Gut microbiota affects obesity susceptibility in mice through gut metabolites.

Introduction: It is well-known that different populations and animals, even experimental animals with the same rearing conditions, differ in their susceptibility to obesity. The disparity in gut microbiota could potentially account for the variation in susceptibility to obesity. However, the precise impact of gut microbiota on gut metabolites and its subsequent influence on susceptibility to obesity remains uncertain. Methods: In this study, we established obesity-prone (OP) and obesity-resistant (OR) mouse models by High Fat Diet (HFD). Fecal contents of cecum were examined using 16S rDNA sequencing and untargeted metabolomics. Correlation analysis and MIMOSA2 analysis were used to explore the association between gut microbiota and intestinal metabolites. Results: After a HFD, gut microbiota and gut metabolic profiles were significantly different between OP and OR mice. Gut microbiota after a HFD may lead to changes in eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), a variety of branched fatty acid esters of hydroxy fatty acids (FAHFAs) and a variety of phospholipids to promote obesity. The bacteria g_Akkermansia (Greengene ID: 175696) may contribute to the difference in obesity susceptibility through the synthesis of glycerophosphoryl diester phosphodiesterase (glpQ) to promote choline production and the synthesis of valyl-tRNA synthetase (VARS) which promotes L-Valine degradation. In addition, gut microbiota may affect obesity and obesity susceptibility through histidine metabolism, linoleic acid metabolism and protein digestion and absorption pathways.

Responses of keratinocytes to Trichophyton mentagrophyte infection based on whole transcriptome analysis.

BACKGROUND: Dermatophytosis is an intractable superficial mycosis in humans and animals mainly caused by Trichophyton mentagrophytes (T. mentagrophytes), with a global prevalence of about 20%. Keratinocytes are the most abundant participants in skin immunity, and they also play a role in the first-line defence against T. mentagrophytes. However, no studies of keratinocyte responses against T. mentagrophytes infection based on the whole transcriptome have been reported. OBJECTIVES: Here, we systematically analysed changes in keratinocytes infected with T. mentagrophytes using whole transcriptome sequencing technology. METHODS: The phenotypic changes in keratinocytes after infection with 1 × 105 conidia/mL T. mentagrophytes were observed by light microscopy, scanning electron microscopy, transmission electron microscopy and terminal deoxynucleotidyl transferase dUTP nick end labeling. RNA-sequencing (RNA-seq), small RNA-seq technology and related bioinformatics methods were used to systematically analyse the whole transcriptome changes in keratinocytes upon T. mentagrophytes stimulation. RESULTS: We found that T. mentagrophytes infection caused morphological changes, membrane damage, the formation of irregular organelles and keratinocyte apoptosis. A total of 204 differentially expressed (DE) circular RNAs (circRNAs), 868 DE long noncoding RNAs (lncRNAs), 2973 DE mRNAs and 209 DE micro RNAs (miRNAs) were identified between noninfected and T. mentagrophytes-infected keratinocytes. The expression level of selected RNAs was validated by quantitative real-time polymerase chain reaction (qRT-PCR). Functional enrichment analysis revealed that the parental genes of DE circRNAs were related to cell response, cell death and establishment of the skin barrier. Genes targeted by miRNA were involved in regulating the initiation of the immune response. Based on the expression level of circRNAs, lncRNAs, mRNAs and miRNAs, circRNA-miRNA-mRNA competing endogenous (ceRNA) networks comprised of 159 DE miRNAs, 141 DE circRNAs and 2307 DE mRNAs, and lncRNA-miRNA-mRNA ceRNA networks comprised of 790 DE lncRNAs, 190 DE miRNAs and 2663 DE mRNAs were constructed. The reliability of two selected ceRNA networks was verified using qRT-PCR. Further functional enrichment analysis revealed that the DE mRNAs interacting with circRNAs and lncRNAs in the ceRNA network mainly participated in fungal recognition, inflammation, the innate immune response and the death of keratinocytes. CONCLUSIONS: Our findings might provide new evidence on the pathogenesis of T. mentagrophytes-induced dermatophytosis, which is essential for identifying new therapeutic targets for dermatophytosis treatment.

