Protein-coupled Receptor Signaling Pathways Research Articles

Time-course study of genetic changes in periodontal ligament regeneration after tooth replantation in a mouse model

This research focused on analyzing gene expression changes in the periodontal ligament (PDL) after tooth re-plantation to identify key genes and pathways involved in healing and regeneration. Utilizing a mouse model, mRNA was extracted from the PDL at various intervals post-replantation for RNA sequencing analysis, spanning from 3 to 56 days. The results revealed significant shifts in gene expression, particularly notable on day 28, supported by hierarchical clustering and principal component analysis. Gene ontology (GO) enrichment analysis highlighted an upregulation in olfactory receptor and G protein-coupled receptor signaling pathways at this time point. These findings were validated through reverse transcription-quantitative PCR (RT-qPCR), with immunochemical staining localizing olfactory receptor gene expression to the PDL and surrounding tissues. Moreover, a scratch assay indicated that olfactory receptor genes might facilitate wound healing in human PDL fibroblasts. These results underscore the importance of the 28-day post-transplant phase as a potential “tipping point” in PDL healing and regeneration. In conclusion, this research sheds light on the potential role of olfactory receptor genes in PDL regeneration, providing a foundation for developing new therapeutic approaches in tooth replantation and transplantation, with broader implications for regenerative medicine in oral health.

Time-course study of genetic changes in periodontal ligament regeneration after tooth replantation in a mouse model

This research focused on analyzing gene expression changes in the periodontal ligament (PDL) after tooth re-plantation to identify key genes and pathways involved in healing and regeneration. Utilizing a mouse model, mRNA was extracted from the PDL at various intervals post-replantation for RNA sequencing analysis, spanning from 3 to 56 days. The results revealed significant shifts in gene expression, particularly notable on day 28, supported by hierarchical clustering and principal component analysis. Gene ontology (GO) enrichment analysis highlighted an upregulation in olfactory receptor and G protein-coupled receptor signaling pathways at this time point. These findings were validated through reverse transcription-quantitative PCR (RT-qPCR), with immunochemical staining localizing olfactory receptor gene expression to the PDL and surrounding tissues. Moreover, a scratch assay indicated that olfactory receptor genes might facilitate wound healing in human PDL fibroblasts. These results underscore the importance of the 28-day post-transplant phase as a potential “tipping point” in PDL healing and regeneration. In conclusion, this research sheds light on the potential role of olfactory receptor genes in PDL regeneration, providing a foundation for developing new therapeutic approaches in tooth replantation and transplantation, with broader implications for regenerative medicine in oral health.

Unraveling the Roles of Neuropeptides in the Chemosensation of the Root-Knot Nematode Meloidogyne javanica.

The identification of novel drug targets in plant-parasitic nematodes (PPNs) is imperative due to the loss of traditional nematicides and a lack of replacements. Chemosensation, which is pivotal for PPNs in locating host roots, has become a focus in nematode behavioral research. However, its underlying molecular basis is still indistinct in such a diverse group of PPNs. To characterize genes participating in chemosensation in the Javanese root-knot nematode Meloidogyne javanica, RNA-sequencing of the second-stage juveniles (J2s) treated with tomato root exudate (TRE) for 1 h and 6 h was performed. Genes related to chemosensation in M. javanica mainly responded to TRE treatment at 1 h. Moreover, a gene ontology (GO) analysis underscored the significance of the neuropeptide G protein-coupled receptor signaling pathway. Consequently, the repertoire of putative neuropeptides in M. javanica, including FMRFamide-like peptides (FLPs), insulin-like peptides (ILPs), and neuropeptide-like peptides (NLPs), were outlined based on a homology analysis. The gene Mjflp-14a, harboring two neuropeptides, was significantly up-regulated at 1 h TRE treatment. Through peptide synthesis and J2 treatment, one of the two neuropeptides (MjFLP-14-2) was proven to influence the J2 chemotaxis towards tomato root tips. Overall, our study reinforces the potential of nematode neuropeptides as novel targets and tools for root-knot nematode control.

Molecular Mechanism of Pasteurized Akkermansia muciniphila in Alleviating Type 2 Diabetes Symptoms.

