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PLoS Genetics May 2024Whether single-cell RNA-sequencing (scRNA-seq) captures the same biological information as single-nucleus RNA-sequencing (snRNA-seq) remains uncertain and likely to be...
Whether single-cell RNA-sequencing (scRNA-seq) captures the same biological information as single-nucleus RNA-sequencing (snRNA-seq) remains uncertain and likely to be context-dependent. Herein, a head-to-head comparison was performed in matched normal-adenocarcinoma human lung samples to assess biological insights derived from scRNA-seq versus snRNA-seq and better understand the cellular transition that occurs from normal to tumoral tissue. Here, the transcriptome of 160,621 cells/nuclei was obtained. In non-tumor lung, cell type proportions varied widely between scRNA-seq and snRNA-seq with a predominance of immune cells in the former (81.5%) and epithelial cells (69.9%) in the later. Similar results were observed in adenocarcinomas, in addition to an overall increase in cell type heterogeneity and a greater prevalence of copy number variants in cells of epithelial origin, which suggests malignant assignment. The cell type transition that occurs from normal lung tissue to adenocarcinoma was not always concordant whether cells or nuclei were examined. As expected, large differential expression of the whole-cell and nuclear transcriptome was observed, but cell-type specific changes of paired normal and tumor lung samples revealed a set of common genes in the cells and nuclei involved in cancer-related pathways. In addition, we showed that the ligand-receptor interactome landscape of lung adenocarcinoma was largely different whether cells or nuclei were evaluated. Immune cell depletion in fresh specimens partly mitigated the difference in cell type composition observed between cells and nuclei. However, the extra manipulations affected cell viability and amplified the transcriptional signatures associated with stress responses. In conclusion, research applications focussing on mapping the immune landscape of lung adenocarcinoma benefit from scRNA-seq in fresh samples, whereas snRNA-seq of frozen samples provide a low-cost alternative to profile more epithelial and cancer cells, and yield cell type proportions that more closely match tissue content.
PubMed: 38814983
DOI: 10.1371/journal.pgen.1011301 -
ELife May 2024Silencing pathways prevent transposable element (TE) proliferation and help to maintain genome integrity through cell division. Silenced genomic regions can be...
Silencing pathways prevent transposable element (TE) proliferation and help to maintain genome integrity through cell division. Silenced genomic regions can be classified as either euchromatic or heterochromatic, and are targeted by genetically separable epigenetic pathways. In plants, the RNA-directed DNA methylation (RdDM) pathway targets mostly euchromatic regions, while CMT DNA methyltransferases are mainly associated with heterochromatin. However, many epigenetic features - including DNA methylation patterning - are largely indistinguishable between these regions, so how the functional separation is maintained is unclear. The linker histone H1 is preferentially localized to heterochromatin and has been proposed to restrict RdDM from encroachment. To test this hypothesis, we followed RdDM genomic localization in an mutant by performing ChIP-seq on the largest subunit, NRPE1, of the central RdDM polymerase, Pol V. Loss of H1 resulted in NRPE1 enrichment predominantly in heterochromatic TEs. Increased NRPE1 binding was associated with increased chromatin accessibility in , suggesting that H1 restricts NRPE1 occupancy by compacting chromatin. However, RdDM occupancy did not impact H1 localization, demonstrating that H1 hierarchically restricts RdDM positioning. H1 mutants experience major symmetric (CG and CHG) DNA methylation gains, and by generating an double mutant, we demonstrate these gains are largely independent of RdDM. However, loss of NRPE1 occupancy from a subset of euchromatic regions in corresponded to the loss of methylation in all sequence contexts, while at ectopically bound heterochromatic loci, NRPE1 deposition correlated with increased methylation specifically in the CHH context. Additionally, we found that H1 similarly restricts the occupancy of the methylation reader, SUVH1, and polycomb-mediated H3K27me3. Together, the results support a model whereby H1 helps maintain the exclusivity of heterochromatin by preventing encroachment from other competing pathways.
