensis genomic DNA (2.5 fg). There was no nonspecific detection with blood samples and sewage samples, giving the tests a high practical application value. For example, in on-site and epidemic areas, the RT-RPA was used for rapid screening and the RPA-CRISPR/Cas12a assay was used for more accurate diagnosis.

ABSTRACT Apostichopus japonicus (sea cucumber) is one of the most valuable aquaculture species in China; however, different diseases can limit its economic development. Recently, a novel disease, body vesicular syndrome (BVS), was observed in A. japonicus aquaculture. Diseased animals displayed no obvious phenotypic characteristics; however, after boiling at the postharvest stage, blisters, lysis, and body ruptures appeared. In this study, a multiomics strategy incorporating analysis of the gut microbiota, the pond microbiome, and A. japonicus genotype was established to investigate BVS. Detailed analyses of differentially expressed proteins (DEPs) and metabolites suggested that changes in cell adhesion structures, caused by disordered fatty acid β-oxidation mediated by vitamin B5 deficiency, could be a putative BVS mechanism. Furthermore, intestinal dysbacteriosis due to microbiome variations in pond water was considered a potential reason for vitamin B5 deficiency. Our BVS index, based on biomarkers identified from the A. japonicus gut microbiota, was a useful tool for BVS diagnosis. Finally, vitamin B5 supplementation was successfully used to treat BVS, suggesting an association with BVS etiology. IMPORTANCE Body vesicular syndrome (BVS) is a novel disease in sea cucumber aquaculture. As no phenotypic features are visible, BVS is difficult to confirm during aquaculture and postharvest activities, until animals are boiled. Therefore, BVS could lead to severe economic losses compared with other diseases in sea cucumber aquaculture. In this study, for the first time, we systematically investigated BVS pathogenesis and proposed an effective treatment for the condition. Moreover, based on the gut microbiota, we established a noninvasive diagnostic method for BVS in sea cucumber.

The potentially pathogenic species of the genus Vibrio pose a threat to both humans and animals, creating medical burdens and economic losses to the mariculture industry. Improvements in surveillance and diagnosis are needed to successfully manage vibriosis outbreaks. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) can provide rapid diagnosis and has been widely used in the identification of Vibrio spp. The main weakness of this technology is the limited number of strains and species of Vibrio in the existing commercial database. Here, we develop a new in-house database named PVBase containing 790 main spectra projections (MSP) of ten Vibrio species that come from various regions of China and include abundant clinical and environmental strains. PVBase was validated through a blind test of 65 Vibrio strains. The identification accuracy and scoring of Vibrio strains was greatly improved through the addition of PVBase. Identification accuracy increased from 73.4 to 100%. The number of strains with identification scores above 2.2 increased from 53.1% to 96.9% and 53.1% of strains had an identification score above 2.59. Moreover, perfect discrimination was obtained when using all of the MSPs created for the Vibrio species, even for very closely related species such as V. cholerae, V. albensis, and V. mimicus or V. alginolyticus, V. parahaemolyticus, and V. harveyi. In addition, we used phyloproteomic analysis to study whether there are differences in protein fingerprints of different regions or pathogenic strains. We found that MSP characteristics of Vibrio species were not related to their region or source. With the construction of PVBase, the identification efficiency of potentially pathogenic Vibrio species has been greatly improved, which is an important advance for epidemic prevention and control, and aquaculture disease detection.

