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Noninvasive Nasopharyngeal Proteomics Identify Coagulation Abnormalities in SARS-CoV-2

ABSTRACT: Vaccines/drugs that could utilize the mucosal immune system to produce antibodies in the nose should help stop the coronavirus at the point of entry. Therefore, Identification of biomarkers in nasal microenvironment associated with viral clearance, vaccine implementation, and easily measurable correlates of protection, can allow monitoring of infection-induced immunity and facilitate novel drug/vaccine development. Our study proposes to use mass spectrometry-based proteomics to reflect the host response to COVID-19 infection in the nasopharyngeal environment and to identify any new biomarkers that can be used for designing non-invasive prognostic tools and treatments at the point of entry of the virus.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Nasal Cavity, Epithelial Cell

DISEASE(S): Covid-19

SUBMITTER: Wanying Cao

LAB HEAD: Lina Abi Mosleh

PROVIDER: PXD029143 | Pride | 2022-07-21

REPOSITORIES: Pride

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Project description:Active genes on the X chromosome of Drosophila males are upregulated by the Male-specific lethal (MSL) complex containing five MSL proteins and two non-coding roX RNAs. To probe the targeting mechanism, we have solved the structure of the MSL3 chromodomain, designed point mutations in key residues that disrupt putative methyl-lysine recognition, and tested their effect on full length MSL3 function. Transgenic males expressing these site-directed point mutants or MSL3short, a naturally occurring MSL3 form lacking the chromodomain, are unhealthy and developmentally delayed. Genomewide analyses of the binding patterns of these mutants support a two-step model: the first step is chromodomain-independent association with “chromatin entry sites” carrying GA-rich MSL recognition elements (MREs). The second step involves spreading from entry sites to the majority of active genes on the X. Either deleting or introducing point mutations in the MSL3 chromodomain disrupts this second step. In vitro studies demonstrate that chromodomain mutants have diminished interaction with recombinant nucleosomes methylated at H3K36. We propose that MSL spreading depends, to a large extent, on the integrity of the MSL3 chromodomain to interact with lysine-methylated nucleosomes. Chromatin immunoprecipitation using TAP-tag was performed as described previously (Alekseyenko et al., 2006). The immunoprecipitated material was eluted from the beads by adding 10 ?L (100 U) of AcTEV protease into 450 ?L of TEV buffer followed by incubation at 25°C for 1 h. To reverse the cross-links, samples were brought to 0.3M NaCl and 1% SDS and incubated at 65°C for 12 h. The samples were then extracted with phenol/chloroform/isoamyl alcohol followed by chloroform, and precipitated by ethanol in the presence of glycogen. The resulting precipitated DNA and the input DNA were amplified using a DNA linker as described (Strutt et al., 1997). LM-PCR (25 cycles) was performed using Platinum Pfu (Invitrogen). The resulting DNA was labeled and hybridized to arrays by NimbleGen Systems, Inc. Two-channel tiling arrays containing 388,000 probes were designed based on FlyBase 3.2. Chromosomes X and 19.6Mb of 2L were covered. The ChIP sample of interest was hybridized on one channel and genomic DNA was used as the reference on the other channel. Each ChIP-chip experiment was performed in duplicate.

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Noninvasive Nasopharyngeal Proteomics Identify Coagulation Abnormalities in SARS-CoV-2

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