Project description:Porcine respiratory and reproductive syndrome virus (PRRSV) is a virus infecting swine and causes swine abortion. Previously, non-structural protein 11 (Nsp11) from PRRSV was shown to have inhibitory function to type I IFN signaling. In this project, we want to see in addition to type I IFN, whether other cellular pathways are influenced by Nsp11 systemtically. A cell line stably expressing PRRSV Nsp11 was established, designated as MARC-Nsp11 cells, and an RNA microarray was conducted using these cells and WT MARC-145 cells MARC-145 and MARC-Nsp11 cells were seeded one day prior to experiments and total cellular RNAs were extracted using Trizol (Invitrogen) and purified by RNeasy mini kit (Qiagen). The quantity and quality of RNA were determined using an Align 2100 bioanalyzer (Agilent Technologies, Palo, Alto, CA, USA), and the RNA integrity was determined above 7. The RNA samples were then subjected to microarray using Human Gene 1.0 ST arrays (Affymetrix UK Ltd, High Wycombe, UK) at the Keck Biotechnology Center, University of Illinois, Urbana, IL). The microarray was repeated twice in duplicates each.
Project description:Purpose: The goals of this study are to monitor the evolution pattern of Shaan virus in depending host cells by viral transcriptome sequencing analysis of MARC145, A549, HEK293, and HRT18 cells infected with Shaan virus. Methods: The original isolate of Shaan virus (B16-40, Genbank accession no. MG230624.1) was passaged in MARC-145 cells. Low-passaged virus was obtained from the virus 4 times serially passaged in MARC-145 cells. The shaan virus which was 44 times serially passaged in MARC-145 cells was used for high-passaged virus. The prepared low- (p4) and high passaged shaan virus (p44) in MARC-145 cells were inoculated in triplicate to different host cells, HEK-293, A549, and HRT18 cell lines at M.O.I of 0.5 for 2 hours. The infected cells were incubated with the maintenance medium for each cell line for 24hrs. The infected cell pellets were resuspended to 250µl with fresh medium, to extract RNA for the high-throughput sequencing. The RNA samples were sequenced with illumine TruSeq Strand Total RNA LT kit and illumine NovaSeq6000 plaform form Macrogen, Inc (Seoul, Korea) for high throughput sequencing. The raw reads were trimmed with BBDuk and mapped the isolate Bat-ParaV/B16-40 (Genebank accession number. MG230624.1) with Bowtie 2 using Geneious program 2021.2.2. Transcript expression level in cells infected with shaan virus were calculated based on annotation on a reference shaan virus. Result: The total reads counts were between 50,285,454 and 76,298,278. The reference mapping of the trimmed reads with a reference genome (Genbank accession no. MG230624.1) showed different coverage values. The mapped reads were normalized and expressed as Transcript per Million (TPM) value. There were no noticeable differences of TPM values of each gene between the cells infected with low- and high-passaged shaan virus. However, the nucleocapsid (N) and matrix (M) gene-associated transcripts were shown to be differently measured among the host cells. Conclusion: In this regard, we tried to investigate certain selected mutation patterns by host switching using shaan virus isolate in MARC-145 cells. Therefore, this study provided potential evidence for host-specific selective mutation patterns by cell types as well as host of cells for shaan virus evolution.
Project description:Heat stress is one of the primary abiotic stresses that limit crop production . Grape is a popular cultivated fruit with high economic value throughout the world, and whose growth and development is often influenced by high temperature. Alternative splicing (AS) is a widespread mechanism increasing transcriptome complexity and proteome diversity. We conducted high temperature treatments (35oC, 40oC and 45oC) on grapevines (Vitis vinifera), and assessed proteomic and transcriptomic (especially AS)changes in leaves. We found that nearly 70% of the genes were alternatively spliced under high temperature. Intron retention (IR), exon skipping (ES) and alternative donor/acceptor sites were markedly induced under different high temperatures. IR was the most abundant up- and down-regulated AS event; moreover, IR events at 40 and 45oC were far higher than those at 35oC. These results indicated AS, especially IR, is an important posttranscriptional regulatory during grape leaf responses to high temperature. Proteomic analysis showed that protein levels of the RNA binding proteins SR45, SR30, and SR34, and the nuclear ribonucleic protein U1A in grape leaves gradually rose as ambient temperature increased. The results also revealed why AS events occurred more frequently under high temperature in grape leaves. After integrating transcriptomic and proteomic data, we found that HSPs and some important transcript factors such as MBF1c and HSFA2 were mainly involved in heat tolerance in grape through up-regulating transcriptional and translational levels, and were especially modulated by AS. The results provide the first simultaneous evidence for grape leaf responses to high temperature at transcriptional, posttranscriptional and translational levels.
Project description:We report the RNAseq technologies for high-throughput profiling of accessible chromatin regions and differentially expressed genes after cold treatment in Vitis amurensis, respectively. By combining the RNAseq results, we built putative transcriptional regulatory networks involving in cold responses in grape, and found some new transcription factors that may participate in cold responses in grape.
Project description:Widiastuti2010 - Genome-scale metabolic
network Zymomonas mobilis (iZM363)
This model is described in the article:
Genome-scale modeling and in
silico analysis of ethanologenic bacteria Zymomonas
mobilis.
Widiastuti H, Kim JY, Selvarasu S,
Karimi IA, Kim H, Seo JS, Lee DY.
Biotechnol. Bioeng. 2011 Mar; 108(3):
655-665
Abstract:
Bioethanol has been recognized as a potential alternative
energy source. Among various ethanol-producing microbes,
Zymomonas mobilis has acquired special attention due to its
higher ethanol yield and tolerance. However, cellular
metabolism in Z. mobilis remains unclear, hindering its
practical application for bioethanol production. To elucidate
such physiological characteristics, we reconstructed and
validated a genome-scale metabolic network (iZM363) of Z.
mobilis ATCC31821 (ZM4) based on its annotated genome and
biochemical information. The phenotypic behaviors and metabolic
states predicted by our genome-scale model were highly
consistent with the experimental observations of Z. mobilis ZM4
strain growing on glucose as well as NMR-measured intracellular
fluxes of an engineered strain utilizing glucose, fructose, and
xylose. Subsequent comparative analysis with Escherichia coli
and Saccharomyces cerevisiae as well as gene essentiality and
flux coupling analyses have also confirmed the functional role
of pdc and adh genes in the ethanologenic activity of Z.
mobilis, thus leading to better understanding of this natural
ethanol producer. In future, the current model could be
employed to identify potential cell engineering targets,
thereby enhancing the productivity of ethanol in Z.
mobilis.
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MODEL1507180057.
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