Pregnant women are at high risk of developing subclinical hypothyroidism (SCH), and anti-thyroid peroxidase antibody (TPOAb) positivity can further inhibit thyroxine synthesis. Emerging evidence indicates that intestinal flora can modulate metabolic and immune homeostasis. The characteristics of intestinal flora of TPOAb-positive women with SCH in their second trimester of pregnancy have not been reported. This single-center prospective observational cohort study investigated gut microbial composition and metabolic function using sequencing of the 16S rRNA gene in fecal samples from 75 TPOAb-positive women with SCH and 90 TPOAb-negative women with SCH during their second trimester of pregnancy. Women were treated with no levothyroxine (LT4), low-dose LT4 (≤50ug/d), or high-dose LT4 (>50ug/d). Taxonomic analysis showed Firmicutes and Bacteroidetes were the dominant phyla, followed by Actinobacteria and Proteobacteria. Faecalibacterium, Bacteroides, Prevotella 9, Bifidobacterium, Subdoligranulum, Lachnospira, and Megamonas were the predominant genera. The intestinal flora of TPOAb-positive women with SCH who received no LT4 was characterized by bacterial amplicon sequence variants (ASVs)/operational taxonomic units (OTUs) enriched in the genus Subdoligranulum. The intestinal flora of TPOAb-positive women with SCH who received low-dose or high-dose LT4 were characterized by bacterial ASVs/OTUs depleted of the species Ruminococcus sp._or Bacteroides massiliensis, respectively. A total of 19 metabolic functions of intestinal flora, mainly involving lipid and amino acid metabolism, discriminated TPOAb-positive and TPOAb-negative women with SCH. Our study suggests that there are differences in the composition and metabolic function of intestinal flora of TPOAb-positive and TPOAb-negative women with SCH treated with different doses of LT4 in the second trimester of pregnancy. The findings provide insight into intestinal flora as novel targets for the treatment of TPOAb-positive women with SCH during pregnancy.

BackgroundCurrently, methylotrophic yeasts (e.g., Pichia pastoris, Ogataea polymorpha, and Candida boindii) are subjects of intense genomics studies in basic research and industrial applications. In the genus Ogataea, most research is focused on three basic O. polymorpha strains-CBS4732, NCYC495, and DL-1. However, the relationship between CBS4732, NCYC495, and DL-1 remains unclear, as the genomic differences between them have not be exactly determined without their high-quality complete genomes. As a nutritionally deficient mutant derived from CBS4732, the O. polymorpha strain CBS4732 ura3Δ (named HU-11) is being used for high-yield production of several important proteins or peptides. HU-11 has the same reference genome as CBS4732 (noted as HU-11/CBS4732), because the only genomic difference between them is a 5-bp insertion.ResultsIn the present study, we have assembled the full-length genome of O. polymorpha HU-11/CBS4732 using high-depth PacBio and Illumina data. Long terminal repeat retrotransposons (LTR-rts), rDNA, 5′ and 3′ telomeric, subtelomeric, low complexity and other repeat regions were exactly determined to improve the genome quality. In brief, the main findings include complete rDNAs, complete LTR-rts, three large duplicated segments in subtelomeric regions and three structural variations between the HU-11/CBS4732 and NCYC495 genomes. These findings are very important for the assembly of full-length genomes of yeast and the correction of assembly errors in the published genomes of Ogataea spp. HU-11/CBS4732 is so phylogenetically close to NCYC495 that the syntenic regions cover nearly 100% of their genomes. Moreover, HU-11/CBS4732 and NCYC495 share a nucleotide identity of 99.5% through their whole genomes. CBS4732 and NCYC495 can be regarded as the same strain in basic research and industrial applications.ConclusionThe present study preliminarily revealed the relationship between CBS4732, NCYC495, and DL-1. Our findings provide new opportunities for in-depth understanding of genome evolution in methylotrophic yeasts and lay the foundations for the industrial applications of O. polymorpha CBS4732, NCYC495, DL-1, and their derivative strains. The full-length genome of O. polymorpha HU-11/CBS4732 should be included into the NCBI RefSeq database for future studies of Ogataea spp.