Genome analysis of the mpox (formerly monkeypox) virus and characterization of core/variable regions.

Since smallpox was eradicated in 1980, the monkeypox virus (MPXV) has emerged as the most threatening orthopoxvirus in the world. In this study, we conducted a comprehensive analysis of the currently published complete genome sequences of the monkeypox virus. The core/variable regions were identified through core-pan analysis of MPXV. Besides single-nucleotide polymorphisms, our study also revealed that specific genes, multi-copy genes, repeat sequences, and recombination fragments are primarily distributed in the variable region. This result suggests that variable regions are not only more susceptible to single-base mutations, but also to events such as gene loss or gain, as well as recombination. Taken together, our results demonstrate the genomic characteristics of the core/variable regions of MPXV, and contribute to our understanding of the evolution of MPXV.

Comparative genomic analysis of alloherpesviruses: Exploring an available genus/species demarcation proposal and method.

The family Alloherpesviridae contains herpesviruses of fish and amphibians. Due to the significant economic losses to aquaculture that herpesviruses can cause, the primary areas of research interest are concerning their pathogenesis and prevention. Despite alloherpesvirus genomic sequences becoming more widely accessible, methods regarding their genus/species classification are still relatively unexplored. In the present study, the phylogenetic relationships between 40 completely sequenced alloherpesviruses were illustrated by the viral proteomic tree (ViPTree), which was divided into three monophyletic groups, namely Cyprinivirus, Ictalurivirus and Batrachovirus. Additionally, average nucleotide identity (ANI) and average amino acid identity (AAI) analyses were performed across all available sequences and clearly displayed species boundaries with the threshold value of ANI/AAI set at 90%. Subsequently, core-pan analysis uncovered 809 orthogroups and 11 core genes shared by all 40 alloherpesvirus genome sequences. For the former, a 15 percent identity depicts a clear genus boundary; for the latter, 8 of them may be qualified for phylogenetic analysis based on amino acid or nucleic acid sequences after being verified using maximum likelihood (ML) or neighbor-joining (NJ) phylogenetic trees. Finally, although the dot plot analysis was valid for the members within Ictalurivirus, it was unsuccessful for Cyprinivirus and Batrachovirus. Taken together, the comparison of individual methodologies provides a wide range of alternatives for alloherpesviruses classification under various circumstances.

Comprehensive analysis of transcriptome and metabolome analysis reveal new targets of Glaesserella parasuis glucose-specific enzyme IIBC (PtsG).

The ptsG (hpIIBCGlc) gene, belonging to the glucose-specific phosphotransferase system, encodes the bacterial glucose-specific enzyme IIBC. In this study, the effects of a deletion of the ptsG gene were investigated by metabolome and transcriptome analyses. At the transcriptional level, we identified 970 differentially expressed genes between ΔptsG and sc1401 (Padj<0.05) and 2072 co-expressed genes. Among these genes, those involved in methane metabolism, amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism, pyruvate metabolism, phosphotransferase system (PTS), biotin metabolism, Two-component system and Terpenoid backbone biosynthesis showed significant changes in the ΔptsG mutant strain. Metabolome analysis revealed that a total of 310 metabolites were identified, including 20 different metabolites (p < 0.05). Among them, 15 metabolites were upregulated and 5 were downregulated in ΔptsG mutant strain. Statistical analysis revealed there were 115 individual metabolites having correlation, of which 89 were positive and 26 negative. These metabolites include amino acids, phosphates, amines, esters, nucleotides, benzoic acid and adenosine, among which amino acids and phosphate metabolites dominate. However, not all of these changes were attributable to changes in mRNA levels and must also be caused by post-transcriptional regulatory processes. The knowledge gained from this lays the foundation for further study on the role of ptsG in the pathogenic process of Glaesserella parasuis (G.parasuis).

Comparative genomic analysis reveals new evidence of genus boundary for family Iridoviridae and explores qualified hallmark genes.