Type 2 diabetes (T2DM) significantly diminishes people's quality of life and imposes a substantial economic burden. This pathological progression is intimately linked with specific gut microbiota, such as Akkermansia muciniphila. Pasteurized A. muciniphila (P-AKK) has been defined as a novel food by the European Food Safety Authority and exhibited significant hypoglycemic activity. However, current research on the hypoglycemic activity of P-AKK is limited to the metabolic level, neglecting systematic exploration at the pathological level. Consequently, its material basis and mechanism of action for hypoglycemia remain unclear. Drawing upon this foundation, we utilized high-temperature killed A. muciniphila (H-K-AKK) with insignificant hypoglycemic activity as the control research object. Assessments were conducted at pathological levels to evaluate the hypoglycemic functions of both P-AKK and H-K-AKK separately. Our study unveiled for the first time that P-AKK ameliorated symptoms of T2DM by enhancing the generation of glucagon-Like Peptide 1 (GLP-1), with pasteurized A. muciniphila total proteins (PP) being a pivotal component responsible for this activity. Utilizing SDS-PAGE, proteomics, and molecular docking techniques, we deeply analyzed the material foundation of PP. We scientifically screened and identified a protein weighing 77.85 kDa, designated as P5. P5 enhanced GLP-1 synthesis and secretion by activating the G protein-coupled receptor (GPCR) signaling pathway, with free fatty acid receptor 2 (FFAR-2) being identified as the pivotal target protein for P5's physiological activity. These findings further promote the widespread application of P-AKK in the food industry, laying a solid theoretical foundation for its utilization as a beneficial food ingredient or functional component.

Triclocarban and triclosan promote breast cancer progression in vitro and in vivo via activating G protein-coupled estrogen receptor signaling pathways

Triclocarban (TCC) and triclosan (TCS) have been detected ubiquitously in human body and evoked increasing concerns. This study aimed to reveal the induction risks of TCC and TCS on triple negative breast cancer through non-genomic GPER-mediated signaling pathways. Molecular simulation indicated that TCC exhibited higher GPER binding affinity than TCS theoretically. Calcium mobilization assay displayed that TCC/TCS activated GPER signaling pathway with the lowest observed effective concentrations (LOEC) of 10 nM/100 nM. TCC and TCS also upregulated MMP-2/9, EGFR, MAPK3 but downregulated MAPK8 via GPER-mediated signaling pathway. Proliferation assay showed that TCC/TCS induced 4 T1 breast cancer cells proliferation with the LOEC of 100 nM/1000 nM. Wound-healing and transwell assays showed that TCC/TCS promoted 4 T1 cells migration in a concentration-dependent manner with the LOEC of 10 nM. The effects of TCC on breast cancer cells proliferation and migration were stronger than TCS and both were regulated by GPER. TCC/TCS induced migratory effects were more significantly than proliferative effect. Mechanism study showed that TCC/TCS downregulated the expression of epithelial marker (E-cadherin) but upregulated mesenchymal markers (snail and N-cadherin), which was reversed by GPER inhibitor G15. These biomarkers results indicated that TCC/TCS-induced 4 T1 cells migration was a classic epithelial to mesenchymal transition mechanism regulated by GPER signaling pathway. Orthotopic tumor model verified that TCC promoted breast cancer in-situ tumor growth and distal tissue metastasis via GPER-mediated signaling pathway at human-exposure level of 10 mg/kg/d. TCC-induced tissue metastasis of breast cancer was more significantly than in-situ tumor growth. Overall, we demonstrated for the first time that TCC/TCS could activate the GPER signaling pathways to induce breast cancer progression.

The Identification of a Novel Nucleomodulin MbovP467 of Mycoplasmopsis bovis and Its Potential Contribution in Pathogenesis.