Abstract This study aimed to explore the effects of artemisinin (ART) on the milk microbiome and metabolites of dairy cow. A total of 12 mid-lactation Holstein dairy cows with similar parity, days in milk were randomly divided into 2 groups receiving either a total mixed ration (TMR) as the control group or this TMR and 120 g/d/head ART as the ART group. The milk samples were collected weekly to determine the contents, and end-of-trial (week 8) milk samples were used to identify microbial species and metabolite profiles by 16S rRNA sequencing and LC–MS analyses, respectively. We observed that the milk fat content significantly increased by ART treatment (P 0.05). Compared with its abundance in the control (CON) group, Firmicutes was significantly decreased, whereas Proteobacteria was significantly increased. Furthermore, in the ART group, the relative abundances of the genera Aerococcus, Staphylococcus, Corynebacterium_1 and Facklamia were significantly lower (P

Carbohydrate-binding modules (CBMs), as an important auxiliary module, play a key role in degrading soluble alginate by alginate lyase, but the function on alginate gel has not been elucidated. Recently, we reported alginate lyase VxAly7B containing a CBM32 and a polysaccharide lyase family 7 (PL7). To investigate the specific function of CBM32, we characterized the full-length alginate lyase VxAly7B (VxAly7B-FL) and truncated mutants VxAly7B-CM (PL7) and VxAly7B-CBM (CBM32). Both VxAly7B-FL and native VxAly7B can spontaneously cleavage between CBM32 and PL7. The substrate-binding capacity and activity of VxAly7B-CM to soluble alginate were 0.86- and 1.97-fold those of VxAly7B-FL, respectively. Moreover, CBM32 could accelerate the expansion and cleavage of alginate gel beads, and the degradation rate of VxAly7B-FL to alginate gel beads was threefold that of VxAly7B-CM. Results showed that CBM32 is not conducive to the degradation of soluble alginate by VxAly7B but is helpful for binding and degradation of insoluble alginate gel. This study provides new insights into the function of CBM32 on alginate gel, which may inspire the application strategy of CBMs in insoluble substrates.

Over the last three years, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related health crisis has claimed over six million lives and caused USD 12 trillion losses to the global economy. SARS-CoV-2 continuously mutates and evolves with a high basic reproduction number (R0), resulting in a variety of clinical manifestations ranging from asymptomatic infection to acute respiratory distress syndrome (ARDS) and even death. To gain a better understanding of coronavirus disease 2019 (COVID-19), it is critical to investigate the components that cause various clinical manifestations. Single-cell sequencing has substantial advantages in terms of identifying differentially expressed genes among individual cells, which can provide a better understanding of the various physiological and pathological processes. This article reviewed the use of single-cell transcriptomics in COVID-19 research, examined the immune response disparities generated by SARS-CoV-2, and offered insights regarding how to improve COVID-19 diagnosis and treatment plans.

Accumulated evidence shows that complex microbial communities resides in the healthy human urinary tract and can change in urological disorders. However, there lacks a comprehensive profiling of the genitourinary microbiota in healthy cohort. Here, we performed 16S rRNA gene sequencing of midstream urine specimens from 1,172 middle-aged and elderly healthy individuals. The core microbiota included 6 dominant genera (mean relative abundance >5%), including Prevotella, Streptococcus, Lactobacillus, Gardnerella, Escherichia-Shigella, and Veillonella, and 131 low-abundance genera (0.01–5%), displaying a distinct microbiome profiles to that of host-matched gut microbiota. The composition and diversity of genitourinary microbiome (GM) were distinct between genders and may fluctuate with ages. Several urotypes were identified by the stratification of microbiome profiles, which were mainly dominated by the six most predominant genera. The prevalence of urotypes was disparate between genders, and the male sample additionally harbored other urotypes dominated by Acinetobacter, Corynebacterium, Staphylococcus, or Sphingomonas. Peptoniphilus, Ezakiella, and Porphyromonas were co-occurred and co-abundant, and they may play crucial roles as keystone genera and be associated with increased microbial diversity. Our results delineated the microbial structure and diversity landscape of the GM in healthy middle-aged and elderly adults and provided insights into the influence of gender and age to it.

