Biology subjects

Liang, M.

Publications and source records attributed to Liang, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

In Vivo Tensor-Valued Diffusion MRI Reveals Isotropic-Anisotropic Kurtosis Mismatch in a Rat MCAO Stroke Model

BackgroundTensor-valued diffusion MRI (dMRI) enables the separation of total kurtosis into isotropic and anisotropic components, offering improved specificity over conventional Diffusion Kurtosis Imaging (DKI). In this study, we introduce a novel framework for detecting anisotropic-isotropic kurtosis mismatch and evaluate its utility in characterizing stroke lesions. MethodsWe performed tensor-valued dMRI in a rat model of middle cerebral artery occlusion (MCAO). Metrics including anisotropic kurtosis (MKA) and isotropic kurtosis (MKI) were quantified to assess microstructural tissue heterogeneity. Histological validation was conducted using co-registered tissue sections. ResultsWe observed significant mismatches between mean diffusivity (MD), mean kurtosis (MK), as well as specifically in MKA and MKI in ischemic regions. Notably, MKA and MKI exhibited statistically significant alterations compared to contralateral regions. Moreover, these metrics strongly correlated with pathological findings. The mismatch highlighted microstructurally distinct subregions within the lesion. ConclusionsThis is the first study to propose an anisotropic-isotropic kurtosis mismatch framework in vivo. Tensor-valued dMRI may offer a more specific imaging biomarker for stroke, with potential for improving lesion subtyping and guiding therapeutic decisions.

biophysics↗

bioRxiv2025

Inferring non-coding RNA regulatory network from transcriptomic data and curated databases

Non-coding RNAs (ncRNAs) have played an indispensable role in regulating gene expression and cellular processes. However, the complex regulatory circuits between ncRNAs and coding genes remain understudied. Major challenges for inferring ncRNA regulatory network (NRN) on a transcriptome-wide scale include high dimensionality of both ncRNA and coding gene expression data, their context-dependent interaction, and lack of validation. We hereby propose a comprehensive analytical framework, namely Construction and Analysis of non-coding RNA regulatory NETwork (CAR-NET), specifically designed to infer NRN from transcriptomic data and curated databases on experimentally validated ncRNA-gene interactions and disease-related ncRNAs. At its core is a novel computationally efficient Bayesian network structure learning method that leverages the semi-bipartite graph structure of NRN for dimension reduction, edge orientation and global network search. We also developed and applied algorithms to perform differential network, subnetwork and pathway analysis of the inferred network. We showed the strength of CAR-NET in simulations and three case studies, encompassing lncRNA regulation in brain development, miRNA differential regulation in renal cell carcinoma and regulation by different classes of ncRNAs in different cell lines using bulk or single cell RNA-seq data. An R-shiny app (https://github.com/kehongjie/CAR-NET) was developed to implement the method(s) with interactive GUI.

bioinformatics↗

bioRxiv2025

Single cell mapping of the metabolic landscape of skin fibrosis in systemic sclerosis

AbstractTissue resident cells undergo metabolic reprogramming during fibrotic tissue remodeling to meet their changing metabolic demands required for extracellular matrix production and phenotypic transitions in fibrosis. However, the metabolic reprogramming in fibrotic tissues has not yet been explored at single cell level with spatial resolution. Moreover, the spatial organization of metabolic niches in fibrotic tissues remains understudied. To address these gaps, we used imaging mass cytometry (IMC) and characterized the metabolic regulome, indicative of the activity in several key metabolic pathways in systemic sclerosis (SSc) as a prototypic systemic fibrotic disease. We identified a distinct metabolically active profile with high activity of glycolysis, TCA/OXPHOS, hypoxia and ROS signaling in fibroblasts, endothelial cells and macrophages in SSc patients with progressive skin fibrosis. These metabolic profiles are associated with expression of markers of profibrotic activation. Metabolically active fibroblasts might shape their microenvironment to induce a similar metabolic phenotype in neighboring endothelial cells and macrophages, facilitating profibrotic interactions. Consistently, specific interactions between metabolically defined, activated cell subsets are associated with the extent or progression of skin fibrosis. Thus, interfering with these metabolic niches might provide therapeutic opportunities in fibrotic diseases.

