RNA-Seq-Based Analysis of Cortisol-Induced Differential Gene Expression Associated with Piscirickettsia salmonis Infection in Rainbow Trout (Oncorhynchus mykiss) Myotubes

Salmonid rickettsial septicemia (SRS) is the major infectious disease of the Chilean salmonid aquaculture industry caused by Piscirickettsia salmonis. Intensive farming conditions generate stress and increased susceptibility to diseases, being skeletal muscle mainly affected. However, the interplay between pathogen infection and stress in muscle is poorly understood. In this study, we perform an RNA-seq analysis on rainbow trout myotubes that are pretreated for 3 h with cortisol (100 ng/mL) and then infected with P. salmonis strain LF-89 for 8 h (MOI 50). Twelve libraries are constructed from RNA samples (n = 3 per group) and sequenced on Illumina HiSeq 4000. A total of 704,979,454 high-quality reads are obtained, with 70.25% mapped against the reference genome. In silico DETs include 175 total genes—124 are upregulated and 51 are downregulated. GO enrichment analysis reveals highly impacted biological processes related to apoptosis, negative regulation of cell proliferation, and innate immune response. These results are validated by RT-qPCR of nine candidate transcripts. Furthermore, cortisol pretreatment significantly stimulated bacterial gene expression of ahpC and 23s compared to infection. In conclusion, for the first time, we describe a transcriptomic response of trout myotubes infected with P. salmonis by inducing apoptosis, downregulating cell proliferation, and intrinsic immune-like response that is differentially regulated by cortisol.

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Multi-species transcriptome meta-analysis of the response to retinoic acid in vertebrates and comparative analysis of the effects of retinol and retinoic acid on gene expression in LMH cells

BMC Genomics ◽

10.1186/s12864-021-07451-2 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Clemens Falker-Gieske ◽

Andrea Mott ◽

Sören Franzenburg ◽

Jens Tetens

Keyword(s):

Gene Expression ◽

Retinoic Acid ◽

Cellular Response ◽

Homo Sapiens ◽

Meta Analysis ◽

Early Response ◽

Rna Seq ◽

Transcriptomic Response ◽

Time Points ◽

Lmh Cells

Abstract Background Retinol (RO) and its active metabolite retinoic acid (RA) are major regulators of gene expression in vertebrates and influence various processes like organ development, cell differentiation, and immune response. To characterize a general transcriptomic response to RA-exposure in vertebrates, independent of species- and tissue-specific effects, four publicly available RNA-Seq datasets from Homo sapiens, Mus musculus, and Xenopus laevis were analyzed. To increase species and cell-type diversity we generated RNA-seq data with chicken hepatocellular carcinoma (LMH) cells. Additionally, we compared the response of LMH cells to RA and RO at different time points. Results By conducting a transcriptome meta-analysis, we identified three retinoic acid response core clusters (RARCCs) consisting of 27 interacting proteins, seven of which have not been associated with retinoids yet. Comparison of the transcriptional response of LMH cells to RO and RA exposure at different time points led to the identification of non-coding RNAs (ncRNAs) that are only differentially expressed (DE) during the early response. Conclusions We propose that these RARCCs stand on top of a common regulatory RA hierarchy among vertebrates. Based on the protein sets included in these clusters we were able to identify an RA-response cluster, a control center type cluster, and a cluster that directs cell proliferation. Concerning the comparison of the cellular response to RA and RO we conclude that ncRNAs play an underestimated role in retinoid-mediated gene regulation.

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RNA-Seq for Bacterial Gene Expression

Current Protocols in Nucleic Acid Chemistry ◽

10.1002/cpnc.55 ◽

2018 ◽

Vol 73 (1) ◽

pp. e55 ◽

Cited By ~ 4

Author(s):

Line Dahl Poulsen ◽

Jeppe Vinther

Keyword(s):

Gene Expression ◽

Rna Seq ◽

Bacterial Gene ◽

Bacterial Gene Expression

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Gill Transcriptome Sequencing and De Novo Annotation of Acanthogobius ommaturus in Response to Salinity Stress

Genes ◽

10.3390/genes11060631 ◽

2020 ◽

Vol 11 (6) ◽

pp. 631

Author(s):

Zhicheng Sun ◽

Fangrui Lou ◽

Yuan Zhang ◽

Na Song

Keyword(s):

