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Megalencephalic leukoencephalopathy with subcortical cyst (MLC) is a rare leukodystrophy primarily caused by mutations in two genes: MLC1, encoding a membrane protein of unknown function, and GlialCAM, a cell adhesion molecule. Although MLC1 has been implicated in downregulating signaling pathways, its molecular mechanisms remain elusive. Recently, the orphan G-protein-coupled receptor GPRC5B was identified as a novel interactor of both GlialCAM and MLC1, with dominant heterozygous mutations found in MLC patients, suggesting that GlialCAM and MLC1 may regulate cell signaling via GPRC5B. Here, we show that GPRC5B exhibits constitutive activity, which is inhibited by MLC1, likely through interference with GPRC5B oligomerization. Conversely, GlialCAM enhances β-arrestin 2 recruitment, leading to its own mislocalization from cell-cell junctions. MLC-associated GPRC5B mutants show enhanced maturation and increased stability at the plasma membrane, retain normal constitutive activity and responsiveness to MLC1 and GlialCAM but display increased affinity for GlialCAM and localize to cell-cell junctions in its presence. Notably, coexpression of GlialCAM with these mutants does not induce GlialCAM mislocalization. We propose a model in which finely tuned interactions among GPRC5B, GlialCAM, and MLC1 regulate receptor signaling. These findings provide the first biochemical evidence of GlialCAM and MLC1 modulating GPRC5B activity, suggesting a biochemical explanation for the gain-of-function phenotype observed in GPRC5B MLC mutants. Importantly, our work supports the potential of targeting GPRC5B as a therapeutic strategy in MLC. Show less

Atherosclerosis is driven by chronic inflammation of the vascular wall, in which macrophages play a central role. The orphan G protein-coupled receptor GPRC5B is expressed in vascular cells and macrophages and is upregulated during monocyte-to-macrophage differentiation. It has been shown to activate NFκB-dependent inflammatory pathways in adipose tissue and glomeruli. Here, we investigated the impact of GPRC5B on macrophage infiltration and the progression of atherosclerotic plaque development in vivo. Bone marrow from heterozygous GPRC5B-transgenic C57BL/6 mice and wild-type controls was transplanted into lethally irradiated LDL receptor-deficient mice. Animals were fed a Western-type diet for 16 weeks, after which atherosclerotic lesions in the aortic sinus were analyzed. Mice receiving GPRC5B-transgenic bone marrow showed no significant differences in serum lipids or cardiac mass indices. However, they exhibited significantly increased macrophage infiltration within atherosclerotic plaques and a non-significant trend toward larger and more complex lesions. GPRC5B overexpression in bone marrow-derived monocyte/macrophage lineage cells promotes a more inflammatory plaque phenotype, characterized by enhanced macrophage infiltration. These findings highlight GPRC5B as a potential modulator of plaque progression and suggest it may represent a novel therapeutic target in vascular inflammation and atherosclerosis. Show less

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare inherited white matter disorder. Initially, a "classic" phenotype has been characterized, presenting early-onset macrocephaly, cerebellar ataxia, mild spasticity, and a distinctive neuroimaging pattern of diffuse white matter abnormalities with subcortical cysts. An "improving" phenotype has also been described, featuring milder or absent neurological signs and a remitting pattern on neuroimaging. Mutations in four genes, MLC1, HEPACAM, GPRC5B and AQP4 have been associated with MLC. We describe clinical and genetic features of a cohort of genetically confirmed Italian MLC patients, representing the largest Italian cohort reported to date. We conducted a retrospective, multicenter, observational study. Patients were included based on clinical and neuroimaging features consistent with MLC, along with a confirmed genetic diagnosis. Data were collected using a standardized database and included demographic, clinical, neuroimaging, neurophysiological, and genetic information. Thirty-three patients from eight Italian centers were enrolled. Twenty-seven harbored biallelic MLC1 variants (23 distinct mutations, including three novel variants), while six had three distinct heterozygous HEPACAM variants. All MLC1-mutated patients exhibited the "classic" phenotype, frequently accompanied by orthopedic, gastrointestinal, and respiratory comorbidities. HEPACAM-mutated patients were consistent with the "improving" phenotype. No patients harbored mutations in GPRC5B or AQP4. Our findings expand the mutational spectrum of MLC1, further characterize the disease phenotype, and provide valuable insights into its presence in Italy. They also underscore management needs of individuals with MLC, highlighting the importance of multidisciplinary care. Show less