cell biology↗

bioRxiv2025

Directed Chemical Evolution via Navigating Molecular Encoding Space

Deep-learning techniques have significantly advanced small-molecule drug discovery. However, a critical gap remains between representation learning and small molecule generations, limiting their effectiveness in developing new drugs. We introduce Ouroboros, a unified framework that integrates molecular representation learning with generative modeling, enabling efficient chemical space exploration using pre-trained molecular encodings. By reframing molecular generation as a process of encoding space compression and decompression, Ouroboros resolves the challenges associated with iterative molecular optimization and facilitates directed chemical evolution within the encoding space. Comprehensive experimental tests demonstrate that Ouroboros significantly outperforms conventional approaches across multiple drug discovery tasks, including ligand-based virtual screening, chemical property prediction, and multi-target inhibitor design and optimization. Unlike task-specific models in traditional approaches, Ouroboros leverages a unified framework to achieve superior performance across diverse applications. Ouroboros offers a novel and highly scalable protocol for rapid chemical space exploration, fostering a potential paradigm shift in AI-driven drug discovery.

bioinformatics↗

bioRxiv2025

Mapping spatially-resolved transcriptomes in systemic sclerosis

Systemic sclerosis (SSc) is a prototypical fibrotic disease with high mortality and limited treatment options. Despite advances in single-cell RNA sequencing (scRNA-seq), the comprehensive understanding of cellular heterogeneity and cell-cell interaction within the fibrogenesis microenvironment remains limited. We generated spatially resolved transcriptome maps from healthy and SSc skin and built a scRNA-seq atlas to map the single-cell data to spatial space. This enabled us to identify a fibrotic niche, enriched with fibroblasts and macrophages, which is significantly expanded in SSc and correlated with clinical outcome. We revealed disease-specific cell states of fibroblasts and macrophages, and evaluated their spatial dependency on other cell types. We identified selective expression of ACKR3 in fibroblast progenitors that diminishes with SSc progression, which may serve to regulate CXCL12/CXCR4-mediated macrophage recruitment and fibrotic remodeling. Together, we provided an in-depth description at cellular and spatial levels of fine-tuned regulatory events occurring in SSc, offering spatiotemporal insights. One Sentence SummaryIntegrated spatial omics provide insight into the cellular and transcriptional landscape in spatially distinct microenvironments, which may drive fibrosis progression in SSc.

immunology↗

bioRxiv2025

From spots to stripes: Evolution of pigmentation patterns in monkeyflowers via modulation of a reaction-diffusion system and its prepatterns

The reaction-diffusion (RD) system is widely assumed to account for many complex, self- organized pigmentation patterns in natural organisms. However, the specific configurations of such RD networks and how RD systems interact with positional information (i.e., prepatterns) that may specify the initiation conditions for the RD operation remain largely unknown. Here, we introduced a three-substance RD system underlying the formation of repetitive pigment spots and stripes in Mimulus flowers. It consists of an R2R3-MYB activator (NEGAN), an R3-MYB inhibitor (RTO), and a coactivator represented by two paralogous bHLH proteins. Through fine- scale genetic analyses, transgenic experiments, and computer simulations, we identified the causal loci contributing to the evolutionary transition from sparsely dispersed spots to longitudinal stripes. Genetic changes at these loci modulate the prepatterns of the activator and coactivator expression and the promoter activities of the inhibitor and one of the coactivator paralogs. Our findings highlight the importance of prepatterns towards a realistic description of RD systems in natural organisms, and reveal the genetic mechanism generating pattern variation through modulation of the kinetics of the RD system and its prepatterns.

developmental biology↗

bioRxiv2024

Spatially informed phenotyping by cyclic-in-situ-hybridization identifies novel fibroblast populations and their pathogenic niches in systemic sclerosis