Members of the family Iridoviridae (iridovirids) are globally distributed and trigger adverse economic and ecological impacts on aquaculture and wildlife. Iridovirids taxonomy has previously been studied based on a limited number of genomes, but this is not suitable for the current and future virological studies as more iridovirids are emerging. In our study, 57 representative iridovirids genomes were selected from a total of 179 whole genomes available on NCBI. Then 18 core genes were screened out for members of the family Iridoviridae. Average amino acid sequence identity (AAI) analysis indicated that a cut-off value of 70% is more suitable for the current iridovirids genome database than ICTV-defined 50% threshold to better clarify viral genus boundaries. In addition, more subgroups were divided at genus level with the AAI threshold of 70%. This observation was further confirmed by genomic synteny analysis, codon usage preference analysis, genome GC content and length analysis, and phylogenic analysis. According to the pairwise comparison analysis of core genes, 9 hallmark genes were screened out to conduct preliminary identification and investigation at the genus level of iridovirids in a more convenient and economical manner.

Corrigendum: The Microbiota and It's Correlation With Metabolites in the Gut of Mice With Nonalcoholic Fatty Liver Disease.

[This corrects the article DOI: 10.3389/fcimb.2022.870785.].

The Microbiota and It's Correlation With Metabolites in the Gut of Mice With Nonalcoholic Fatty Liver Disease.

In recent years, nonalcoholic fatty liver disease (NAFLD) has become the most common liver disease in the world. As an important model animal, the characteristics of gut microbiota alteration in mice with NAFLD have been studied but the changes in metabolite abundance in NAFLD mice and how the gut microbiota affects these intestinal metabolites remain unclear. In this experiment, a mouse model for NAFLD was established by a high-fat diet. The use of 16S rDNA technology showed that while there were no significant changes in the alpha diversity in the cecum of NAFLD mice, the beta diversity changed significantly. The abundance of Blautia, Unidentified-Lachnospiraceae, Romboutsia, Faecalibaculum, and Ileibacterium increased significantly in NAFLD mice, while Allobaculum and Enterorhabdus decreased significantly. Amino acids, lipids, bile acids and nucleotide metabolites were among the 167 significantly different metabolites selected. The metabolic pathways of amino acids, SFAs, and bile acids were significantly enhanced, while the metabolic pathways of PUFAs, vitamins, and nucleotides were significantly inhibited. Through correlation and MIMOSA2 analysis, it is suggested that gut microbiota does not affect the changes of lipids and bile acids but can reduce thiamine, pyridoxine, and promote L-phenylalanine and tyramine production. The findings of this study will help us to better understand the relationship between gut microbiota and metabolites in NAFLD.

Deletion of two-component system QseBC weakened virulence of Glaesserella parasuis in a murine acute infection model and adhesion to host cells.

The widespread two-component system (TCS), QseBC, involves vital virulence regulators in Enterobacteriaceae and Pasteurellaceae. Here we studied the function of QseBC in Glaesserella parasuis. A ΔqseBC mutant was constructed using a Glaesserella parasuis serovar 11 clinical strain SC1401 by natural transformation. Immunofluorescence was used to evaluate cellular adhesion, the levels of inflammation and apoptosis. The ability of ΔqseBC and ΔqseC mutant strains to adhere to PAM and MLE-12 cells was significantly reduced. Additionally, by focusing on the clinical signs, H&E, and IFA for inflammation and apoptosis, we found that the ΔqseBC mutant weakened virulence in the murine models. Together, these findings suggest that QseBC plays an important role in the virulence of Glaesserella parasuis.

Impact of intracellular response regulator QseB in quorum sensing regulatory network in a clinical isolate SC1401 of Glaesserella parasuis.