Metabolic syndrome (MetS) is a recognized risk factor for prostate cancer (PCa), yet the precise biological mechanisms driving this association remain poorly understood. Unraveling these molecular pathways is essential for developing targeted interventions to improve patient outcomes. In this study, we analyzed NHANES (2005-2014) data to examine associations between MetS and PCa outcomes, finding that MetS was significantly associated with higher PCa risk (OR = 1.52), all-cause mortality (HR = 1.53), and cancer-specific mortality (HR = 2.17). Through integrated multi-omics, weighted gene co-expression network analysis, and machine learning, we identified the orphan receptor GPRC5B as a critical hub gene downregulated in both conditions. Single-cell transcriptomic analysis further confirmed that GPRC5B is predominantly expressed in endothelial cells. Mechanistically, GPRC5B loss was found to hyperactivate p38 MAPK signaling through a specific dual mechanism: increasing phosphorylation of upstream MKK3/6 kinases while concurrently suppressing the negative feedback phosphatase DUSP1. This synergistic dysregulation drove enhanced endothelial proliferation, migration, and tube formation in vitro. In vivo, endothelial GPRC5B deficiency significantly accelerated tumor growth and neovascularization, phenotypes that were effectively reversed by the p38 inhibitor SB202190. Clinical specimens corroborated reduced GPRC5B expression and increased microvessel density in MetS-associated PCa. Collectively, our findings establish endothelial GPRC5B downregulation as a key molecular driver promoting pathological angiogenesis via the MKK3/6-DUSP1-p38 axis, suggesting that targeting this signaling cascade offers a promising therapeutic strategy for managing MetS-associated PCa aggression. Show less

This study aimed to identify breast cancer-specific circulating tumor DNA (ctDNA) methylation markers that correspond to tissue DNA methylation. Using The Cancer Genome Atlas (TCGA) database, we selected breast cancer-specific DNA methylation markers. The methylation and expression patterns of candidate genes were analyzed in breast cancer cell lines and tissue samples. We also assessed the methylation status in ctDNA obtained from breast cancer patients and examined associations with the clinicopathological features. Among candidate genes with breast cancer-specific methylation patterns, USP44, ZNF454, and GPRC5B were selected. The methylation status and expression of selected genes varied by molecular subtype of cancer in the cell line. In tissue samples, expression of all three genes was generally lower in breast cancer than in controls. ctDNA methylation patterns showed no significant change before and after treatment for each candidate gene. Correlations between gene expression and DNA methylation status or clinicopathological characteristics in cancer tissues differed among genes. Further studies are needed for clinical application of liquid biopsy using methylation analysis for ctDNA according to individual characteristics for breast cancer. Show less

Volume-regulated anion channels (VRACs) are central to cell volume homeostasis. They mediate swelling-induced efflux of chloride and organic osmolytes to drive regulatory volume decrease. In the brain, VRACs have been proposed to play a key role in astrocytic volume regulation. Genetic defects in astrocytic VRAC modulating proteins (MLC1, GlialCAM, Aquaporin-4, GPRC5B) cause the leukodystrophy Megalencephalic leukoencephalopathy with subcortical cysts (MLC), characterized by chronic white matter edema and myelin vacuolization. Disrupted VRAC activity in MLC-patient-derived lymphoblasts and primary astrocytes from MLC mice further supports a pathogenic link between defective VRAC activity and MLC. Here, we studied the physiological and pathological consequences of astrocyte-specific removal of the essential VRAC subunit LRRC8A. In contrast to established MLC mouse models, astrocyte specific Lrrc8a knockout mice had normal brain water content, no myelin vacuolization, and preserved expression of MLC-related proteins. At a late age they developed a mildly ataxic gait and displayed increased severity of kainate-induced seizures. Two-photon imaging in acute brain slices revealed that astrocytes lacking LRRC8A show normal volume recovery and chloride dynamics upon high potassium-induced cell swelling. Together, these findings demonstrate that astrocyte LRRC8A is not essential for volume regulation in situ and that its loss alone is insufficient to cause the chronic white matter edema typical of MLC. The mild neurological deficits indicate a physiological role for astrocyte LRRC8A, but MLC pathology likely arises from broader dysregulation of the astrocytic protein complex coordinating ion and water homeostasis. Show less