Spatially non-resolved transcriptomic data identified functionally distinct populations of fibroblasts in health and disease. However, in-depth transcriptional profiling in situ at single-cell resolution has not been possible so far. Here, we studied fibroblast populations in the skin of SSc patients and healthy individuals using cyclic in situ hybridization (cISH) as a novel approach for spatially-resolved transcriptional phenotyping with subcellular resolution. cISH deconvoluted the heterogeneity of 20,979 cells including 3,764 fibroblasts (FB). BANKSY-based spatially-informed clustering identified nine FB subpopulations, with SFRP2+ RetD FB and CCL19+ nonPV FB as novel subpopulations that reside in specific cellular niches and display unique gene expression profiles. SFRP2+ RetD FB and CCL19+ nonPV FB as well as COL8A1+ FB, display altered frequencies in SSc skin and play specific, disease-promoting roles for extracellular matrix release and leukocyte recruitment as revealed by their transcriptional profile, their cellular interactions and ligand-receptor analyses. The frequencies of COL8A1+ FB and their interactions with monocytic cells and B cells are associated with progression of skin fibrosis in SSc. In summary, our spatially-resolved transcriptomic approach identified novel fibroblast subpopulations deregulated in SSc skin with specific pathogenic roles, some of which may potentially serve as biomarkers for progression of skin fibrosis.

immunology↗

bioRxiv2024

Global and genetic regulation of gene expression in human endothelial and vascular smooth muscle cells

BackgroundThe understanding of genetic and epigenetic regulation of gene expression in endothelial and vascular smooth muscle cells remains fragmented with limited experimental validation. MethodsChromatin conformation (Micro-C), DNA methylation (RRBS), chromatin accessibility (ATAC-seq), and transcriptome profiles (RNA-seq) were mapped in human induced pluripotent stem cell (hiPSC)-derived, isogenic endothelial and vascular smooth muscle cells (iECs and iVSMCs). CTCF and RAD21 were depleted to assess the functional relevance of chromatin architecture, and genome editing was used to evaluate the allelic effect of a blood pressure-associated single nucleotide polymorphism (SNP). ResultsSignificant correlations were identified between gene expression levels and chromatin interactions, chromatin accessibility, and DNA methylation in iECs and iVSMCs, with chromatin interactions showing the strongest association. Chromatin contact regions displayed distinct epigenetic landscapes depending on the types of regulatory element interactions involved. Perturbation of CTCF and RAD21 revealed their differential regulatory effects, particularly on the expression of genes overlapping chromatin contacts, with RAD21 exhibiting a broader regulatory impact. SNPs associated with several vascular traits were enriched in chromatin loops or accessible regions in iECs or iVSMCs. Precise genome editing demonstrated allele-dependent effects of SNP rs9833313 on the expression of SHOX2 located 247.4 kbp away but within the same chromatin loops as the SNP. ConclusionThis study provides an extensive epigenetic landscape of vascular cells that may drive novel research on the role of genetic and epigenetic mechanisms of vascular function and disease as demonstrated by our targeted experiments.

genomics↗

bioRxiv2024

A single-cell map of hypertension

Hypertension is a leading risk factor for disease burden and death worldwide. Several organ systems are involved in the development of hypertension, which contributes to stroke, heart disease, and kidney disease. Despite the broad health relevance, our understanding of the molecular landscape in hypertension is limited and lags other major diseases. Here we report an extensive analysis of the molecular landscape in hypertension and its end-organ damage and uncover novel mechanisms linking human genetic variants to the development of these diseases. We obtained single-nucleus RNA-seq (612,984 nuclei), single-nucleus ATAC-seq (179,637 nuclei), or spatial transcriptome data from five organs (hypothalamus, kidney, heart, 3rd order mesenteric artery, middle cerebral artery) in three mouse and rat models under twelve experimental conditions. More than one third of all hypertension research in animal models involves these three models. We identified both model-specific and convergent responses in cell types, genes, and pathways. By integrating our data with human genomic data, we partitioned the blood pressure and end-organ damage traits into cell type-specific transcriptional contributions and cell types common across multiple traits. Using genomic editing in animal models and human induced pluripotent stem cells, we extended key findings and identified new mechanisms linking human genetic variants to the development of hypertension and related renal injury. We anticipate that our rich data sets and findings will broadly drive forward the research of hypertension and hypertensive end-organ damage. Our approach of integrating multi-model and multi-tissue single-cell analysis with human genetic data and in vivo and in vitro genome editing can be applied to investigate other complex traits.

