Summary:
HCaRG increases renal cell migration by a TGF-alpha autocrine loop mechanism.
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View SamplesHEK293 cells were transfected with control plasmid (pcDNAI/Neo;Invitrogen) or with the plasmid encoding HCaRG. Stable transfectants were synchronized and grown in the presence of 10% FBS for 48 h. Total RNAs were purified with the mini RNeasy kit (Qiagen).
HCaRG increases renal cell migration by a TGF-alpha autocrine loop mechanism.
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View SamplesC. elegans has served as a laboratory model organism due to its ease of manipulation and the availability of both forward and reverse genetics. In recent years, efforts to study host-pathogen interactions in C. elegans have increased. For example, analysis of infections by bacteria such as Pseudomonas, Salmonella or Serratia has revealed the existence of innate immune pathways in C. elegans that are also conserved in vertebrates. To date, there has been no natural virus infection reported in C. elegans or C. briggsae. Here we describe evidence of natural virus infection in wild isolates of both C. elegans and C. briggsae. Two highly divergent but related RNA viruses in the family Nodaviridae, tentatively named Orsay nodavirus and Santeuil nodavirus, were detected and their genomes partially sequenced. Infected worm lysates passed through 0.2 um filters could be used to infect uninfected worms, which could be further passaged for many generations. Furthermore, the viruses were subject to processing by the RNAi machinery as evidenced by the detection of virally derived small RNAs. Infection of mutant worms defective in small RNA pathways yielded more robust levels of viral RNA as compared to infection of isogenic N2 reference worms. These data demonstrate that nodaviruses are natural parasites of nematodes in the wild. Further study of the interactions between these viruses and nematodes is likely to provide insight into the natural ecology of nematodes and may reveal novel innate immune mechanisms that respond to viral infection. Overall design: Two small RNA libraries (18-30 nt) from nodavirus-infected and cured C. elegans wild isolate JU1580 were sequenced on the Illumina Genome Analyzer II platform. Samples were treated with tobacco acid pyrophosphatase to allow cloning of small RNA molecules with 5'-triphosphates. Each sample was labelled with a unique four base pair barcode and libraries were multiplexed together with a third library (not included in this submission). The multiplexed libraries were sequenced in triplicate.
Natural and experimental infection of Caenorhabditis nematodes by novel viruses related to nodaviruses.
Subject
View SamplesThe supply of soluble silicon (Si) to plants has been associated with many benefits that remain poorly explained and often contested. In this work, the effect of Si was studied on wheat plants under both control and pathogen stress (Blumeria graminis f.sp. tritici (Bgt)) conditions by conducting an exhaustive transcriptomic analysis (55,000 genes) aimed at comparing the differential response of plants under four treatments. The response to the supply of Si on control (uninfected) plants was limited to 47 genes providing little evidence of regulation of a specific metabolic process. Plants reacted to inoculation with Bgt by an up-regulation of many genes linked to stress and metabolic processes and a down-regulation of genes linked to photosynthesis. Supplying Si to inoculated plants largely prevented disease development, a phenotypic response that translated into a nearly perfect reversal of genes regulated by the effect of Bgt alone. These results suggest that Si plays a limited role on a plants metabolism in absence of stress, even in the case of a high-Si accumulating monocot such as wheat. On the other hand, the benefits of Si, in the form of biotic stress alleviation, were remarkably aligned with a counter-response to transcriptomic changes induced by the pathogen Bgt.
A comprehensive transcriptomic analysis of the effect of silicon on wheat plants under control and pathogen stress conditions.
No sample metadata fields
View SamplesHepatitis C virus (HCV) infection is a global health problem. A number of studies have implicated a direct role of cellular lipid metabolism in the HCV life cycle and inhibitors of the mevalonate pathway have been demonstrated to result in an antiviral state within the host cell. Transcriptome profiling was also conducted on Huh-7 human hepatoma cells bearing subgenomic HCV replicons with and without treatment with 25-hydroxycholesterol (25-HC), an inhibitor of the mevalonate pathway that alters lipid metabolism, to assess metabolic determinants of pro- and antiviral states within the host cell.
Transcriptional profiling of the effects of 25-hydroxycholesterol on human hepatocyte metabolism and the antiviral state it conveys against the hepatitis C virus.
No sample metadata fields
View SamplesUncontrolled microglial activation may lead to development of inflammation-induced brain damage. Here we uncover a ribosome-based mechanism/check point involved in control of the innate immune response and microglial activation. Using an in vivo model-system for analysis of the dynamic translational state of microglial ribosomes with mRNAs as input and newly synthesized peptides as an output, we find a marked dissociation of microglia mRNA and protein networks following innate immune challenge. Highly up-regulated and ribosome-associated mRNAs were not translated resulting in two distinct microglial molecular signatures, a highly specialized pro-inflammatory mRNA and immunomodulatory/homeostatic protein signature. We find that this is due to specific translational suppression of highly expressed mRNAs through a 3UTR-mediated mechanism involving the RNA binding protein SRSF3. This discovery suggests avenues for therapeutic modulation of innate immune response in resident microglia.
