Project description:The cynomolgus macaque (Macaca fascicularis) is an extensively utilised nonhuman primate model for biomedical research due to its biological, behavioural, and genetic similarities to humans. Genomic information of cynomolgus macaque is vital for research in various fields; however, there is presently a shortage of genomic information on the Malaysian cynomolgus macaque. This study aimed to sequence, assemble, annotate, and profile the Peninsular Malaysian cynomolgus macaque transcriptome derived from three tissues (lymph node, spleen, and thymus) using RNA sequencing (RNA-Seq) technology. A total of 174,208,078 paired end 70 base pair sequencing reads were obtained from the Illumina Hi-Seq 2500 sequencer. The overall mapping percentage of the sequencing reads to the M. fascicularis reference genome ranged from 53-63%. Categorisation of expressed genes to Gene Ontology (GO) and KEGG pathway categories revealed that GO terms with the highest number of associated expressed genes include Cellular process, Catalytic activity, and Cell part, while for pathway categorisation, the majority of expressed genes in lymph node, spleen, and thymus fall under the Global overview and maps pathway category, while 266, 221, and 138 genes from lymph node, spleen, and thymus were respectively enriched in the Immune system category. Enriched Immune system pathways include Platelet activation pathway, Antigen processing and presentation, B cell receptor signalling pathway, and Intestinal immune network for IgA production. Differential gene expression analysis among the three tissues revealed 574 differentially expressed genes (DEG) between lymph and spleen, 5402 DEGs between lymph and thymus, and 7008 DEGs between spleen and thymus. Venn diagram analysis of expressed genes revealed a total of 2,630, 253, and 279 tissue-specific genes respectively for lymph node, spleen, and thymus tissues. This is the first time the lymph node, spleen, and thymus transcriptome of the Peninsular Malaysian cynomolgus macaque have been sequenced via RNA-Seq. Novel transcriptomic data will further enrich the present M. fascicularis genomic database and provide future research potentials, including novel transcript discovery, comparative studies, and molecular markers development.
Project description:We used RNA-seq to investigate gene expression changes in sheep mammary gland and spleen tissue after experimental infection with Mycoplasma agalactiae (strain PG2). Sheep (3 per each group) were given an intra-mammary infection with 10^9 cfu infectious organisms or PBS as control. The animals were euthanized 15 days post infection to obtain the samples. Two replicates of mammary gland and spleen tissue per animal were used for Illumina RNA-sequencing.
Project description:To investigate specific miRNA expression profiles of Marek's disease virus (MDV)-infected samples, we performed deep sequencing for miRNAs in four small RNA libraries, including MDV-infected tumorous spleen, MD lymphoma from liver, and non-infected spleen and lymphocytes from controls. A total of 7.76x106, 6.36x106, 6.36x106, and 7.60x106 counts were obtained in four libraries, respectively. The sequences were blasted with chicken and MDV genomes and miRBase 16.0 to identify known and novel miRNAs. In total, 187 and 16 known mature miRNAs were identified in the chicken and MDV, respectively. Deep sequencing detected 942 novel chicken miRNA candidates, of which 646 were in tumorous spleen. These results indicate that MDV infection induced new host miRNA candidates and increased diversity of miRNAs. Of 942 miRNA candidates, 276 of 533 were verified by customized microarray, and 17 of them were further confirmed by qPCR. Four samples examined: MDV-infected tumorous spleen, MD lymphoma from liver, Non-infected spleen, Non-infected lymphocytes
Project description:Newcastle disease virus (NDV) was detected by reverse transcriptase PCR (RT-PCR) from total RNA isolated from a chicken spleen of a backyard flock in Jordan. The complete coding genome sequence of NDV/chicken/Jordan/J11-spleen/2018 was obtained with MiSeq (Illumina) sequencing. Phylogenetic analysis of the concatenated coding sequences classified the virus as class II subgenotype VIIi.
Project description:<h4>Background</h4>Schistosomiasis is a chronic, debilitating infectious disease caused by members of the genus Schistosoma. Previous findings have suggested a relationship between infection with Schistosoma spp. and alterations in the liver and spleen of infected animals. Recent reports have shown the regulatory role of noncoding RNAs, such as long noncoding RNAs (lncRNAs), in different biological processes. However, little is known about the role of lncRNAs in the mouse liver and spleen during Schistosoma japonicum infection.<h4>Methods</h4>In this study, we identified and investigated lncRNAs using standard RNA sequencing (RNA-Seq). The biological functions of the altered expression of lncRNAs and their target genes were predicted using bioinformatics. Ten dysregulated lncRNAs were selected randomly and validated in reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) experiments.<h4>Results</h4>Our study identified 29,845 and 33,788 lncRNAs from the liver and spleen, respectively, of which 212 were novel lncRNAs. We observed that 759 and 789 of the lncRNAs were differentially expressed in the respective organs. The RT-qPCR results correlated well with the sequencing data. In the liver, 657 differentially expressed lncRNAs were predicted to target 2548 protein-coding genes, whereas in the spleen 660 differentially expressed lncRNAs were predicted to target 2673 protein-coding genes. Moreover, functional annotation showed that the target genes of the differentially expressed lncRNAs were associated with cellular processes, metabolic processes, and binding, and were significantly enriched in metabolic pathways, the cell cycle, ubiquitin-mediated proteolysis, and pathways in cancer.<h4>Conclusions</h4>Our study showed that numerous lncRNAs were differentially expressed in S. japonicum-infected liver and spleen compared to control liver and spleen; this suggested that lncRNAs may be involved in pathogenesis in the liver and spleen during S. japonicum infection.