Osteosarcoma, the most common primary malignant bone tumor with poor prognosis, underscores the need for a deeper understanding of its molecular mechanisms. Recent studies have highlighted the importance of RNA modifications, including 5-methylcytosine (m5C), in cancer progression, yet the m5C modification landscape in osteosarcoma remains unexplored. Here, we performed transcriptome-wide profiling of m5C modifications in osteosarcoma using meRIP-seq and RNA-seq, analyzing four pairs of osteosarcoma and adjacent normal tissues. Furthermore, through conjunction analyses of meRIP-seq and RNA-seq data, we identified 637 genes with significant changes in both the m5C modification and mRNA levels. Among these, GPRC5B emerged as a key prognostic gene, with its high expression and m5C hypermethylation significantly associated with poor survival in osteosarcoma patients. Functional experiments demonstrated that GPRC5B suppresses apoptosis and promotes osteosarcoma cell proliferation and migration. Mechanistically, NSUN2-mediated m5C modification upregulates GPRC5B expression, and the anti-apoptotic effects of NSUN2 are primarily dependent on its ability to modulate GPRC5B m5C modification and expression. Knockdown of GPRC5B partially rescues the anti-apoptotic effects of NSUN2, highlighting the critical role of GPRC5B in osteosarcoma survival. Our study identified an m5C-dependent NSUN2-GPRC5B regulatory axis, providing insights into osteosarcoma progression and revealing its therapeutic potential. Show less

GATA family transcription factors are somatically variable (SV) in esophageal adenocarcinomas (EAC) and inducible by simulated reflux. Our study examines the mechanisms whereby GATA family members (GATA4, GATA6, and the atypical TRPS-1) influence oncogenesis during the Barrett's esophagus (BE) metaplasia-dysplasia transition preceding EAC. RNAseq analyses of esophageal cell lines and lesion-derived adult stem cells (ASCs) in conjunction shRNA- or CRISPR-facilitated gene silencing, together with reanalysis of The Cancer Genome Atlas data, spatial transcriptomics, and organ-on-a-chip studies were used. Although a gastroesophageal reflux disease history positively correlated with GATA4/6 somatically variable and a columnar-associated gene signature (ANPEP/GATA4) in The Cancer Genome Atlas EAC cases, it negatively associated with a squamous lineage-linked signature (TP63/SOX15) containing TRPS1. In experimental data, opposing effects on regulators of squamous and columnar lineage identity were uncovered between TRPS1 and classical GATA factors (GATA4/6). Interrogation of this GATA "fulcrum" defined further genes (CGN, IL6R, and GPRC5B) targeted for TRPS1-mediated suppression or GATA4/6 activation. A novel spatial transcriptomic signature of BE-associated high-grade dysplasia (HGD) captured GATA fulcrum action, through GPRC5B expression. Functionally, GPRC5B was found to be low-pH-responsive, to increase proliferative and colony formation rates, and when overexpressed facilitate a hyperproliferative HGD-like transformation of BE-ASCs. Using an organ-on-a-chip platform, cellular overgrowth, reduced luminal villus structures, lower goblet cell numbers, and loss of intestine-associated marker gene expression (TFF3/MUC2) were observed following GPRC5B overexpression in BE-ASCs, mirroring HGD. This study identifies critical GATA factor-mediated processes underlying cellular phenotype in the BE-HGD-EAC transition and identifies GATA-inducible GPRC5B as a functional marker and possible driver of progression through HGD to EAC. Show less

Melatonin (MLT) can improve mitophagy, thereby ameliorating cognitive deficits in Alzheimer's disease (AD) patients. Hence, our research focused on the potential value of MLT-related genes (MRGs) in AD through bioinformatic analysis. First, the key cells in the single-cell dataset GSE138852 were screened out based on the proportion of annotated cells and Fisher's test between the AD and control groups. The differentially expressed genes (DEGs) in the key cell and GSE5281 datasets were identified, and the MRGs in GSE5281 were selected via weighted gene coexpression network analysis. After intersecting two sets of DEGs and MRGs, we performed Mendelian randomization analysis to identify the MRGs causally related to AD. Biomarkers were further ascertained through receiver operating characteristic curve (ROC) and expression analysis in GSE5281 and GSE48350. Furthermore, gene set enrichment analysis, immune infiltration analysis and correlation analysis with metabolic pathways were conducted, as well as construction of a regulator network and molecular docking. According to the Fisher test, oligodendrocytes were regarded as key cells due to their excellent abundance in the GSE138852 dataset, in which there were 281 DEGs between the AD and control groups. After overlapping with 3,490 DEGs and 550 MRGs in GSE5281, four genes were found to be causally related to AD, namely, G protein-coupled receptor, family C, group 5, member B (GPRC5B), Methyltransferase-like protein 7 A (METTL7A), NF-κB inhibitor alpha (NFKBIA) and RAS association domain family 4(RASSF4). Moreover, GPRC5B, NFKBIA and RASSF4 were deemed biomarkers, except for METTL7A, because of their indistinctive expression between the AD and control groups. Biomarkers might be involved in oxidative phosphorylation, adipogenesis and heme metabolism. Moreover, T helper type 17 cells, natural killer cells and CD56dim natural killer cells were significantly correlated with biomarkers. Transcription factors (GATA2, POU2F2, NFKB1, etc.) can regulate the expression of biomarkers. Finally, we discovered that all biomarkers could bind to MLT with a strong binding energy. Our study identified three novel biomarkers related to MLT for AD, namely, GPRC5B, NFKBIA and RASSF4, providing a novel approach for the investigation and treatment of AD patients. Show less

