ABSTRACT: On our microbial screening effort. We identified promising activity. Chasing the activity we annotated the molecules along with some computational docking studies.
Project description:Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. A major class of regulatory proteins is microbial allosteric transcription factors (aTFs), but aTF–inducer pairs are currently limited by those that naturally occur. Altering inducer specificity in these proteins is difficult because mutations that affect inducer binding may also disrupt allostery. Here, we engineer an aTF, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol or sucralose. We employ computational protein design, single-residue saturation mutagenesis, or random mutagenesis, along with multiplex assembly, and identify initial hits via a two-stage enrichment screen. Following activity maturation, we identify LacI variants with specificity to and induction by these new inducers comparable to that of wild-type LacI and its inducer, IPTG. The ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits.
Project description:The most commonly used genome annotation processes are to a great extent based on computational methods. However, those can only predict genes that have been described earlier or that have sequence signatures indicative of a gene function. We reported a synonymous proteogenomic approach for experimentally improving microbial genome annotation based on label-free quantitative MS/MS. The approach was exemplified by analysis of cell extracts from in vitro cultured enterotoxigenic Escherichia coli (ETEC) strain TW10598, as part of an effort to create a new reference ETEC genome sequence. The proteomic analysis yielded identification of 2,060 proteins, out of which 274 proteins were originally described as hypothetical. For 84% of the identified proteins we have provided description of their relative quantitative levels, among others, for 20 abundantly expressed ETEC virulence factors. Proteogenomic mapping supported the existence of four protein-coding genes that had not been annotated, and led to correction of translation start positions of another nine.
Project description:Sensing of microbial products by innate immune cells skew their transcriptional program to optimize anti-microbial defences. Chromatin remodeling by histone deacetylases (HDACs) plays a fundamental role in tailoring gene expression. HDAC inhibitors are among the most promising anti-cancer drugs and possess intrinsic anti-inflammatory properties. Yet, the influence of HDAC inhibition on innate immune responses to microbial infection is unknown. Here we show that HDAC inhibitors repress the expression of less than 10% of the genes expressed at baseline in BM-derived macrophages. In sharp contrast, HDAC inhibitors strongly interfere with transcriptome remodeling induced by LPS and Pam3CSK4, affecting the expression of 30-70% of genes modulated by microbial stimuli. Strikingly, HDAC inhibitors target the expression of numerous genes involved in anti-microbial host defences, encoding for microbial sensors, cytokines, chemokines, growth factors and their receptors, adhesion and signaling molecules, and molecules involved in antigen processing and presentation. At the molecular level, HDAC inhibitors do not impair mitogen-activated protein kinase, NF-kB, interferon-related factor signal and STAT1 transduction pathways, but inhibit NF-kB p65 recruitment to the promoter region of HDAC inhibitor-sensitive genes. HDAC inhibitors also inhibit the response of mouse and human DCs, splenocytes and whole blood to a broad range of microbial products and microorganisms. In agreement with these in vitro findings, HDAC inhibitors increase bacterial burden and sensitize mice to sub-lethal infection with Klebsiella pneumoniae and Candida albicans. Conversely, HDAC inhibitors confer protection in models of Pam3CSK4-induced fulminant toxic shock and severe sepsis following cecal ligation and puncture. Overall, these data substantiate the concept of immunomodulation by HDAC inhibitors, and suggest that these drugs could represent efficacious adjunctive therapy of severe sepsis. Mus musculus cells were grown in presence of LPS or LPS + TSA and pam or pam + TSA and hybrydised against a cRNA pool UMRR (from Mus musculus cells).
Project description:The aim of this work was to unveil the molecular mechanisms by which Streptomyces respond to a ROS intracellular imbalance and the effect of such response on the biosynthesis of secondary metabolites. The study was focused on the industrial actinomycete S. natalensis ATCC 27448 producer of the polyene pimaricin - an antifungal agent widely used in the food industry and promising for antiviral activity and stimulation of immune response.
Project description:<p>Diffuse Intrinsic Pontine Glioma (DIPG) is a universally fatal childhood cancer. Here, we performed a chemical screen in patient-derived DIPG cell cultures along with RNAseq expression analysis and integrated computational modeling to identify potentially effective therapeutic strategies. Panobinostat, among the more promising agents identified, demonstrated efficacy in pontine orthotopic xenograft models of both H3K27M and histone WT DIPG. These data suggest the potential utility of specific drug combinations and provides evidence of in vivo treatment efficacy of the multi-histone deacetylase inhibitor panobinostat. We are depositing to dbGaP deep sequencing whole exome data for 22 patient tumor samples and 13 matched normals, along with RNAseq data for 12 patient tumor samples and 6 normal pediatric brain tissue samples. In addition, we are depositing 22 RNAseq samples from DIPG cell lines before and after panobinostat treatment.</p>
Project description:Understanding and quantifying the effects of environmental factors influencing the variation of abundance and diversity of microbial communities was a key theme of ecology. For microbial communities, there were two factors proposed in explaining the variation in current theory, which were contemporary environmental heterogeneity and historical events. Here, we report a study to profile soil microbial structure, which infers functional roles of microbial communities, along the latitudinal gradient from the north to the south in China mainland, aiming to explore potential microbial responses to external condition, especially for global climate changes via a strategy of space-for-time substitution. Using a microarray-based metagenomics tool named GeoChip 5.0, we showed that microbial communities were distinct for most but not all of the sites. Using substantial statistical analyses, exploring the dominant factor in influencing the soil microbial communities along the latitudinal gradient. Substantial variations were apparent in nutrient cycling genes, but they were in line with the functional roles of these genes. 300 samples were collected from 30 sites along the latitudinal gradient, with 10 replicates in every site
Project description:Inhibitory receptors (IR) and inhibitory ligands function as critical regulators of immune responses by tempering T cell activity to microbial infections and cancers. In humans, several types of persisting viruses such as HIV, HBV and HCV, as well as cancers exploit IR signaling by upregulating IR ligands, resulting in suppression of T cell function (i.e., exhaustion). This allows escape from immune surveillance and continuation of disease. By screening a collection of bioactive small molecules, we identified and validated compounds that restore cytokine production and enhance proliferation of exhausted T cells. Analysis of our top hit ingenol mebutate, a protein kinase C inducing diterpene ester, revealed a role for this molecule in overriding the suppressive signaling cascade mediated by IR signaling on T cells.
Project description:The discovery of effective senolytics offers a promising approach for treating many age-related diseases. In this study, we employed a phenotypic drug discovery approach, combining drug screening and drug design, to identify and develop novel senolytic agents based on the flavonoid fisetin. We successfully developed two novel flavonoid analogs, SR29384 and SR31133, which demonstrated significantly enhanced senolytic activities compared to fisetin. These analogs showed broad-spectrum efficacy in eliminating various senescent cell types, reducing tissue senescence, extending healthspan in mice, and prolonging lifespan in Drosophila. Through RNA sequencing, machine learning, and computational screening, our mechanistic studies suggest that these novel flavonoid senolytics may target PARP1, BCL2L1, and CDK2 to induce senescent cell death.
Project description:We present a novel mass spectrometry-based high-throughput workflow and an open-source computational and data resource to reproducibly identify and quantify HLA-associated peptides. Collectively, the resources support the generation of HLA allele-specific peptide assay libraries consisting of consensus fragment ion spectra, and the analysis of quantitative digital maps of HLA peptidomes generated from a range of biological sources by SWATH mass spectrometry (MS). This study represents the first community-based effort to develop a robust platform for the reproducible and quantitative measurement of the entire repertoire of peptides presented by HLA molecules, an essential step towards the design of efficient immunotherapies.