Protein-coupled Receptor Signaling Pathways Research Articles

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 < 0.05 and fold change > 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 > 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.

A Comprehensive Weighted Gene Co-expression Network Analysis Uncovers Potential Targets in Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is one of the most common microvascular complications of diabetes. It has always been difficult to explore novel biomarkers and therapeutic targets of DKD. We aimed to identify new biomarkers and further explore their functions in DKD. The weighted gene co-expression network analysis (WGCNA) method was used to analyze the expression profile data of DKD, obtain key modules related to the clinical traits of DKD, and perform gene enrichment analysis. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the mRNA expression of the hub genes in DKD. Spearman's correlation coefficients were used to determine the relationship between gene expression and clinical indicators. Fifteen gene modules were obtained via WGCNA analysis, among which the green module had the most significant correlation with DKD. Gene enrichment analysis revealed that the genes in this module were mainly involved in sugar and lipid metabolism, regulation of small guanosine triphosphatase (GTPase) mediated signal transduction, G protein-coupled receptor signaling pathway, peroxisome proliferator-activated receptor (PPAR) molecular signaling pathway, Rho protein signal transduction, and oxidoreductase activity. The qRT-PCR results showed that the relative expression of nuclear pore complex-interacting protein family member A2 (NPIPA2) and ankyrin repeat domain 36 (ANKRD36) was notably increased in DKD compared to the control. NPIPA2 was positively correlated with the urine albumin/creatinine ratio (ACR) and serum creatinine (Scr) but negatively correlated with albumin (ALB) and hemoglobin (Hb) levels. ANKRD36 was positively correlated with the triglyceride (TG) level and white blood cell (WBC) count. NPIPA2 expression is closely related to the disease condition of DKD, whereas ANKRD36 may be involved in the progression of DKD through lipid metabolism and inflammation, providing an experimental basis to further explore the pathogenesis of DKD.

Genome-wide gene by environment study of time spent in daylight and chronotype identifies emerging genetic architecture underlying light sensitivity.

Light is the primary stimulus for synchronizing the circadian clock in humans. There are very large interindividual differences in the sensitivity of the circadian clock to light. Little is currently known about the genetic basis for these interindividual differences. We performed a genome-wide gene-by-environment interaction study (GWIS) in 280 897 individuals from the UK Biobank cohort to identify genetic variants that moderate the effect of daytime light exposure on chronotype (individual time of day preference), acting as "light sensitivity" variants for the impact of daylight on the circadian system. We identified a genome-wide significant SNP mapped to the ARL14EP gene (rs3847634; p < 5 × 10-8), where additional minor alleles were found to enhance the morningness effect of daytime light exposure (βGxE = -.03, SE = 0.005) and were associated with increased gene ARL14EP expression in brain and retinal tissues. Gene-property analysis showed light sensitivity loci were enriched for genes in the G protein-coupled glutamate receptor signaling pathway and genes expressed in Per2+ hypothalamic neurons. Linkage disequilibrium score regression identified Bonferroni significant genetic correlations of greater light sensitivity GWIS with later chronotype and shorter sleep duration. Greater light sensitivity was nominally genetically correlated with insomnia symptoms and risk for post-traumatic stress disorder (PTSD). This study is the first to assess light as an important exposure in the genomics of chronotype and is a critical first step in uncovering the genetic architecture of human circadian light sensitivity and its links to sleep and mental health.

A metabotropic glutamate receptor affects the growth and development of Schistosoma japonicum.

Schistosomiasis is a zoonotic parasitic disease caused by schistosome infection that severely threatens human health. Therapy relies mainly on single drug treatment with praziquantel. Therefore, there is an urgent need to develop alternative medicines. The glutamate neurotransmitter in helminths is involved in many physiological functions by interacting with various cell-surface receptors. However, the roles and detailed regulatory mechanisms of the metabotropic glutamate receptor (mGluR) in the growth and development of Schistosoma japonicum remain poorly understood. In this study, we identified two putative mGluRs in S. japonicum and named them SjGRM7 (Sjc_001309, similar to GRM7) and SjGRM (Sjc_001163, similar to mGluR). Further validation using a calcium mobilization assay showed that SjGRM7 and SjGRM are glutamate-specific. The results of in situ hybridization showed that SjGRM is mainly located in the nerves of both males and gonads of females, and SjGRM7 is principally found in the nerves and gonads of males and females. In a RNA interference experiment, the results showed that SjGRM7 knockdown by double-stranded RNA (dsRNA) in S. japonicum caused edema, chassis detachment, and separation of paired worms in vitro. Furthermore, dsRNA interference of SjGRM7 could significantly affect the development and egg production of male and female worms in vivo and alleviate the host liver granulomas and fibrosis. Finally, we examined the molecular mechanisms underlying the regulatory function of mGluR using RNA sequencing. The data suggest that SjGRM7 propagates its signals through the G protein-coupled receptor signaling pathway to promote nervous system development in S. japonicum. In conclusion, SjGRM7 is a potential target for anti-schistosomiasis. This study enables future research on the mechanisms of action of Schistosomiasis japonica drugs.