Increasing evidence indicated that 6mA could respond to environmental stressors in multicellular eukaryotes. As 6mA distribution and function differ significantly in multicellular and unicellular organisms, whether and how 6mA in unicellular eukaryotes responds to environmental stress remains elusive.

Abstract Background Numerous studies have shown that stress induction and genetic engineering can effectively increase lipid accumulation, but lead to a decrease of growth in the majority of microalgae. We previously found that elevated CO2 concentration increased lipid productivity as well as growth in Phaeodactylum tricornutum, along with an enhancement of the oxidative pentose phosphate pathway (OPPP) activity. The purpose of this work directed toward the verification of the critical role of glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme in the OPPP, in lipid accumulation in P. tricornutum and its simultaneous rapid growth rate under high-CO2 (0.15%) cultivation. Results In this study, G6PDH was identified as a target for algal strain improvement, wherein G6PDH gene was successfully overexpressed and antisense knockdown in P. tricornutum, and systematic comparisons of the photosynthesis performance, algal growth, lipid content, fatty acid profiles, NADPH production, G6PDH activity and transcriptional abundance were performed. The results showed that, due to the enhanced G6PDH activity, transcriptional abundance and NAPDH production, overexpression of G6PDH accompanied by high-CO2 cultivation resulted in a much higher of both lipid content and growth in P. tricornutum, while knockdown of G6PDH greatly decreased algal growth as well as lipid accumulation. In addition, the total proportions of saturated and unsaturated fatty acid, especially the polyunsaturated fatty acid eicosapentaenoic acid (EPA; C20:5, n-3), were highly increased in high-CO2 cultivated G6PDH overexpressed strains. Conclusions The successful of overexpression and antisense knockdown of G6PDH well demonstrated the positive influence of G6PDH on algal growth and lipid accumulation in P. tricornutum. The improvement of algal growth, lipid content as well as polyunsaturated fatty acids in high-CO2 cultivated G6PDH overexpressed P. tricornutum suggested this G6PDH overexpression-high CO2 cultivation pattern provides an efficient and economical route for algal strain improvement to develop algal-based biodiesel production.

In December 2019, the world awoke to a new betacoronavirus strain named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Betacoronavirus consists of A, B, C and D subgroups. Both SARS-CoV and SARS-CoV-2 belong to betacoronavirus subgroup B. In the present study, we divided betacoronavirus subgroup B into the SARS1 and SARS2 classes by six key insertions and deletions (InDels) in betacoronavirus genomes, and identified a recently detected betacoronavirus strains RmYN02 as a recombinant strain across the SARS1 and SARS2 classes, which has potential to generate a new strain with similar risk as SARS-CoV and SARS-CoV-2. By analyzing genomic features of betacoronavirus, we concluded: (1) the jumping transcription and recombination of CoVs share the same molecular mechanism, which inevitably causes CoV outbreaks; (2) recombination, receptor binding abilities, junction furin cleavage sites (FCSs), first hairpins and ORF8s are main factors contributing to extraordinary transmission, virulence and host adaptability of betacoronavirus; and (3) the strong recombination ability of CoVs integrated other main factors to generate multiple recombinant strains, two of which evolved into SARS-CoV and SARS-CoV-2, resulting in the SARS and COVID-19 pandemics. As the most important genomic features of SARS-CoV and SARS-CoV-2, an enhanced ORF8 and a novel junction FCS, respectively, are indispensable clues for future studies of their origin and evolution. The WIV1 strain without the enhanced ORF8 and the RaTG13 strain without the junction FCS “RRAR” may contribute to, but are not the immediate ancestors of SARS-CoV and SARS-CoV-2, respectively.

