Protocol for isolating specific C. elegans neuron types for bulk and single-cell RNA sequencing.

The C. elegans nervous system is compact and well described, ideal for identifying genetic programs that drive neuron-specific development, function, and connectivity. Here, we present a protocol for isolating specific neuron types for gene expression profiling by bulk or single-cell RNA sequencing.

Cell-type-specific and disease-associated expression quantitative trait loci in the human lung.

Common genetic variants confer substantial risk for chronic lung diseases, including pulmonary fibrosis. Defining the genetic control of gene expression in a cell-type-specific and context-dependent manner is critical for understanding the mechanisms through which genetic variation influences complex traits and disease pathobiology. To this end, we performed single-cell RNA sequencing of lung tissue from 66 individuals with pulmonary fibrosis and 48 unaffected donors.

Cell type-specific and disease-associated eQTL in the human lung.

Common genetic variants confer substantial risk for chronic lung diseases, including pulmonary fibrosis (PF). Defining the genetic control of gene expression in a cell-type-specific and context-dependent manner is critical for understanding the mechanisms through which genetic variation influences complex traits and disease pathobiology. To this end, we performed single-cell RNA-sequencing of lung tissue from 67 PF and 49 unaffected donors.

Neutrophil trafficking to the site of infection requires Cpt1a-dependent fatty acid β-oxidation.

Cellular metabolism influences immune cell function, with mitochondrial fatty acid β-oxidation and oxidative phosphorylation required for multiple immune cell phenotypes. Carnitine palmitoyltransferase 1a (Cpt1a) is considered the rate-limiting enzyme for mitochondrial metabolism of long-chain fatty acids, and Cpt1a deficiency is associated with infant mortality and infection risk. This study was undertaken to test the hypothesis that impairment in Cpt1a-dependent fatty acid oxidation results in increased susceptibility to infection.

Synergistic action of WDR5 and HDM2 inhibitors in SMARCB1-deficient cancer cells.

Rhabdoid tumors (RT) are rare and deadly pediatric cancers driven by loss of , which encodes the SNF5 component of the SWI/SNF chromatin remodeler. Loss of is associated with a complex set of phenotypic changes including vulnerability to inhibitors of protein synthesis and of the p53 ubiquitin-ligase HDM2. Recently, we discovered small molecule inhibitors of the 'WIN' site of WDR5, which in MLL-rearranged leukemia cells decrease the expression of a set of genes linked to protein synthesis, inducing a translational choke and causing p53-dependent inhibition of proliferation.

WIN site inhibition disrupts a subset of WDR5 function.

WDR5 nucleates the assembly of histone-modifying complexes and acts outside this context in a range of chromatin-centric processes. WDR5 is also a prominent target for pharmacological inhibition in cancer. Small-molecule degraders of WDR5 have been described, but most drug discovery efforts center on blocking the WIN site of WDR5, an arginine binding cavity that engages MLL/SET enzymes that deposit histone H3 lysine 4 methylation (H3K4me).

A single-cell atlas of mouse lung development.

Lung organogenesis requires precise timing and coordination to effect spatial organization and function of the parenchymal cells. To provide a systematic broad-based view of the mechanisms governing the dynamic alterations in parenchymal cells over crucial periods of development, we performed a single-cell RNA-sequencing time-series yielding 102,571 epithelial, endothelial and mesenchymal cells across nine time points from embryonic day 12 to postnatal day 14 in mice. Combining computational fate-likelihood prediction with RNA in situ hybridization and immunofluorescence, we explore lineage relationships during the saccular to alveolar stage transition.

Multiple interactions of the oncoprotein transcription factor MYC with the SWI/SNF chromatin remodeler.

The SNF5 subunit of the SWI/SNF chromatin remodeling complex has been shown to act as a tumor suppressor through multiple mechanisms, including impairing the ability of the oncoprotein transcription factor MYC to bind chromatin. Beyond SNF5, however, it is unknown to what extent MYC can access additional SWI/SNF subunits or how these interactions affect the ability of MYC to drive transcription, particularly in SNF5-null cancers. Here, we report that MYC interacts with multiple SWI/SNF components independent of SNF5.

Combinatorial Transcriptional Profiling of Mouse and Human Enteric Neurons Identifies Shared and Disparate Subtypes In Situ.

