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In JoVE (1)
- RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells
Other Publications (3)
Articles by Suman Chaudhary in JoVE
RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells
Dilyara Cheranova1, Margaret Gibson1, Suman Chaudhary1, Li Qin Zhang1, Daniel P. Heruth1, Dmitry N. Grigoryev1, Shui Qing Ye1
1Children's Mercy Hospital and Clinics, School of Medicine, University of Missouri-Kansas City
This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.
Published February 13, 2013. Keywords: Genetics, Molecular Biology, Immunology, Medicine, Genomics, Proteins, RNA-seq, Next Generation DNA Sequencing, Transcriptome, Transcription, Thrombin, Endothelial cells, high-throughput, DNA, genomic DNA, RT-PCR, PCR
Other articles by Suman Chaudhary on PubMed
Genetic Characterization of Human Immunodeficiency Virus Type 1 Tat Before and After Highly Active Antiretroviral Therapy
AIDS Research and Human Retroviruses. Oct, 2004 | Pubmed ID: 15585102
Two HIV-infected individuals were followed for changes in the first exon of Tat. Plasma virus collected at 2 months after commencement of highly active antiretroviral therapy (HAART) was compared with the virus collected before initiation of HAART. This short-term therapy significantly reduced the plasma viral burden and also helped modest CD4 recovery in the blood. One of the two individuals (ACG) showed only one form of the Tat before commencement of HAART whereas five different forms (including a form identical to the pre-HAART sample) were found in the post-HAART sample. Some of the post-HAART clones showed a difference of 12.8% suggesting that the source of virus replication might be a reservoir. In the other patients multiple variants of the virus were found within each time point and also between two time points. This patient also showed a debilitating mutation (25-C/R) in three clones suggesting that some of the post-HAART viral forms were not viable in this patient. The dS/dN ratios in the patients were >2 suggesting lack of positive selection.
HIV-1 Gp120 Induces Expression of IL-6 Through a Nuclear Factor-kappa B-dependent Mechanism: Suppression by Gp120 Specific Small Interfering RNA
PloS One. 2011 | Pubmed ID: 21712995
In addition to its role in virus entry, HIV-1 gp120 has also been implicated in HIV-associated neurocognitive disorders. However, the mechanism(s) responsible for gp120-mediated neuroinflammation remain undefined. In view of increased levels of IL-6 in HIV-positive individuals with neurological manifestations, we sought to address whether gp120 is involved in IL-6 over-expression in astrocytes. Transfection of a human astrocyte cell line with a plasmid encoding gp120 resulted in increased expression of IL-6 at the levels of mRNA and protein by 51.3Â±2.1 and 11.6Â±2.2 fold respectively; this effect of gp120 on IL-6 expression was also demonstrated using primary human fetal astrocytes. A similar effect on IL-6 expression was observed when primary astrocytes were treated with gp120 protein derived from different strains of X4 and R5 tropic HIV-1. The induction of IL-6 could be abrogated by use of gp120-specific siRNA. Furthermore, this study showed that the NF-ÎºB pathway is involved in gp120-mediated IL-6 over-expression, as IKK-2 and IKKÎ² inhibitors inhibited IL-6 expression by 56.5% and 60.8%, respectively. These results were also confirmed through the use of NF-ÎºB specific siRNA. We also showed that gp120 could increase the phosphorylation of IÎºBÎ±. Furthermore, gp120 transfection in the SVGA cells increased translocation of NF-ÎºB from cytoplasm to nucleus. These results demonstrate that HIV-1 gp120-mediated over-expression of IL-6 in astrocytes is one mechanism responsible for neuroinflammation in HIV-infected individuals and this is mediated by the NF-ÎºB pathway.
Correlation Between CD4 T Cell Counts and Virus Compartmentalization in Genital and Systemic Compartments of HIV-infected Females
Virology. Sep, 2011 | Pubmed ID: 21745672
The majority of infection by the human immunodeficiency virus (HIV-1) across the world occurs by heterosexual transmission and is likely mediated by virus present in genital secretions. In spite of this, infection is followed by clinical markers of the virus present in blood, which may not be representative of the virus involved in transmission. In fact, several studies have demonstrated that the genital tract represents a unique compartment for the virus. We assessed the relationship between immune system integrity, represented by CD4+ T cell counts, and the maintenance of viral compartmentalization between plasma and vaginal fluid virus in treatment naÃ¯ve women from the Dominican Republic infected by the heterosexual transmission route. We cloned and sequenced cell free virus from plasma and genital fluid samples from six women to assess viral evolution, phylogenetic relatedness, and calculated co-receptor use for the C2V3 region of the envelope. Our analyses demonstrated plasma and vaginal fluid virus compartments remained intact only in samples from women with CD4+ T cell counts over 350 cells/Î¼l. The majority of viral forms were predicted to use the CCR5 co-receptor, although several dual tropic forms were also identified. None of the clones were found to use the CXCR4 co-receptor even though many of the patients showed severe disease. Our findings lend further support to the role of an intact immune system in maintaining compartmentalization across blood and genital quasispecies and provide a compelling rationale to specifically consider genital tract viral forms in therapeutic and vaccine research.