Mycoplasmopsis bovis is a causative agent of crucial diseases in both dairy and beef cattle leading to substantial economic losses. However, limited control measures for M. bovis-related diseases exist due to a lack of understanding about the virulence factors of this pathogen, a common challenge in mycoplasma research. Consequently, this study aimed to characterize a novel nucleomodulin as a virulence-related factor of M. bovis. Employing bioinformatic tools, we initially predicted MbovP467 to be a secreted protein with a nuclear localization signal based on SignalP scores and the cNLS (Nuclear Localization Signal) Mapper, respectively. Subsequently, the MbovP467 gene was synthesized and cloned into a pEGFP plasmid with EGFP labeling to obtain a recombinant plasmid (rpEGFP-MbovP467) and then was also cloned in pET-30a with a consideration for an Escherichia coli codon bias and expressed and purified for the production of polyclonal antibodies against the recombinant MbovP467 protein. Confocal microscopy and a Western blotting assay confirmed the nuclear location of MbovP467 in bovine macrophages (BoMacs). RNA-seq data revealed 220 up-regulated and 20 down-regulated genes in the rpEGFP-MbovP467-treated BoMac group compared to the control group (pEGFP). A GO- and KEGG-enrichment analysis identified associations with inflammatory responses, G protein-coupled receptor signaling pathways, nuclear receptor activity, sequence-specific DNA binding, the regulation of cell proliferation, IL-8, apoptotic processes, cell growth and death, the TNF signaling pathway, the NF-κB signaling pathway, pathways in cancer, and protein families of signaling and cellular processes among the differentially expressed up-regulated mRNAs. Further experiments, investigating cell viability and the inflammatory response, demonstrated that MbovP467 reduces BoMac cell viability and induces the mRNA expression of IL-1β, IL-6, IL-8, TNF-α, and apoptosis in BoMac cells. Further, MbovP467 increased the promoter activity of TNF-α. In conclusion, this study identified a new nucleomodulin, MbovP467, for M. bovis, which might have an important role in M. bovis pathogenesis.

Abstract 126: Connectome Gradient Dysfunction Contributes to White Matter Hyperintensity-Related Cognitive Decline

Introduction: Although White matter hyperintensity (WMH) is closely associated with cognitive decline, the precise neurobiological mechanisms underlying this relationship are not fully elucidated. Connectome studies have identified a primary-to-transmodal gradient in functional brain networks that support the spectrum from sensation to cognition. However, whether this gradient structure is altered as WMH progresses and how this alteration is associated with WMH-related cognitive decline remain unknown. Methods: A total of 758 individuals with WMH were enrolled from two medical centers and one community. All these participants completed cognitive assessment and resting-state functional MRI (rs-fMRI). The functional connectome gradient was reconstructed based on rs-fMRI by using a gradient decomposition framework. Interrelations amongst regional WMH, functional gradient measures, and specific cognitive domains were explored. Results: The larger WMH volume, the narrower range ( r = -0.099, p = 0.006) and smaller variance ( r = -0.121, p < 0.001) of the primary-to-transmodal gradient, as well as the poorer executive function ( r = -0.135, p = 0.001) and information processing speed ( r = -0.224, p = 0.001). A narrower range ( r = 0.131, p = 0.001) and smaller variance ( r = 0.136, p = 0.001) of the primary-to-transmodal gradient corresponded to poorer executive function. In particular, the relationship between frontal/occipital WMH and executive function was partly mediated by gradient range/variance of the primary-to-transmodal gradient (the 95% confidence interval did not contain the value 0). WMH-related gradient alterations were associated with gene expression levels involved in the G protein-coupled receptor (GPCR) signaling pathway (FDR q -value < 10 -4 ) and chemical synaptic transmission (FDR q -value < 10 -6 ). Discussion: We found that the primary-to-transmodal gradient dysfunction contributes to WMH-related cognitive decline. The detrimental effect of frontal/occipital WMH on executive function was mediated by decreased differentiation of the connectivity pattern between the primary and transmodal areas. The neurobiological mechanisms may be related to the GPCR signaling pathway and chemical synaptic transmission.

Reshaping the yeast galactose regulon via GPCR signaling cascade

Dynamically regulated systems are preferable to control metabolic pathways for an improved strain performance with better productivity. Here, we harnessed to the G protein-coupled receptor (GPCR) signaling pathway to reshape the yeast galactose regulon. The galactose-regulated (GAL) system was coupled with the GPCR signaling pathway for mating pheromone via a synthetic transcription factor. In this study, we refabricated the dynamic range, sensitivity, and response time of the GAL system to α factor by modulating the key components of the GPCR signaling cascade. A series of engineered yeasts with self-secretion of α factor were constructed to achieve quorum-sensing behaviors. In addition, we also repurposed the GAL system to make it responsive to heat shock. Taken together, our work showcases the great potential of synthetic biology in creating user-defined metabolic controls. We envision that the plasticity of our genetic design would be of significant interest for the future fabrication of novel gene expression systems.

