Bioinformatic Analysis Reveals the Distinct Role of 5’UTR-specific m6A RNA Modification in Mice Developing Cerebral Cortices

N6-methyladenosine (m6A) abundantly exists in the cerebral cortex, and is emerging as an essential factor in cortical development and function. As the m6A binding site appears to be dynamically methylated in different RNA regions at the temporal-specific developing stage, it is of value to distinguish the unique character of region- and temporal-specific m6A. Herein, we analyzed the status of temporal-specific m6A within RNA 5’ untranslated region (5’UTR) using m6A-methylated sequencing data and transcriptomic sequencing data from 12.5-13-day embryonic cerebral cortices and 14-day postnatal ones. We identified sorts of RNAs that are uniquely m6A-methylated in the 5’UTR region and sorted them into specific neurological processes. Compared with 3’UTR-m6A-methylated RNAs, 5’UTR-m6A-methylated RNAs showed unique functions and mechanisms in regulating cortical development, especially through the pathway of mRNA transport and surveillance. Moreover, the 5’UTR-specific m6A was associated with neurological disorders as well. The FoxO signaling pathway was then focused by these pathogenic 5’UTR-m6A-methylated RNAs, and explored to be involved in the determination of neurological disorders. Additionally, the 5’UTR-m6A-modification patterns and transcriptional patterns play independent but cohesive roles in the developing cortices. Our study emphasizes the importance of 5’UTR-specific m6A in the developing cortex and provides an informative reference for future studies of 5’UTR-specific m6A in normal cortical development and neurological disorders.

Detection of structural variations and fusion genes in breast cancer samples using third-generation sequencing

Background: Structural variations (SVs) are common genetic alterations in the human genome that could cause different phenotypes and various diseases including cancer. However, the detection of structural variations using the second-generation sequencing was limited by its short read-length which in turn restrained our understanding of structural variations. Methods: In this study, we developed a 28-gene panel for long-read sequencing and employed it to both Oxford Nanopore Technologies and Pacific Biosciences platforms. We analyzed structural variations in the 28 breast cancer-related genes through long-read genomic and transcriptomic sequencing of tumor, para-tumor and blood samples in 19 breast cancer patients. Results: Our results showed that some somatic SVs were recurring among the selected genes, though the majority of them occurred in the non-exonic region. We found evidence supporting the existence of hotspot regions for SVs, which extended our previous understanding that they exist only for single nucleotide variations. Conclusions: In conclusion, we employed long-read genomic and transcriptomic sequencing in identifying SVs from breast cancer patients and proved that this approach holds great potential in clinical application.

Th17 cell differentiation induced by cytopathogenic biotype BVDV-2 in bovine PBLCs

Background Bovine viral diarrhea virus (BVDV) is a major pathogen that causes bovine viral diarrhea/mucosal disease (BVD-MD), which has become a global infectious disease due to its wide spread and the lack of effective treatment. The process of BVDV infection is complex. Once infected, host immune cells are activated and modulated. As a major immune cell, peripheral blood lymphocyte cells (PBLCs) are the primary target of BVDV. In order to further understand the mechanism of BVDV- host interaction, the expression profiles of host lymphocytes mRNAs associated with BVDV infection were investigated by transcriptomic sequencing analysis. Results The transcriptomic sequencing analysis was performed on bovine PBLCs infected with CP BVDV-2 GS2018 after 12 h of infection. Gene expression profiling demonstrated that 1052 genes were differentially expressed in GS2018 infected PBLCs compared with the control group. Of these genes, 485 genes were up-regulated and 567 were down-regulated. The 19 differential expressed genes (DEGs) were selected for validation using quantitative real-time PCR and the results were consistent with the results of RNA-Seq. Gene ontology enrichment and KEGG pathway analysis showed that 1052 DEGs were significantly enriched in 16 pathways, including cytokine-cytokine receptor interaction, IL17, PI3K-Akt, MAPK and TNF signaling pathway. PPI network analysis showed that IL17A, IFN-γ and TNF-α interacted with various proteins and may play crucial roles in BVDV-2 infection. Of note, we confirmed that GS2018 induced Th17 cell differentiation in PBLCs and persistently increased the expression levels of IL17A. In turn, the replication of GS2018 was inhibited by IL17A. Conclusion In this study, the transcription changes of DEGs related to host immune responses in bovine PBLCs were caused by CP BVDV-2 infection. In particular, the effector molecules IL17A of Th17 cells were significantly up-regulated, which inhibited viral replication. These results will contribute to exploration and further understanding of the host immune response mechanism and interaction between host and BVDV-2.

Transcriptome Analysis of Neuroendocrine Regulation of Ovine Hypothalamus-Pituitary-Ovary Axis during Ovine Anestrus and the Breeding Season

Most sheep are seasonal estrus, and they breed in autumn when the days get shorter. Seasonal estrus is an important factor that affects the productivity and fertility of sheep. The key point to solve this problem is to explore the regulation mechanism of estrus in sheep. Therefore, in this study, transcriptomic sequencing technology was used to identify differentially expressed mRNAs in the hypothalamus, pituitary and ovary of Small Tail Han sheep (year-round estrus) and tan sheep (seasonal estrus) among luteal, proestrus and estrus stages. There were 256,923,304,156 mRNAs being identified in the hypothalamus, pituitary and ovary, respectively. Functional analysis showed that the photosensor, leucine and isoleucine biosynthesis pathways were enriched significantly. It is speculated that photoperiod may initiate estrus by stimulating the corresponding pathways in hypothalamus. ODC1, PRLH, CRYBB2, SMAD5, OPN1SW, TPH1 are believed to be key genes involved in the estrogen process. In conclusion, this study expanded the database of indigenous sheep breeds, and also provided new candidate genes for future genetic and molecular studies on the seasonal estrus trait in sheep.