Signal Transduction ◽

Salinity Stress ◽

De Novo ◽

Enrichment Analysis ◽

Gill Tissue ◽

Control Group ◽

Rna Seq ◽

Illumina Hiseq ◽

Kegg Pathways ◽

Pathways Analysis

Acanthogobius ommaturus is a euryhaline fish widely distributed in coastal, bay and estuarine areas, showing a strong tolerance to salinity. In order to understand the mechanism of adaptation to salinity stress, RNA-seq was used to compare the transcriptome responses of Acanthogobius ommaturus to the changes of salinity. Four salinity gradients, 0 psu, 15 psu (control), 30 psu and 45 psu were set to conduct the experiment. In total, 131,225 unigenes were obtained from the gill tissue of A. ommaturus using the Illumina HiSeq 2000 platform (San Diego, USA). Compared with the gene expression profile of the control group, 572 differentially expressed genes (DEGs) were screened, with 150 at 0 psu, 170 at 30 psu, and 252 at 45 psu. Additionally, among these DEGs, Gene Ontology (GO) analysis indicated that binding, metabolic processes and cellular processes were significantly enriched. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis detected 3, 5 and 8 pathways related to signal transduction, metabolism, digestive and endocrine systems at 0 psu, 30 psu and 45 psu, respectively. Based on GO enrichment analysis and manual literature searches, the results of the present study indicated that A. ommaturus mainly responded to energy metabolism, ion transport and signal transduction to resist the damage caused by salinity stress. Eight DEGs were randomly selected for further validation by quantitative real-time PCR (qRT-PCR) and the results were consistent with the RNA-seq data.

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Transcriptome Analyses Revealed Adaptive-Responses in Livers of Mice Treated With Hua-Feng-Dan and Its “Guide Drug” Yaomu

10.21203/rs.3.rs-143356/v1 ◽

2021 ◽

Author(s):

Jia-Jia Liu ◽

Ya Zhang ◽

Shang-Fu Xu ◽

Feng Zhang ◽

Jing-Shan Shi ◽

...

Keyword(s):

Gene Expression ◽

Liver Toxicity ◽

Cholesterol Metabolism ◽

Enrichment Analysis ◽

Stroke Recovery ◽

Pathway Enrichment Analysis ◽

High Dose ◽

Hepatic Gene Expression ◽

Rna Seq ◽

Adaptive Responses

Abstract BackgroundHua-Feng-Dan is a patent Chinese medicine for stroke recovery and is effective against Parkinson’s disease models with modulatory effects on gut microbiota, but its effects on hepatic gene expression are unknown. This study used RNA-Seq to profile hepatic gene expression by Hua-Feng-Dan and its “Guide Drug” Yaomu.MethodsMice received orally Hua-Feng-Dan 1.2 g/kg, Yaomu 0.1-0.3 g/kg, or vehicle for 7 days. Liver pathology was examined, and total RNA was isolated for RNA-Seq. The bioinformatics, including GO and KEGG pathway enrichment analysis, two-dimensional clustering, Ingenuity Pathways Analysis (IPA), and Illumina BaseSpace Correlation Engine were used to analyze differentially expressed genes (DEGs). qPCR was performed to verify selected genes.ResultsHua-Feng-Dan and Yaomu did not produce liver toxicity as evidenced by histopathology and serum ALT and AST. GO Enrichment revealed Hua-Feng-Dan affected lipid homeostasis, protein folding and cell adhesion. KEGG showed activated cholesterol metabolism, bile secretion and PPAR signaling pathways. DEGs were identified by DESeq2 with p < 0.05 compared to controls. Hua-Feng-Dan produced 806 DEGs, Yaomu-0.1 had 235, and Yaomu-0.3 had 92 DEGs. qPCR on selected genes largely verified RNA-Seq results. IPA upstream regulator analysis revealed activation of MAPK and adaptive responses. Yaomu-0.1 had similar effects, but Yaomu-0.3 had little effects. Hua-Feng-Dan-induced DEGs were highly correlated with the GEO database of chemical-induced adaptive transcriptome changes in the liver. ConclusionHua-Feng-Dan at clinical dose did not produce liver pathological changes but induced metabolic and signaling pathway activations. Low dose of its Guide Drug Yaomu produced similar changes to a lesser extent, but high dose of Yaomu had little effects. The effects of Hua-Feng-Dan on liver transcriptome changes may produce adaptive responses to program the liver to produce beneficial or detrimental (over-dosed) pharmacological effects.