A greater understanding of the genes involved in antibiotic resistance in Mycobacterium tuberculosis (Mtb) is necessary for the design of improved therapies. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been previously utilized in mycobacteria to identify novel drug targets by the demonstration of gene essentiality. The work presented here shows that it can also be usefully applied to the study of non-essential genes involved in antibiotic resistance. The expression of an ADP-ribosyltransferase (Arr) involved in rifampicin resistance in Mycobacterium smegmatis was silenced using CRISPRi and the impact on rifampicin susceptibility was measured. Gene silencing resulted in a decrease in the minimum inhibitory concentration (MIC) similar to that previously reported in an arr deletion mutant. There is contradictory evidence for the toxicity of Streptococcus pyogenes dCas9 (dCas9Spy) in the literature. In this study the expression of dCas9Spy in M. smegmatis showed no impact on viability. Silencing was achieved with concentrations of the aTc inducer lower than previously described and with shorter induction times. Finally, designing small guide RNAs (sgRNAs) that target transcription initiation, or the early stages of elongation had the most impact on rifampicin susceptibility. This study demonstrates that CRISPRi based gene silencing can be as impactful as gene deletion for the study of non-essential genes and further contributes to the knowledge on the design and induction of sgRNAs for CRISPRi. This approach can be applied to other non-essential antimicrobial resistance genes such as drug efflux pumps.

How to achieve protein diversity by genome and transcriptome processing is essential for organismal complexity and adaptation. The present work identifies that the macronuclear genome of Halteria grandinella

Genotypic based detection methods using specific target sites in the pathogen genome can complement phenotypic identification. We report the development of species-specific antisense peptide nucleic acid (PNA) combined with selective and differential enrichment growth conditions for Salmonella treatment and detection. An antisense PNA oligomer targeting the Salmonella ftsZ gene and conjugated with a cell-penetrating peptide ((KFF)3K) was exploited to probe bacteria cultured in three different growth media (Muller Hinton broth (MHB), Rappaport-Vassiliadis Soya Peptone Broth (RVS, Oxoid), and in-house modified Rappaport-Vassiliadis Soya Peptone Broths (mRVSs). Also, water and milk artificially contaminated with bacteria were probed. Antisense PNA provided detectable changes in Salmonella growth and morphology in all media and artificially contaminated matrices except RVS. Salmonella was detected as elongated cells. On the contrary, treated Escherichia coli did not elongate, providing evidence of differentiation and selectivity for Salmonella. Similarly, Salmonella probed with mismatched PNAs did not elongate. Antisense oligomers targeted ftsZ mRNA in combination with selective growth conditions can provide a detection strategy for viable Salmonella in a single reaction, and act as a potential tool for bacteria detection in real food and environmental samples.

Abstract Background Vibrio parahaemolyticus is as an important food-borne pathogen circulating in China. Since 1996, the core serotype has become O3:K6, which has specific genetic markers. This serotype causes the majority of outbreaks worldwide. Until now, nearly 21 serotypes were considered as serovariants of O3:K6. Among these, O4:K68, O1:K25 and O1:KUT have caused pandemic outbreaks. O4:K8, a serovariant of O3:K6, has become the second most dominant serotype circulating in China after O3:K6. In this study, we report the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze and characterize 146 V. parahaemolyticus isolates belonging to 23 serotypes. Results Upon mass spectral analysis, isolates belonging to O4:K8 formed a distinct group among the five main pandemic groups (O3:K6, O4:K8, O4:K68, O1:K25 and O1:KUT). Two major protein peaks (m/z 4383 and 4397) were significantly different between serotype O4:K8 and the four other pandemic strains. Both of these peaks were present in 32 out of 36 O4:K8 isolates, but were absent in 105 out of 110 non-O4:K8 isolates. These peaks were also absent in all 74 pandemic serotypes (O3:K6, O4:K68, O1:K25 and O1:KUT). Conclusion Our results highlight the threat of O4:K8 forming a distinct group, which differs significantly from pandemic serotypes on the proteomic level. The use of MALDI-TOF MS has not been reported before in a study of this nature. Mass spectrum peaks at m/z 4383 and 4397 may be specific for O4:K8. However, we cannot conclude that MALDI-TOF MS can be used to serotype V. parahaemolyticus.

