While the health effects of the colonization of the reproductive tracts of mammals by bacterial communities are widely known, there is a dearth of knowledge specifically in relation to giant panda microbiomes. In order to investigate the vaginal and uterine bacterial diversity of healthy giant pandas, we used high-throughput sequence analysis of portions of the 16S rRNA gene, based on samples taken from the vaginas (GPV group) and uteri (GPU group) of these animals. Results showed that the four most abundant phyla, which contained in excess of 98% of the total sequences, were Proteobacteria (59.2% for GPV and 51.4% for GPU), Firmicutes (34.4% for GPV and 23.3% for GPU), Actinobacteria (5.2% for GPV and 14.0% for GPU) and Bacteroidetes (0.3% for GPV and 10.3% for GPU). At the genus level, Escherichia was most abundant (11.0%) in the GPV, followed by Leuconostoc (8.7%), Pseudomonas (8.0%), Acinetobacter (7.3%), Streptococcus (6.3%) and Lactococcus (6.0%). In relation to the uterine samples, Janthinobacterium had the highest prevalence rate (20.2%), followed by Corynebacterium (13.2%), Streptococcus (19.6%), Psychrobacter (9.3%), Escherichia (7.5%) and Bacteroides (6.2%). Moreover, both Chao1 and abundance-based coverage estimator (ACE) species richness indices, which were operating at the same sequencing depth for each sample, demonstrated that GPV had more species richness than GPU, while Simpson and Shannon indices of diversity indicated that GPV had the higher bacterial diversity. These findings contribute to our understanding of the potential influence abnormal reproductive tract microbial communities have on negative pregnancy outcomes in giant pandas.

ObjectiveThe role of N6-methyladenosine (m6A) in tumour immune microenvironment (TIME) remains understudied. Here, we elucidate function and mechanism of YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) in colorectal cancer (CRC) TIME.DesignClinical significance of YTHDF1 was assessed in tissue microarrays (N=408) and TCGA (N=526) cohorts.YTHDF1function was determined in syngeneic tumours, intestine-specificYthdf1knockin mice, and humanised mice. Single-cell RNA-seq (scRNA-seq) was employed to profile TIME. Methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNA sequencing (RNA-seq) and ribosome sequencing (Ribo-seq) were used to identify YTHDF1 direct targets. Vesicle-like nanoparticles (VNPs)-encapsulatedYTHDF1-siRNA was used forYTHDF1silencing in vivo.ResultsYTHDF1expression negatively correlated with interferon-γ gene signature in TCGA-CRC. Concordantly, YTHDF1 protein negatively correlated with CD8+T-cell infiltration in independent tissue microarrays cohorts, implying its role in TIME. Genetic depletion ofYthdf1augmented antitumour immunity in CT26 (MSS-CRC) and MC38 (MSI-H-CRC) syngeneic tumours, whileYthdf1knockin promoted an immunosuppressive TIME facilitating CRC in azoxymethane-dextran sulphate-sodium orApcMin/+models. scRNA-seq identified reduction of myeloid-derived suppressor cells (MDSCs), concomitant with increased cytotoxic T cells inYthdf1knockout tumours. Integrated MeRIP-seq, RNA-seq and Ribo-seq revealed p65/Rela as a YTHDF1 target. YTHDF1 promoted p65 translation to upregulate CXCL1, which increased MDSC migration via CXCL1-CXCR2 axis. Increased MSDCs in turn antagonised functional CD8+T cells in TIME. Importantly, targeting YTHDF1 by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) or VNPs-siYTHDF1boosted anti-PD1 efficacy in MSI-H CRC, and overcame anti-PD1 resistance in MSS CRC.ConclusionYTHDF1 impairs antitumour immunity via an m6A-p65-CXCL1/CXCR2 axis to promote CRC and serves as a therapeutic target in immune checkpoint blockade therapy.

Porcine epidemic diarrhea virus (PEDV), which re-emerged in China since 2010, has swept across the whole country leading to tremendous economic losses. In this study, a total of 645 diarrhea samples collected from 156 pig farms in Sichuan and Guizhou province during 2014-2018 were tested for PEDV. We found that samples from 47.66% (84/156) of the farms were positive for PEDV with an overall detection rate of 35.81% (231/645). Fifty-two strains were selected for full-length S gene analyses, and these strains were classified into three subgroups, an S-INDEL subgroup (G1c), and two non-S-INDEL subgroups (G2b, AJ1102-like and G2c), accounting for 15.38% (8/52), 23.08% (12/52) and 59.62% (31/52) of the total analysed strains, respectively. We found these three subgroups of PEDV coexisted in Sichuan province, and the S-INDEL strain was detected in Guizhou. Further antigenic variation analysis of the neutralizing epitopes (S10, COE, SS2, SS6 and 2C10) on the spike protein revealed that the S-INDEL and non-S-INDEL strains shared similar variation features in COE and SS6, but exhibited distinct variation patterns in the S10 domain. Unique variation patterns on N-glycosylation sites in the S protein were also observed for the S-INDEL and non-S-INDEL strains. Moreover, nine strains (three from each subgroup) were subjected to full-genome characterization.Complete genome phylogeny showed an inconsistent tree topology for genotyping, with two G2c strains grouped into the GII-b (AH2012-like) genogroup and the remaining seven strains including three S-INDEL strains grouped into the GII-c genogroup. Further recombination analyses indicated that six of the GII-c strains probably originated from intra-genogroup recombinations. Notably, three newly emerged S-INDEL strains with novel recombination patterns were first identified. Together, our data revealed a new status of PEDV in southwest China, which can increase understanding of the prevalence, genetic characteristics and evolutionary profiles of circulating PEDV strains in China.

Infectious bronchitis virus (IBV) can cause a highly contagious and acute respiratory disease in poultry. MicroRNAs (miRNAs) have emerged as a class of crucial regulators for gene expression and are involved in the regulation of virus defence and immunological processes. To understand miRNA regulation in chickens in response to IBV infection, high-throughput sequencing was performed to compare the small RNA libraries from the kidneys of chicken infected with SCK2, SCDY2 and LDT3-A. By comparing these data to healthy chickens, a total of 58 differentially expressed (DE) miRNAs were identified. The DE miRNAs were further classified into five miRNA expression patterns (up or down regulation compared to control). Using Gene Ontology (GO) enrichment prediction, the DE miRNAs were shown to be mostly associated with metabolic processes, catalytic activities, gene expression, binding activities and immune responses. Seven highly expressed miRNAs (gga-miR-30d, gga-miR-1454, gga-miR-7b, gga-miR-215-5p, gga-miR-1a-3p, gga-miR-3538 and gga-miR-2954) were selected for miRNA-mRNA conjoint analysis. Furthermore, the miRNAs inversely correlated with the corresponding target gene mRNAs. These seven miRNAs were considered to play an important role in IBV-host interactions and the differing virulence of IBV strains. This is the first demonstration that infection with different virulent IBVs elicits different expression of miRNAs in chicken kidneys; this expression also seems to be associated with the virulence of IBV. These results are significant for the study of immune responses to infection with different virulent IBVs mediated by miRNAs as well as the interaction between the chicken host and IBV.