Proopiomelanocortin and its potential functions in the pathological synovium of patients with osteoarthritis.

Osteoarthritis (OA) is a chronic degenerative disease of the joints, adversely affecting the quality of life for the patients. To better understand the mechanisms underlying the pathological changes in osteoarthritis and identify the key genes associated with osteoarthritis pathogenesis, we utilized a comprehensive bioinformatics approach to analyze the transcriptome between osteoarthritis synovial and control samples with public microarray datasets. First, the GSE82107 microarray dataset containing ten osteoarthritis synovial and seven control samples were selected from the Gene Expression Omnibus (GEO) database. A total of 52 overlapped differentially expressed genes (DEGs) in GSE82107 and OA-associated genes in the Comparative Toxicogenomics Database were identified. These OA-associated DEGs were further incorporated into a protein-protein interaction (PPI) network. Gene proopiomelanocortin (POMC) was identified in the largest cluster of PPI network with Cytoscape. GO and KEGG analyses suggested that these genes were associated with multiple functions. Other GEO datasets of osteoarthritis synovial tissues, including GSE55235 and GSE55457, were used to validate the expression level of POMC. Quantitative polymerase chain reaction and western blot analyses were also used to test the expression levels of POMC in our osteoarthritis samples. We found POMC was positively associated with transporter complex, ion channel activity, and G protein-coupled receptor signaling pathway. This study highlighted the OA-associated gene POMC, and its related biological pathways, suggesting it served as a potential treatment target in osteoarthritis.

Network pharmacology -based study on the mechanism of traditional Chinese medicine in the treatment of glioblastoma multiforme

BackgroundGlioblastoma multiforme (GBM) is one of the most common primary malignant brain tumors. Yi Qi Qu Yu Jie Du Fang (YYQQJDF) is a traditional Chinese medicine (TCM) prescription for GBM. The present study aimed to use a network pharmacology method to analyze the underlying mechanism of YQQYJDF in treating GBM.MethodsGBM sample data, active ingredients and potential targets of YQQYJDF were obtained from databases. R language was used to screen differentially expressed genes (DEGs) between GBM tissues and normal tissues, and to perform enrichment analysis and weighted gene coexpression network analysis (WGCNA). The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database was used to perform a protein‒protein interaction (PPI) analysis. A Venn diagram was used to obtain the core target genes of YQQYJDF for GBM treatment. Molecular docking was used to verify the binding between the active ingredient molecules and the proteins corresponding to the core target genes. Cell proliferation assays and invasion assays were used to verify the effect of active ingredients on the proliferation and invasion of glioma cells.ResultsA total of 73 potential targets of YQQYJDF in the treatment of GBM were obtained. Enrichment analyses showed that the biological processes and molecular functions involved in these target genes were related to the activation of the G protein-coupled receptor (GPCR) signaling pathway and the regulation of hypoxia. The neuroactive ligand‒receptor pathway, the cellular senescence pathway, the calcium signaling pathway, the cell cycle pathway and the p53 signaling pathway might play important roles. Combining the results of WGCNA and PPI analysis, five core target genes and their corresponding four core active ingredients were screened. Molecular docking indicated that the core active ingredient molecules and the proteins corresponding to the core target genes had strong binding affinities. Cell proliferation and invasion assays showed that the core active ingredients of YQQYJDF significantly inhibited the proliferation and invasion of glioma cells (P < 0.01).ConclusionsThe present study predicted the possible active ingredients and targets of YQQYJDF in treating GBM, and analyzed its possible mechanism. These results may provide a basis and ideas for further research.

