Project description:Melanoma is a rare but deadly form of skin cancer, which is often treated with BRAF inhibitors such as Vemurafenib (referred to as PLX4032). Whilst Vemurafenib prolongs the survival of patients, BRAF inhibitor resistance inevitably occurs in most cases. Previous studies demonstrated that metabolic rewiring occurs in BRAF inhibitor resistance and causes dependence on glutamine. To investigate whether this vulnerability could be exploited with clinically relevant drugs, we used the BRAF inhibitor, Vemurafenib, and the glutaminase imhibitor, CB839 to treat A375-derived melanoma xenografted tumors. We showed that whilst CB839 did not significantly affect the growth of A375-derived tumors compared to those given a vehicle, the addition of CB839 to Vemurafenib treatment had a significant anti-tumor effect. Tumors were taken at the endpoint (Max tumor length 15mm) from the 6 mice in each treatment group and cut into fragments and stored in RNAlater for RNAseq analysis. RNA extraction was performed on 1-3 fragments per tumor to make up 200-300mg of tissue.
Project description:Genome-wide identification of transcription factor (TF) binding sites in the genome of the fission yeast Schizosaccharomyces pombe. The ChIP-nexus method was used. TFs included were: Cbf11-TAP and Cbf12-TAP (and their DBM mutants with impaired DNA binding), TAP-Mga2, and Fkh2-TAP (as an irrelevant control TF). IPs from an untagged WT strain were also analyzed. Cbf11-related IPs were performed from exponential cultures, while Cbf12-related IPs were performed from stationary cultures. YES complex medium was used for all cultivations.
Project description:We performed high numbers of replicates of CUT&RUN LoV-U against H3K4me3, β-catenin, and the negative control IgG in human colorectal cancer HCT116 cells over two independent rounds of experiments to discover the complete set of binding events.
Project description:Lipid intermediates derived from sphingolipid metabolism are crucial regulators of mitochondrial function from yeast to humans. Among these intermediates, trans-2-hexadecenal (t-2hex) within the sphingolipid degradation pathway exhibits remarkable efficiency in inducing mitochondria-mediated cell death. In yeast cell cultures, the addition of t-2-hex triggers complete disintegration of the mitochondrial network, leading to subsequent cell death. This effect is particularly pronounced in yeast cells lacking the activity of the t-2-hex degrading enzyme, Hfd1. However, the molecular mechanisms of t-2-hex induction of mitochondrial dysfunction are completely unknown. In this project, we want to exploit the unprecedented power of yeast genetics to unveil novel genetic determinants involved in t-2-hex's pro-apoptotic function. To accomplish this, we employed the SATAY method, which combines saturated transposon mutagenesis with high-throughput sequencing to functionally explore the yeast genome. In our screening approach, hfd1 mutant cells harboring a plasmid-encoded inducible MiniDs transposon were induced by galactose, resulting in extensive integration of the transposon throughout the yeast genome. Cells with the plasmid excised and the transposon genomically integrated were pooled together, creating a high-density transposon library comprising approximately 2.3E+06 independent insertion mutants. Subsequently, the pooled mutant library was subjected to treatment with the mitochondria-mediated death inducer, t-2-hexadecenal. As a control, cells were also incubated with the solvent dimethyl sulfoxide (DMSO), in which hexadecenal is dissolved. Following the treatments, cells were collected for genomic DNA extraction and digestion, using restriction enzymes with frequent four-base pair recognition sites. The resulting library fragments were circularized using T4 DNA ligase, and the transposon-genome junctions were selectively amplified through PCR with outward-facing primers specific to the transposon. Finally, the pooled and purified amplicons were subjected to massive sequencing on an Illumina MiSeq platform. The obtained sequences were then aligned to the reference genome of Saccharomyces cerevisiae, allowing for the mapping of transposon insertions and the calculation of transposon counts per gene. This project enabled the identification of genes required for the resistance and toxicity to t-2hex.
Project description:Identification of cell types in the interphase between muscle and tendon by single-nuclei RNA-seq of three human semitendinous muscle-tendon biopsies. With special focus on the myotendinous junction specific myonuclei, transcripts were identified and confirmed to myotendinous junction with immunofluorescence.
Project description:To identify possible molecular mechanisms with a role in root branching initiation in S. moellendorffii, we employed the developmental root branching assay for an RNA-seq experiment and sampled root tips on each day from 0 to 5 d after the first branching (time point 0), in which 300 µm apical parts were sampled to enrich for the meristematic region, while non-meristematic root regions were sampled separately
Project description:Identification of copy number alterations of HPV-positive and HPV-negative vulvar squamous cell carcinomas (VSCC) and vulvar intraepithelial neoplasias (VIN), with special focus on VIN with and without VSCC, the latter group being defined as VIN with no VSCC development during >10 year follow-up.
Project description:Protein oxidation results from the reaction of amino-acid side chains with reactive oxygen species (ROS) and is mostly irreversible. In non-photosynthetic tissues, mitochondria are a main source of ROS, whereas plastids are the major source in photosynthetic tissues. Oxidized proteins suffer from decreased structural integrity and even loss of function, and their accumulation ultimately leads to cytotoxic aggregates. In mammals, this correlates with aging and is linked to several age-related pathologies. Mammalian proteolytic pathways for clearance of oxidized proteins are under intensive research, while mechanistic insights into this process in plants is scarce. AARE enzymes are ATP-independent serine proteases presumably acting on oxidized proteins and operating in a dual exo-/endopeptidase mode. They are found in all domains of life. Here, we investigated AARE enzymes in the moss Physcomitrella and the angiosperm Arabidopsis and identified three homologous nuclear genes in Physcomitrella (PpAARE1-3) and a single nuclear gene in Arabidopsis (AtAARE). Surprisingly, we observed triple localization of the proteins AtAARE and PpAARE1 to plastids, mitochondria and the cytosol in vivo, likely conserved across the plant lineage. This represents an ATP-independent possibility for degradation of oxidized proteins in the major source organelles of ROS in plants, which is distinct to mammals. Combinatorial knockout plants and protein interaction analysis revealed specific interactions of the moss AARE isoforms and functions in progressive aging. Analysis of an AtAARE T-DNA mutant further suggests conservation of AARE function in age-related development.
Project description:Human adipose and bone marrow-derived mesenchymal stem cells were cultured either on collagenase biomaterial (CardioCel®) or normal tissue culture plastic over 48 hours in standard culture conditions, in serum-free medium.
Project description:We performed ChIP-seq for the meiotic strand exchange protein DMC1, which marks an early stage in the meiotic recombination pathway, and the chromosome axis protein ASY1, which promotes interhomolog synapsis and recombination in plants, using tissue collected from immature pre-emergence spikes from wild type bread wheat cultivar Chinese Spring plants. To investigate connections between meiotic recombination and chromatin states in wheat, we also performed ChIP-seq for euchromatic (H3K4me3) and constitutive heterochromatic (H3K9me2 and H3K27me1) marks, and mapped genome-wide nucleosome occupancy via micrococcal nuclease sequencing (MNase-seq) using leaf tissue from Chinese Spring.