Project description:This project contains proteomic (LC-MS/MS) data from 27 samples. The samples are as follows: (1) Major ampullate glands dissolved in 8M Urea (3 replicates); (2) Major ampullate silk fibers dissolved in 8M Urea (3 replicates); (3) Major ampullate silk fibers dissolved in Hexafluoroisopropanol (3 replicates); (4) Major ampullate silk fibers dissolved in 9M Lithium Bromide (3 replicates); (5) Major ampullate silk fibers dissolved in 2M Urea (3 replicates); (6) Major ampullate silk fibers dissolved in 4M Urea (3 replicates); (7) Major ampullate silk fibers dissolved in 8M Urea (3 replicates); (8) Major ampullate silk fibers first dissolved in Formic acid and then in 2M Urea (2 replicates); (9) Major ampullate silk fibers first dissolved in Formic acid and then in 4M Urea (2 replicates); (10) Major ampullate silk fibers first dissolved in Formic acid and then in 8M Urea (2 replicates);
Project description:Spider silk research has largely focused on spidroins, proteins that are the primary components of spider silk fibers. Although a number of spidroins have been characterized, other types of proteins associated with silk synthesis are virtually unknown. Previous comparison of tissue-specific RNAseq libraries identified 647 predicted genes that were differentially expressed in silk glands of the Western black widow, Latrodectus hesperus. Only ~5% of these transcripts encode spidroins and the remaining predicted genes presumably encode other proteins associated with silk production. Here, we used proteomic analysis of multiple silk glands and dragline silk fiber to investigate the translation of the differentially expressed genes. We find 48 proteins encoded by the differentially expressed transcripts in L. hesperus major ampullate, minor ampullate, and tubuliform silk glands, and detect 16 SST encoded proteins in major ampullate silk fibers. The observed proteins include known silk-related proteins, but most are uncharacterized, with no annotation. These unannotated proteins likely include novel silk associated proteins. Major ampullate and minor ampullate glands have the highest overlap of identified proteins, consistent with their shared, distinctive ampullate shape and the overlapping functions of major ampullate and minor ampullate silks. Our study substantiates and prioritizes predictions from differential expression analysis of spider silk gland transcriptomes.
Project description:Spider silk research has largely focused on spidroins, proteins that are the primary components of spider silk fibers. Although a number of spidroins have been characterized, other types of proteins associated with silk synthesis are virtually unknown. Previous comparison of tissue-specific RNAseq libraries identified 647 predicted genes that were differentially expressed in silk glands of the Western black widow, Latrodectus hesperus. Only ~5% of these transcripts encode spidroins and the remaining predicted genes presumably encode other proteins associated with silk production. Here, we used proteomic analysis of multiple silk glands and dragline silk fiber to investigate the translation of the differentially expressed genes. We find 48 proteins encoded by the differentially expressed transcripts in L. hesperus major ampullate, minor ampullate, and tubuliform silk glands, and detect 16 SST encoded proteins in major ampullate silk fibers. The observed proteins include known silk-related proteins, but most are uncharacterized, with no annotation. These unannotated proteins likely include novel silk associated proteins. Major ampullate and minor ampullate glands have the highest overlap of identified proteins, consistent with their shared, distinctive ampullate shape and the overlapping functions of major ampullate and minor ampullate silks. Our study substantiates and prioritizes predictions from differential expression analysis of spider silk gland transcriptomes.
Project description:Spider silk proteins are synthesized in the silk-producing glands, where the spidroins are produced, stored and processed into a solid fiber from a crystalline liquid solution. Despite great interest in the spider silk properties, that make this material suitable for biomedical and biotechnological applications, the mechanism of formation and spinning of the silk fibers has not been fully elucidated; and no combination of proteomic and transcriptomic study has been carried out so far in the spider silk-producing glands. Nephila clavipes is an attractive orb-web spider to investigate the spinning process of silk production, given the properties of strength, elasticity and biocompatibility of their silk fibers. Thus, considering that the combination of proteomic and transcriptomic analysis may reveal an extensive repertoire of novel proteins involved in the silk spinning process, and in order to facilitate and enable proteomics in this non-model organism, the current study aims to construct a high quality reference mRNA-derived protein database that could be used to identify tissue specific expression patterns in spider silk glands. Next-generation sequencing has offered a powerful and cost-efficient technique for the generation of transcriptomic datasets in non-model species using diverse platforms such as the Illumina HiSeq, Roche 454, Pacific Biosystems, and Applied Biosystems SOLiD; In the current study, the Illumina HiSeq 2000 platform will be used to generate a N. clavipes spider silk glands transcriptome-based protein database. The transcriptome data generated in this study will provide a comprehensive and valuable genomic resource for future research of the group of spider silk-producing glands, in order to improve our understanding of the overall mechanism of action involved in production, secretion, storage, transport, protection and conformational changes of spidroins during the spinning process, and prey capture; and the results may be relevant for scientists in material Science, biology, biochemistry, and environmental scientists.