physiology↗

bioRxiv2024

C9orf72 expansion creates the unstable folate-sensitive fragile site FRA9A

The hyper-unstable Chr9p21 locus, harbouring the interferon gene cluster, oncogenes and C9orf72, is linked to multiple diseases. C9orf72 (GGGGCC)n expansions (C9orf72Exp) are associated with incompletely penetrant amyotrophic lateral sclerosis, frontotemporal dementia and autoimmune disorders. C9orf72Exp patients display hyperactive cGAS-STING-linked interferon immune and DNA damage responses, but the source of immuno-stimulatory or damaged DNA is unknown. Here, we show C9orf72Exp in pre-symptomatic and ALS-FTD patient cells and brains cause the folate- sensitive chromosomal fragile site, FRA9A. FRA9A centers on >33kb of C9orf72 as highly-compacted chromatin embedded in an 8.2Mb fragility zone spanning 9p21, encompassing 46 genes, making FRA9A one of the largest fragile sites. C9orf72Exp cells show chromosomal instability, heightened global- and Chr9p-enriched sister-chromatid exchanges, truncated-Chr9s, acentric-Chr9s and Chr9-containing micronuclei, providing endogenous sources of damaged and immunostimulatory DNA. Cells from one C9orf72Exp patient contained highly-rearranged FRA9A-expressing Chr9 with Chr9-wide dysregulated gene expression. Somatic C9orf72Exp repeat instability and chromosomal fragility are sensitive to folate-deficiency. Age-dependent repeat instability, chromosomal fragility, and chromosomal instability can be transferred to CNS and peripheral tissues of transgenic C9orf72Exp mice, implicating C9orf72Exp as the source. Our results highlight unappreciated effects of C9orf72 expansions that trigger vitamin-sensitive chromosome fragility, adding structural variations to the disease-enriched 9p21 locus, and likely elsewhere. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/620312v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@154b23borg.highwire.dtl.DTLVardef@1814a6dorg.highwire.dtl.DTLVardef@13d713eorg.highwire.dtl.DTLVardef@d5d994_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2024

Chromatin interaction maps of human arterioles reveal new mechanisms for the genetic regulation of blood pressure

Arterioles are small blood vessels located just upstream of capillaries in nearly all tissues. The constriction and dilation of arterioles regulate tissue perfusion and are primary determinants of systemic blood pressure (BP). Abnormalities in arterioles are central to the development of major diseases such as hypertension, stroke, and microvascular complications of diabetes. Despite the broad and essential role of arterioles in physiology and disease, current knowledge of the functional genomics of arterioles is largely absent, partly because it is challenging to obtain and analyze human arteriole samples. Here, we report extensive maps of chromatin interactions, single-cell expression, and other molecular features in human arterioles and uncover new mechanisms linking human genetic variants to gene expression in vascular cells and the development of hypertension. Compared to large arteries, arterioles exhibited a higher proportion of pericytes which were strongly associated with BP traits. BP-associated single nucleotide polymorphisms (SNPs) were enriched in chromatin interaction regions in arterioles, particularly through enhancer SNP-promoter interactions, which were further linked to gene expression specificity across tissue components and cell types. Using genomic editing in animal models and human induced pluripotent stem cells, we discovered novel mechanisms linking BP-associated noncoding SNP rs1882961 to gene expression through long-range chromatin contacts and revealed remarkable effects of a 4-bp noncoding genomic segment on hypertension in vivo. We anticipate that our rich data and findings will advance the study of the numerous diseases involving arterioles. Moreover, our approach of integrating chromatin interaction mapping in trait-relevant tissues with SNP analysis and in vivo and in vitro genome editing can be applied broadly to bridge the critical gap between genetic discoveries and physiological understanding.

genomics↗

bioRxiv2024

Liprin-α/RIM complex regulates the dynamic assembly of presynaptic active zones via liquid-liquid phase separation