Chikungunya fever (CHIKF) is an acute infectious disease that is mediated by the mosquito-transmitted chikungunya virus (CHIKV). People infected with CHIKV may experience high fever, severe joint pain, skin rash, and headache. In recent years, this disease has become a global public health problem. However, there is no licensed vaccine available for CHIKV. Accumulating research data have provided novel approaches and new directions for the development of CHIKV vaccines. Our review focuses on recent progress in CHIKV vaccine studies. The potential vaccine candidates are classified into seven types: inactivated vaccine, subunit vaccine, live-attenuated vaccine, recombinant virus-vectored vaccine, virus-like particle vaccine, chimeric vaccine, and nucleic acid vaccine. These studies will provide important insights into the future development of CHIKV vaccines.

ABSTRACT Chronic rhinosinusitis (CRS) is a heterogeneous condition characterized by persistent sinus inflammation and microbial dysbiosis. This study aimed to identify clinically relevant subgroups of CRS patients based on distinct microbial signatures, with a comparison to the commonly used phenotypic subgrouping approach. The underlying drivers of these distinct microbial clusters were also investigated, together with associations with epithelial barrier integrity. Sinus biopsy specimens were collected from CRS patients (n = 23) and disease controls (n = 8). The expression of 42 tight junction genes was evaluated using quantitative PCR together with microbiota analysis and immunohistochemistry for measuring mucosal integrity and inflammation. CRS patients clustered into two distinct microbial subgroups using probabilistic modelling Dirichlet (DC) multinomial mixtures. DC1 exhibited significantly reduced bacterial diversity and increased dispersion and was dominated by Pseudomonas, Haemophilus, and Achromobacter. DC2 had significantly elevated B cells and incidences of nasal polyps and higher numbers of Anaerococcus, Megasphaera, Prevotella, Atopobium, and Propionibacterium. In addition, each DC exhibited distinct tight junction gene and protein expression profiles compared with those of controls. Stratifying CRS patients based on clinical phenotypic subtypes (absence or presence of nasal polyps [CRSsNP or CRSwNP, respectively] or with cystic fibrosis [CRSwCF]) accounted for a larger proportion of the variation in the microbial data set than with DC groupings. However, no significant differences between CRSsNP and CRSwNP cohorts were observed for inflammatory markers, beta-dispersion, and alpha-diversity measures. In conclusion, both approaches used for stratifying CRS patients had benefits and pitfalls, but DC clustering provided greater resolution when studying tight junction impairment. Future studies in CRS should give careful consideration to the patient subtyping approach used. IMPORTANCE Chronic rhinosinusitis (CRS) is a major human health problem that significantly reduces quality of life. While various microbes have been implicated, there is no clear understanding of the role they play in CRS pathogenesis. Another equally important observation made for CRS patients is that the epithelial barrier in the sinonasal cavity is defective. Finding a robust approach to subtype CRS patients would be the first step toward unravelling the pathogenesis of this heterogeneous condition. Previous work has explored stratification based on the clinical presentation of the disease (with or without polyps), inflammatory markers, pathology, or microbial composition. Comparisons between the different stratification approaches used in these studies have not been possible due to the different cohorts, analytical methods, or sample sites used. In this study, two approaches for subtyping CRS patients were compared, and the underlying drivers of the heterogeneity in CRS were also explored.