Project description:Human blood vessel organoids (hBVOs) derived from human pluripotent stem cells have emerged as a novel system to understand human vascular development, model disorders, and develop regenerative therapies. However, it was unclear which molecular states constitute hBVOs and how cells differentiate and self-organize within hBVOs in vitro and after transplantation. Here we reconstruct hBVO development over a time course using single-cell transcriptomics. Data includes day 3, 4, 5, 6, 7, 14 and 21 of hBVO differentiation, as well as 2 months post-transplantation of 14-day hBVOs into immunocompromised mice. We observe progenitor states that bifurcate into endothelial and mural fates, and find that hBVOs do not acquire definitive arterio-venous endothelial identities in vitro. Chromatin accessibility profiling at days 3, 4 and 7 identifies gene regulatory network (GRN) features associated with endothelial and mural fate decisions. Transcriptome-coupled lineage recording reveals multipotent progenitor states within BVOs. These data provide the first comprehensive cell state atlas of BVO development.
Project description:Human mesothelial cells (LP9/TERT-1) were exposed to low and high (15 and 75 μm2/cm2 dish) equal surface area concentrations of crocidolite asbestos, nonfibrous talc, fine titanium dioxide (TiO2), or glass beads for 8 or 24 h. RNA was then isolated for Affymetrix microarrays, GeneSifter analysis and QRT-PCR. Gene changes by asbestos were concentration- and time-dependent. At low nontoxic concentrations, asbestos caused significant changes in mRNA expression of 29 genes at 8 h and 205 genes at 24 h, whereas changes in mRNA levels of 236 genes occurred in cells exposed to high concentrations of asbestos for 8 h. Human primary pleural mesothelial cells also showed the same patterns of increased gene expression by asbestos. Nonfibrous talc at low concentrations in LP9/TERT-1 mesothelial cells caused increased expression of 1 gene Activating Transcription Factor 3 (ATF3) at 8 h and no changes at 24 h, whereas expression levels of 30 genes were elevated at 8 h at high talc concentrations. Fine TiO2 or glass beads caused no changes in gene expression. In human ovarian epithelial (IOSE) cells, asbestos at high concentrations elevated expression of 2 genes (NR4A2, MIP2) at 8 h and 16 genes at 24 h that were distinct from those elevated in mesothelial cells. Since ATF3 was the most highly expressed gene by asbestos, its functional importance in cytokine production by LP9/TERT-1 cells was assessed using siRNA approaches. Results reveal that ATF3 modulates production of inflammatory cytokines (IL-1β, IL-13, G-CSF) and growth factors (VEGF and PDGF-BB) in human mesothelial cells. Microarrays were performed on samples from 3 independent experiments. All cell types, time points, and mineral types and concentrations were included in all 3 experiments. For each experiment, n=3 dishes were pooled into one sample per treatment group. Each of the pooled samples was analyzed on a separate array, i.e., n=3 arrays per condition (3 independent biological replicates). We tested the hypothesis that alteration in gene expression in human cells correlate with mineral pathogenicity. We used GeneSifter program to analyze our data and pairwise analysis showed that number of gene changes correlate with toxicity of pathogenic minerals. While non-pathogenic minerals glass beads and fine TiO2 treatment to cell resulted in no gene change, crocidolite asbestos caused maximum number of gene changes followed by talc.
Project description:KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) is highly immunosuppressive and resistant to targeted therapies, immune checkpoint blockade and engineered T cells. In this study, we performed a systematic high throughput combinatorial drug screen and identified a synergistic interaction between the MEK inhibitor trametinib and the multi- kinase inhibitor nintedanib. Using single cell RNA sequencing and immunophenotyping, we show that the combination therapy reprograms the immunosuppressive microenvironment and primes cytotoxic and memory T cells to infiltrate the tumors, thereby sensitizing mesenchymal PDAC to PD-L1 inhibition.
Project description:In this study we sought to determine the effect of overexpressing the SUMOylation E2 conjugase Ubc9 on the response of murine Neural Stem Cells (NSCs) to oxygen-glucose-deprivation and restoration of oxygen/glucose (OGD/ROG). We established stably-expandable lines of NSCs from the subventricular zones (SVZ) of adult wild-type mice (WT NSCs) and Ubc9-overexpressing mice (Ubc9 NSCs) and profiled their transcriptional changes in response to OGD/ROG as well as in response to differentiation.
Project description:The molecular machine that synthesizes RNA in Eucarya and Archaea, RNA polymerase, is composed of 11 or 12 subunits M-^V 9 or 10 that form the core holoenzyme, and a heterodimer formed from subunits E and F that associates with the core.<br><br>In this study we used a recombinant archaeal MbRpoE/F heterodimer to capture cellular mRNA and a custom Agilent microarray to determine which mRNA it binds. Transcripts bound by the heterodimer were identified through competitive hybridization of the total RNA obtained from Methanococcoides burtonii and the RNA obtained through the selection of the transcripts that interact with the MbRpoE/F heterodimer bound to the column.