We report our results of RNA-seq analysis on freshly isolated, sorted subsets of cytotoxic lymphocytes Overall design: RNA was isolated from sorted cells. Libraries were created using standard Illumina reagents and analyzed using a HiSeq2500.
ARID5B regulates metabolic programming in human adaptive NK cells.
Specimen part, Subject
View SamplesRegulatory T cells (Tregs) are key brakes on the VAT inflammation that regulates local and systemic metabolic tenor. The cytokine, IL-33, expands and sustains the unique Treg population residing within VAT. Making use of single-cell RNA sequencing, we identified the major IL-33 producers in VAT to be particular mSC subtypes, related to but distinct from adipocyte progenitor cells. We further characterize these subsets by individually isolating them and performing bulk-RNA sequencing. We explored modulation of the VAT-mSC (VmSC) landscape with physiologic variables such as age and sex, as well as pathogenic states like obesity. We uncovered a VAT Treg:stromal-cell negative regulatory loop that keeps the potent effect of IL-33 under rein. Overall design: Gene expression profiles of VmSC subtypes from young male and female mice. 2-4 mice were pooled for each biological replicate and at least 2 biological replicates were obtained per VmSC subtype.
Distinct immunocyte-promoting and adipocyte-generating stromal components coordinate adipose tissue immune and metabolic tenors.
Specimen part, Cell line, Subject
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Cytomegalovirus infection drives adaptive epigenetic diversification of NK cells with altered signaling and effector function.
Specimen part, Subject
View SamplesThe mechanisms underlying human natural killer (NK) cell phenotypic and functional heterogeneity are unknown. Here, we have described the emergence of diverse subsets of human NK cells selectively lacking expression of signaling proteins following cytomegalovirus (CMV) infection. The absence of B and myeloid cell-related signaling protein expression in these NK cell subsets correlated with promoter DNA hypermethylation. Genome-wide DNA methylation patterns were strikingly similar between CMV-associated adaptive NK cells and cytotoxic effector T cells, but differed from those of canonical NK cells. Functional interrogation demonstrated altered cytokine responsiveness in adaptive NK cells that was linked to reduced expression of the transcription factor PLZF. Furthermore, subsets of adaptive NK cells demonstrated significantly reduced functional responses to activated autologous T cells. The present results uncover a spectrum of epigenetically unique adaptive NK cell subsets that diversify in response to viral infection and have distinct functional capabilities compared to canonical NK cell subsets.
Cytomegalovirus infection drives adaptive epigenetic diversification of NK cells with altered signaling and effector function.
Specimen part, Subject
View SamplesWe report the transcriptome analysis of epidermal CD8 tissue resident memory T (TRM) cells from healthy human skin. Specifically, epidermal CD8+CD103+CD49a+ and CD8+CD103+CD49- TRM cells from healthy human skin were sorted by FACS. Differential gene expression analysis revealed functional dichotomy of epidermal CD8+CD103+CD49a+ and CD8+CD103+CD49- TRM cells. Overall design: Analysis of differentially expressed genes between epidermal CD8+CD103+CD49a+ and CD8+CD103+CD49- T cells from healthy human skin, biological replicates (n=7) (healthy skin donors).
CD49a Expression Defines Tissue-Resident CD8<sup>+</sup> T Cells Poised for Cytotoxic Function in Human Skin.
Specimen part, Subject
View SamplesNeuronal function critically depends on coordinated subcellular distribution of mRNAs. Disturbed mRNA processing and axonal transport has been found in spinal muscular atrophy and could be causative for dysfunction and degeneration of motoneurons. Despite the advances made in characterizing the transport mechanisms of several axonal mRNAs, an unbiased approach to identify the axonal repertoire of mRNAs in healthy and degenerating motoneurons has been lacking. Here we used compartmentalized microfluidic chambers to investigate the somatodendritic and axonal mRNA content of cultured motoneurons by microarray analysis. In axons, transcripts related to protein synthesis and energy production were enriched relative to the somatodendritic compartment. Knockdown of Smn, the protein deficient in spinal muscular atrophy, produced a large number of transcript alterations in both compartments. Transcripts related to immune functions, including MHC class I genes, and with roles in RNA splicing were upregulated in the somatodendritic compartment. On the axonal side, transcripts associated with axon growth and synaptic activity were downregulated. These alterations provide evidence that subcellular localization of transcripts with axonal functions as well as regulation of specific transcripts with nonautonomous functions is disturbed in Smn-deficient motoneurons, most likely contributing to the pathophysiology of spinal muscular atrophy.