Presynaptic scaffold proteins, including liprin-, RIM, and ELKS, are pivotal to the assembly of the active zone and regulating the coupling of calcium signals and neurotransmitter release, yet the underlying mechanism remains poorly understood. Here, we determined the crystal structure of the liprin-2/RIM1 complex, revealing a multifaceted intermolecular interaction that drives the liprin-/RIM assembly. Neurodevelopmental disease-associated mutations block the formation of the complex. Disrupting this interaction in neurons impairs synaptic transmission and reduces the readily releasable pool of synaptic vesicles. Super-resolution imaging analysis supports a role for liprin- in recruiting RIM1 to the active zone, presumably by promoting the liquid-liquid phase separation (LLPS) of RIM1. Strikingly, the liprin-/RIM interaction modulates the competitive distribution of ELKS1 and voltage-gated calcium channels (VGCCs) in RIM1 condensates. Disrupting the liprin-/RIM interaction significantly decreased VGCC accumulation in the condensed phase and rendered release more sensitive to the slow calcium buffer EGTA, suggesting an increased physical distance between VGCC and vesicular calcium sensors. Together, our findings provide a plausible mechanism of the liprin-/RIM complex in regulating the coupling of calcium channels and primed synaptic vesicles via LLPS for efficient synaptic transmission and uncover the pathological implication of liprin- mutations in neurodevelopmental disorders.

neuroscience↗

bioRxiv2024

Tim-3 promotes viral infection by suppressing USP25-TRAF3-IRF7 signaling pathway

Tim-3, an immune checkpoint inhibitor, plays key roles in maintaining immune homeostasis and is involved in viral evasion. However, the precise role of Tim-3 in viral infection remains to be determined. USP25 is one of the deubiquitinating enzymes that serves to initiate antiviral immunity by deubiquitinating TRAF3 and triggering the anti-viral signaling pathway. Here we found that Tim-3 specific knockout in myeloid cells leads to enhanced anti-viral immunity in mice with VSV encephalitis by increasing type I interferon response. Mechanistically, Tim-3 inhibits the expression of USP25 via STAT1, interacts with USP25 but does not regulate its post-translational modification, and as a result, Tim-3 inhibits USP25 mediated deubiquitination of TRAF3, promotes k48-linked ubiquitination and degradation of TRAF3, and then inhibits the phosphorylation of IRF7, finally down-regulates the interferon response. These findings provide new insights into the function of Tim-3 in antiviral immunity and its related clinical significance.

immunology↗

bioRxiv2024

Inhibition of Endothelial Lipase by MEDI5884 Normalizes Phosphatidylinositol Levels in Coronary Artery Disease Patients

BackgroundEndothelial lipase (EL) promotes high-density lipoproteins (HDL) phospholipid degradation, increases catabolism of HDL and is an attractive target for the potential treatment for cardiovascular disease. Inhibition of EL using a monoclonal neutralizing antibody, MEDI5884, demonstrated increased quantity and function of HDL. Determinants of anti-atherosclerotic function of HDL comprise the interplay of various components of HDL structure-activity relationship: size, shape and composition (lipid and protein). Previous studies have shown that single doses of MEDI5884 administered to healthy nonhuman primates (NHPs) and healthy subjects resulted in a dose- dependent increase in plasma phospholipids (PL) and that plasma PI levels in placebo treated healthy subjects are significantly increased relative to CAD subjects participating in clinical trials NCT03001297 and NCT03351738, respectively. MethodsHerein, we characterized using LC-MS/MS the plasma lipidome of NHPs, heathy subjects and subjects with coronary artery disease (CAD) following MEDI5884 administration. ResultsMEDI5884 treated NHPs resulted in a prominent increase in phosphatidylinositols (PI) and cholesteryl esters (CE). Treatment with MEDI5884 restores near-normal levels of PI in CAD patients. PI increases in both healthy subjects and CAD patients were dose-dependent, correlated with exposure and saturated at approximately 200 mg MEDI5884 subcutaneous (SC) dose in CAD patients. Comparison of pharmacodynamic (PD) effects of repeat SC 200 mg doses of MEDI5884 in CAD patients revealed greater and more rapid increases in PI levels compared to HDL-C and HDL phospholipid (HDL-PL). The increase in PI species was inversely correlated with decreases in free EL mass levels. ConclusionsPI has previously been shown to possess anti-atherosclerotic properties and led to increases in HDL cholesterol (HDL-C) and reverse cholesterol transport (RCT). The mechanism by which CE levels increase as the result of MEDI5884 administration can be attributed to the observed increase in both substrates of the lecithin-cholesterol acyltransferase (LCAT) reaction: phosphatidylcholine/phosphatidylethanolamine (PC/PE) and cholesterol as the consequence of EL inhibition. Further characterization of the underlying biological mechanisms responsible for the decrease of the PI biomarker in CAD patient population relative to healthy subjects as well as in conjunction with pharmacological intervention by MEDI5884 may reveal more information on this clinically-relevant biomarker and potential role in CAD.

