Project description:The early stages of the immune response against aberrant tissues such a tumors are technically difficult to study and therefore little understood. To analyze the innate immune response towards modified self-tissue we expressed a dominant-active version of the Ras oncogene in Drosophila imaginal discs and salivary glands. Signs of an immune response are observed on wings of adult flies, which show spots of melanization and in larvae, where the salivary glands attract blood cells. In line with the strong response mounted against salivary glands, they are found to express metalloproteases and apoptotic markers making them both accessible and recognizable for the immune system. The response against the glands bears several hallmarks of an encapsulation response including flattening of plasmatocytes, differentiation of lamellocytes, attachment of crystal cells and melanization at the final stage. RNA sequencing of the fat body, the primary producer of immune effectors, reveals a pattern of induced genes, which overlaps the one previously found upon wasp infestation but shows also several specific features. Evidence for the functional importance of one of the induced genes is presented proving the power of our approach to identify regulators of the response against modified self. Transcriptome analysis of the immune response in the fat body after expression of dominant-active RAS in the wing imaginal discs and salivary glands. Comparison of fat bodies from 3 control and 3 RAS-expressing larvae. For each sample fat bodies from 40 individuals were dissected. Differential gene expression files [output of Cuffdiff] are linked below: ResultsContr123vsRas123Individual.xls: Output file of Cuffdiff. Differential gene expression analysis, three control compared to individual Ras induced; replicate analysis using Cuffdiff. ResultsContr123vsRas123Multiple.xls: Output file of Cuffdiff. Differential gene expression analysis, three control compared to three Ras induced; replicate analysis using Cuffdiff. ResultsContr123vsRas12Multiple.xls: Output file of Cuffdiff. Differential gene expression analysis, three control compared to two Ras induced; replicate analysis using Cuffdiff.
Project description:We use mRNA-seq to transcriptionally profile larval salivary gland tissue from Drosophila third instar larvae. These data provide insights into tissue physiology and can be used to identify tissue specific transcripts. Salivary glands were dissected from 200 wandering third instar larvae and the associated fat body was removed.Salivary glands were transferred to Graces unsupplemented medium on ice prior to RNA extraction with TRIzol reagent. mRNA-seq samples were prepared from 10 ug of total RNA and subject to Illumina based sequencing.
Project description:We have developed genetically modified Ae. aegypti mosquitoes that activate the conserved antiviral JAK/STAT pathway in the fat body tissue, by overexpressing either the receptor Dome or the Janus kinase Hop by the blood feeding-induced vitellogenin (Vg) promoter. Transgene expression inhibits infection with several dengue virus (DENV) serotypes in the midgut as well as systemically and in the salivary glands. The impact of the transgenes Dome and Hop on mosquito longevity was minimal, but it resulted in a compromised fecundity when compared to wild-type mosquitoes. Overexpression of Dome and Hop resulted in profound transcriptome regulation in the fat body tissue as well as the midgut tissue, pinpointing several expression signatures that reflect mechanisms of DENV restriction. Our transcriptome studies and reverse genetic analyses suggested that enrichment of DENV restriction factor and depletion of DENV host factor transcripts likely accounts for the DENV inhibition, and they allowed us to identify novel factors that modulate infection. Interestingly, the fat body-specific activation of the JAK/STAT pathway did not result in any enhanced resistance to Zika virus (ZIKV) or chikungunya virus (CHIKV) infection, thereby indicating a possible specialization of the pathway’s antiviral role.