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Identification of aberrantly methylated differentially expressed genes in melanoma

10.21203/rs.2.24149/v1 ◽

2020 ◽

Author(s):

Wei Han ◽

Guo-liang Shen

Keyword(s):

Gene Expression ◽

Cell Proliferation ◽

Differentially Expressed Genes ◽

Enrichment Analysis ◽

Differentially Expressed ◽

Regulation Of Transcription ◽

Biological Processes ◽

Hub Genes ◽

Positive Regulation ◽

Upregulated Genes

Abstract Background: Skin Cutaneous Melanoma (SKCM) is known as an aggressive malignant cancer, which could be directly derived from melanocytic nevi. However, the molecular mechanisms underlying malignant transformation of melanocytes and melanoma tumor progression still remain unclear. Increasing researches showed significant roles of epigenetic modifications, especially DNA methylation, in melanoma. This study focused on identification and analysis of methylation-regulated differentially expressed genes (MeDEGs) between melanocytic nevus and malignant melanoma in genome-wide profiles. Methods: The gene expression profiling datasets (GSE3189 and GSE114445) and gene methylation profiling datasets (GSE86355 and GSE120878) were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) and differentially methylated genes (DMGs) were identified via GEO2R. MeDEGs were obtained by integrating the DEGs and DMGs. Then, functional enrichment analysis of MeDEGs were performed. STRING and Cytoscape were used to describe protein-protein interaction(PPI) network. Furthermore, survival analysis was implemented to select the prognostic hub genes. Finally, we conducted gene set enrichment analysis (GSEA) of hub genes. Results: We identified 237 hypomethylated, upregulated genes and 182 hypermethylated, downregulated genes. Hypomethylation-upregulated genes were enriched in biological processes of the oxidation-reduction process, cell proliferation, cell division, phosphorylation, extracellular matrix disassembly and protein sumoylation. Pathway enrichment showed selenocompound metabolism, small cell lung cancer and lysosome. Hypermethylation-downregulated genes were enriched in biological processes of positive regulation of transcription from RNA polymerase II promoter, cell adhesion, cell proliferation, positive regulation of transcription, DNA-templated and angiogenesis. The most significantly enriched pathways involved the transcriptional misregulation in cancer, circadian rhythm, tight junction, protein digestion and absorption and Hippo signaling pathway. After PPI establishment and survival analysis, seven prognostic hub genes were CKS2, DTL, KIF2C, KPNA2, MYBL2, TPX2 and FBL. Moreover, the most involved hallmarks obtained by GSEA were E2F targets, G2M checkpoint and mitotic spindle. Conclusions: Our study identified potential aberrantly methylated-differentially expressed genes participating in the process of malignant transformation from nevus to melanoma tissues based on comprehensive genomic profiles. Transcription profiles of CKS2, DTL, KIF2C, KPNA2, MYBL2, TPX2 and FBL provided clues of aberrantly methylation-based biomarkers, which might improve the development of precise medicine.

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Comparative analysis of sequencing technologies platforms for single-cell transcriptomics

10.1101/463117 ◽

2018 ◽

Cited By ~ 1

Author(s):

Kedar Nath Natarajan ◽

Zhichao Miao ◽

Miaomiao Jiang ◽

Xiaoyun Huang ◽

Hongpo Zhou ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Single Cells ◽

K562 Cells ◽

Library Preparation ◽

Rna Seq ◽

Illumina Hiseq ◽

Technical Variability ◽

Sequencing Technologies ◽

Sequencing Platforms

AbstractAll single-cell RNA-seq protocols and technologies require library preparation prior to sequencing on a platform such as Illumina. Here, we present the first report to utilize the BGISEQ-500 platform for scRNA-seq, and compare the sensitivity and accuracy to Illumina sequencing. We generate a scRNA-seq resource of 468 unique single-cells and 1,297 matched single cDNA samples, performing SMARTer and Smart-seq2 protocols on mESCs and K562 cells with RNA spike-ins. We sequence these libraries on both BGISEQ-500 and Illumina HiSeq platforms using single- and paired-end reads. The two platforms have comparable sensitivity and accuracy in terms of quantification of gene expression, and low technical variability. Our study provides a standardised scRNA-seq resource to benchmark new scRNA-seq library preparation protocols and sequencing platforms.