A comprehensive weighted gene co-expression network analysis uncovers potential targets in diabetic kidney disease

Abstract Background and Objectives Diabetic kidney disease (DKD) is one of the most common microvascular complications of diabetes. It has always been difficult to explore novel biomarkers and therapeutic targets of DKD. We aimed to identify new biomarkers and further explore their functions in DKD. Methods The weighted gene co-expression network analysis (WGCNA) method was used to analyze the expression profile data of DKD, obtain key modules related to the clinical traits of DKD, and perform gene enrichment analysis. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the mRNA expression of the hub genes in DKD. Spearman’s correlation coefficients were used to determine the relationship between gene expression and clinical indicators. Results Fifteen gene modules were obtained via WGCNA analysis, among which the green module had the most significant correlation with DKD. Gene enrichment analysis revealed that the genes in this module were mainly involved in sugar and lipid metabolism, regulation of small guanosine triphosphatase (GTPase) mediated signal transduction, G protein-coupled receptor signaling pathway, peroxisome proliferator-activated receptor (PPAR) molecular signaling pathway, Rho protein signal transduction, and oxidoreductase activity. The qRT-PCR results showed that the relative expression of nuclear pore complex-interacting protein family member A2 (NPIPA2) and ankyrin repeat domain 36 (ANKRD36) was notably increased in DKD compared to the control. NPIPA2 was positively correlated with the urine albumin/creatinine ratio (ACR) and serum creatinine (Scr) but negatively correlated with albumin (ALB) and hemoglobin (Hb) levels. ANKRD36 was positively correlated with the triglyceride (TG) level and white blood cell (WBC) count. Conclusion NPIPA2 expression is closely related to the disease condition of DKD, whereas ANKRD36 may be involved in the progression of DKD through lipid metabolism and inflammation, providing an experimental basis to further explore the pathogenesis of DKD.

Network toxicology and molecular docking analyses on strychnine indicate CHRM1 is a potential neurotoxic target

BackgroundImproper use of strychnine can cause death. The aim of this study was to identify and evaluate toxic mechanisms of action associated with active compounds in strychnine using a network toxicology approach, and explore potential pathogenic targets.MethodsIn the present study, strychnine target and central nervous system-related gene set were established using the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and four disease gene databases (Genecards, OMIM, PharmGkb, TTD). An “ingredient-target” interactive active network map was constructed using Cytoscape software (version 3.8.0). Functional enrichment analysis was performed based on the hub genes. A protein-protein interaction network was constructed using STRING database. The pharmacokinetics (ADMET) properties of strychnine were evaluated using SwissADME tool. Molecular docking was performed using Autodock Vina to explore the interactions between the active compounds and the target protein.ResultsFive strychnine toxicity-related components and a gene set of 40 genes were obtained. GO and KEGG analyses showed that Strychnine acts on the central nervous system through G protein-coupled receptor signaling pathway. Analysis of “ADMET” related parameters showed a high gastrointestinal tract absorption of (S)-stylopine and isobrucine and the compounds could cross the blood brain barrier. CHRM1 was selected as a key gene in strychnine toxicity. Molecular docking results showed that the co-crystalized ligands did not form hydrogen bond with CHRM1. (S)-stylopine had the highest binding affinity (binding energy = − 8.5 kcal/mol) compared with the other two compounds.ConclusionNetwork toxicology and molecular docking reveal the toxicity mechanisms of strychnine active compounds. The findings showed that CHRM1 is a potential neurotoxic target. (S)-stylopine showed stronger neurotoxic effect compared with the other ligands.

Comprehensive in silico prioritization of pathogenic nsSNPs in human β-adducin gene towards finding its relation with cancer

The adducin family consists of three closely related proteins ADD1 (α-isoform), ADD2 (β-isoform) and ADD3 (γ-isoform) with similar domain structures. Adducins exist as heterodimers or tetramers composed of either α/β or α/γ heterodimers. Experimental evidences implicate the involvement of ADD2 variants in hypertension, renal dysfunction, impaired locomotor function and learning abilities. Non-synonymous SNPs (nsSNPs) are potentially damaging in nature as they results in amino acid substitutions. In human, a total 476 nsSNPs of ADD2 gene are reported in Ensembl and in the present work, computational prioritization has revealed 27 nsSNPs as highly deleterious and destabilizing in nature. Pan-cancer transcriptomic analyses reveal differential ADD2 expression in adrenal, breast, colon, lung, ovary, pancreas, testis, thyroid, and uterine cancer. In several cancers, overexpression of ADD2 is also found to be associated with lower overall survival rate and more patients at risk. It was also found that several nsSNPs of ADD2 had direct, proximal, distal and network rewriting PTM effects in different cancers and thereby account for potential structural and functional alterations. The coding mutations in ADD2 can affect overall gene expression in colon adenocarcinoma, melanoma, gastric cancer, and uterine cancer. The hub proteins, from the PPI network of these differentially expressed genes, are involved in positive regulation of acute inflammatory response to antigenic stimulus, acute phase response, G protein-coupled receptor signaling pathway. The present study is hypothetically driven and performed using public databases. Further experimental works are necessary to understand the role of ADD2 nsSNPs in tumor development and metastatic transition.