Until recently, Clostridium difficile phages were limited to Myoviruses and Siphoviruses of medium genome length (32–57 kb). Here we report the finding of phiCD5763, a Siphovirus with a large extrachromosomal circular genome (132.5 kb, 172 ORFs) and a large capsid (205.6 ± 25.6 nm in diameter) infecting MLST Clade 1 strains of C. difficile. Two subgroups of big phage genomes similar to phiCD5763 were identified in 32 NAPCR1/RT012/ST-54 C. difficile isolates from Costa Rica and in whole genome sequences (WGS) of 41 C. difficile isolates of Clades 1, 2, 3, and 4 from Canada, USA, UK, Belgium, Iraq, and China. Through comparative genomics we discovered another putative big phage genome in a non-NAPCR1 isolate from Costa Rica, phiCD2955, which represents other big phage genomes found in 130 WGS of MLST Clade 1 and 2 isolates from Canada, USA, Hungary, France, Austria, and UK. phiCD2955 (131.6 kb, 172 ORFs) is related to a previously reported C. difficile phage genome, phiCD211/phiCDIF1296T. Detailed genome analyses of phiCD5763, phiCD2955, phiCD211/phiCDIF1296T, and seven other putative C. difficile big phage genome sequences of 131–136 kb reconstructed from publicly available WGS revealed a modular gene organization and high levels of sequence heterogeneity at several hotspots, suggesting that these genomes correspond to biological entities undergoing recombination. Compared to other C. difficile phages, these big phages have unique predicted terminase, capsid, portal, neck and tail proteins, receptor binding proteins (RBPs), recombinases, resolvases, primases, helicases, ligases, and hypothetical proteins. Moreover, their predicted gene load suggests a complex regulation of both phage and host functions. Overall, our results indicate that the prevalence of C. difficile big bacteriophages is more widespread than realized and open new avenues of research aiming to decipher how these viral elements influence the biology of this emerging pathogen.

Dietary intake affects the structure and function of microbes in host intestine. However, the succession of gut microbiota in response to changes in macronutrient levels during a long period of time remains insufficiently studied. Here, we determined the succession and metabolic products of intestinal microbiota in grass carp (Ctenopharyngodon idellus) undergoing an abrupt and extreme diet change, from fish meal to Sudan grass (Sorghum sudanense). Grass carp hindgut microbiota responded rapidly to the diet shift, reaching a new equilibrium approximately within 11 days. In comparison to animal-diet samples, Bacteroides, Lachnospiraceae and Erysipelotrichaceae increased significantly while Cetobacterium decreased significantly in plant-diet samples. Cetobacterium was negatively correlated with Bacteroides, Lachnospiraceae and Erysipelotrichaceae, while Bacteroides was positively correlated with Lachnospiraceae. Predicted glycoside hydrolase and polysaccharide lyase genes in Bacteroides and Lachnospiraceae from the Carbohydrate-Active enZymes (CAZy) database might be involved in degradation of the plant cell wall polysaccharides. However, none of these enzymes was detected in the grass carp genome searched against dbCAN database. Additionally, a significant decrease of short chain fatty acids levels in plant-based samples was observed. Generally, our results suggest a rapid adaption of grass carp intestinal microbiota to dietary shift, and that microbiota are likely to play an indispensable role in nutrient turnover and fermentation.

ABSTRACT In this work, we show that Clostridium difficile phage ϕC2 transduces erm(B), which confers erythromycin resistance, from a donor to a recipient strain at a frequency of 10−6 per PFU. The transductants were lysogenic for ϕC2 and contained the erm(B) gene in a novel transposon, Tn6215. This element is 13,008 bp in length and contains 17 putative open reading frames (ORFs). It could also be transferred at a lower frequency by filter mating. IMPORTANCE Clostridium difficile is a major human pathogen that causes diarrhea that can be persistent and difficult to resolve using antibiotics. C. difficile is potentially zoonotic and has been detected in animals, food, and environmental samples. C. difficile genomes contain large portions of horizontally acquired genetic elements. The conjugative elements have been reasonably well studied, but transduction has not yet been demonstrated. Here, we show for the first time transduction as a mechanism for the transfer of a novel genetic element in C. difficile. Transduction may also be a useful tool for the genetic manipulation of C. difficile.