Topics: Heterochromatin; Euchromatin; DNA Methylation; Histones; Arabidopsis; Arabidopsis Proteins; Epigenesis, Genetic
PubMed: 38814684
DOI: 10.7554/eLife.89353 -
ELife May 2024Neurexins play diverse functions as presynaptic organizers in various glutamatergic and GABAergic synapses. However, it remains unknown whether and how neurexins are...
Neurexins play diverse functions as presynaptic organizers in various glutamatergic and GABAergic synapses. However, it remains unknown whether and how neurexins are involved in shaping functional properties of the glycinergic synapses, which mediate prominent inhibition in the brainstem and spinal cord. To address these issues, we examined the role of neurexins in a model glycinergic synapse between the principal neuron in the medial nucleus of the trapezoid body (MNTB) and the principal neuron in the lateral superior olive (LSO) in the auditory brainstem. Combining RNAscope with stereotactic injection of AAV-Cre in the MNTB of neurexin1/2/3 conditional triple knockout mice, we showed that MNTB neurons highly express all isoforms of neurexins although their expression levels vary remarkably. Selective ablation of all neurexins in MNTB neurons not only reduced the amplitude but also altered the kinetics of the glycinergic synaptic transmission at LSO neurons. The synaptic dysfunctions primarily resulted from an impaired Ca sensitivity of release and a loosened coupling between voltage-gated Ca channels and synaptic vesicles. Together, our current findings demonstrate that neurexins are essential in controlling the strength and temporal precision of the glycinergic synapse, which therefore corroborates the role of neurexins as key presynaptic organizers in all major types of fast chemical synapses.
Topics: Animals; Mice, Knockout; Glycine; Mice; Trapezoid Body; Synaptic Transmission; Neural Cell Adhesion Molecules; Superior Olivary Complex; Brain Stem; Synapses; Neurons; Cell Adhesion Molecules, Neuronal; Nerve Tissue Proteins; Neurexins; Calcium-Binding Proteins
PubMed: 38814174
DOI: 10.7554/eLife.94315 -
Arteriosclerosis, Thrombosis, and... May 2024Pericoronary epicardial adipose tissue (EAT) is a unique visceral fat depot that surrounds the adventitia of the coronary arteries without any anatomic barrier. Clinical...
BACKGROUND
Pericoronary epicardial adipose tissue (EAT) is a unique visceral fat depot that surrounds the adventitia of the coronary arteries without any anatomic barrier. Clinical studies have demonstrated the association between EAT volume and increased risks for coronary artery disease (CAD). However, the cellular and molecular mechanisms underlying the association remain elusive.
METHODS
We performed single-nucleus RNA sequencing on pericoronary EAT samples collected from 3 groups of subjects: patients undergoing coronary bypass surgery for severe CAD (n=8), patients with CAD with concomitant type 2 diabetes (n=8), and patients with valvular diseases but without concomitant CAD and type 2 diabetes as the control group (n=8). Comparative analyses were performed among groups, including cellular compositional analysis, cell type-resolved transcriptomic changes, gene coexpression network analysis, and intercellular communication analysis. Immunofluorescence staining was performed to confirm the presence of CAD-associated subclusters.
RESULTS
Unsupervised clustering of 73 386 nuclei identified 15 clusters, encompassing all known cell types in the adipose tissue. Distinct subpopulations were identified within primary cell types, including adipocytes, adipose stem and progenitor cells, and macrophages. macrophages and adipocytes were significantly expanded in CAD. In comparison to normal controls, both disease groups exhibited dysregulated pathways and altered secretome in the primary cell types. Nevertheless, minimal differences were noted between the disease groups in terms of cellular composition and transcriptome. In addition, our data highlight a potential interplay between dysregulated circadian clock and altered physiological functions in adipocytes of pericoronary EAT. ANXA1 and SEMA3B were identified as important adipokines potentially involved in functional changes of pericoronary EAT and CAD pathogenesis.
CONCLUSIONS
We built a complete single-nucleus transcriptomic atlas of human pericoronary EAT in normal and diseased conditions of CAD. Our study lays the foundation for developing novel therapeutic strategies for treating CAD by targeting and modifying pericoronary EAT functions.
