Explore the vast range of titles available.

Myeloid neoplasms are clonal hematopoietic stem cell disorders driven by the sequential acquisition of recurrent genetic lesions. Truncating mutations in the chromatin remodeler ASXL1 (ASXL1 MT ) are associated with a high-risk disease phenotype with increased proliferation, epigenetic therapeutic resistance, and poor survival outcomes. We performed a multi-omics interrogation to define gene expression and chromatin remodeling associated with ASXL1 MT in chronic myelomonocytic leukemia (CMML). ASXL1 MT are associated with a loss of repressive histone methylation and increase in permissive histone methylation and acetylation in promoter regions. ASXL1 MT are further associated with de novo accessibility of distal enhancers binding ETS transcription factors, targeting important leukemogenic driver genes. Chromatin remodeling of promoters and enhancers is strongly associated with gene expression and heterogenous among overexpressed genes. These results provide a comprehensive map of the transcriptome and chromatin landscape of ASXL1 MT CMML, forming an important framework for the development of novel therapeutic strategies targeting oncogenic cis interactions. ‘Mutations in the chromatin remodeler ASXL1 (ASXL1 MT ) are associated with poor clinical outcome, however, their impact on chromatin dynamics remains unexplored. Here the authors use a multi-omics approach for chronic myelomonocytic leukemia (CMML) and investigate the transcriptome and chromatin landscape of ASXL1 MT CMML.

Voltage-gated ion channels (VGICs) orchestrate electrical activities that drive mechanical functions in contractile tissues such as the heart and gut. In turn, contractions change membrane tension and impact ion channels. VGICs are mechanosensitive, but the mechanisms of mechanosensitivity remain poorly understood. Here, we leverage the relative simplicity of NaChBac, a prokaryotic voltage-gated sodium channel from Bacillus halodurans , to investigate mechanosensitivity. In whole-cell experiments on heterologously transfected HEK293 cells, shear stress reversibly altered the kinetic properties of NaChBac and increased its maximum current, comparably to the mechanosensitive eukaryotic sodium channel Na V 1.5. In single-channel experiments, patch suction reversibly increased the open probability of a NaChBac mutant with inactivation removed. A simple kinetic mechanism featuring a mechanosensitive pore opening transition explained the overall response to force, whereas an alternative model with mechanosensitive voltage sensor activation diverged from the data. Structural analysis of NaChBac identified a large displacement of the hinged intracellular gate, and mutagenesis near the hinge diminished NaChBac mechanosensitivity, further supporting the proposed mechanism. Our results suggest that NaChBac is overall mechanosensitive due to the mechanosensitivity of a voltage-insensitive gating step associated with the pore opening. This mechanism may apply to eukaryotic VGICs, including Na V 1.5.

Considerable efforts have been made to characterize active enhancer elements, which can be annotated by accessible chromatin and H3 lysine 27 acetylation (H3K27ac). However, apart from poised enhancers that are observed in early stages of development and putative silencers, the functional significance of cis -regulatory elements lacking H3K27ac is poorly understood. Here we show that macroH2A histone variants mark a subset of enhancers in normal and cancer cells, which we coined ‘macro-Bound Enhancers’, that modulate enhancer activity. We find macroH2A variants localized at enhancer elements that are devoid of H3K27ac in a cell type-specific manner, indicating a role for macroH2A at inactive enhancers to maintain cell identity. In following, reactivation of macro-bound enhancers is associated with oncogenic programs in breast cancer and their repressive role is correlated with the activity of macroH2A2 as a negative regulator of BRD4 chromatin occupancy. Finally, through single cell epigenomic profiling of normal mammary stem cells derived from mice, we show that macroH2A deficiency facilitates increased activity of transcription factors associated with stem cell activity. MacroH2A histone variants are shown to mark a subset of enhancers in both normal and cancer cells. In mice, macroH2A deficiency is shown to facilitate increased activity of transcription stem cell-associated transcription factors.

This study was designed to evaluate the roles of microRNAs (miRNAs) in slow transit constipation (STC). All human tissue samples were from the of the colon. Expression of 372 miRNAs was examined in a discovery cohort of four patients with STC versus three age/sex-matched controls by a quantitative PCR array. Upregulated miRNAs were examined by quantitative reverse transcription qPCR (RT-qPCR) in a validation cohort of seven patients with STC and age/sex-matched controls. The effect of a highly differentially expressed miRNA on a custom human smooth muscle cell line was examined by RT-qPCR, electrophysiology, traction force microscopy, and ex vivo by lentiviral transduction in rat organotypic cultures. The expression of 13 miRNAs was increased in STC samples. Of those miRNAs, four were predicted to target , the gene that encodes the Na channel Na 1.5. The expression of mRNA was decreased in STC samples. Let-7f significantly decreased Na current density in vitro in human smooth muscle cells. In rat organotypic cultures, overexpression of let-7f resulted in reduced frequency and amplitude of contraction. A small group of miRNAs is upregulated in STC, and many of these miRNAs target the SCN5A-encoded Na channel Na 1.5. Within this set, a novel Na 1.5 regulator, let-7f, resulted in decreased Na 1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest Na 1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC.