Diverging mRNA and Protein Networks in Activated Microglia Reveal SRSF3 Suppresses Translation of Highly Upregulated Innate Immune Transcripts.
Treatment
View SamplesIntravenous Immunoglobulin (IVIg) is widely used as an immunomodulatory therapy. We have recently demonstrated that IVIg protects against airway hyper-reactivity (AHR) and inflammation in mouse models of allergic airway disease (AAD), associated with induction of Foxp3+ regulatory T cells (Treg). Using DEREG (DEpletion of REGulatory T cell) mice, in which endogenous Treg can be ablated with Diphtheria toxin (DTx) treatment, we demonstrate that IVIg generates a de novo population of induced Treg (iTreg) in the absence of endogenous Treg. IVIg-generated iTreg were sufficient for inhibition of ovalbumin-induced AHR in an antigen-driven murine model of AAD. In the absence of endogenous Treg, IVIg failed to confer protection against AHR and airway inflammation. Adoptive transfer of purified IVIg-generated iTreg prior to antigen challenge effectively prevented airway inflammation and AHR in an antigen-specific manner.
Peripherally Generated Foxp3<sup>+</sup> Regulatory T Cells Mediate the Immunomodulatory Effects of IVIg in Allergic Airways Disease.
Specimen part
View SamplesDifferentiation of naive CD4+ T cells into T-helper (Th) effector subsets is critical for protection against pathogens. Together, E-protein transcription factors and the inhibitor-of-DNA binding (Id) proteins are important arbiters of T cell development, but their role in the differentiation of Th1 and Tfh cells is not well understood. Th1 cells show robust Id2 expression compared to Tfh cells, and RNAi depletion of Id2 increased Tfh cell frequencies and germinal center responses, while impairing Th1 cell accumulation during viral infection. Further, Th1 cell differentiation was blocked by genetic ablation of Id2, leading to E-protein dependent accumulation of effector cells with 78% of Th1-associated genes showing diminished expression and a concurrent enrichment of the Tfh gene-expression program. The Tfh-defining transcriptional repressor Bcl6 bound to the Id2 locus inhibiting expression, providing a mechanism by which bimodal expression of Id2 in Tfh and Th1 cells can be established. Thus, Id2 is critical in enforcing the reciprocal development of Th1 and Tfh cell fates.
Id2 reinforces TH1 differentiation and inhibits E2A to repress TFH differentiation.
Age, Specimen part
View SamplesSilicon (Si) has long been known to play a major physiological role in certain organisms, including some sponges and many diatoms and higher plants, leading to the recent identification of multiple proteins responsible for silicon transport in a range of algal and plant species. In mammals, despite several convincing studies suggesting that silicon is an important factor in bone development and connective tissue health, there is a critical lack of understanding in biochemical pathways that enable silicon homeostasis. Here we report the identification of a mammalian efflux silicon transporter, namely Slc34a2 (also known as NaPiIIb), which was upregulated in the kidneys of rats following chronic dietary silicon deprivation. When heterologously expressed in Xenopus laevis oocytes, the protein displayed marked silicon transport activity, specifically efflux, comparable to plant OsLsi2 transfected in the same fashion and independent of sodium and/or phosphate influx. This is the first evidence for a specific active transporter protein for silicon in mammals and suggests an important role for silicon in vertebrates.
Identification of a mammalian silicon transporter.
Sex, Age, Specimen part
View SamplesNK cell development, maturation, and activation by cytokines is driven by alterations in gene expression mediated by activation and repression or transcriptional programs. In particular, we have extensively studied the role of STAT3 in human NK cells. This was based in part on a method we developed for in vitro expansion of large numbers of highly active NK cells using a genetically-modified feeder cell expressing 4-1BBL and membrane-bound IL-21. To dissect the various gene expression profiles induced by IL-21 from the various other signals received from the feeder cell, we purified peripheral NK cells from 4 healthy subjects (naïve, N), expanded NK cells for 14 days using CSTX002 feeder cells (expanded, E), and extracted RNA from the cells without (Neg) or after (Pos) the cells were activated with IL-21. We then performed RNA sequencing on each sample. Overall design: NK cells were purified from buffy coats obtained from 4 normal healthy blood-bank donors using RosetteSep NK for negative depletion of other cell subsets. NK cells were expanded by weekly stimulation with irradiated CSTX002 feeder cells. Naïve or expanded NK cells were stimulated for 30 minutes with 20 ng/ml recombinant human IL-21. Total RNA was prepared using the Total RNA Purification Plus Kit (Norgen Biotek, Ontario, ON, Canada). Libraries were prepared using the TruSeq RNA Sample Preparation Kit (Illumina Inc., San Diego, CA). 60–80 million paired-end 150 bp sequence reads per library were generated using the Illumina HiSeq4000 platform.
Efficient and Robust NK-Cell Transduction With Baboon Envelope Pseudotyped Lentivector.
Specimen part, Disease, Treatment, Subject
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