Two component systems (TCS) mediate specific responses to different conditions and/or pressures. In the quorum sensing Glaesserella parasuis (QSE) BC TCS, qseB, as a response regulator, is closely related to the transcriptional regulation of multiple downstream genes. In this study, the effects of qseB gene deletion, which encodes the response regulator of population density sensing in G. parasuis, were studied through biological characteristics and metabolomic analysis. Based on previous research, we further explored the virulence of ΔqseB mutant strains through cell morphology, adhesion and invasion. The ΔqseB mutant and parent strains were sequenced by metabolome and combined with the previous transcriptome sequencing results for joint analysis. This study aims to clarify the regulatory effect of QseB on the virulence of G. parasuis and lay the foundation for revealing the pathogenic mechanism of G. parasuis. We detected 476 different metabolites, of which 30 metabolites (6.3%) had a significant difference in abundance between SC1401 and ΔqseB (p < 0.05). We conducted a comparative analysis of pathway enrichment on the transcriptome and metabolome, and found that the two omics participate in seven metabolic pathways together. The top 10 KEGG pathways with the largest number of genes and metabolites identified in this experiment are ABC transporters, Biosynthesis of secondary metabolites, Cysteine and methionine metabolism, Purine metabolism, Pyrimidine metabolism, Metabolic pathways, and Nicotinate and nicotinamide metabolism. Analysis of metabolome sequencing results showed that differential metabolites were also enriched in metabolic pathways, such as Purine metabolism, cGMP-PKG signaling pathway and cAMP signaling pathway, which were not found in transcriptome sequencing data. The internal coloration of the mutant strain ΔqseB was uneven, and the adhesion and invasion ability of PAM cell lines were significantly reduced. We speculate that QseB may affect the adhesion and invasion ability of Glaesserella parasuis by influencing substance transport and signal transduction.

ANI analysis of poxvirus genomes reveals its potential application to viral species rank demarcation.

Average nucleotide identity (ANI) is a prominent approach for rapidly classifying archaea and bacteria by recruiting both whole genomic sequences and draft assemblies. To evaluate the feasibility of ANI in virus taxon demarcation, 685 poxviruses were assessed. Prior to the analysis, the fragment length and threshold of the ANI value were optimized as 200 bp and 98 per cent, respectively. After ANI analysis and network visualization, the resulting sixty-one species (ANI species rank) were clustered and largely consistent with the groupings found in National Center for Biotechnology Information Virus [within the International Committee on Taxonomy of Viruses (ICTV) Master Species List]. The species identities of thirty-four other poxviruses (excluded by the ICTV Master Species List) were also identified. Subsequent phylogenetic analysis and Guanine-Cytosine (GC) content comparison done were found to support the ANI analysis. Finally, the BLAST identity of concatenated sequences from previously identified core genes showed 91.8 per cent congruence with ANI analysis at the species rank, thus showing potential as a marker gene for poxviruses classification. Collectively, our results reveal that the ANI analysis may serve as a novel and efficient method for poxviruses demarcation.

Landscape of keratinocytes transcriptome alterations in response to Trichophyton mentagrophytes infection.

Dermatophytosis is an intractable superficial fungal infection of keratinized structures, with approximately 20% incidence in humans. Alterations of keratinocytes in the pathogenesis of dermatophytosis at the transcriptome level remain unclear. To understand and characterize such responses, keratinocytes were infected with Trichophyton mentagrophytes. After infection with 1 × 105 conidia/mL T. mentagrophytes for 24 h, the adherence of fungal hyphae to keratinocytes and the damage caused to cell morphology and structure were observed by light microscopy and transmission electron microscopy, respectively. Levels of pro-inflammatory cytokines IL-1α, IL-1ß, TNFα, and IL-8 significantly increased after infection. RNA-seq and bioinformatic analyses revealed that 766 genes were significantly whereas 2207 genes were repressed in the T. mentagrophyte-infected cells. Some of the differentially expressed genes (DEGs) were related to inflammation, immune responses, wound healing, metabolism, and oxidative stress. GO and KEGG pathway enrichment analyses revealed that DEGs and pathways involved in inflammatory response, immune response, and pathogen-induced dysfunction were significantly enriched in the infected cells. Furthermore, gene set enrichment analysis revealed that higher expression gene sets were mainly involved in immune responses, whereas lower expression gene sets were related to cell component organization or biogenesis and transporter activity. Furthermore, protein-protein interaction network and function analyses revealed that JUN, TP53, FOS, MYC, and HSP90AA1 play a key role in immune responses. Overall, our study systematically uncovered the transcriptome-level response of keratinocytes to T. mentagrophyte and provided insights into dermatophytosis treatment.

Characteristics of intestinal microbiota in C57BL/6 mice with non-alcoholic fatty liver induced by high-fat diet.

