Notes

Intestinal Stromal Tumors-A Tiny Assessment.
Receptor tyrosine kinases (RTKs) are transmembrane receptors of great clinical interest due to their role in disease. Historically, therapeutics targeting RTKs have been identified using in vitro kinase assays. Due to frequent development of drug resistance, however, there is a need to identify more diverse compounds that inhibit mutated but not wild-type RTKs. Here, we describe MaMTH-DS (mammalian membrane two-hybrid drug screening), a live-cell platform for high-throughput identification of small molecules targeting functional protein-protein interactions of RTKs. We applied MaMTH-DS to an oncogenic epidermal growth factor receptor (EGFR) mutant resistant to the latest generation of clinically approved tyrosine kinase inhibitors (TKIs). We identified four mutant-specific compounds, including two that would not have been detected by conventional in vitro kinase assays. One of these targets mutant EGFR via a new mechanism of action, distinct from classical TKI inhibition. Our results demonstrate how MaMTH-DS is a powerful complement to traditional drug screening approaches.We report new lipid-based, high-density, environmentally sensitive (HIDE) probes that accurately and selectively image endo-lysosomes and their dynamics at super-resolution for extended times. Treatment of live cells with the small molecules DiIC16TCO or DiIC16'TCO followed by in situ tetrazine ligation reaction with the silicon-rhodamine dye SiR-Tz generates the HIDE probes DiIC16-SiR and DiIC16'-SiR in the endo-lysosomal membrane. These new probes support the acquisition of super-resolution videos of organelle dynamics in primary cells for more than 7 min with no detectable change in endosome structure or function. Using DiIC16-SiR and DiIC16'-SiR, we describe direct evidence of endosome motility defects in cells from patients with Niemann-Pick Type-C disease. In wild-type fibroblasts, the probes reveal distinct but rare inter-endosome kiss-and-run events that cannot be observed using confocal methods. Our results shed new light on the role of NPC1 in organelle motility and cholesterol trafficking.An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.Lineage-specific gene expression is modulated by a balance between transcriptional activation and repression during animal development. Knowledge about enhancer-centered transcriptional activation has advanced considerably, but silencers and their roles in normal development remain poorly understood. Here, we performed chromatin interaction analyses of Polycomb repressive complex 2 (PRC2), a key inducer of transcriptional gene silencing, to uncover silencers, their molecular identity and associated chromatin connectivity. Systematic analysis of cis-regulatory silencer elements reveals their chromatin features and gene-targeting specificity. Deletion of certain PRC2-bound silencers in mice results in transcriptional derepression of their interacting genes and pleiotropic developmental phenotypes, including embryonic lethality. While some PRC2-bound elements function as silencers in pluripotent cells, they can transition into active tissue-specific enhancers during development, highlighting their regulatory versatility. Our study characterizes the molecular profile of silencers and their associated chromatin architectures, and suggests the possibility of targeted reactivation of epigenetically silenced genes.The majority of the human genome does not encode proteins. Many of these noncoding regions contain important regulatory sequences that control gene expression. To date, most studies have focused on activators such as enhancers, but regions that repress gene expression-silencers-have not been systematically studied. We have developed a system that identifies silencer regions in a genome-wide fashion on the basis of silencer-mediated transcriptional repression of caspase 9. We found that silencers are widely distributed and may function in a tissue-specific fashion. These silencers harbor unique epigenetic signatures and are associated with specific transcription factors. click here Silencers also act at multiple genes, and at the level of chromosomal domains and long-range interactions. Deletion of silencer regions linked to the drug transporter genes ABCC2 and ABCG2 caused chemo-resistance. Overall, our study demonstrates that tissue-specific silencing is widespread throughout the human genome and probably contributes substantially to the regulation of gene expression and human biology.The influx and efflux of cells and antigens to and from the draining lymph nodes largely take place through the subcapsular, cortical and medullary sinus systems. Recent analyses in mice and humans have revealed unexpected diversity in the lymphatic endothelial cells, which form the distinct regions of the sinuses. As a semipermeable barrier, the lymphatic endothelial cells regulate the sorting of lymph-borne antigens to the lymph node parenchyma and can themselves serve as antigen-presenting cells. The leukocytes entering the lymph node via the sinus system and the lymphocytes egressing from the parenchyma migrate through the lymphatic endothelial cell layer. The sinus lymphatic endothelial cells also orchestrate the organogenesis of lymph nodes, and they undergo bidirectional signalling with other sinus-resident cells, such as subcapsular sinus macrophages, to generate a unique lymphatic niche. In this Review, we consider the structural and functional basis of how the lymph node sinus system coordinates immune responses under physiological conditions, and in inflammation and cancer.Analysis of RNA-protein complexes is central to understanding the molecular circuitry governing cellular processes. In recent years, several proteome-wide studies have been dedicated to the identification of RNA-binding proteins. Here, we describe in detail R-DeeP, an approach built on RNA dependence, defined as the ability of a protein to engage in protein complexes only in the presence of RNA, involving direct or indirect interaction with RNA. This approach provides-for the first time, to our knowledge-quantitative information on the fraction of a protein associated with RNA-protein complexes. R-DeeP is independent of any potentially biased purification procedures. It is based on cellular lysate fractionation by density gradient ultracentrifugation and subsequent analysis by proteome-wide mass spectrometry (MS) or individual western blotting. The comparison of lysates with and without previous RNase treatment enables the identification of differences in the apparent molecular weight and, hence, the size of the complexes.
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