The objective of this study was to determine whether constipation-predominant irritable bowel syndrome (IBS-C) is associated with changes in intestinal barrier and secretory function. A total of 19 IBS-C patients and 18 healthy volunteers (all females) underwent saccharide excretion assay (0.1 g C mannitol and 1 g lactulose), measurements of duodenal and colonic mucosal barrier (transmucosal resistance (TMR), macromolecular and Escherichia coli Bio-Particle translocation), mucosal secretion (basal and acetylcholine (Ach)-evoked short-circuit current (Isc)), in vivo duodenal mucosal impedance, circulating endotoxins, and colonic tight junction gene expression. There were no differences in the in vivo measurements of barrier function between IBS-C patients and healthy controls: cumulative excretion of C mannitol (0-2 h mean (s.e.m.); IBS-C: 12.1 (0.9) mg vs. healthy: 13.2 (0.8) mg) and lactulose (8-24 h; IBS-C: 0.9 (0.5) mg vs. healthy: 0.5 (0.2) mg); duodenal impedance IBS-C: 729 (65) Ω vs. healthy: 706 (43) Ω; plasma mean endotoxin activity level IBS-C: 0.36 (0.03) vs. healthy: 0.35 (0.02); and in colonic mRNA expression of occludin, zonula occludens (ZO) 1-3, and claudins 1-12 and 14-19. The ex vivo findings were consistent, with no group differences: duodenal TMR (IBS-C: 28.2 (1.9) Ω cm vs. healthy: 29.8 (1.9) Ω cm ) and colonic TMR (IBS-C: 19.1 (1.1) Ω cm vs. healthy: 17.6 (1.7) Ω cm ); fluorescein isothiocyanate (FITC)-dextran (4 kDa) and E. coli Bio-Particle flux. Colonic basal Isc was similar, but duodenal basal Isc was lower in IBS-C (43.5 (4.5) μA cm ) vs. healthy (56.9 (4.9) μA cm ), P=0.05. Ach-evoked ΔIsc was similar. Females with IBS-C have normal colonic barrier and secretory function. Basal duodenal secretion is decreased in IBS-C.

ObjectiveThis study was designed to evaluate the roles of microRNAs (miRNAs) in slow transit constipation (STC).DesignAll human tissue samples were from the muscularis externa of the colon. Expression of 372 miRNAs was examined in a discovery cohort of four patients with STC versus three age/sex-matched controls by a quantitative PCR array. Upregulated miRNAs were examined by quantitative reverse transcription qPCR (RT-qPCR) in a validation cohort of seven patients with STC and age/sex-matched controls. The effect of a highly differentially expressed miRNA on a custom human smooth muscle cell line was examined in vitro by RT-qPCR, electrophysiology, traction force microscopy, and ex vivo by lentiviral transduction in rat muscularis externa organotypic cultures.ResultsThe expression of 13 miRNAs was increased in STC samples. Of those miRNAs, four were predicted to target SCN5A, the gene that encodes the Na+ channel NaV1.5. The expression of SCN5A mRNA was decreased in STC samples. Let-7f significantly decreased Na+ current density in vitro in human smooth muscle cells. In rat muscularis externa organotypic cultures, overexpression of let-7f resulted in reduced frequency and amplitude of contraction.ConclusionsA small group of miRNAs is upregulated in STC, and many of these miRNAs target the SCN5A-encoded Na+ channel NaV1.5. Within this set, a novel NaV1.5 regulator, let-7f, resulted in decreased NaV1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest NaV1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC.

The snATAC + snRNA platform allows epigenomic profiling of open chromatin and gene expression with single-cell resolution. The most critical assay step is to isolate high-quality nuclei to proceed with droplet-base single nuclei isolation and barcoding. With the increasing popularity of multiomic profiling in various fields, there is a need for optimized and reliable nuclei isolation methods, mainly for human tissue samples. Herein we compared different nuclei isolation methods for cell suspensions, such as peripheral blood mononuclear cells (PBMC, n = 18) and a solid tumor type, ovarian cancer (OC, n = 18), derived from debulking surgery. Nuclei morphology and sequencing output parameters were used to evaluate the quality of preparation. Our results show that NP-40 detergent-based nuclei isolation yields better sequencing results than collagenase tissue dissociation for OC, significantly impacting cell type identification and analysis. Given the utility of applying such techniques to frozen samples, we also tested frozen preparation and digestion (n = 6). A paired comparison between frozen and fresh samples validated the quality of both specimens. Finally, we demonstrate the reproducibility of scRNA and snATAC + snRNA platform, by comparing the gene expression profiling of PBMC. Our results highlight how the choice of nuclei isolation methods is critical for obtaining quality data in multiomic assays. It also shows that the measurement of expression between scRNA and snRNA is comparable and effective for cell type identification.