pharmacology and toxicology↗

bioRxiv2024

Physiological role and mechanisms of action for a long noncoding haplotype region

Most common sequence variants associated with human traits are in noncoding regions of the genome, form haplotypes with other noncoding variants, and exhibit small effect sizes in the general population. Determining the physiological roles and mechanisms of action for these noncoding variants, particularly large haplotypes containing multiple variants, is both critical and challenging. To address this challenge, we developed an approach that integrates physiological studies in genetically engineered and phenotypically permissive animal models, precise editing of large haplotypes in human induced pluripotent stem cells (hiPSCs), and targeted chromatin conformation analysis. We applied this approach to examine the blood pressure associated rs1173771 locus, which includes a haplotype containing 11 single nucleotide polymorphisms (SNPs) spanning 17.4 kbp. Deleting the orthologous noncoding region in the genome of the Dahl salt-sensitive rat attenuated the salt-induced increase in systolic blood pressure by nearly 10 mmHg. This attenuation of hypertension appeared to be mediated by upregulation of the adjacent gene Npr3 (natriuretic peptide receptor 3) in arteries, enhancing vasodilation. The blood pressure-elevating and -lowering haplotypes were precisely reconstituted in hiPSCs using an efficient, two-step genome editing technique. The blood pressure-elevating haplotype decreased NPR3 expression in endothelial cells and vascular smooth muscle cells derived from the edited, isogenic hiPSCs. The influence of the haplotype was partially recapitulated by the sentinel SNP rs1173771. Additionally, the blood pressure-elevating haplotype showed significantly greater chromatin interactions with the NPR3 promoter region. This study illustrates the feasibility of ascertaining the physiological roles and mechanisms of action for large noncoding haplotypes. Our efficient, integrated, and targeted approach can be applied to investigate other noncoding variants.

genetics↗

bioRxiv2024

Ferroptosis mediates the progression of hyperuricemic nephropathy by activating RAGE signaling

Hyperuricemic nephropathy (HN) represents a prevalent complication arising from hyperuricemia, typified by tubular dysfunction, inflammation, and progressive renal fibrosis, whose pathogenic mechanisms remain enigmatic. Ferroptosis, a newly elucidated iron-dependent form of regulated cell death, plays a role in various disease states. However, its involvement in HN has seldom been explored. In this study, we observed indications of ferroptosis in the renal tissues of urate oxidase knockout (UOX-/-) mice, a model of hyperuricemia, as evidenced by increased iron deposition and reduced expression of glutathione peroxidase 4 (GPX4). These findings suggest a substantial role of ferroptosis in the pathogenesis of HN. To further explore this hypothesis, UOX-/- mice were administered Ferrostatin-1, a known inhibitor ferroptosis. This treatment markedly ameliorated tubular injury, necrosis, and inflammatory cell infiltration, mitigated renal fibrosis, reinstated the expression of proteins associated with ferroptosis in renal tissues, and reduced iron overload, lipid peroxidation, and mitochondrial damage in UOX-/- mice. Additionally, we found that receptor for advanced glycation end products (RAGE) propagates ferroptosis-induced renal injury, inflammation and fibrosis, albeit without directly facilitating ferroptosis. Finally, ferroptosis and RAGE upregulation were validated in renal tissues of patients with hyperuricemia-related kidney disease. Collectively, our research elucidates the critical contribution of ferroptosis to HN pathogenesis, indicating that therapeutic strategies targeting ferroptosis and the related RAGE signaling may offer novel therapeutic approaches for managing this condition.