Identification of key genes affecting sperm motility in chicken based on whole-transcriptome sequencing

Sperm motility is an important index for the evaluation of semen quality. Improving sperm motility is important to improve reproductive performance, promote breeding process, and reduce production cost. However, the molecular mechanisms regulating sperm motility in chickens remain unclear. In this study, histological observation and whole-transcriptome analysis were performed on testicular tissue of chickens with high and low sperm motility. Histological observations showed that roosters with high sperm motility exhibited better semen quality than those with low sperm motility. In addition, the germinal epithelial cells of roosters with low sperm motility were loosely arranged and contained many vacuoles. RNA-seq results revealed the expression of 23,033 mRNAs, 2,893 lncRNAs, and 515 miRNAs in chicken testes. Among them, there were 417 differentially expressed mRNAs (DEmRNAs), 106 differentially expressed lncRNAs (DElncRNAs), and 15 differentially expressed miRNAs (DEmiRNAs) between high and low sperm motility testes. These differentially expressed genes were involved in the G protein-coupled receptor signaling pathway, cilia structure, Wnt signaling, MAPK signaling, GnRH signaling, and mTOR signaling. By integrating the competitive relationships between DEmRNAs, DElncRNAs, and DEmiRNAs, we identified the regulatory pathway of MSTRG.3077.3/MSTRG.9085.1-gga-miR-138-5p-CADM1 and MSTRG.2290.1-gga-miR-142-3p-GNAQ/PPP3CA as crucial in the modulation of chicken sperm motility. This study provides new insights into the function and mechanism of ceRNAs in regulating sperm motility in chicken testes.

Phosphorylation motif dictates GPCR C-terminal domain conformation and arrestin interaction

Arrestin-dependent G protein-coupled receptor (GPCR) signaling pathway is regulated by the phosphorylation state of GPCR's C-terminal domain, but the molecular bases of arrestin:receptor interaction are to be further illuminated. Here we investigated the impact of phosphorylation on the conformational features of the C-terminal region from three rhodopsin-like GPCRs, the vasopressin V2 receptor (V2R), the growth hormone secretagogue or ghrelin receptor type 1a (GHSR), and the β2-adernergic receptor (β2AR). Using phosphomimetic variants, we identified pre-formed secondary structure elements, or short linear motifs (SLiMs), that undergo specific conformational transitions upon phosphorylation. Of importance, such conformational transitions appear to favor arrestin-2 binding. Hence, our results suggest a model in which the phosphorylation-dependent structuration of the GPCR C-terminal regions would modulate arrestin binding and therefore signaling outcomes in arrestin-dependent pathways.

Effect of Juanbi Qianggu Formula on biological behaviors of fibroblast-like synoviocytes in rheumatoid arthritis by regulating FGFR1 signaling pathway based on network pharmacology and cell function experiments

This study aimed to explore the molecular mechanism of Juanbi Qianggu Formula(JBQGF), an empirical formula formulated by the prestigious doctor in traditional Chinese medicine, in the treatment of rheumatoid arthritis based on network pharmacology and cell function experiments. The main active components and targets of JBQGF were obtained through Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and Encyclopedia of Traditional Chinese Medicine(ETCM), and the core targets underwent functional enrichment analysis and signaling pathway analysis. Cytoscape 3.6.0 was used to construct a visualized &quot;active component-target-signaling pathway&quot; network of JBQGF. After screening, nine potential pathways of JBQGF were obtained, mainly including G protein-coupled receptor signaling pathway and tyrosine kinase receptor signaling pathway. As previously indicated, the fibroblast growth factor receptor 1(FGFR1) signaling pathway was highly activated in active fibroblast-like synoviocytes(FLS) in rheumatoid arthritis, and cell and animal experiments demonstrated that inhibition of the FGFR1 signaling pathway could significantly reduce joint inflammation and joint destruction in collagen-induced arthritis(CIA) rats. In terms of the tyrosine kinase receptor signal transduction pathway, the analysis of its target genes revealed that FGFR1 might be a potential target of JBQGF for rheumatoid arthritis treatment. The biological effect of JBQGF by inhibiting FGFR1 phosphorylation was preliminarily verified by Western blot, Transwell invasion assay, and pannus erosion assay, thereby inhibiting matrix metalloproteinase 2(MMP2) and receptor activator of nuclear factor-κB ligand(RANKL) and suppressing the invasion of fibroblasts in rheumatoid arthritis and erosive effect of pannus bone. This study provides ideas for searching potential targets of rheumatoid arthritis treatment and TCM drugs through network pharmacology.