T-type ... currents have been detected in cells from the external muscular layers of gastrointestinal smooth muscles and appear to contribute to the generation of pacemaker potentials in interstitial cells of Cajal from those tissues. However, the ... channel α subunit responsible for these currents has not been determined. We established that the α subunit of the ... channel is expressed in single myocytes and interstitial cells of Cajal using reverse transcription and polymerase chain reaction from whole tissue, laser capture microdissected tissue and single cells isolated from the mouse jejunum. Whole-cell voltage clamp recordings demonstrated that a nifedipine and ... resistant, mibefradil-sensitive current is present in myocytes dissociated from the jejunum. Electrical recordings from the circular muscle layer demonstrated that mibefradil reduced the frequency and initial rate of rise of the electrical slow wave. Gene targeted knockout of both alleles of the cacna1h gene, which encodes the ... channel subunit, resulted in embryonic lethality because of death of the homozygous knockouts prior to E13.5 days in utero. We conclude that a channel with the pharmacological and molecular characteristics of the ... channel subunit is expressed in interstitial cells of Cajal and myocytes from the mouse jejunum, and that ionic conductances through the ... channel contribute to the upstroke of the pacemaker potential. Furthermore, the survival of mice that do not express the ... channel protein is dependent on the genetic background and targeting approach used to generate the knockout mice. (ProQuest: ... denotes formulae/symbols omitted.)

T-type Ca(2+) currents have been detected in cells from the external muscular layers of gastrointestinal smooth muscles and appear to contribute to the generation of pacemaker potentials in interstitial cells of Cajal from those tissues. However, the Ca(2+) channel alpha subunit responsible for these currents has not been determined. We established that the alpha subunit of the alpha(1H) Ca(2+) channel is expressed in single myocytes and interstitial cells of Cajal using reverse transcription and polymerase chain reaction from whole tissue, laser capture microdissected tissue and single cells isolated from the mouse jejunum. Whole-cell voltage clamp recordings demonstrated that a nifedipine and Cd(2+) resistant, mibefradil-sensitive current is present in myocytes dissociated from the jejunum. Electrical recordings from the circular muscle layer demonstrated that mibefradil reduced the frequency and initial rate of rise of the electrical slow wave. Gene targeted knockout of both alleles of the cacna1h gene, which encodes the alpha(1H) Ca(2+) channel subunit, resulted in embryonic lethality because of death of the homozygous knockouts prior to E13.5 days in utero. We conclude that a channel with the pharmacological and molecular characteristics of the alpha(1H) Ca(2+) channel subunit is expressed in interstitial cells of Cajal and myocytes from the mouse jejunum, and that ionic conductances through the alpha(1H) Ca(2+) channel contribute to the upstroke of the pacemaker potential. Furthermore, the survival of mice that do not express the alpha(1H) Ca(2+) channel protein is dependent on the genetic background and targeting approach used to generate the knockout mice.

T‐type Ca 2+ currents have been detected in cells from the external muscular layers of gastrointestinal smooth muscles and appear to contribute to the generation of pacemaker potentials in interstitial cells of Cajal from those tissues. However, the Ca 2+ channel α subunit responsible for these currents has not been determined. We established that the α subunit of the α 1H Ca 2+ channel is expressed in single myocytes and interstitial cells of Cajal using reverse transcription and polymerase chain reaction from whole tissue, laser capture microdissected tissue and single cells isolated from the mouse jejunum. Whole‐cell voltage clamp recordings demonstrated that a nifedipine and Cd 2+ resistant, mibefradil‐sensitive current is present in myocytes dissociated from the jejunum. Electrical recordings from the circular muscle layer demonstrated that mibefradil reduced the frequency and initial rate of rise of the electrical slow wave. Gene targeted knockout of both alleles of the cacna1h gene, which encodes the α 1H Ca 2+ channel subunit, resulted in embryonic lethality because of death of the homozygous knockouts prior to E13.5 days in utero . We conclude that a channel with the pharmacological and molecular characteristics of the α 1H Ca 2+ channel subunit is expressed in interstitial cells of Cajal and myocytes from the mouse jejunum, and that ionic conductances through the α 1H Ca 2+ channel contribute to the upstroke of the pacemaker potential. Furthermore, the survival of mice that do not express the α 1H Ca 2+ channel protein is dependent on the genetic background and targeting approach used to generate the knockout mice.