Understudied orphan GPCRs lack identified natural ligands, yet understanding their function is critical for therapeutic development. GPRC5B is a brain-enriched, retinoic acid (RA)-induced orphan GPCR. While RA is used to treat severe acne, chronic exposure is associated with depression, likely due to its inhibition of adult hippocampal neurogenesis. Here, we tested whether GPRC5B plays a role in the molecular and cellular mechanisms underlying RA-induced depressive-like behaviors in mice by suppressing adult hippocampal neurogenesis. We found that Gprc5b knockout (KO) mice were resilient to RA-induced behavioral effects and that RA increased GPRC5B expression in the hippocampal neurogenic subgranular zone. This correlated with RA-mediated inhibition of adult hippocampal neurogenesis, an effect absent in Gprc5b KO mice, which also exhibited a larger pool of proliferative neuronal stem cells. Collectively, these findings suggest that GPRC5B mediates RA-induced anti-neurogenic effects and depressive-like behaviors. Show less

In vitro studies have implicated orphan receptor GPRC5B in β cell survival, proliferation, and insulin secretion, but its relevance for glucose homeostasis in vivo is largely unknown. Using tamoxifen-inducible, β cell-specific GPRC5B-KO mice (Ins-G5b-KOs), we show here that loss of GPRC5B does not affect β cell function in the lean state but results in strongly reduced insulin secretion and disturbed glucose tolerance in mice subjected to high-fat diet for 16 weeks. Flow cytometry and single-cell expression analyses in islets from obese mice show a reduced β cell abundance and a less mature β cell phenotype in Ins-G5b-KOs. Expression of β cell-specific transcription factor MafA is reduced both on the RNA and protein level, as are transcripts of MafA target genes. Mechanistically, we show that phosphorylation of cAMP response element-binding protein (CREB), a major regulator of MafA expression, is reduced in islets of obese Ins-G5b-KOs, and we show that this phenotype precedes the downregulation of MafA and MafA target genes. Taken together, GPRC5B helps to maintain mature β cell function in obesity through cAMP/CREB-dependent regulation of MafA expression. Show less

Macrophages express numerous G protein-coupled receptors (GPCRs) that regulate adhesion, migration, and activation, but the function of orphan receptor GPRC5B in macrophages is unknown. Both resident peritoneal and bone marrow-derived macrophages from myeloid-specific GPRC5B-deficient mice show increased migration and phagocytosis, resulting in improved bacterial clearance in a peritonitis model. In other models such as myocardial infarction, increased myeloid cell recruitment has adverse effects. Mechanistically, we found that GPRC5B physically interacts with GPCRs of the prostanoid receptor family, resulting in enhanced signaling through the prostaglandin E receptor 2 (EP2). In GPRC5B-deficient macrophages, EP2-mediated anti-inflammatory effects are diminished, resulting in hyperactivity. Using in silico modelling and docking, we identify residues potentially mediating GPRC5B/EP2 dimerization and show that their mutation results in loss of GPRC5B-mediated facilitation of EP2 signaling. Finally, we demonstrate that decoy peptides mimicking the interacting sequence are able to reduce GPRC5B-mediated facilitation of EP2-induced cAMP signaling in macrophages. Show less

