Project description:M3-Seq is a single-cell RNA-sequencing platform for bacteria that pairs combinatorial cell indexing with post hoc rRNA depletion. We use M3-Seq to profile hundreds of thousands of bacterial cells and reveal rare populations of bacteria that include bet-hedging strategies, prophage induction, and phage-infected cells in E. coli and B. subtilis.
Project description:Recent whole-genome sequencing of large populations of the same bacterial species has revealed significant disparity among genes in the frequency of single nucleotide polymorphisms (SNPs). For example, a previous analysis of invasive serotype M3 group A streptococci (GAS) found the highest frequency of SNPs in the gene (ropB) encoding the regulator of proteinase B (RopB). This finding led us to hypothesize that RopB polymorphisms contribute to altered GAS host-pathogen interactions. Sequencing of ropB in 171 invasive serotype M3 GAS strains from a surveillance study identified 19 distinct ropB alleles. Inactivation of the ropB gene in strains producing distinct RopB variants had dramatically different effects on GAS global gene expression. Further, analysis of laboratory-generated isoallelic GAS strains differing only by a single amino acid replacement in RopB confirmed that the variant protein affected the transcript level of the gene encoding streptococcal proteinase B, a major RopB-regulated virulence factor. Comparison of parental, RopB-inactivated, and RopB isoallelic strains in mouse infection models demonstrated that RopB polymorphisms significantly influence GAS virulence and disease manifestations. These studies detail a paradigm in which unbiased, whole-genome sequence analysis of populations of clinical bacterial isolates creates new avenues of productive investigation into the pathogenesis of common human infections. This study examined the effects of RopB inactivaiton on two distinct serotype M3 group A streptococcal strains with distinct forms of the RopB protein. RopB was inactivated in a strain with a wild-type RopB allele (strain MGAS10870) and in a strain with a RopB allele containing a C85Y polymorphism (strain MGAS9937). The wild-type and RopB inactivated strains were grown in duplicate to the early stationary growth phase in standard laboratory medium (THY). Total RNA was isolated, converted to cDNA, and hybridized to a custom-made Affymetrix GeneChip.
Project description:Muscarinic acetylcholine receptor M3 (M3) and its downstream effector Gq/11 are critical drug development targets given their involvement in numerous physiological processes and diseases. Although a cryo-electron microscopy study previously defined the structure of the M3-miniGq complex, the lack of information on the intracellular loop 3 (ICL3) of M3 and α-helical domain (AHD) of Gαq has made it difficult to comprehend the molecular mechanism of M3-Gq coupling fully. Here, we present the molecular mechanism underlying the dynamic interactions between the wild-type full-length M3 and heterotrimeric Gq using hydrogen-deuterium exchange mass spectrometry and NanoLuc Binary Technology-based cell systems. This study suggests potential binding interfaces between M3 and Gq in pre-assembled and fully active nucleotide-free complexes. In addition to well-known binding interfaces, we observed the interaction of long ICL3 with Gβγ. Furthermore, M3 ICL3 negatively affected M3-Gq coupling, and the Gαq AHD underwent unique conformational changes during M3-Gq coupling. Therefore, we propose a comprehensive molecular mechanism of M3-Gq coupling by analyzing previously well-defined binding interfaces and neglected regions, such as M3 ICL3 and the Gαq AHD.
Project description:We compared differential gene expression in ST399/EMM-77, invasive ST28/EMM-1 and non-invasive ST28/EMM-1, 4 isolates in each group.
Project description:Recent whole-genome sequencing of large populations of the same bacterial species has revealed significant disparity among genes in the frequency of single nucleotide polymorphisms (SNPs). For example, a previous analysis of invasive serotype M3 group A streptococci (GAS) found the highest frequency of SNPs in the gene (ropB) encoding the regulator of proteinase B (RopB). This finding led us to hypothesize that RopB polymorphisms contribute to altered GAS host-pathogen interactions. Sequencing of ropB in 171 invasive serotype M3 GAS strains from a surveillance study identified 19 distinct ropB alleles. Inactivation of the ropB gene in strains producing distinct RopB variants had dramatically different effects on GAS global gene expression. Further, analysis of laboratory-generated isoallelic GAS strains differing only by a single amino acid replacement in RopB confirmed that the variant protein affected the transcript level of the gene encoding streptococcal proteinase B, a major RopB-regulated virulence factor. Comparison of parental, RopB-inactivated, and RopB isoallelic strains in mouse infection models demonstrated that RopB polymorphisms significantly influence GAS virulence and disease manifestations. These studies detail a paradigm in which unbiased, whole-genome sequence analysis of populations of clinical bacterial isolates creates new avenues of productive investigation into the pathogenesis of common human infections.
Project description:Mantle cell lymphoma gene expression profiling array VFN-M3. The dataset contains data from 10 samples hybridized on Illumina HumanHT-12 array. -patient's primary lymphoma cells obtained from leukemized blood (P6-PBMC1, P6-PBMC2) -non-malignant B-cells isolated from peripheral blood of 5 healthy donors (VFN-M3-CTRL1, VFN-M3-CTRL2) -lymphoma cell line named UPF7U established from the patient's primary lymphoma cells after 166 and 226 days of in vitro cultivation (UPF7U-D166, UPF7U-D226) -cells established by xenotransplantation of UPF7U cell line isolated ex vivo from subcutaneously growing lymphoma (UPF7U-SC) -patient-derived xenograft (PDX) cells named VFN-M3 established by xenotransplantation of primary lymphoma cells into immunodeficient mice isolated ex vivo from the lymph node like tumor (VFN-M3-LN3) -patient-derived xenograft (PDX) cells named VFN-M3 established by xenotransplantation of primary lymphoma cells into immunodeficient mice isolated ex vivo from subcutaneously growing lymphoma (VFN-M3-SC1, VFN-M3-SC3). Samples were sorted using CD19-microbeads (Miltenyi). For xenotransplantation, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice (referred to as NSG mice) were used.