Project description:Assessment of the effect of cold ischaemic time on single cell RNAseq data from human tissues using 10x Genomics 3' single cell RNA sequencing. This project contains data for spleen, oesophagus epithelium and lung parenchyma, three tissues that had previously been reported to have differential sensitivity to ischaemia. Samples were collected into Hypothermasol FRS hypothermic preservation media and dissociated fresh (as soon as possible) or at 12h, 24h, 72h post onset of cold ischaemia in the donor. Single cell and bulk RNA sequencing data was generated at each time point and whole genome sequencing was carried out for each donor.
Project description:Natural killer (NK) cells are innate cytotoxic lymphoid cells (ILCs) involved in the killing of infected and tumor cells. Among human and mouse NK cells from the spleen and blood, we previously identified by single-cell RNA sequencing (scRNAseq) two similar major subsets, NK1 and NK2. Using the same technology, we report here the identification, by single-cell RNA sequencing (scRNAseq), of three NK cell subpopulations in human bone marrow. Pseudotime analysis identified a subset of resident CD56<sup>bright</sup> NK cells, NK0 cells, as the precursor of both circulating CD56<sup>dim</sup> NK1-like NK cells and CD56<sup>bright</sup> NK2-like NK cells in human bone marrow and spleen under physiological conditions. Transcriptomic profiles of bone marrow NK cells from patients with acute myeloid leukemia (AML) exhibited stress-induced repression of NK cell effector functions, highlighting the profound impact of this disease on NK cell heterogeneity. Bone marrow NK cells from AML patients exhibited reduced levels of CD160, but the CD160<sup>high</sup> group had a significantly higher survival rate.
Project description:RATIONALE:The etiology of sarcoidosis is unknown, but microbial agents are suspected as triggers. OBJECTIVES:We sought to identify bacterial, fungal, or viral lineages in specimens from patients with sarcoidosis enriched relative to control subjects using metagenomic DNA sequencing. Because DNA from environmental contamination contributes disproportionately to samples with low authentic microbial content, we developed improved methods for filtering environmental contamination. METHODS:We analyzed specimens from subjects with sarcoidosis (n?=?93), control subjects without sarcoidosis (n?=?72), and various environmental controls (n?=?150). Sarcoidosis specimens consisted of two independent sets of formalin-fixed, paraffin-embedded lymph node biopsies, BAL, Kveim reagent, and fresh granulomatous spleen from a patient with sarcoidosis. All specimens were analyzed by bacterial 16S and fungal internal transcribed spacer ribosomal RNA gene sequencing. In addition, BAL was analyzed by shotgun sequencing of fractions enriched for viral particles, and Kveim and spleen were subjected to whole-genome shotgun sequencing. MEASUREMENTS AND MAIN RESULTS:In one tissue set, fungi in the Cladosporiaceae family were enriched in sarcoidosis compared with nonsarcoidosis tissues; in the other tissue set, we detected enrichment of several bacterial lineages in sarcoidosis but not Cladosporiaceae. BAL showed limited enrichment of Aspergillus fungi. Several microbial lineages were detected in Kveim and spleen, including Cladosporium. No microbial lineage was enriched in more than one sample type after correction for multiple comparisons. CONCLUSIONS:Metagenomic sequencing revealed enrichment of microbes in single types of sarcoidosis samples but limited concordance across sample types. Statistical analysis accounting for environmental contamination was essential to avoiding false positives.
Project description:Targeted noninvasive control of the nervous system and end-organs may enable safer and more effective treatment of multiple diseases compared to invasive devices or systemic medications. One target is the cholinergic anti-inflammatory pathway that consists of the vagus nerve to spleen circuit, which has been stimulated with implantable devices to improve autoimmune conditions such as rheumatoid arthritis. Here we report that daily noninvasive ultrasound (US) stimulation targeting the spleen significantly reduces disease severity in a mouse model of inflammatory arthritis. Improvements are observed only with specific parameters, in which US can provide both protective and therapeutic effects. Single cell RNA sequencing of splenocytes and experiments in genetically-immunodeficient mice reveal the importance of both T and B cell populations in the anti-inflammatory pathway. These findings demonstrate the potential for US stimulation of the spleen to treat inflammatory diseases.