The paper is aimed to screen the target molecules of miR-12 and to further explore the mechanism of GAS5 action in prostate cancer. The expression of GPRC5B in prostate cancer cell lines LNCaP, VCaP, 22RV1, DU145, and PC3 was measured by quantitative real-time PCR with reverse transcription (RT-qPCR) and variations in GPRC5B expression were analyzed after down-regulating GAS5 or silencing miR-12. CCK8 and plate clone experiments were performed to detect changes in proliferative activity and colony-forming capacity of prostate cancer cells after down-regulating GPRC5B. After transfection of prostate cancer cells with sh-GAS5 and/or miR-12 inhibitor, the changes in GPRC5B expression were evaluated with RT-qPCR and Western blotting. Our results showed that GPRC5B was highly expressed in prostate cancer cell lines. Down-regulating of GAS5 decreased GPRC5B expression, while silencing miR-12 increased it. CCK8 and plate clone experiments showed that expression of GPRC5B increased proliferative activity and clone formation ability of prostate cancer cells. RT-qPCR and Western blotting revealed that miR-12 inhibited the promoting effect of GAS5 on GPRC5B expression. Thus, GPRC5B is directly bound to miR-12. GAS5 promotes proliferation, migration, and invasion of prostate cancer cells and participates in malignant progression of tumors by suppressing miR-12-mediated regulation of GPRC5B expression. Show less

Studies verified that sphingosine kinase 1 (SPHK1)/sphingosine 1-phosphate receptors (S1PRs) and platelet-derived growth factor receptors (PDGFRs) play important roles in tumor occurrence and progression. However, the expression and clinical value of SPHK1/S1PRs and PDGFRs in colon adenocarcinoma (COAD) remains unclear. This study aimed to explore the expression of SPHK1/S1PRs and PDGFRs in COAD and further investigate their roles in predicting the prognosis of patients with COAD. SPHK1/S1PRs and PDGFRs expression in tissues from patient with COAD were analyzed using The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Kaplan-Meier survival analysis was used to evaluate the prognostic roles of SPHK1/S1PRs and PDGFRs in patients with COAD. Spearman's correlation analysis was performed to assess the relationship between SPHK1/S1PRs and PDGFRs in COAD. Then, χ2 test was performed to analyze the correlation between SPHK1/S1PR3/PDGFRB and clinicopathological characteristics of the patients. Additionally, possible signaling pathways co-regulated by S1PR3 and PDGFRB were predicted using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses. Least absolute shrinkage and selection operator (LASSO) regression was used to identify hub genes that co-regulated S1PR3 and PDGFRB expression. A prognostic model based on hub genes was constructed for patients with COPD. Finally, the relationship between the hub genes and tumor immune cell infiltration was investigated. The expression levels of SPHK1 and PDGFRB were significantly upregulated in COAD patient tissues (P < 0.001 and P < 0.001, respectively). Moreover, Kaplan-Meier analysis showed that patients with COAD with high expression levels of SPHK1 and S1PR3 had shorter overall survival (OS) than those with low expression levels (P = 0.013 and P = 0.005, respectively). Spearman's correlation analysis verified a strong positive correlation (P < 0.001, r = 0.790) between the expression of S1PR3 and PDGFRB. In addition, we found that high SPHK1 and PDGGRB expression levels were associated with perineural invasion (P < 0.001 and P = 0.011, respectively). High expression of S1PR3 and PDGGRB was prominently associated with N stage (P = 0.002 and P = 0.021, respectively). High levels of SPHK1, S1PR3, and PDGFRB were associated with lymph node invasion. (P = 0.018, P = 0.004, and P = 0.001, respectively). GO and KEGG results revealed that S1PR3 and PDGFRB may participate in COAD cell extracellular matrix organization and cellular signal transduction. Five hub genes, SFRP2, GPRC5B, RSPO3, FGF14, and TCF7L1, were identified using LASSO regression. Survival analysis showed that the OS in the high-risk group was remarkably shorter than that in the low-risk group. The results indicated that tumor immune cells were significantly increased in the high-risk group compared to those in the low-risk group. S1PR3 and PDGFRB may be important markers for predicting lymphatic metastasis and poor prognosis in patients with COAD. The underlying mechanisms may involve immune cell infiltration. Show less