Antidepressant effect and molecular mechanism of notoginsenoside R_1 based on network pharmacology and animal experiments

To provide experimental basis and theoretical guidance for further research on the molecular mechanism of notoginsenoside R_1(NGR_1) in the treatment of depression, the present study analyzed the potential mechanism of NGR_1 in the treatment of depression through network pharmacology and verified it by molecular docking and animal experiments. PharmMapper, SwissTargetPrediction, and GeneCards were used to predict the related targets of both NGR_1 and depression to obtain the potential targets of NGR_1 in the treatment of depression. The database for annotation, visualization and integrated discovery(DAVID) was used for GO functional annotation and KEGG pathway enrichment analysis to screen the possible mechanisms of NGR_1 exerting antidepressant effect. Cytoscape 3.9.0 was adopted to construct a protein-protein interaction(PPI) network, and the topological analysis was performed to obtain the core targets. The binding activity of NGR_1 to core targets was tested by molecular docking. The depression model was prepared by injecting lipopolysaccharide(LPS) into the lateral ventricle in mice, and intervened with NGR_1. The antidepressant effect of NGR_1 was detected by behavioral tests and RT-qPCR. The results showed that by network pharmacology, 56 common targets of NGR_1 and depression were predicted, and GO enrichment analysis determined 13 related biological processes, mainly involving G protein-coupled receptor signaling pathway, positive regulation of transcription from RNA polymerase Ⅱ promoter, cytokine-mediated signaling pathway, gene expression, apoptosis, cell proliferation, and signal transduction. In addition, KEGG pathway enrichment analysis identified ten potential pathways, including neuroactive ligand-receptor interaction signaling pathway, lipid and atherosclerosis signaling pathway, cAMP signaling pathway, PI3 K-AKT signaling pathway, and lipid and atherosclerosis signaling pathway. PPI analysis revealed that the core targets included CASP3, VEGFA, IGF1, STAT3, MAPK1, PPARG, MTOR, MAPK14, NR3 C1 and AR, and molecular docking demonstrated that NGR_1 had desirable binding activity to these target proteins. In animal experiments, the results showed that NGR_1 improved the disease behavior of depressed mice, significantly inhibited the neuroinflammatory response(reducing the mRNA expression of Iba-1, TNF-α, IL-1β, and IL-6), and regulated the mRNA expression of lipid and atherosclerosis signaling pathway-related targets(CASP3, STAT3, MAPK1 and MAPK14). This indicated that the antidepressant mechanism of NGR_1 may be related to the regulation of lipid and atherosclerosis signaling pathway. In conclusion, network pharmacology was used to reveal the core targets and pathways of NGR_1, and some of them were verified in animal experiments, which provided the basis for in-depth exploration on the mechanism of NGR_1 in the treatment of depression.

Molecular mechanism of Sishen pills in the treatment of diarrheal diabetic enteropathy based on network pharmacology.

This study aimed to explore the effectiveness and safety of Sishen pills for the treatment of diarrheal diabetic enteropathy (DDE).The Traditional Chinese Medicine (TCM) Systems Pharmacology and BATMAN-TCM databases were used to determine the chemical composition of Sishen pills and thus predict information on protein targets. We searched for potential targets of DDE in the GeneCards, DrugBank, Therapeutic Target (TTD), and DisGeNET databases. Using the intersection of the drug and disease targets, protein–protein interaction (PPI) networks and molecular interaction modules were constructed, and key targets were screened. The intersecting gene targets were imported into the Metascape database to conduct Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The core targets and active ingredients were then docked at the molecular level.Sishen pills contain 70 active ingredients, 463 targets, and 566 disease targets. A module analysis of the targets revealed that the module was mainly related to adrenergic receptor activity, the adenosine phosphate kinase signaling pathway, and the G protein-coupled receptor signaling pathway. The GO and KEGG pathway enrichment results indicated that the protein genes regulated by Sishen pills were mainly enriched in the response to lipopolysaccharides, the AMPK signaling pathway, the JAK-STAT signaling pathway, and other signaling pathways. The molecular docking results showed that the core active compounds exhibited good binding activity with the predicted targets.Sishen pills can regulate the immune function of the body through anti-inflammatory and antibacterial effects for the treatment of DDE.