PubMed: 38813696
DOI: 10.1161/ATVBAHA.124.320923 -
International Journal of Nanomedicine 2024As a major cause of low back pain, intervertebral disc degeneration is an increasingly prevalent chronic disease worldwide that leads to huge annual financial losses.... (Review)
Review
As a major cause of low back pain, intervertebral disc degeneration is an increasingly prevalent chronic disease worldwide that leads to huge annual financial losses. The intervertebral disc consists of the inner nucleus pulposus, outer annulus fibrosus, and sandwiched cartilage endplates. All these factors collectively participate in maintaining the structure and physiological functions of the disc. During the unavoidable degeneration stage, the degenerated discs are surrounded by a harsh microenvironment characterized by acidic, oxidative, inflammatory, and chaotic cytokine expression. Loss of stem cell markers, imbalance of the extracellular matrix, increase in inflammation, sensory hyperinnervation, and vascularization have been considered as the reasons for the progression of intervertebral disc degeneration. The current treatment approaches include conservative therapy and surgery, both of which have drawbacks. Novel stimuli-responsive delivery systems are more promising future therapeutic options than traditional treatments. By combining bioactive agents with specially designed hydrogels, scaffolds, microspheres, and nanoparticles, novel stimuli-responsive delivery systems can realize the targeted and sustained release of drugs, which can both reduce systematic adverse effects and maximize therapeutic efficacy. Trigger factors are categorized into internal (pH, reactive oxygen species, enzymes, etc.) and external stimuli (photo, ultrasound, magnetic, etc.) based on their intrinsic properties. This review systematically summarizes novel stimuli-responsive delivery systems for intervertebral disc degeneration, shedding new light on intervertebral disc therapy.
Topics: Humans; Intervertebral Disc Degeneration; Drug Delivery Systems; Animals; Nanoparticles; Hydrogels; Intervertebral Disc; Reactive Oxygen Species
PubMed: 38813390
DOI: 10.2147/IJN.S463939 -
Frontiers in Molecular Biosciences 2024Prohibitins are the central regulatory element of cellular homeostasis, especially by modulating the response at different levels: Nucleus, mitochondria and membranes.... (Review)
Review
Prohibitins are the central regulatory element of cellular homeostasis, especially by modulating the response at different levels: Nucleus, mitochondria and membranes. Their localization and interaction with various proteins, homons, transcription and nuclear factors, and mtDNA indicate the globality and complexity of their pleiotropic properties, which remain to be investigated. A more detailed deciphering of cellular metabolism in relation to prohibitins under normal conditions and in various metabolic diseases will allow us to understand the precise role of prohibitins in the signaling cascades of PI3K/Akt, Raf/MAP/ERK, STAT3, p53, and others and to fathom their mutual influence. A valuable research perspective is to investigate the role of prohibitins in the molecular and cellular interactions between the two major players in the pathogenesis of obesity-adipocytes and macrophages - that form the basis of the meta-inflammatory response. Investigating the subtle intercellular communication and molecular cascades triggered in these cells will allow us to propose new therapeutic strategies to eliminate persistent inflammation, taking into account novel molecular genetic approaches to activate/inactivate prohibitins.
PubMed: 38813101
DOI: 10.3389/fmolb.2024.1322687 -
Frontiers in Immunology 2024IL-2Rα knock out (KO) mice have been instrumental to discovering the immunoregulatory properties of IL-2Rα. While initially thought of only as a stimulatory cytokine,...
INTRODUCTION
IL-2Rα knock out (KO) mice have been instrumental to discovering the immunoregulatory properties of IL-2Rα. While initially thought of only as a stimulatory cytokine, IL-2 and IL-2Rα KO mice revealed that this cytokine-receptor system controls immune responses through restimulation-induced cell death and by promoting the survival of T regulatory cells. Although described mostly in the context of lymphocytes, recent studies by our laboratory showed that IL-2R is expressed in smooth muscle cells. Given this finding, we sought to use IL-2Rα KO to determine the function of this receptor in vascular smooth muscle cells. Surprisingly, we found that IL-2Rα KO vascular smooth muscle cells had detectable IL-2Rα.
