Project description:miRNA profiling comparing modified (COS = Cytokine (low dose), Oxygen (physiological), Stimulation (low frequency electrical)) or standard (STD) culture condtions for 72hrs prior to harvest at day 8 post differentiation. Secondary experiment to assess additional impact of dexamethasone when given in COS conditions. 5 arrays were labeled with same RNA in each channel (self-self hybridisations) to establish parameters for detecting real change for each probe on the arrays.
Project description:Spike-in heavy labeled protein lysate was generated by culturing SCN slices for 6 week in the precense of heavy arginine and lysine. SCN slices where harvested at 5 diffrent time points during one circadian cycle. The SCN slices where lysed and spiked with heavy SCN lysate. The samples were processed with the FASP protocol and and proteins quantified by LC-MS/MS using the MaxQuant software. To identify proteins with a circadian change in abundance with use JTK-cycle.
Project description:Purpose of our study is to determine the status of synaptosomal microRNAs in Alzheimer's disease and their roles in Alzheimer's progression.
Project description:Histone lysine demethylase (KDMs) are involved in the dynamic regulation of gene expression by reversible regulation of the methylation levels on lysine residues in histone tails. Among the KDMs, the jumonji (JmjC)-domain-containing KDMs (KDM2-7) are Fe(II), 2- OG (α-ketoglutarate) and molecular oxygen-dependent enzymes that employ an oxygenase mechanism to demethylate specific methylation states at various histone sites. KDMs play a critical role in several biological processes such as cell differentiation, inflammation, cancer progression and resistance. Achieving selectivity over the different families of KDMs has been a major challenge. Here we report potent and selective KDM5 covalent inhibitors designed to target a cysteine residue only present in the KDM5 sub-family. In vitro assays show that compounds are selective for the KDM5 sub-family, showing potencies in the low nanomolar range, with higher affinity for KDM5A/B. The covalent binding to the targeted proteins was proved by MS. A kinetic approach was studied in order to describe the components of overall inhibitor potency (reversible binding and chemical reactivity), showing a time-dependent decrease of IC50 values for irreversible inhibition. Additional 2-OG competition assays show that compounds were non 2-OG competitive and target engagement and ChIPs-seq assays showed that the compounds inhibited the KDM5 members in cells in the low micromolar and they induce a global increase of the H3K4Me3 mark.
Project description:We describe a chemical method to label and purify 4-thiouridine (s4U) -containing RNA. We demonstrate that methanethiolsulfonate (MTS) reagents form disulfide bonds with s4U more efficiently than the commonly used HPDP-biotin, leading to higher yields and less biased enrichment. This increase in efficiency allowed us to use s4U-labeling to study global microRNA (miRNA) turnover in proliferating cultured human cells without perturbing global miRNA levels or the miRNA processing machinery. This improved chemistry will enhance methods that depend on tracking different populations of RNA such as 4-thiouridine-tagging to study tissue-specific transcription and dynamic transcriptome analysis (DTA) to study RNA turnover. s4U metabolic labeling of RNA in 293T cells, followed by biochemical enrichment of labeled RNA with two biotinylation reagents, RNAs >200nt and miRNAs in separate experiments
Project description:Healthy and Alzheimer's diseased mice were treated with/without Bacteroides fragilis gut bacteria. Frontal cortex and hippocampus brain tissue were analyzed for metabolites using untargeted LC-MS.
Project description:Numerous neurological disorders, including Alzheimer's disease, display a sex-biased prevalence. To identify molecular correlates of this sex bias, we investigated sex-differences in molecular pathology in the hippocampus using the 5XFAD mouse model of Alzheimer's disease during early stages of disease progression (1, 2, and 4 months of age).
Project description:A long-standing question in developmental and reproductive biology is when the mammalian embryo becomes sufficiently distinct from its oocyte precursor. Myriads of studies examined the messenger RNAs that change during the oocyte-to-embryo transition, whereas proteins have been much less studied, in spite of their greater vicinity to phenotype. In the present study we modified the widely used embryo culture medium KSOM (PMID 12470333, PMID 10859270) to make it apt for our application. We replaced the serum albumin with polyvinylpyrrolidone and also replaced the natural Arginine and Lysine with their “heavy” isotopic variants Arginine 13C 15N and Lysine 13C 15N. Fertilized oocytes were retrieved from oviducts of gonadotropin-primed B6C3F1 females mated to CD1 males, and cultured at 37 degrees Celsius under 5% CO2 in KSOM containing 0.3 mM Arginine 13C 15N and 0.2 mM Lysine 13C 15N, which are the regular concentrations of these two amino acids in KSOM medium (PMID 12470333; PMID 10859270). After 4 days of culture, the embryos of the isotopic group had undergone blastocyst formation just like the control embryos cultured in normal medium. Samples of approx. 500 “heavy”-labeled blastocysts were collected zona-free and subjected to mass spectrometric analysis. The median labeling rate was 83%, ranging from 0% in proteins that did not incorporate any Arginine 13C 15N and Lysine 13C 15N, to 100% in proteins that were completely labeled. Our study demonstrates that a commonly used, chemically defined medium can be adapted for Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) and combined with high-resolution mass spectrometry, in a preimplantation embryo setting. This allows to tackle long-standing questions in developmental and reproductive biology, such as the identification of putative maternal (0% labeled), putative embryonic (100% labeled) or shared proteins in live mammalian embryos.
Project description:The mouse lncRNA Braveheart (Bvht) as a non-coding transcript has been found to act in trans to regulate cardiovascular lineage commitment. However, the mechanism of Bvht action is still not clear. lncRNAs have been shown to regulate gene expression though cooperating with protein partners. Recently, we experimentally determine the secondary structure of Bvht containing a novel structural motif AGIL. AGIL motif deletion (BvhtdAGIL) in mouse embryonic stem cells prevents the transition from mesoderm cells to cardiac progenitors. To identify proteins that interact with the Bvht AGIL motif, we used a human protein microarray platform (Human ProtoArray, Life Technology). Full-length Bvht and BvhtdAGIL transcripts were generated by in vitro transcription and labeled with Cy5. 50pmol Cy5-labeled RNAs were individually incubated with the protein microarray.
Project description:Using highly-parallel RNA-sequencing on samples from rTg4510 and J20 mice (n = 121 mice), collected at four different time points, we profiled transcriptional changes in the entorhinal cortex paralleling the progression of Alzheimer's disease-associated pathology.