Project description:To understand how GIPC3 exerts its effects on cuticular plate dimensions, we examined the GIPC3 protein-interaction network in hair cells. We immunoaffinity purified GIPC3 from crosslinked chicken inner ear extracts that were enriched for stereocilia, but still contain large amounts of hair-cell cytoplasmic proteins (Morgan et al., 2016). We carried out two separate experiments, each with ~1000 chicken ears, where we stabilized protein complexes using primary amine-reactive homo-bifunctional N-hydroxysuccimide ester crosslinkers that are thiol-cleavable and hence reversible (Mattson et al., 1993). In one experiment, we used dithiobis(succinimidyl propionate) (DSP), a membrane-permeable crosslinker that crosslinks extracellular and intracellular complexes; in the other experiment, we used 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), which is membrane impermeant and thus only stabilizes extracellular and transmembrane complexes. We prepared soluble extracts of crude, crosslinked stereocilia and used these fractions for identifying GIPC3-interacting proteins.
Project description:Enzymatic Methyl-seq (EM-seq) is an enzyme-based method giving us reliable methylome data from small amounts of purified DNA. However, it is unclear whether EM-seq library can be constructed from crude cell lysate containing genomic DNA. We evaluated the quality of EM-seq libraries directly prepared from crude cell lysate.
Project description:Genome-wide association studies in multiple sclerosis (MS) identified a polymorphism (rs6897932) located in the coding region of the alpha chain of the cytokine receptor interleukin 7 receptor (IL7R) as a component that increases susceptibility to develop the disease. This single nucleotide polymorphism (SNP) affects the splicing of the primary transcript leading to genotype-defined transcript ratios encoding either a full length membrane spanning form or a soluble receptor chain. Genotyping at the IL7R locus reveals that the region can be described by four haplotypes. Interestingly, only one out of three haplotypes harbouring the associated SNP is positively associated with MS whereas the other two do not show association. The minor allele containing haplotype shows a reduced susceptibility to develop MS. We hypothesized that additional functional or phenotypic differences exist between individuals homozygous for haplotypes shown to have either positive, negative, or neutral effect, on susceptibility to develop MS. Gene expression profiles of CD4+ T cells from MS individuals before and after stimulation with IL7 were recorded. Haplotype-specific gene signatures were found indicating small alterations in IL7/IL7R signal processing/sensitivity through JAK/STAT and p38/MAPK14. We can not exclude that the obtained signatures result from differences within the CD4+ T cell compartment that, in fact, should be seen as a consequence of systemic haplotype-specific processing of homeostatic and proliferation signals transmitted through IL7/IL7R. Samples of CD4+ cells were obtained from 7 MS patients (homozygous for Hap1 (3), Hap2 (2), Hap3 (2)). CD4+ cells were collected from peripheral blood, frozen and stored in liquid nitrogen. All samples were thawed and CD4+ cells were purified by magnetic bead separation. Purity and viability of cells was analyzed by Fluorescence Activated Cell Sorter (FACS). Total cellular RNA were extracted with TRIzol reagent and analyzed with the Human Gene 1.0 ST Array (affymetrix). IL7R haplotypes and susceptibility to develop MS: Hap1 homozygous <-> Risk <-> positive effect on MS susceptibility Hap2 homozygous <-> Hap2 <-> neutral effect on MS susceptibility Hap3 homozygous <-> Prot <-> neutral effect on MS susceptibility [Note: Haplotype nomenclature subject to revision.]
Project description:Screening of 14 novel proteins derived from Klebsiella pneumoniae MGH 78578 identified prior via screening of cDNA libraries. The full-length proteins were attached using a specific HaloTag to their corresponding ligand surface, HaloLink. Screening was performed using two different polyclonal antibodies to Klebsiella pneumoniae (Acris AP00792PU-N and Abcam ab20947) and detection achieved by Goat polyclonal to rabbit IgG conjugated with Chromeo-546 (Abcam ab60317). In order to assess their potential immungenic nature and rank the proteins investigated, comparative analysis using already described antigens from K. pneumoniae were used in the assay. Each microarray was seperated into different incubation chambers using the 16-well ProPlate (Grace Biolabs) multi-well gaskets. As positive references ompA and mdh were used. For negative control gapA was used and the crude lysates of the expression host (Acella E.coli) and buffer were spotted as well.Samples and controls were spotted with five replicates each. Incubation was performed using different antibodies reactive to K. pneumoniae.
Project description:We identified the abundance of isoforms of miRNAs encoded by human adenovirus 5 in total cytoplasmic RNA fractions of infected cells as well as in fractions containing the purified RNA-induced silencing complex (RISC).
Project description:Genome-wide association studies in multiple sclerosis (MS) identified a polymorphism (rs6897932) located in the coding region of the alpha chain of the cytokine receptor interleukin 7 receptor (IL7R) as a component that increases susceptibility to develop the disease. This single nucleotide polymorphism (SNP) affects the splicing of the primary transcript leading to genotype-defined transcript ratios encoding either a full length membrane spanning form or a soluble receptor chain. Genotyping at the IL7R locus reveals that the region can be described by four haplotypes. Interestingly, only one out of three haplotypes harbouring the associated SNP is positively associated with MS whereas the other two do not show association. The minor allele containing haplotype shows a reduced susceptibility to develop MS. We hypothesized that additional functional or phenotypic differences exist between individuals homozygous for haplotypes shown to have either positive, negative, or neutral effect, on susceptibility to develop MS. Gene expression profiles of CD4+ T cells from MS individuals before and after stimulation with IL7 were recorded. Haplotype-specific gene signatures were found indicating small alterations in IL7/IL7R signal processing/sensitivity through JAK/STAT and p38/MAPK14. We can not exclude that the obtained signatures result from differences within the CD4+ T cell compartment that, in fact, should be seen as a consequence of systemic haplotype-specific processing of homeostatic and proliferation signals transmitted through IL7/IL7R.