METHODS
We used multiple gene and protein-based methods to determine why IL-2Rα KO vascular smooth muscle cells exhibited IL-2Rα protein. These methods included: genomic sequencing, assessing cells and tissues for evidence of maternal microchimerism, and determining the half-life of IL-2Rα protein.
RESULTS
Our studies demonstrated the following: (1) in addition to the cell surface, IL-2Rα is localized to the nucleus; (2) the genetic deletion of IL-2Rα is intact in IL-2Rα KO mice; (3) both IL-2Rα KO and WT tissues show evidence of maternal microchimerism, the likely source of IL-2Rα (4) IL-2Rα is transmitted between cells; (5) IL-2Rα has a long half-life; and (6) nuclear IL-2Rα contributes to the regulation of cell proliferation and size.
CONCLUSION
Our findings suggest that the phenotype of complete IL-2Rα loss is more severe than demonstrated by IL-2Rα KO mice, and that IL-2Rα plays a here-to-fore unrecognized role in regulating cell proliferation in non-lymphoid cells.
Topics: Animals; Mice, Knockout; Mice; Female; Interleukin-2 Receptor alpha Subunit; Cell Nucleus; Chimerism; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Mice, Inbred C57BL; Lymphocytes
PubMed: 38812502
DOI: 10.3389/fimmu.2024.1369818 -
Frontiers in Bioscience (Landmark... May 2024Gastric cancer (GC) is frequently diagnosed at advanced stages, when cancer cells have already metastasized. Therefore, patients with GC have a low survival rate and...
BACKGROUND
Gastric cancer (GC) is frequently diagnosed at advanced stages, when cancer cells have already metastasized. Therefore, patients with GC have a low survival rate and poor prognosis even after treatment.
METHODS
We downloaded GC-related RNA sequencing (RNA-Seq) data, copy number variation (CNV) data, and clinical data for bioinformatics analysis to screen prognostic genes of GC. Single-sample gene set enrichment analysis and survival analyses were performed on the RNA-Seq data, and differential and correlation analyses were conducted on the CNV data to obtain CNV-driven differentially expressed genes (DEGs). Prognostic genes were identified through univariate Cox analyses of the CNV-driven DEGs, combined with the clinical data. () was finally selected for verification after functional and survival analyses of the prognostic genes.
RESULTS
expression was lower in paracancer tissue than in GC tissue, and lower in GES-1 gastric epithelial cells than in GC cells. The cell culture supernatants from -knockdown GC cells were collected and used to culture human umbilical vein endothelial cells (HUVECs). It was observed that enhanced the activity, metastasis, invasion, and angiogenesis of GC cells; promoted the epithelial-mesenchymal transition (EMT) of GC cells; and impacted the Ras-associated protein 1 (Rap1)/mitogen-activated protein kinase (MAPK) pathway. To further explore the involvement of the Rap1/MAPK pathway in GC development, a pathway activator was added to GC cells with knockdown of expression. This pathway activator not only enhanced the activity, invasion, and migration of GC cells but also promoted the EMT and blood vessel formation.
CONCLUSIONS
regulates the angiogenesis and EMT of GC cells through the Rap1/MAPK pathway, thus influencing the onset and progression of GC.
Topics: Stomach Neoplasms; Humans; Epithelial-Mesenchymal Transition; Neovascularization, Pathologic; Cell Line, Tumor; Prognosis; Gene Expression Regulation, Neoplastic; MAP Kinase Signaling System; Human Umbilical Vein Endothelial Cells; Shelterin Complex; Male; Female; Telomere-Binding Proteins; DNA Copy Number Variations; Cell Movement; rap1 GTP-Binding Proteins; Angiogenesis
PubMed: 38812308
DOI: 10.31083/j.fbl2905177 -
Scientific Reports May 2024Epithelial tissue forms and maintains a critical barrier function in the body. A novel culture design aimed at promoting uniform maturation of epithelial cells using...