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Sexual dimorphism of the pulmonary transcriptome in neonatal hyperoxic lung injury: identification of angiogenesis as a key pathway

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00230.2017 ◽

2017 ◽

Vol 313 (6) ◽

pp. L991-L1005 ◽

Cited By ~ 13

Author(s):

Cristian Coarfa ◽

Yuhao Zhang ◽

Suman Maity ◽

Dimuthu N. Perera ◽

Weiwu Jiang ◽

...

Keyword(s):

Gene Expression ◽

Lung Injury ◽

Mrna Level ◽

Sexually Dimorphic ◽

Rna Seq ◽

Illumina Hiseq ◽

Data Set ◽

Vascular Growth ◽

Hyperoxic Lung Injury ◽

High Concentrations

Bronchopulmonary dysplasia (BPD) is characterized by impaired alveolar secondary septation and vascular growth. Exposure to high concentrations of oxygen (hyperoxia) contributes to the development of BPD. The male sex is considered an independent risk factor for the development of BPD. The reasons underlying sexually dimorphic outcomes in premature neonates are not known. We hypothesized that sex-specific modulation of biological processes in the lung under hyperoxic conditions contributes to sex-based differences. Neonatal male and female mice (C57BL/6) were exposed to hyperoxia [95% [Formula: see text], postnatal day (PND) 1–5: saccular stage of lung development] and euthanized on PND 7 or 21. Pulmonary gene expression was studied using RNA-Seq on the Illumina HiSeq 2500 platform. Analysis of the pulmonary transcriptome revealed differential sex-specific modulation of crucial pathways such as angiogenesis, response to hypoxia, inflammatory response, and p53 pathway. Candidate genes from these pathways were validated at the mRNA level by qPCR. Analysis also revealed sex-specific differences in the modulation of crucial transcription factors. Focusing on the differential modulation of the angiogenesis pathway, we also showed sex-specific differential activation of Hif-1α-regulated genes using ChIP-qPCR and differences in expression of crucial genes ( Vegf, VegfR2, and Phd2) modulating angiogenesis. We demonstrate the translational relevance of our findings by showing that our murine sex-specific differences in gene expression correlate with those from a preexisting human BPD data set. In conclusion, we provide novel molecular insights into differential sex-specific modulation of the pulmonary transcriptome in neonatal hyperoxic lung injury and highlight angiogenesis as one of the crucial differentially modulated pathways.

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Induction of foxp3 during the Crosstalk between Antigen Presenting Like-Cells MHCII+CD83+ and Splenocytes CD4+IgM− in Rainbow Trout

Biology ◽

10.3390/biology10040324 ◽

2021 ◽

Vol 10 (4) ◽

pp. 324

Author(s):

Byron Morales-Lange ◽

Ivan Nombela ◽

María Del Mar Ortega-Villaizán ◽

Mónica Imarai ◽

Paulina Schmitt ◽

...

Keyword(s):

Gene Expression ◽

Rainbow Trout ◽

Immune Cells ◽

Mhc Ii ◽

Different Types ◽

Piscirickettsia Salmonis ◽

Antigen Presenting ◽

Treg Phenotype ◽

Stimulation Of

In fish, the spleen is one of the major immune organs in the animal, and the splenocytes could play a key role in the activation and modulation of the immune response, both innate and adaptive. However, the crosstalk between different types of immune cells in the spleen has been poorly understood. In this work, an in vitro strategy is carried out to obtain and characterize mononuclear splenocytes from rainbow trout, using biomarkers associated with lymphocytes (CD4 and IgM) and antigen-presenting cells (CD83 and MHC II). Using these splenocytes, co-cultures of 24 and 48 h are used to determine the gene expression of master transcriptional factors that coordinate the polarization of T cells (t-bet, gata3, and foxp3). The results show a proportional upregulation of foxp3 (compared to t-bet and gata3) in co-cultures (at 24 h) of IFNγ-induced splenocytes with and without stimulation of Piscirickettsia salmonis proteins. In addition, foxp3 upregulation was established in co-cultures with IFNγ-induced cells and in cells only stimulated previously with P. salmonis proteins at 48 h of co-culture. These results show a potential communication between antigen-presenting-like cells and lymphocyte in the spleen, which could be induced towards a Treg phenotype.