Plumage color is a key trait for identifying waterfowl breeds with significant economic importance. A white-feathered group has recently emerged within the native Matahu duck population, presenting an opportunity for breeding new lines. However, the genetic basis for this plumage variation is still unknown, necessitating further research. This study aims to identify the genetic mechanisms underlying the emergence of white-feathered individuals in the Matahu duck population through combined genome and transcriptome analysis, providing insights for selective breeding and the development of new white-feathered lines. In this study, a total of 1344 selected genes and 1406 significantly differentially expressed genes were identified through selection signal analysis and transcriptomic analysis, respectively. The functional enrichment of these genes revealed several key signaling pathways, including those related to cGMP-PKG, cAMP, PI3K-Akt, and MAPK. Furthermore, important candidate genes involved in melanin biosynthesis, such as Show less

Pterygium is a frequent eye surface condition that is characterized by a high rate of proliferation, fibrovascular development, cellular migration, corneal infiltration, and angiogenesis. We investigated that ex vivo primary pterygium and conjunctival cell cultures were generated to analyze the effect of trehalose on cellular proliferation. After trehalose treatment, we performed microarray analysis to evaluate changes in the mRNA profile. We analyzed gene ontology (GO) and KEGG pathways to identify hub genes that changed expression levels after treatment and were associated with pterygium development. We selected three genes to verify their expression levels using qRT-PCR. The study also evaluated the impact of trehalose treatment on cell migration through a wound-healing assay. Our results suggested that pterygium cell proliferation was inhibited in a dose-dependent manner by trehalose. 2354 DEG were identified in pterygium and conjunctiva cells treated with trehalose compared to untreated groups. Functional enrichment analysis showed that differentially expressed mRNAs are involved in proliferation, vasculature development, and cell migration. We identified ten hub genes including upregulated (RANBP3L, SLC5A3, RERG, ANKRD1, DHCR7, RAB27B, GPRC5B, MSMO1, ASPN, DRAM1) and downregulated (TNC, PTGS2, GREM2, NPTX1, NR4A1, HMOX1, CXCL12, IL6, MYH2, TXNIP). Microarray analysis and functional investigations suggest that trehalose affects the pathogenesis of pterygium by modifying the expression of genes involved in crucial pathways related to cell function. Show less

We aimed to explore the potential link of maternal age at menarche (mAAM) gene polymorphisms with risk of the fetal growth restriction (FGR). This case (FGR)-control (FGR free) study included 904 women (273 FGR and 631 control) in the third trimester of gestation examined/treated in the Departments of Obstetrics. For single nucleotide polymorphism (SNP) multiplex genotyping, 50 candidate loci of mAAM were chosen. The relationship of mAAM SNPs and FGR was appreciated by regression procedures (logistic/model-based multifactor dimensionality reduction [MB-MDR]) with subsequent in silico assessment of the assumed functionality pithy of FGR-related loci. Three mAAM-appertain loci were FGR-linked to genes such as Show less

The leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC) is characterized by infantile-onset macrocephaly and chronic edema of the brain white matter. With delayed onset, patients typically experience motor problems, epilepsy and slow cognitive decline. No treatment is available. Classic MLC is caused by bi-allelic recessive pathogenic variants in Show less

Stroke embolic source have an unknown origin in 30-40% of cases. Mechanical thrombectomy for acute large vessel occlusion stroke has provided us with a method to directly retrieve the thrombi from patients for analysis. By collecting stroke-causing thrombi from known sources, we can then use high-throughput RNA sequencing (RNAseq) technology to directly measure the gene expression signatures of these clots. This may allow us to identify genetic markers to predict the cause of cryptogenic embolism. This is a prospective study in which RNAseq was used to analyze cerebral thrombi retrieved by mechanical thrombectomy devices in acute ischemic stroke patients. Samples were separated into two groups based on known stroke thrombus etiology, including Carotid group (patients with ipsilateral >70% carotid stenosis) and Atrial fibrillation (AF) group (patients with atrial fibrillation). Gene expression was compared by RNAseq analysis between the groups. From October 2016 to September 2017, 8 thrombi (4 in Carotid group, 4 in Afib group) were included in this study. There were 131 genes that were significantly up- or down-regulated between the two groups defined as a false discovery rate ≤ 0.05 and a fold change ≥ 2. Twenty-six genes were selected as candidate gene biomarkers based on the criteria in the methods section. Candidate genes HSPA1B, which encodes a heatshock protein, and GPRC5B, which encodes a G-protein, showed the greatest fold differences in expression between the two groups. This study has shown that RNA sequencing of acute ischemic stroke thrombi is feasible and indentified potential novel biomarkers for identifying stroke-causing thrombi origin, especially in cryptogenic stroke. Show less