Epithelial tissue forms and maintains a critical barrier function in the body. A novel culture design aimed at promoting uniform maturation of epithelial cells using liquid materials is described. Culturing Madin-Darby canine kidney (MDCK) cells at the liquid-liquid interface yielded reduced migration and stimulated active cell growth. Similar to solid-liquid interfaces, cells cultured on a fibronectin-coated liquid-liquid interface exhibited active migration and growth, ultimately reaching a confluent state. These cells exhibited reduced stress fiber formation and adopted a cobblestone-like shape, which led to their even distribution in the culture vessel. To inhibit stress fiber formation and apoptosis, the exposure of cells on liquid-liquid interfaces to Y27632, a specific inhibitor of the Rho-associated protein kinase (ROCK), facilitated tight junction formation (frequency of ZO-2-positive cells, F = 0.73). In Y27632-exposed cells on the liquid-liquid interface, the value obtained by subtracting the standard deviation of the ratio of nucleus densities in each region that compartmentalized a culture vessel from 1, denoted as H, was 0.93 ± 0.01, indicated even cell distribution in the culture vessel at t = 72 h. The behavior of epithelial cells on liquid-liquid interfaces contributes to the promotion of their uniform maturation.
Topics: Dogs; Epithelial Cells; Animals; Madin Darby Canine Kidney Cells; Cell Movement; Tight Junctions; Cell Proliferation; Cell Culture Techniques; Amides; Pyridines; Apoptosis; rho-Associated Kinases; Stress Fibers; Cell Differentiation
PubMed: 38811617
DOI: 10.1038/s41598-024-63115-7 -
Nature Communications May 2024The mammalian SWI/SNF-like BAF complexes play critical roles during animal development and pathological conditions. Previous gene deletion studies and characterization...
The mammalian SWI/SNF-like BAF complexes play critical roles during animal development and pathological conditions. Previous gene deletion studies and characterization of human gene mutations implicate that the complexes both repress and activate a large number of genes. However, the direct function of the complexes in cells remains largely unclear due to the relatively long-term nature of gene deletion or natural mutation. Here we generate a mouse line by knocking in the auxin-inducible degron tag (AID) to the Smarca4 gene, which encodes BRG1, the essential ATPase subunit of the BAF complexes. We show that the tagged BRG1 can be efficiently depleted by osTIR1 expression and auxin treatment for 6 to 10 h in CD4 + T cells, hepatocytes, and fibroblasts isolated from the knock-in mice. The acute depletion of BRG1 leads to decreases in nascent RNAs and RNA polymerase II binding at a large number of genes, which are positively correlated with the loss of BRG1. Further, these changes are correlated with diminished accessibility at DNase I Hypersensitive Sites (DHSs) and p300 binding. The acute BRG1 depletion results in three major patterns of nucleosome shifts leading to narrower nucleosome spacing surrounding transcription factor motifs and at enhancers and transcription start sites (TSSs), which are correlated with loss of BRG1, decreased chromatin accessibility and decreased nascent RNAs. Acute depletion of BRG1 severely compromises the Trichostatin A (TSA) -induced histone acetylation, suggesting a substantial interplay between the chromatin remodeling activity of BRG1 and histone acetylation. Our data suggest BRG1 mainly plays a direct positive role in chromatin accessibility, RNAPII binding, and nascent RNA production by regulating nucleosome positioning and facilitating transcription factor binding to their target sites.
Topics: Animals; Transcription Factors; DNA Helicases; Nuclear Proteins; Mice; Nucleosomes; Indoleacetic Acids; RNA Polymerase II; Fibroblasts; Gene Knock-In Techniques; Hepatocytes; E1A-Associated p300 Protein; Transcriptional Activation; Transcription, Genetic; Histones; Deoxyribonuclease I; Chromatin; Humans
PubMed: 38811575
DOI: 10.1038/s41467-024-48911-z