Project description:Recently, cell therapy has emerged as a promising treatment option for various disorders. Given the intricate mechanisms of action (MOA) and heterogenous distribution in target tissues inherent to cell therapy, it is necessary to develop more sophisticated, unbiased approaches to evaluate the distribution of administered cells and the molecular changes at a microscopic level. This study introduces a label-free approach for assessing the tissue distribution of administered human mesenchymal stem cells (hMSCs) and their MOA, leveraging spatially resolved transcriptomics (ST) analysis. The hMSCs were introduced into a mouse model with lung fibrosis, followed by the manipulation of ST to visualize the spatial distribution of hMSCs within the tissue. This was achieved by capitalizing on interspecies transcript differences between human and mouse. Furthermore, the method allowed for the examination of molecular changes associated with the spatial distribution of hMSCs. Therefore, our method has the potential to serve as an effective tool for various cell-based therapeutic agents.
Project description:Idiopathic pulmonary fibrosis is a debilitating disease leading ultimately to death without existing treatment. Here we profiled the proteome of 30 IPF cores and 10 control cores. The goal was to validate findings of snRNA-seq and new informatics means to mine the data (UNAGI) and evaluate in silico drugs that could present efficiency against IPF. The samples were extracted using the MPLEx method, peptides were reduced, alkylated, and digested with trypsin and 5 ul of 0.1 ug/ul were injected on LC-MS/MS on a Lumos instrument. The analysis was performed in DDA label free mode.
Project description:Chromatin immunoprecipitation (ChIP) has been a cornerstone for epigenetic analyses over the last decades, but even coupled to sequencing approaches (ChIP-seq), it is ultimately limited to one protein at a time. In a complementary effort, we here combined ChIP with label-free quantitative (LFQ) mass spectrometry (ChIP-MS) to interrogate local chromatin compositions. We demonstrate the versality of our approach at telomeres, with transcription factors, in tissue and by dCas9-driven locus-specific enrichment.
Project description:Chromatin immunoprecipitation (ChIP) has been a cornerstone for epigenetic analyses over the last decades, but even coupled to sequencing approaches (ChIP-seq), it is ultimately limited to one protein at a time. In a complementary effort, we here combined ChIP with label-free quantitative (LFQ) mass spectrometry (ChIP-MS) to interrogate local chromatin compositions. We demonstrate the versality of our approach at telomeres, with transcription factors, in tissue and by dCas9-driven locus-specific enrichment.
Project description:To establish the plasmodesmata core proteome, the identification and the relative amount of proteins in different cellular fractions, namely, the plasmodesmata, plasma membrane, total cell extract, microsomal and cell wall fractions, were determined using label-free quantification method. Four to six biological replicates for each fraction were used for quantification.
Project description:Using Mycoplasma pneumoniae as a model organism, we conditionally depleted the two essential ATP-dependent proteases (Lon and FtsH) of this bacterium, by engineering three strains carrying a Lon and/or FtsH inducible expression locus. An integrative comparative study combining label-free shotgun proteomics and RNA-seq allowed us to decipher the global cellular response to Lon and FtsH depletion and to define protease substrates in this genome-reduced organism.
Project description:Here we report a metabolic labeling method to map mRNA N6-methyladenosine (m6A) modification transcriptome-wide at base resolution, termed m6A-label-seq. The cells were fed with Se-allyl-L-selenohomocysteine, an analog of methoine, which serves as the precursor of methylation enzyme cofactor, so that cellular RNAs were continuously deposited with N6-allyladenosine (a6A) at supposed m6A sites. We enriched a6A-containing mRNAs and sequenced their a6A sites which are identical to m6A sites, based on iodination-induced misincorporation during reverse transcription.