Introduction: As a representation of the gut microbiota, fecal and cecal samples are most often used in human and animal studies, including in non-alcoholic fatty liver disease (NAFLD) research. However, due to the regional structure and function of intestinal microbiota, whether it is representative to use cecal or fecal contents to study intestinal microbiota in the study of NAFLD remains to be shown. Methods: The NAFLD mouse model was established by high-fat diet induction, and the contents of the jejunum, ileum, cecum, and colon (formed fecal balls) were collected for 16S rRNA gene analysis. Results: Compared with normal mice, the diversity and the relative abundance of major bacteria and functional genes of the ileum, cecum and colon were significantly changed, but not in the jejunum. In NAFLD mice, the variation characteristics of microbiota in the cecum and colon (feces) were similar. However, the variation characteristics of intestinal microbiota in the ileum and large intestine segments (cecum and colon) were quite different. Discussion: Therefore, the study results of cecal and colonic (fecal) microbiota cannot completely represent the results of jejunal and ileal microbiota.

Genomic analysis of Poxviridae and exploring qualified gene sequences for phylogenetics.

The members of the Poxviridae family are globally distributed all over the world and can cause infectious diseases. Although genome sequences are publicly available for representative isolates of all genera, studies on the criteria for genome-based classification within the Poxviridae family have rarely been reported. In our study, 60 Poxviridae genomes were re-annotated using Prokka. By using BLAST filtration and MCScanX, synteny and similarity of whole genomic amino acid sequences were visualized. According to the analysis pattern, the Chordopoxvirinae and Entomopoxvirinae subfamilies can be subdivided into five and two categories respectively, which is consistent with the phylogenetic tree constructed based on whole genomic amino acid sequences and Poxvirus core genes. Finally, four genes (Early transcription factor, DNA-directed RNA polymerase, RNA polymerase-associated transcription-specificity factor and DNA-dependent RNA polymerase) were selected from Poxvirus core genes by substitution saturation analysis and phylogenetic tree verification. Phylogenetic trees constructed based on single gene and concatenated sequences of the four selected genes showed that the classification of subgroups was consistent with the phylogenetic trees based on genome. Conclusion: a new method based on the similarity of whole genomic amino acid sequences was proposed for Poxviridae taxon demarcation, and the use of the four selected qualified genes will help make phylogenic identification of newly discovered Poxviridae isolates more convenient and accurate.

The Insights of Genomic Synteny and Codon Usage Preference on Genera Demarcation of Iridoviridae Family.

The members of the family Iridoviridae are large, double-stranded DNA viruses that infect various hosts, including both vertebrates and invertebrates. Although great progress has been made in genomic and phylogenetic analyses, the adequacy of the existing criteria for classification within the Iridoviridae family remains unknown. In this study, we redetermined 23 Iridoviridae core genes by re-annotation, core-pan analysis and local BLASTN search. The phylogenetic tree based on the 23 re-annotated core genes (Maximum Likelihood, ML-Tree) and amino acid sequences (composition vector, CV-Tree) were found to be consistent with previous reports. Furthermore, the information provided by synteny analysis and codon usage preference (relative synonymous codon usage, correspondence analysis, ENC-plot and Neutrality plot) also supports the phylogenetic relationship. Collectively, our results will be conducive to understanding the genera demarcation within the Iridoviridae family based on genomic synteny and component (codon usage preference) and contribute to the existing taxonomy methods for the Iridoviridae family.

Genomic analysis of Ranavirus and exploring alternative genes for phylogenetics.