Project description:We report the transcriptome profile of one sequenced sample of mRNA isolated from pooled (20 from each genotype) abdomen fly extracts enriched in fat body content of fat body-specific Sdc RNAi knockdown and control flies Abdominal fat body mRNA profiles of 4-6-day old control and fat body-specific Sdc RNAi knockdown were generated by deep sequencing using Illumina HiSeq 2500
Project description:We report the transcriptome profile of one sequenced sample of mRNA isolated from pooled (20 from each genotype) abdomen fly extracts enriched in fat body content of fat body-specific Sdc RNAi knockdown and control flies
Project description:Transcriptome analysis of partially degraded and fragmented RNA samples from body fluids Global gene expression profiling has shown great promise in high-throughput biomarker discovery for early disease detection in body fluids such as saliva, which is accessible, cost-effective, and non-invasive. However, this goal has not been fully realized because saliva, like many clinical samples, contains partially fragmented and degraded RNAs that are difficult to amplify and detect with prevailing technologies. Here, using nanogram scale salivary RNA as a proof-of-principle example, we describe our progress with a novel poly-A tail independent mRNA amplification strategy combined with the Affymetrix GeneChip Exon arrays. We defined a Salivary Exon Core Transcriptome (SECT) with highly similar expression profiles in healthy individuals verified by quantitative PCR. Informatics analysis of SECT provided important mechanistic insight to their potential origin and function. Finally we demonstrated the diagnostic potential of true exon level expression profiling approach with salivary exon biomarkers that accurately discriminated gender in healthy individuals.
Project description:Obesity and associated metabolic outcomes define the metabolic syndrome. Interestingly, an under-appreciated fact is that body fat distribution, rather than total body fat amount, is a key determinant of metabolic disease. Indeed, in contrast to upper-body obesity, lower-body fat accumulation inversely correlates with metabolic risks. Understanding processes regulating upper- vs. lower-body fat expansion is paramount to predict (and prevent) these risks. We combine functional, proteomics, transcriptomics and epigenomics analyses to identify chromatin-associated mechanisms of adipose depot-specific fat expansion. Here, we analyze by RNA-seq the transcriptome of adipocytes differentiated in vitro from human gluteal (lower-body) and abdominal subcutaneous (upper-body) depots-derived adipose stem cells. We aim to identify adipose depot-specific and temporal differences in the up- or down-regulation of gene expression, and relate these differences to changes in chromatin states.
Project description:Persistent plant viruses multiply and circulate inside insect vectors following the route of midgut-hemolymph-salivary gland. Currently, how viruses interact with insect vectors after they are released into hemolymph is not entirely clear. In this study, we found that the hemolymph and fat body (HF) contained the highest RSV levels. Proteomic analysis on RSV-free and RSV-infected HF identified 156 differentially expressed proteins (DEPs), with the majority of them participating in metabolism, transportation, and detoxification.
Project description:We evaluated the utility of serum and salivary microRNAs (miRNAs) to serve as sensitive and specific peripheral biomarkers of possible mTBI. Levels of miRNAs were obtained stranded small RNA-sequencing. Functional outcomes were evaluated using a computerized assessment system that measured cognitive performance, body sway, and combined cognitive performance and body sway during dual task completion. We identified a subset of salivary and serum miRNAs that showed robust utility at predicting TBI likelihood and demonstrated quantitative associations with head impacts as well as cognitive and balance measures.
Project description:Transcriptome analysis of partially degraded and fragmented RNA samples from body fluids Global gene expression profiling has shown great promise in high-throughput biomarker discovery for early disease detection in body fluids such as saliva, which is accessible, cost-effective, and non-invasive. However, this goal has not been fully realized because saliva, like many clinical samples, contains partially fragmented and degraded RNAs that are difficult to amplify and detect with prevailing technologies. Here, using nanogram scale salivary RNA as a proof-of-principle example, we describe our progress with a novel poly-A tail independent mRNA amplification strategy combined with the Affymetrix GeneChip Exon arrays. We defined a Salivary Exon Core Transcriptome (SECT) with highly similar expression profiles in healthy individuals verified by quantitative PCR. Informatics analysis of SECT provided important mechanistic insight to their potential origin and function. Finally we demonstrated the diagnostic potential of true exon level expression profiling approach with salivary exon biomarkers that accurately discriminated gender in healthy individuals. We analyzed saliva from 18 healthy subjects (7 males, 11 females) using the Affymetrix Human Exon 1.0 ST platform. Array data was processed by Affymetrix Exon Array Computational Tool. No techinical replicates were performed.