Coding RNA expression profile and transcription factor analysis of H.pylori-associated chronic atrophic gastritis

PurposeAtrophic gastritis, one of the processes leading to gastric cancer (GC), is closely related to Helicobacter pylori (HP) infection. This study aimed to understand how HP causes chronic inflammation that leads to ulcers and stomach problems. MethodsTwenty-eight CAG patients were included in the study (9 HP-infected and 19 HP-uninfected). Endoscopy, histopathology, and high-throughput mRNA sequencing were performed. Differentially expressed genes (DEGs) were validated via qRT-PCR. ResultsPrincipal component analysis (PCA) results showed that more than 88.9 ​% of the samples were classified into the HP (+) group. A total of 157 DEGs were identified, of which 38 were up-regulated and 119 were down-regulated. The DEGs were mainly enriched in the biological process (BP) terms associated with immune system process, adaptive immune response, G protein-coupled receptor signaling pathway, as well as point to numerous key pathways, including fat digestion and absorption, retinol metabolism, steroid hormone biosynthesis, ascorbate and aldarate metabolism, and chemical carcinogenesis. APOA1, APOA4, FOXP3, NR1H4, ABCG5, ACTA1, CCL19, CCR7, CYP3A4, and PDCD had the highest degrees in protein–protein interaction network as the hub genes; they were also included into the transcription factor (TF)-target network except for PDCD. APOA1 and CYP3A4 were extremely significantly up-regulated in HP (+) CAG patients compared with the HP (−) CAG patients, while FOXP3, CCR7 and CCL19 were significantly down-regulated. ConclusionThe expression of APOA1, CYP3A4, FOXP3, CCR7, and CCL19 are the potential indicators for CAG to GC development, being the biomarkers to predict progression of CAG and poor prognosis of GC.

Comparative phylogenetic analysis and transcriptomic profiling of Dengue (DENV-3 genotype I) outbreak in 2021 in Bangladesh

BackgroundThe next-generation sequencing (NGS) technology facilitates in-depth study of host-pathogen metatranscriptome. We, therefore, implicated phylodynamic and transcriptomic approaches through NGS technology to know/understand the dengue virus (DENV) origin and host response with dengue fever.MethodsIn this study, blood serum RNA was extracted from 21 dengue patients and 3 healthy individuals. Total transcriptomic data were analyzed for phylogenetic, phylodynamic, differential express gene (DEG), and gene ontology (GO) using respective bioinformatics tools.ResultsThe viral genome sequence revealed dengue viral genome size ranges 10647 to 10707 nucleotide. Phylogenetic and phylodynamic analysis showed that the 2021 epidemic isolates were DENV-3 genotype-I and maintained as a new clade in compared to 2019 epidemic. Transcriptome analysis showed a total of 2686 genes were DEG in dengue patients compared to control with a q-value < 0.05. DESeq2 plot counts function of the top 24 genes with the smallest q-values of differential gene expression of RNA-seq data showed that 11 genes were upregulated, whereas 13 genes were downregulated. GO analysis showed a significant upregulation (p = < 0.001) in a process of multicellular organismal, nervous system, sensory perception of chemical stimulus, and G protein-coupled receptor signaling pathways in the dengue patients. However, there were a significant downregulation (p = < 0.001) of intracellular component, cellular anatomical entity, and protein-containing complex in dengue patients. Most importantly, there was a significant increase of a class of immunoregulatory proteins in dengue patients in compared to the controls, with increased GO of immune system process. In addition, upregulation of toll receptor (TLR) signaling pathways were found in dengue patients. These TLR pathways were particularly involved for the activation of innate system coupled with adaptive immune system that probably involved the rapid elimination of dengue virus infected cells. These differentially expressed genes could be further investigated for target based prophylactic interventions for dengue.ConclusionThis is a first report describing DENV complete genomic features and differentially expressed genes in patients in Bangladesh. These genes may have diagnostic and therapeutic values for dengue infection. Continual genomic surveillance is required to further investigate the shift in dominant genotypes in relation to viral pathogenesis.

Identification of key factors driving inflammation-induced sensitization of muscle sensory neurons.

