Biology subjects

Weiskirchen, R.

Publications and source records attributed to Weiskirchen, R..

3 recordsLinked to original sources

bioRxiv2024

SIngle cell level Genotyping Using scRna Data (SIGURD)

MotivationBy accounting for variants within measured transcripts, it is possible to evaluate the status of somatic variants using single-cell RNA-sequencing (scRNA-seq) and to characterize their clonality. However, the sparsity (very few reads per transcript) or bias in protocols (favoring 3 ends of the transcripts) makes the chance of capturing somatic variants very unlikely. This can be overcome by targeted sequencing or the use of mitochondrial variants as natural barcodes for clone identification. Currently, available computational tools focus on genotyping, but do not provide functionality for combined analysis of somatic and mitochondrial variants and functional analysis such as characterization of gene expression changes in detected clones. ResultsHere, we propose SIGURD, which is an R-based pipeline for the clonal analysis of single-cell RNA-seq data. This allows the quantification of clones by leveraging both somatic and mitochondrial variants. SIGURD also allows for functional analysis after clonal detection: association of clones with cell populations, detection of differentially expressed genes across clones and association of somatic and mitochondrial variants. Here, we demonstrate the power of SIGURD by analyzing single-cell data of colony-forming cells derived from patients with myeloproliferative neoplasms. AvailabilityCode and tutorial of SIGURD are available at GitHub https://github.com/CostaLab/sigurd. Contactivan.costa@rwth-aachen.de, skoschmieder@ukaachen.de Supplementary InformationSupplementary data are available online.

bioinformatics↗

bioRxiv2023

TGF-β1 inhibits cholesterol metabolism in hepatocytes to facilitate cell death, EMT and signals for HSC activation.

Background and Aims: Transforming growth factor-{beta}1 (TGF-{beta}1) plays important roles in chronic liver diseases, including metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD involves various biological processes including dysfunctional cholesterol metabolism and contributes to progression to metabolic dysfunction-associated steatohepatitis (MASH) and hepatocellular carcinoma (HCC). However, the reciprocal regulation of TGF-{beta}1 signaling and cholesterol metabolism in MASLD is yet unknown. Methods: Changes in transcription of genes associated with cholesterol metabolism were assessed by RNA-Seq of murine hepatocyte cell line (AML12) and mouse primary hepatocytes (MPH) treated with TGF-{beta}1. Functional assays were performed on AML12 cells (untreated, TGF-{beta}1 treated, or subjected to cholesterol enrichment (CE) or depletion (CD)), and on mice injected with adeno-associated virus 8 (AAV8)-Control/TGF-{beta}1. Results: TGF-{beta}1 inhibited mRNA expression of several cholesterol metabolism regulatory genes, including rate-limiting enzymes of cholesterol biosynthesis in AML12 cells, MPHs, and AAV8-TGF-{beta}1-treated mice. Total cholesterol levels and lipid droplet accumulation in AML12 cells and liver tissue were also reduced upon TGF-{beta}1 treatment. Smad2/3 phosphorylation following 2 h TGF-{beta}1 treatment persisted after CE or CD and was mildly increased following CD, while TGF-{beta}1-mediated AKT phosphorylation (30 min) was inhibited by CE. Furthermore, CE protected AML12 cells from several effects mediated by 72 h incubation with TGF-{beta}1, including EMT, actin polymerization, and apoptosis. CD mimicked the outcome of long term TGF- {beta}1 administration, an effect that was blocked by an inhibitor of the type I TGF-{beta} receptor. Additionally, the supernatant of CE- or CD-treated AML12 cells inhibited or promoted, respectively, the activation of LX-2 hepatic stellate cells. Conclusions: TGF-{beta}1 inhibits cholesterol metabolism while cholesterol attenuates TGF-{beta}1 downstream effects in hepatocytes.

cell biology↗

bioRxiv2023

Transformation of primary murine peritoneal mast cells by constitutive KIT activation as a result of lost Cdkn2a/Arf expression

Mast cells (MCs) are immune cells of the myeloid lineage distributed in tissues throughout the body. Phenotypically, they are a heterogeneous group characterized by different protease repertoires stored in secretory granules and differential presence of receptors. To adequately address aspects of MC biology either primary MCs isolated from human or mouse tissue or different human MC lines, like HMC-1.1 and -1.2, or rodent MC lines like L138.8A or RBL-2H3 are frequently used. Nevertheless, cellular systems to study MC functions are very limited. We have generated a murine connective tissue-like MC line, termed PMC-306, derived from primary peritoneal MCs (PMCs), which spontaneously transformed. We analyzed PMC-306 cells regarding MC surface receptor expression, effector functions and respective signaling pathways, and found that the cells reacted very similar to primary wildtype (WT) PMCs. In this regard, stimulation with MAS-related G-protein-coupled receptor member B2 (MRGPRB2) ligands induced respective signaling and effector functions. Furthermore, PMC-306 cells revealed significantly accelerated cell cycle progression, which however was still dependent on IL-3 and stem cell factor (SCF). Phenotypically, PMC-306 cells adopted an immature connective tissue-like MCs appearance. The reason for immortalization most likely is the loss of the two critical cell cycle regulators Cdkn2a/INK4A and Arf/p19, respectively. The loss of Cdkn2a and Arf expression could be mimicked in primary bone marrow-derived mast cells (BMMCs) by SCF supplementation strongly arguing for an involvement of KIT activation in the transformation process. Hence, this new cell line might be a useful tool to study further aspects of PMC function and to address tumorigenic processes associated with MC leukemia.

immunology↗