Heterogeneity of Early Host Response to Infection with Four Low-Pathogenic H7 Viruses with a Different Evolutionary History in the Field

Once low-pathogenic avian influenza viruses (LPAIVs) of the H5 and H7 subtypes from wild birds enter into poultry species, there is the possibility of them mutating into highly pathogenic avian influenza viruses (HPAIVs), resulting in severe epizootics with up to 100% mortality. This mutation from a LPAIV to HPAIV strain is the main cause of an AIV’s major economic impact on poultry production. Although AIVs are inextricably linked to their hosts in their evolutionary history, the contribution of host-related factors in the emergence of HPAI viruses has only been marginally explored so far. In this study, transcriptomic sequencing of tracheal tissue from chickens infected with four distinct LP H7 viruses, characterized by a different history of pathogenicity evolution in the field, was implemented. Despite the inoculation of a normalized infectious dose of viruses belonging to the same subtype (H7) and pathotype (LPAI), the use of animals of the same age, sex and species as well as the identification of a comparable viral load in the target samples, the analyses revealed a heterogeneity in the gene expression profile in response to infection with each of the H7 viruses administered.

Single-Cell RNA Sequencing Defines the Regulation of Spermatogenesis by Sertoli-Cell Androgen Signaling

Androgen receptor (AR) signaling is essential for maintaining spermatogenesis and male fertility. However, the molecular mechanisms by which AR acts between male germ cells and somatic cells during spermatogenesis have not begun to be revealed until recently. With the advances obtained from the use of transgenic mice lacking AR in Sertoli cells (SCARKO) and single-cell transcriptomic sequencing (scRNA-seq), the cell specific targets of AR action as well as the genes and signaling pathways that are regulated by AR are being identified. In this study, we collected scRNA-seq data from wild-type (WT) and SCARKO mice testes at p20 and identified four somatic cell populations and two male germ cell populations. Further analysis identified that the distribution of Sertoli cells was completely different and uncovered the cellular heterogeneity and transcriptional changes between WT and SCARKO Sertoli cells. In addition, several differentially expressed genes (DEGs) in SCARKO Sertoli cells, many of which have been previously implicated in cell cycle, apoptosis and male infertility, have also been identified. Together, our research explores a novel perspective on the changes in the transcription level of various cell types between WT and SCARKO mice testes, providing new insights for the investigations of the molecular and cellular processes regulated by AR signaling in Sertoli cells.

The origin and impeded dissemination of the DNA phosphorothioation system in prokaryotes

Phosphorothioate (PT) modification by the dnd gene cluster is the first identified DNA backbone modification and constitute an epigenetic system with multiple functions, including antioxidant ability, restriction modification, and virus resistance. Despite these advantages for hosting dnd systems, they are surprisingly distributed sporadically among contemporary prokaryotic genomes. To address this ecological paradox, we systematically investigate the occurrence and phylogeny of dnd systems, and they are suggested to have originated in ancient Cyanobacteria after the Great Oxygenation Event. Interestingly, the occurrence of dnd systems and prophages is significantly negatively correlated. Further, we experimentally confirm that PT modification activates the filamentous phage SW1 by altering the binding affinity of repressor and the transcription level of its encoding gene. Competition assays, concurrent epigenomic and transcriptomic sequencing subsequently show that PT modification affects the expression of a variety of metabolic genes, which reduces the competitive fitness of the marine bacterium Shewanella piezotolerans WP3. Our findings strongly suggest that a series of negative effects on microorganisms caused by dnd systems limit horizontal gene transfer, thus leading to their sporadic distribution. Overall, our study reveals putative evolutionary scenario of the dnd system and provides novel insights into the physiological and ecological influences of PT modification.

Immunoglobulin-producing AT2-like cells secrete IgA into the extracellular matrix in pulmonary fibrosis

Pulmonary fibrosis is an interstitial lung disease that can be caused by various factors. Here, we first observed extensive IgA deposition in the extracellular matrix (ECM) of the lungs of mice with pulmonary fibrosis induced by silica inhalation. Consistent with this phenomenon, spatial transcriptomic sequencing of fresh mouse lung tissues from control mice and model mice showed that Igha transcripts were highly expressed in the lesion area. Single-cell RNA sequencing (scRNA-seq) and reconstruction of B cell receptor (BCR) sequences revealed a new cluster of cells with a shared BCR and high expression of genes related to immunoglobulin IgA production. Surprisingly, these clonal cells had more characteristics of AT2 (alveolar epithelial cell type 2) cells than B cells; thus, these cells were named AT2-like cells. Therefore, we propose that secretion of IgA into the ECM by AT2-like cells is an important process that occurs during lung fibrosis.

Detection of structural variations and fusion genes in breast cancer samples using third-generation sequencing

Structural variations (SVs) are common genetic alterations in the human genome that could cause different phenotypes and various diseases including cancer. However, the detection of structural variations using the second-generation sequencing was limited by its short read-length which in turn restrained our understanding of structural variations. In this study, we analyzed structural variations in 28 breast cancer-related genes through long-read genomic and transcriptomic sequencing of tumor, para-tumor and blood samples in 19 breast cancer patients. Our results showed that some somatic SVs were recurring among the selected genes, though the majority of them occurred in the non-exonic region. We found evidence supporting the existence of hotspot regions for SVs, which extended our previous understanding that they exist only for single nucleotide variations. In conclusion, we employed long-read genomic and transcriptomic sequencing in identifying SVs from breast cancer patients and proved that this approach holds great potential in clinical application.