Ranaviruses can infect both captive and wild cold-blooded vertebrates, leading to significant economic and environmental losses. With the cases of ranavirus infection increasing, many ranavirus genomic sequences were published, but little is known about ranavirus taxonomy on a whole-genome level. In this study, 44 ranaviruses core genes were identified in 32 ranaviruses genome sequences by using PanX. The neighbour-joining phylogenetic trees (NJ-tree) based on 44 ranaviruses core genes and 24 iridoviridae core genes and composition vector phylogenetic tree (CV-Tree) based on whole genome were constructed. The three of phylogenetic trees showed that 32 ranavirus isolates can be divided into 4 different subgroups including SGIV-like, EHNV-like, FV3-like and CMTV-like, and subgroups taxonomic position of three phylogenetic trees were consistent. However, the phylogenetic position of ToRV could not be determined if it belongs to FV3-like or CMTV-like group. Subsequently, we carried out dot plot analysis and confirmed that ToRV should belong to CMTV-like group. Based on dot plot analysis and phylogenetic trees, the taxonomic classification of ranaviruses was confirmed. Finally, four genes which are suitable for the construction of phylogenetic tree were selected from ranavirus core genes by recombination analysis, substitution saturation analysis and single-gene phylogenetic analysis. Phylogenetic tree based on concatenated sequences of the four selected genes showed that the classification of subgroups was identical with three of the phylogenetic trees. Conclusion: Our results confirmed taxonomic identification of ranaviruses; the four selected genes used in phylogenetic analysis will make taxonomic identification more convenient and accurate.

ß-Glucan augments IL-1ß production by activating the JAK2/STAT3 pathway in cultured rabbit keratinocytes.

Trichophyton mentagrophytes (T. mentagrophytes) is the main cause of rabbit dermatophytosis. As the main pathogen-associated molecular pattern of T. mentagrophytes, the role of ß-glucan in the pathogenesis of rabbit dermatophytosis remains elusive. Keratinocytes (KC) are the main cellular component and the first defensive line against fungal pathogens in the skin. Therefore, the present study investigated the effects of ß-glucan on rabbit KC from dorsal skin. ß-glucan was found to inhibit KC proliferation by 10% at 20 ug/ml and this concentration was thus considered as optimal. Next, 20 ug/ml ß-glucan stimulation for 24 h significantly increased CXCL8, CXCL11, and IL-1ß secretions in KC. Furthermore, ß-glucan exposure induced the expressions of JAK2 mRNA, STAT3 mRNA, and p-STAT3 protein. Silencing JAK2 expression inhibited p-STAT3 protein expression and ß-glucan-induced IL-1ß secretion. And overexpression of JAK2 further promoted ß-glucan-mediated p-STAT3 protein and IL-1ß productions. These results suggested that ß-glucan-induced CXCL8, CXCL11, and IL-1ß secretions in rabbit KC might be involved in the inflammatory response of T. mentagrophytes infected rabbit dorsal skin. However, only IL-1ß secretion was promoted by the JAK2/STAT3 signaling pathway. In conclusion, this study is a necessary step toward elucidating the mechanisms that underlie skin immune system injury stimulated by ß-glucan.

MicroRNA-216b inhibits heat stress-induced cell apoptosis by targeting Fas in bovine mammary epithelial cells.

Heat stress affects milk yield and quality in lactating dairy cows in summer. Bovine mammary epithelial cells (bMECs) play a key role in milk secretion, and microRNAs (miRNAs) regulate numerous functions of bMEC. Previous reports have verified that miR-216b regulated cell apoptosis through repressing target genes in several cancer cells. So, our purpose was to explore the potential involvement of miR-216b in heat stress-induced cell apoptosis in bMECs. Firstly, the heat stress model was constructed and we found that apoptotic rates of bMECs significantly increased under heat stress. The expression of miR-216b, Bax mRNA, and caspase-3 mRNA was upregulated. However, Bcl-2 mRNA level was detected to differentially downregulated. Overexpression of miR-216b remarkably downregulated the expression of caspase-3 and Bax mRNA and protein, and the mRNA and protein level of Bcl-2 was increased. Inhibition of miR-216b increased the activity of caspase-3 and Bax, and the level of Bcl-2 was inhibited. Moreover, Fas was identified as a target gene of miR-216b through bioinformatic analysis and dual-luciferase reporter assay. Fas activity was significantly inhibited and enhanced respectively after transfecting miRNA mimics and inhibitor. Finally, inhibition of Fas via the small interfering RNA (siRNA) also inhibited cell apoptosis induced by heat stress. Taken together, our results indicated that miR-216b exerted as an anti-apoptotic effect under heat stress in bMECs by targeting Fas.