Project description:Purpose: Identification of transcriptionally active genes in the unculturable community constituent, Smithella, during hexadecane degradation; Differential gene expression analysis of hexadecane-relevant genes acoss three different conditions; Extension of metatranscriptomic datasets to other community constituents to identify interspecies relationships. mRNA profiles were generated for this community across three different conditions (hexadecane-, butyric acid-, caprylic acid-degrading conditions) using a modified version of Nextera and sequenced using Illumina's Miseq platform.
Project description:Yeast protein microarrays were utilized to investigate determinants of S-nitrosylation by biologically relevant low-mass S-nitrosothiols (SNOs). Large numbers of S-nitrosylated yeast proteins were identified after treatment with SNOs, among which those with active-site Cys thiols residing at N termini of alpha-helices or within catalytic loops were particularly prominent. However, S-nitrosylation varied substantially even within these families of proteins (e.g., papain-related Cys-dependent hydrolases and rhodanese/Cdc25 phosphatases), suggesting that neither secondary structure nor intrinsic nucleophilicity of Cys thiols was sufficient to explain specificity. Further analyses revealed a substantial influence of NO-donor stereochemistry and structure on efficiency of S-nitrosylation as well as an unanticipated and important role for allosteric effectors. Thus, high-throughput screening and unbiased proteome coverage reveal multifactorial determinants of S-nitrosylation (which may be overlooked in alternative proteomic analyses), and support the idea that target specificity can be achieved through rational design of S-nitrosothiols Invitrogen yeast Protoarrays for kinase substrate identification (KSI) were treated with S-nitrosothiols and assayed for protein S-nitrosylation by using a modified biotin switch protocol. Slides were scanned and with a Genepix 4000b scanner (Molecular Devices) using Genepix Pro and analyzed by using Prospector Analyzer (Invitrogen). Results were validated using yeast cell lysates and recombinant, purified yeast proteins.
Project description:Systemic lupus erythematosus is progressive, immune complex-mediated autoimmune disease targeting numerous organs. A central feature of the disease is the development of antibodies against nuclear components, including DNA. Antibodies against double-stranded DNA are so characteristic of this disease that their detection constitutes one of the criteria for diagnosis. We examined the formation of immune complexes on the surface of autoantigen microarrays incubated in the sera of 39 inactive and 22 active lupus patients and of 31 control subjects. Three different kinds of nucleic acids, dsDNA, ssDNA and RNA were used as antigens, along with chromatin (nucleosomal extract) and several other reference molecules. The composition with respect to IgG, IgM and complement components C3 and C4 was determined. We find that while IgM and C4 are physiological components of immune complexes formed from nucleic acids, both IgG and C3 are extremely characteristic of lupus patients. Complement C4 deposition changes were not consistent: these increased on ssDNA and RNA, decreased on chromatin and did not change significantly on dsDNA. The presence of IgG and C3 in the immune complexes formed from different nucleic acids was characteristic for both active and inactive lupus patients. Receiver-operating curve statistics indicate that C3 deposition measurements can improve the efficiency of identification of inactive lupus patients. These observations reveal the complexity of immune profile changes accompanying SLE.
Project description:Identification BCL6 target genes in primary germinal center cells and DLBCL cell lines by ChIP-on-chip Three replicates in each type of cells
Project description:Elongin is a hetero-trimeric elongation factor for RNA polymerase (Pol) II transcription that is conserved among metazoa. We solved three structures of human Elongin bound to transcribing Pol II using cryo-EM assisted by crosslinking mass spectrometry. The structures show that Elongin subunit ELOA binds the RPB2 side of Pol II and anchors the ELOB-8 ELOC subunit heterodimer. ELOA contains an N-terminal ‘latch’ that binds between the end of the RPB1 bridge helix and the funnel helices, thereby inducing a conformational change near the Pol II active center. The latch is strictly required for the elongation-stimulatory activity of Elongin, but not for its binding to Pol II, indicating that Elongin functions by allosterically influencing the conformational mobility of the active center. Structural comparisons show that Elongin binding to Pol II is incompatible with association of super elongation complex, the PAF1 complex, and RTF1, which also contains a latch element that stimulates Pol II.
Project description:Systemic lupus erythematosus is progressive, immune complex-mediated autoimmune disease targeting numerous organs. A central feature of the disease is the development of antibodies against nuclear components, including DNA. Antibodies against double-stranded DNA are so characteristic of this disease that their detection constitutes one of the criteria for diagnosis. We examined the formation of immune complexes on the surface of autoantigen microarrays incubated in the sera of 39 inactive and 22 active lupus patients and of 31 control subjects. Three different kinds of nucleic acids, dsDNA, ssDNA and RNA were used as antigens, along with chromatin (nucleosomal extract) and several other reference molecules. The composition with respect to IgG, IgM and complement components C3 and C4 was determined. We find that while IgM and C4 are physiological components of immune complexes formed from nucleic acids, both IgG and C3 are extremely characteristic of lupus patients. Complement C4 deposition changes were not consistent: these increased on ssDNA and RNA, decreased on chromatin and did not change significantly on dsDNA. The presence of IgG and C3 in the immune complexes formed from different nucleic acids was characteristic for both active and inactive lupus patients. Receiver-operating curve statistics indicate that C3 deposition measurements can improve the efficiency of identification of inactive lupus patients. These observations reveal the complexity of immune profile changes accompanying SLE. C3, IgM, C4 and IgG binding in 92 human serum samples were examined using custom-made protein arrays
Project description:Human infection with Mycobacterium tuberculosis results in a continuum of ill-defined, clinical manifestations with stable latent M. tuberculosis infection (LTBI) and severe active disease at the ends. Identifying different states of infection is of importance to tuberculosis (TB) control since risk of developing active disease varies among different asymptomatic states while infectiousness varies among patients with different bacterial burden. We investigated changes in proteome-scale antibody responses during disease progression in a non-human primate model of tuberculosis. We probed M. tuberculosis proteome microarrays with serial sera collected from three infection-outcome groups (active, reactivation, and latent). We found that each infection outcome is associated with characteristic changes in the antibody levels and number of antigenic targets, which suggested an association between antibody responses and bacillary burden. Additional proteome-scale serological profiling of > 400 human TB suspects established that antibody responses are positively associated with bacterial load. Thus tuberculosis-specific antibody levels and number of antigenic targets increases with disease progression.
Project description:Human infection with Mycobacterium tuberculosis results in a continuum of ill-defined, clinical manifestations with stable latent M. tuberculosis infection (LTBI) and severe active disease at the ends. Identifying different states of infection is of importance to tuberculosis (TB) control since risk of developing active disease varies among different asymptomatic states while infectiousness varies among patients with different bacterial burden. We investigated changes in proteome-scale antibody responses during disease progression in a non-human primate model of tuberculosis. We probed M. tuberculosis proteome microarrays with serial sera collected from three infection-outcome groups (active, reactivation, and latent). We found that each infection outcome is associated with characteristic changes in the antibody levels and number of antigenic targets, which suggested an association between antibody responses and bacillary burden. Additional proteome-scale serological profiling of > 400 human TB suspects established that antibody responses are positively associated with bacterial load. Thus tuberculosis-specific antibody levels and number of antigenic targets increases with disease progression.