Background & Aims: The enteric nervous system (ENS) coordinates essential intestinal functions through the concerted action of diverse enteric neurons (ENs). However, integrated molecular knowledge of EN subtypes is lacking. To compare human and mouse ENs, we transcriptionally profiled healthy ENS from adult humans and mice.

WDR5 is a conserved regulator of protein synthesis gene expression.

WDR5 is a highly-conserved nuclear protein that performs multiple scaffolding functions in the context of chromatin. WDR5 is also a promising target for pharmacological inhibition in cancer, with small molecule inhibitors of an arginine-binding pocket of WDR5 (the 'WIN' site) showing efficacy against a range of cancer cell lines in vitro. Efforts to understand WDR5, or establish the mechanism of action of WIN site inhibitors, however, are stymied by its many functions in the nucleus, and a lack of knowledge of the conserved gene networks-if any-that are under its control.

The LRRC8 volume-regulated anion channel inhibitor, DCPIB, inhibits mitochondrial respiration independently of the channel.

There has been a resurgence of interest in the volume-regulated anion channel (VRAC) since the recent cloning of the LRRC8A-E gene family that encodes VRAC. The channel is a heteromer comprised of LRRC8A and at least one other family member; disruption of LRRC8A expression abolishes VRAC activity. The best-in-class VRAC inhibitor, DCPIB, suffers from off-target activity toward several different channels and transporters.

Novel kidney dissociation protocol and image-based flow cytometry facilitate improved analysis of injured proximal tubules.

Flow cytometry studies on injured kidney tubules are complicated by the low yield of nucleated single cells. Furthermore, cell-specific responses such as cell cycle dynamics in vivo have conventionally relied on indirect immunohistochemistry and proximal tubule markers that may be downregulated in injury. Here, we report a new tissue dissociation protocol for the kidney with an early fixation step that greatly enhances the yield of single cells.

Improved proliferation of antigen-specific cytolytic T lymphocytes using a multimodal nanovaccine.

The present study investigated the immunoenhancing property of our newly designed nanovaccine, that is, its ability to induce antigen-specific immunity. This study also evaluated the synergistic effect of a novel compound PBS-44, an α-galactosylceramide analog, in boosting the immune response induced by our nanovaccine. The nanovaccine was prepared by encapsulating ovalbumin (ova) and an adjuvant within the poly(lactic-co-glycolic acid) nanoparticles.

Multiparameter analysis of stimulated human peripheral blood mononuclear cells: A comparison of mass and fluorescence cytometry.

Mass and fluorescence cytometry are quantitative single cell flow cytometry approaches that are powerful tools for characterizing diverse tissues and cellular systems. Here mass cytometry was directly compared with fluorescence cytometry by studying phenotypes of healthy human peripheral blood mononuclear cells (PBMC) in the context of superantigen stimulation. One mass cytometry panel and five fluorescence cytometry panels were used to measure 20 well-established lymphocyte markers of memory and activation.

Methotrexate inhibits NF-κB activity via long intergenic (noncoding) RNA-p21 induction.

Objective: To determine interrelationships between the expression of long intergenic (noncoding) RNA-p21 (lincRNA-p21), NF-κB activity, and responses to methotrexate (MTX) in rheumatoid arthritis (RA) by analyzing patient blood samples and cell culture models.

Methods: Expression levels of long noncoding RNA and messenger RNA (mRNA) were determined by quantitative reverse transcription-polymerase chain reaction. Western blotting and flow cytometry were used to quantify levels of intracellular proteins.

Cell-cycle control of developmentally regulated transcription factors accounts for heterogeneity in human pluripotent cells.

Heterogeneity within pluripotent stem cell (PSC) populations is indicative of dynamic changes that occur when cells drift between different states. Although the role of metastability in PSCs is unclear, it appears to reflect heterogeneity in cell signaling. Using the Fucci cell-cycle indicator system, we show that elevated expression of developmental regulators in G1 is a major determinant of heterogeneity in human embryonic stem cells.

Obesity induced by a high-fat diet is associated with increased immune cell entry into the central nervous system.

Obesity is associated with chronic low-grade inflammation in peripheral tissues caused, in part, by the recruitment of inflammatory monocytes into adipose tissue. Studies in rodent models have also shown increased inflammation in the central nervous system (CNS) during obesity. The goal of this study was to determine whether obesity is associated with recruitment of peripheral immune cells into the CNS.