G protein-coupled receptor class C group 5 member B (GPRC5B) is involved in extracellular glucose sensing, glucose metabolism, and insulin resistance. Many cancers require glucose at high concentrations to survive and grow. We have investigated the association between tumour GPRC5B expression and the prognosis for patients with cancer, including head-and-neck squamous cell carcinoma (HNSCC), using data from The Human Protein Atlas. The 5-year survival rate was significantly reduced in patients with HNSCC, gastric, pancreatic, colorectal, and breast cancers if their tumours exhibited high levels of GPRC5B expression. The role of GPRC5B in glucose metabolism was assessed using six HNSCC cell lines with varying levels of GPRC5B expression. High levels of GPRC5B expression were found to favour rapid cell growth. The viability of an HNSCC cell line with normal and transfected GPRC5B expression was also assessed and no differences were observed under standard culture conditions. However, under glucose-deficient culture conditions, GPRC5B-overexpressing cells exhibited increased viability and reduced apoptosis. The results highlight the association between high GPRC5B expression and poor 5-year survival rates in patients with various cancers, including HNSCC. Furthermore, we have demonstrated that GPRC5B supports cancer cell survival under glucose-depleted conditions and could be a target molecule for cancer therapy. Show less

The epithelial-mesenchymal transition (EMT) is an important process during metastasis in various tumors, including colorectal cancer (CRC). Thus, the study of its characteristics and related genes is of great significance for CRC treatment. In this study, 26 EMT-related gene sets were used to score each sample from The Cancer Genome Atlas program (TCGA) colon adenocarcinoma (COAD) database. Based on the 26 EMT enrichment scores for each sample, we performed unsupervised cluster analysis and classified the TCGA-COAD samples into three EMT clusters. Then, weighted gene co-expression network analysis (WGCNA) was used to investigate the gene modules that were significantly associated with these three EMT clusters. Two gene modules that were strongly positively correlated with the EMT cluster 2 (worst prognosis) were subjected to Cox regression and least absolute shrinkage and selection operator (LASSO) regression analysis. Then, a prognosis-related risk model composed of three hub genes Show less

This study aimed to identify the potential urine biomarkers of vascular dementia (VD) and unravel the disease-associated mechanisms by applying Liquid chromatography tandem-mass spectrometry (LC-MS/MS). LC-MS/MS proteomic analysis was applied to urine samples from 3 groups, including 14 patients with VD, 9 patients with AD, and 21 normal controls (NC). By searching the MS data by Proteome Discoverer software, analyzing the protein abundances qualitatively and quantitatively, comparing between groups, combining bioinformatics analysis using Gene Ontology (GO) and pathway crosstalk analysis using Kyoto Encyclopedia of Genes and Genomes (KEGG), and literature searching, the differentially expressed proteins (DEPs) of VD can be comprehensively determined at last and were further quantified by receiver operating characteristic (ROC) curve methods. The proteomic findings showed quantitative changes in patients with VD compared to patients with NC and AD groups; among 4,699 identified urine proteins, 939 and 1,147 proteins displayed quantitative changes unique to VD vs. NC and AD, respectively, including 484 overlapped common DEPs. Then, 10 unique proteins named in KEGG database (including PLOD3, SDCBP, SRC, GPRC5B, TSG101/STP22/VPS23, THY1/CD90, PLCD, CDH16, NARS/asnS, AGRN) were confirmed by a ROC curve method. Our results suggested that urine proteins enable detection of VD from AD and VC, which may provide an opportunity for intervention. Show less

Brain oedema is a life-threatening complication of various neurological conditions. Understanding molecular mechanisms of brain volume regulation is critical for therapy development. Unique insight comes from monogenic diseases characterized by chronic brain oedema, of which megalencephalic leukoencephalopathy with subcortical cysts (MLC) is the prototype. Variants in MLC1 or GLIALCAM, encoding proteins involved in astrocyte volume regulation, are the main causes of MLC. In some patients, the genetic cause remains unknown. We performed genetic studies to identify novel gene variants in MLC patients, diagnosed by clinical and MRI features, without MLC1 or GLIALCAM variants. We determined subcellular localization of the related novel proteins in cells and in human brain tissue. We investigated functional consequences of the newly identified variants on volume regulation pathways using cell volume measurements, biochemical analysis and electrophysiology. We identified a novel homozygous variant in AQP4, encoding the water channel aquaporin-4, in two siblings, and two de novo heterozygous variants in GPRC5B, encoding the orphan G protein-coupled receptor GPRC5B, in three unrelated patients. The AQP4 variant disrupts membrane localization and thereby channel function. GPRC5B, like MLC1, GlialCAM and aquaporin-4, is expressed in astrocyte endfeet in human brain. Cell volume regulation is disrupted in GPRC5B patient-derived lymphoblasts. GPRC5B functionally interacts with ion channels involved in astrocyte volume regulation. In conclusion, we identify aquaporin-4 and GPRC5B as old and new players in genetic brain oedema. Our findings shed light on the protein complex involved in astrocyte volume regulation and identify GPRC5B as novel potentially druggable target for treating brain oedema. Show less