Sensory neurons embedded in muscle tissue that initiate pain sensations, i.e., nociceptors, are temporarily sensitized by inflammatory mediators during musculoskeletal trauma. These neurons transduce peripheral noxious stimuli into an electrical signal [i.e., an action potential (AP)] and, when sensitized, demonstrate lower activation thresholds and a heightened AP response. We still do not understand the relative contributions of the various transmembrane proteins and intracellular signaling processes that drive the inflammation-induced hyperexcitability of nociceptors. In this study, we used computational analysis to identify key proteins that could regulate the inflammation-induced increase in the magnitude of AP firing in mechanosensitive muscle nociceptors. First, we extended a previously validated model of a mechanosensitive mouse muscle nociceptor to incorporate two inflammation-activated G protein-coupled receptor (GPCR) signaling pathways and validated the model simulations of inflammation-induced nociceptor sensitization using literature data. Then, by performing global sensitivity analyses that simulated thousands of inflammation-induced nociceptor sensitization scenarios, we identified three ion channels and four molecular processes (from the 17 modeled transmembrane proteins and 28 intracellular signaling components) as potential regulators of the inflammation-induced increase in AP firing in response to mechanical forces. Moreover, we found that simulating single knockouts of transient receptor potential ankyrin 1 (TRPA1) and reducing the rates of Gαq-coupled receptor phosphorylation and Gαq subunit activation considerably altered the excitability of nociceptors (i.e., each modification increased or decreased the inflammation-induced fold change in the number of triggered APs compared to when all channels were present). These results suggest that altering the expression of TRPA1 or the concentration of intracellular Gαq might regulate the inflammation-induced increase in AP response of mechanosensitive muscle nociceptors.

Structural Insights into M1 Muscarinic Acetylcholine Receptor Signaling Bias between Gαq and β-Arrestin through BRET Assays and Molecular Docking.

The selectivity of drugs for G protein-coupled receptor (GPCR) signaling pathways is crucial for their therapeutic efficacy. Different agonists can cause receptors to recruit effector proteins at varying levels, thus inducing different signaling responses, called signaling bias. Although several GPCR-biased drugs are currently being developed, only a limited number of biased ligands have been identified regarding their signaling bias for the M1 muscarinic acetylcholine receptor (M1mAChR), and the mechanism is not yet well understood. In this study, we utilized bioluminescence resonance energy transfer (BRET) assays to compare the efficacy of six agonists in inducing Gαq and β-arrestin2 binding to M1mAChR. Our findings reveal notable variations in agonist efficacy in the recruitment of Gαq and β-arrestin2. Pilocarpine preferentially promoted the recruitment of β-arrestin2 (∆∆RAi = -0.5), while McN-A-343 (∆∆RAi = 1.5), Xanomeline (∆∆RAi = 0.6), and Iperoxo (∆∆RAi = 0.3) exhibited a preference for the recruitment of Gαq. We also used commercial methods to verify the agonists and obtained consistent results. Molecular docking revealed that certain residues (e.g., Y404, located in TM7 of M1mAChR) could play crucial roles in Gαq signaling bias by interacting with McN-A-343, Xanomeline, and Iperoxo, whereas other residues (e.g., W378 and Y381, located in TM6) contributed to β-arrestin recruitment by interacting with Pilocarpine. The preference of activated M1mAChR for different effectors may be due to significant conformational changes induced by biased agonists. By characterizing bias towards Gαq and β-arrestin2 recruitment, our study provides insights into M1mAChR signaling bias.

Bioinformatic analysis identifies GPR91 as a potential key gene in brain injury after deep hypothermic low flow

PurposeExplore the transcription change of brain ischemia and reperfusion injury after deep hypothermic low flow. MethodThe data from PRJNA739516 and GSE104036 were obtained for the differentially expressed genes identification, functional enrichment analysis, gene set enrichment analysis, protein-protein interaction construction and hub gene identification. Oxygen and glucose deprivation model was set to validate the hub gene and explore the detailed brain injury mechanism. ResultInterleukin, immunological response, NF-κB signaling pathway, G protein-coupled receptor signaling pathway and NLRP inflammatory are functional pathway were enriched in differentially expressed genes analysis. Sucnr1, Casr, Cxcr4, C5ar1, Tas2r41, Tas2r60 and Hcar2 were identified and verified in the OGD model. Knocking down GPR91 reduces the inflammatory response after OGD and GPR91 may be involved in the inflammatory pre-reaction through the synergistic activation of NF-κB, NLRP3, and IL-1β respectively. ConclusionOur study found that Interleukin, immunological response, NF-κB signaling pathway, G protein-coupled receptor signaling pathway and NLRP inflammatory are all associated with brain ischemia and reperfusion injury after deep hypothermic low flow and GPR91 can activate NF-κB/NLRP3 pathway and trigger the release of IL-1β in this progress.