ecology↗

bioRxiv2024

Exosomal miRNA-124 is involved in the immune regulation of cervical cancer by regulating CD45 alternative splicing

ObjectiveTo investigate the regulatory mechanism of miRNA-124 in tumor immune regulation of cervical cancer. MethodsPeripheral blood samples of cervical cancer patients and transient infection controls were collected to extract exosomal miR-124 and Stem-loop Q-PCR to detect the expression level of miR-124. QPCR, WB, and flow cytometry were used to detect the mRNA and protein expression of Th cell differentiation and function-related genes. Transfection, QPCR, and flow cytometry were used to detect the effects of miR-124 overexpression and silencing on the differentiation of Th immune memory cells. In vitro, cell-killing experiments were performed to detect the tumor-killing activity of TH immune memory cells co-cultured with the Hela cell line, and the downstream target genes of miR-124 were predicted and verified by luciferase. Multiplex PCR was used to detect the genotype of miR-124 SNP in peripheral blood DNA. QPCR and WB were used to detect the mRNA and protein expression and phosphorylation of miR-124 target gene GSK3-PSF-CD45 pathway, and GSK3 inhibitor was used to block the pathway. ResultsWe found that exosomal miRNA-124 was significantly downregulated in cervical cancer tissues and affected Th cell immune function and memory cell generation. The overexpression of miRNA-124 can directly target GSK3, phosphorylate the target gene, and inhibit the expression of GSK3, thereby increasing the expression of CD45, increasing the ability of Th immune cells to recognize and kill the HPV virus, and significantly inhibiting the proliferation and migration of cervical cancer cells.

molecular biology↗

bioRxiv2024

Atomic resolution map of the solvent interactions driving SOD1 unfolding in CAPRIN1 condensates

Biomolecules can be sequestered into membrane-less compartments, referred to as biomolecular condensates. Experimental and computational methods have helped define the physical-chemical properties of condensates. Less is known about how the high macromolecule concentrations in condensed phases contribute "solvent" interactions that can remodel the free-energy landscape of other condensate-resident proteins, altering thermally accessible conformations and, in turn, modulating function. Here, we use solution Nuclear Magnetic Resonance (NMR) spectroscopy to obtain atomic resolution insights into the interactions between the immature form of superoxide dismutase 1 (SOD1), which can mislocalize and aggregate in stress granules, and the RNA-binding protein CAPRIN1, a component of stress granules. NMR studies of CAPRIN1:SOD1, focused on both unfolded and folded SOD1 states in mixed phase and de-mixed CAPRIN1-based condensates, establish that CAPRIN1 shifts the folding equilibrium of SOD1 towards the unfolded state through preferential interactions with the unfolded ensemble, with little change to the structure of the folded conformation. Key contacts between CAPRIN1 and the H80-H120 region of unfolded SOD1 are identified, as well as SOD1 interaction sites near both the arginine-rich and aromatic-rich regions of CAPRIN1. Unfolding of immature SOD1 in the CAPRIN1 condensed phase is shown to be coupled to aggregation, while a more stable zinc-bound, dimeric form of SOD1 is less susceptible to unfolding when solvated by CAPRIN1. Our work underscores the impact of the condensate solvent environment on the conformational states of resident proteins and supports the hypothesis that ALS mutations that decrease metal binding or dimerization function as drivers of aggregation in condensates. Significance StatementBiomolecular condensates concentrate proteins and nucleic acids to regulate and perform key biological functions. Although the material properties of these condensates are well-studied, much less is understood about how the structure and dynamics of proteins within them are affected by the high concentration of biomolecules. In this study we have used NMR spectroscopy to study how the folding equilibrium and structural dynamics of the ALS protein SOD1 are modulated inside a condensate formed by CAPRIN1. Our study reveals that the CAPRIN1 condensed phase biases an immature form of SOD1 towards unfolded states that are susceptible to aggregation and provides insights into why this is the case, while a more mature form of the protein is much less affected.

biophysics↗