GPRC5A is the first member of a new class of orphan receptors coupled to G proteins, which also includes GPRC5B, GPRC5C, and GPRC5D. Since its cloning and identification in the 1990s, substantial progress has been made in understanding the possible functions of this receptor. Show less

Osteoarthritis (OA) is one of the most common chronic diseases in the world. However, current treatment modalities mainly relieve pain and inhibit cartilage degradation, but do not promote cartilage regeneration. In this study, we show that G protein-coupled receptor class C group 5 member B (GPRC5B), an orphan G-protein-couple receptor, not only inhibits cartilage degradation, but also increases cartilage regeneration and thereby is protective against OA. We observed that Show less

Colostrum performs nutritional, anti-inflammatory and anti-infective functions and promotes immune system formation and organ development. The new coronavirus, SARS-CoV-2, has generated concerns about viral transmission through human milk, with a lack of evidence about human milk's protective effects against the infection. This study aimed at analyzing presence of the virus and at identifying the protein expression profile of human colostrum in active and COVID-19-recovered patients. Colostrum samples were collected from women with COVID-19 ( Show less

Aldosterone-producing adenoma (APA) is a common cause of secondary hypertension. This study aimed to explore the lncRNA-miRNA-mRNA competitive endogenous RNA (ceRNA) network to uncover molecular mechanism underlying APA. The mRNA and lncRNA expression data of APA and adjacent adrenal gland (AAG) from GSE60044, GSE64957 and GSE101894 were obtained from the Gene Expression Omnibus (GEO) database to analyse differentially expressed genes (DEGs) and lncRNAs (DElncs). Hub genes were identified by robust rank aggregation (RRA) and protein-protein interaction (PPI) network analysis. The miRcode and miRWalk network tools were used to construct the ceRNA network. 1526 upregulated and 1512 downregulated DEGs were identified, which are mainly enriched in extracellular matrix and Ca Show less

Nanodiscs are emerging to serve as transfer vectors for the insertion of recombinant membrane proteins into membranes of living cells. In combination with cell-free expression technologies, this novel process opens new perspectives to analyze the effects of even problematic targets such as toxic, hard-to-express, or artificially modified membrane proteins in complex cellular environments of different cell lines. Furthermore, transferred cells must not be genetically engineered and primary cell lines or cancer cells could be implemented as well. We have systematically analyzed the basic parameters of the nanotransfer approach and compared the transfer efficiencies from nanodiscs with that from Salipro particles. The transfer of five membrane proteins was analyzed: the prokaryotic proton pump proteorhodopsin, the human class A family G-protein coupled receptors for endothelin type B, prostacyclin, free fatty acids type 2, and the orphan GPRC5B receptor as a class C family member. The membrane proteins were cell-free synthesized with a detergent-free strategy by their cotranslational insertion into preformed nanoparticles containing defined lipid environments. The purified membrane protein/nanoparticles were then incubated with mammalian cells. We demonstrate that nanodiscs disassemble and only lipids and membrane proteins, not the scaffold protein, are transferred into cell membranes. The process is detectable within minutes, independent of the nanoparticle lipid composition, and the transfer efficiency directly correlates with the membrane protein concentration in the transfer mixture and with the incubation time. Transferred membrane proteins insert in both orientations, N-terminus in and N-terminus out, in the cell membrane, and the ratio can be modulated by engineering. The viability of cells is not notably affected by the transfer procedure, and transferred membrane proteins stay detectable in the cell membrane for up to 3 days. Transferred G-protein coupled receptors retained their functionality in the cell environment as shown by ligand binding, induction of internalization, and specific protein interactions. In comparison to transfection, the cellular membrane protein concentration is better controllable and more uniformly distributed within the analyzed cell population. A further notable difference to transfection is the accumulation of transferred membrane proteins in clusters, presumably determined by microdomain structures in the cell membranes. Show less