Subcellular transcriptome alterations in a cell culture model of spinal muscular atrophy point to widespread defects in axonal growth and presynaptic differentiation.
Specimen part
View SamplesPolymorphisms in the interleukin-4 receptor chain (IL-4R) have been linked to asthma incidence and severity, but a causal relationship has remained uncertain. In particular, a glutamine to arginine substitution at position 576 (Q576R) of IL-4R has been associated with severe asthma, especially in African Americans. We show that mice carrying the Q576R polymorphism exhibited intense allergen-induced airway inflammation and remodeling. The Q576R polymorphism did not affect proximal signal transducer and activator of transcription (STAT) 6 activation, but synergized with STAT6 in a gene target and tissue-specific manner to mediate heightened expression of a subset of IL-4 and IL-13responsive genes involved in allergic inflammation. Our findings indicate that the Q576R polymorphism directly promotes asthma in carrier populations by selectively augmenting IL-4Rdependent signaling.
Pathogenicity of a disease-associated human IL-4 receptor allele in experimental asthma.
No sample metadata fields
View SamplesTo address the need to study frozen clinical specimens using next-generation RNA, DNA, chromatin immunoprecipitation (ChIP) sequencing and protein analyses, we developed a biobank work flow to prospectively collect biospecimens from patients with renal cell carcinoma (RCC). We describe our standard operating procedures and work flow to annotate pathologic results and clinical outcomes. We report quality control outcomes, nucleic acid yields of our RCC submissions (N=16) to The Cancer Genome Atlas (TCGA) project, as well as newer discovery platforms by describing mass spectrometry analysis of albumin oxidation in plasma and 6 ChIP sequencing libraries generated from nephrectomy specimens after histone H3 lysine 36 trimethylation (H3K36me3) immunoprecipitation. From June 1, 2010, through January 1, 2013, we enrolled 328 patients with RCC. Our mean (SD) TCGA RNA integrity numbers (RINs) were 8.1 (0.8) for papillary RCC, with a 12.5% overall rate of sample disqualification for RIN <7. Banked plasma had significantly less albumin oxidation (by mass spectrometry analysis) than plasma kept at 25°C (P<.001). For ChIP sequencing, the FastQC score for average read quality was at least 30 for 91-95% of paired-end reads. In parallel, we analyzed frozen tissue by RNA sequencing and after genome alignments, only 0.2-0.4% of total reads failed the default quality check steps of Bowtie2, which was comparable to the disqualification ratio (0.1%) of the 786-O RCC cell line, prepared under optimal RNA isolation conditions. The overall correlation coefficients for gene expression between the Mayo Clinic vs. TCGA tissues ranged from 0.75 to 0.82. These data support the generation of high-quality nucleic acids for genomic analyses from banked RCC. Importantly, the protocol does not interfere with routine clinical care. Collections over defined time points during disease treatment further enhance collaborative efforts to integrate genomic information with outcomes. Overall design: Examination of RNA expression in ccRCC
A Multidisciplinary Biospecimen Bank of Renal Cell Carcinomas Compatible with Discovery Platforms at Mayo Clinic, Scottsdale, Arizona.
No sample metadata fields
View SamplesThe transcriptomic changes induced in the human liver cell line HepG2 by 100M menadione, 200M TBH or 50M H2O2 after treatment for 0.5, 1, 2, 4, 6, 8 and 24h.
Time series analysis of oxidative stress response patterns in HepG2: a toxicogenomics approach.
Cell line
View SamplesPurpose: Epidemiological and intervention studies have attempted to link the health effects of a diet rich in fruits and vegetables with the consumption of polyphenols and their impact in neurodegenerative diseases. Studies have shown that polyphenols can cross the intestinal barrier and reach concentrations in the bloodstream able to exert effects in vivo. However, the effective uptake of polyphenols in the brain is still regarded with some reservations. Here we describe a combination of approaches to examine the putative transport of blackberry-digested polyphenols (BDP) across the blood-brain barrier (BBB) and ultimate evaluation of their beneficial effects.
Blood-brain barrier transport and neuroprotective potential of blackberry-digested polyphenols: an in vitro study.
Sex, Specimen part, Cell line, Race
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