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Characterization of the rat developmental liver transcriptome

Physiological Genomics ◽

10.1152/physiolgenomics.00128.2012 ◽

2013 ◽

Vol 45 (8) ◽

pp. 301-311 ◽

Cited By ~ 13

Author(s):

Richard H. Chapple ◽

Polyana C. Tizioto ◽

Kevin D. Wells ◽

Scott A. Givan ◽

JaeWoo Kim ◽

...

Keyword(s):

Gene Expression ◽

High Throughput Sequencing ◽

Mitochondrial Gene ◽

Probe Design ◽

Rna Seq ◽

Illumina Hiseq ◽

Biologically Relevant ◽

Gradual Process ◽

Intergenic Regions ◽

Sequencing Platforms

Gene regulation and transcriptome studies have been enabled by the development of RNA-Seq applications for high-throughput sequencing platforms. Next generation sequencing is remarkably efficient and avoids many issues inherent in hybridization-based microarray methodologies including the exon-specific dependence of probe design. Biologically relevant transcripts including messenger and regulatory RNAs may now be quantified and annotated regardless of whether they have previously been observed. We used RNA-Seq to investigate global patterns of gene expression in early developing rat liver. Liver samples from timed-pregnant Lewis rats were collected at six fetal and neonatal stages [embryonic day (E)14, E16, E18, E20, postnatal day (P)1, P7], transcripts were sequenced using an Illumina HiSeq 2000, and data analysis was performed with the Tuxedo software suite. Genes and isoforms differing in abundance were queried for enrichment within functionally related gene groups using the Functional Annotation Tool of the DAVID Bioinformatics Database. While hematopoietic gene expression is initiated by E14, hepatocyte maturation is a gradual process involving clusters of genes responsible for response to nutrients and enzymes responsible for glycolysis and fatty acid catabolism. Following birth, a large cluster of differentially abundant genes was enriched for mitochondrial gene expression and cholesterol synthesis indicating that by 1 wk of age, the liver is engaged in lipid sensing and bile production. Clustering results for differentially abundant genes and isoforms were similar with the greatest difference for the E14/E16 comparison. Finally, a bioinformatic approach was used to annotate 1,307 novel liver transcripts assembled from sequences that aligned to intergenic regions of the rat genome.

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Transcriptomics Analysis ofCandida albicansTreated with Huanglian Jiedu Decoction Using RNA-seq

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2016/3198249 ◽

2016 ◽

Vol 2016 ◽

pp. 1-9 ◽

Cited By ~ 5

Author(s):

Qianqian Yang ◽

Lei Gao ◽

Maocan Tao ◽

Zhe Chen ◽

Xiaohong Yang ◽

...

Keyword(s):

Gene Expression ◽

Antifungal Agents ◽

Enrichment Analysis ◽

Trichophyton Mentagrophytes ◽

Inhibition Mechanism ◽

Pathway Enrichment Analysis ◽

Rna Seq ◽

Genes Encoding ◽

Life Threatening ◽

Systemic Infections

Candida albicansis the major invasive fungal pathogen of humans, causing diseases ranging from superficial mucosal infections to disseminated, systemic infections that are often life-threatening. Resistance ofC. albicansto antifungal agents and limited antifungal agents has potentially serious implications for management of infections. As a famous multiherb prescription in China, Huanglian Jiedu Decoction (HLJJD,Orengedokutoin Japan) is efficient againstTrichophyton mentagrophytesandC. albicans. But the antifungal mechanism of HLJDD remains unclear. In this study, by using RNA-seq technique, we performed a transcriptomics analysis of gene expression changes forC. albicansunder the treatment of HLJDD. A total of 6057 predicted protein-encoding genes were identified. By gene expression analysis, we obtained a total of 735 differentially expressed genes (DEGs), including 700 upregulated genes and 35 downregulated genes. Genes encoding multidrug transporters such as ABC transporter and MFS transporter were identified to be significantly upregulated. Meanwhile, by pathway enrichment analysis, we identified 26 significant pathways, in which pathways of DNA replication and transporter activity were mainly involved. These results might provide insights for the inhibition mechanism of HLJDD againstC. albicans.

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