Abstract 5861: Tumor cell-intrinsic factors promoting fibrotic tumor microenvironment in gastric cancer

Abstract Background: Gastric cancer (GC) is one of the leading causes of death in the world. Evidence has shown over the past decade that cancer-associated​ fibroblasts (CAFs) play essential roles in various types of cancers, including GC, such as tumorigenesis, therapy resistance, and immunosuppression, which contribute to the progress of tumors. Although GC tumors are known to be enriched with stroma, the molecular mechanisms of how GC tumors foster the fibrotic milieu is still largely unknown. Here we analyzed a large-scale genomic and transcriptomic dataset of GC to identify GC tumor cell-intrinsic factors for developing fibroblast-rich microenvironments​. Methods: We analyzed GC patient samples with gene expression data and copy number variation profiles from the Asian Cancer Research Group (ACRG) (N = 271). The microenvironment cell populations-counter (MCP-counter) method was used for transcriptome deconvolution to estimate the population of fibroblasts and immune cells in GC tumors. Genes with q value &amp;lt; 0.05 and fold change &amp;gt; 1.5 were defined as differentially expressed genes (DEGs) and identified using the Subio Platform. The copy number amplified genes found in more than 3% of the samples were selected as amplified genes. Pathway and process enrichment analysis and protein-protein interaction analysis were performed using Metascape. Results: We analyzed MCP-counter outputs and observed a fibroblast-enriched population with few infiltrations of anti-tumor immune cells, such as CD8 T cells and cytotoxic T cells in GC patients, suggesting the immunosuppressive role of CAFs in the GC tumor. By analyzing 933 DEGs upregulated in fibroblast-high patients, we found 96 genes with frequent copy number amplification that were not listed on the curated stroma and extracellular matrix-related genes and thus potentially tumor cell-intrinsic genes correlated with the abundance of the stroma. Pathway and protein-interaction analyses revealed that G protein-coupled receptor (GPCR) signaling pathway was enriched in these genes, including HTR2A, AGTR1, FZD1, CALCRL, and NBEA with strong correlations between fibroblast score (Spearman's r &amp;gt; 0.55), implying that these signaling in GC tumor cells contribute to promoting fibroblast-enriched microenvironment. Conclusions: Bioinformatic analyses of a publicly available genomic and transcriptomic dataset revealed a fibroblast-rich and immune-excluded population in GC patients. Several GPCR signaling in tumor cells were strongly correlated with fibroblast abundance in tumors, and these signaling pathways have potential roles in promoting the stroma-rich GC microenvironment. Citation Format: Takashi Semba, Yiling Tong, Feng Wei, Atsuko Yonemura, Tadahito Yasuda, Tomoyuki Uchihara, Huaito Wang, Hideo Baba, Takatsugu Ishimoto. Tumor cell-intrinsic factors promoting fibrotic tumor microenvironment in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5861.

Opsin 3 mediates UVA-induced keratinocyte supranuclear melanin cap formation

Solar ultraviolet (UV) radiation-induced DNA damage is a major risk factor for skin cancer development. UV-induced redistribution of melanin near keratinocyte nuclei leads to the formation of a supranuclear cap, which acts as a natural sunscreen and protects DNA by absorbing and scattering UV radiation. However, the mechanism underlying the intracellular movement of melanin in nuclear capping is poorly understood. In this study, we found that OPN3 is an important photoreceptor in human epidermal keratinocytes and is critical for UVA-mediated supranuclear cap formation. OPN3 mediates supranuclear cap formation via the calcium-dependent G protein-coupled receptor signaling pathway and ultimately upregulates Dync1i1 and DCTN1 expression in human epidermal keratinocytes via activating calcium/CaMKII, CREB, and Akt signal transduction. Together, these results clarify the role of OPN3 in regulating melanin cap formation in human epidermal keratinocytes, greatly expanding our understanding of the phototransduction mechanisms involved in physiological function in skin keratinocytes.