Systems assessment of statins hazard: Integrating in silico prediction, developmental toxicity profile and transcriptomics in zebrafish

Statins are prescribed widely as lipid-lowering agents. However, statins are associated with an increased harmful risk on public health and the ecosystem. Little is known about statins’ toxicity on biological development and the underlying molecular mechanisms. We exposed zebrafish embryos to a series of statins to evaluate their development toxicity. Statins-induced embryonic developmental defects in a concentration-dependent manner. 72 h LC50 values for lovastatin, simvastatin, fluvastatin, atorvastatin, rosuvastatin, and pravastatin were 0.01 μM, 0.04 μM, 1.93 μM, 37.28 μM, 79.29 μM, and 2170 μM, respectively. Moreover, the expression of genes involved in heart contraction, calcium ion binding, transcription factors, nucleus, and G protein-coupled receptor signaling pathway was altered by statins. The early growth response gene (egr4) and transcription factor genes (fosab and fosb) were screened as potential toxicity targets due to their significant upregulation based on protein-protein interaction (PPI) and drug-gene interaction network analysis. Finally, the ecotoxicity profile of statins was predicted by in silico method, and statins were high or moderate risk to aquatic organisms. We provide a systems toxicology strategy to explore the toxicity of statins and illustrate the potential mechanisms of action.

Genome-wide association mapping of nutritional traits for designing superior chickpea varieties.

Micronutrient malnutrition is a serious concern in many parts of the world; therefore, enhancing crop nutrient content is an important challenge. Chickpea (Cicer arietinum L.), a major food legume crop worldwide, is a vital source of protein and minerals in the vegetarian diet. This study evaluated a diverse set of 258 chickpea germplasm accessions for 12 key nutritional traits. A significant variation was observed for several nutritional traits, including crude protein (16.56–24.64/100 g), β-Carotene (0.003–0.104 mg/100 g), calcium (60.69–176.55 mg/100 g), and folate (0.413–6.537 mg/kg). These data, combined with the available whole-genome sequencing data for 318,644 SNPs, were used in genome-wide association studies comprising single-locus and multi-locus models. We also explored the effect of varying the minor allele frequency (MAF) levels and heterozygosity. We identified 62 significant marker-trait associations (MTAs) explaining up to 28.63% of the phenotypic variance (PV), of which nine were localized within genes regulating G protein-coupled receptor signaling pathway, proteasome assembly, intracellular signal transduction, and oxidation–reduction process, among others. The significant effect MTAs were located primarily on Ca1, Ca3, Ca4, and Ca6. Importantly, varying the level of heterozygosity was found to significantly affect the detection of associations contributing to traits of interest. We further identified seven promising accessions (ICC10399, ICC1392, ICC1710, ICC2263, ICC1431, ICC4182, and ICC16915) with superior agronomic performance and high nutritional content as potential donors for developing nutrient-rich, high-yielding chickpea varieties. Validation of the significant MTAs with higher PV could identify factors controlling the nutrient acquisition and facilitate the design of biofortified chickpeas for the future.

Functional analysis and expression profile of human platelets infected by EBV in vitro.

Platelet activation is commonly detected after infection by multiple viruses such as human immunodeficiency virus (HIV), H1N1 influenza, Hepatitis C virus (HCV), Ebola virus (EBV), and Dengue virus (DENV). Non-coding RNAs (ncRNAs) constitute the majority of the human transcribed genome, but the biology of platelet ncRNAs is largely unexplored. In this study, we performed microarray profiling to characterize the expression profile of human platelets infected with EBV in vitro after 2h. A total of 187 long non-coding RNAs (lncRNAs) displayed differences, of which 114 were upregulated and 73 were downregulated; 78 microRNAs (miRNAs) showed differences, including 73 upregulated and 5 downregulated; 808 mRNAs displayed differences, among which 367 were upregulated and 441 were downregulated. Gene ontology (GO) analysis mostly related to G protein-coupled receptor signaling pathway, detection of chemical stimulus involved in sensory perception of smell and regulation of transcription by RNA polymerase II. Pathway analysis showed that the differentially expressed genes were mainly enriched in cell metabolism and immune-related response. A ceRNA network was established based on predicting regulatory pairs in differentially expressed genes, in which hsa-miR-6877-3p had the highest regulatory capability (degree=31), FAM230A was the lncRNA with the highest regulatory capability (degree=28). According to the EBV related miRNA regulation network, it revealed that ebv-miR-BART19-3p had the most target genes and BRWD1, FAM126B, TFRC and JMY were the genes most regulated by EBV-related miRNAs. After overlapping the three networks, we found that the EIFAK2 gene was strongly correlated with autologous ncRNAs, including hsa-miR-1972, hsa-miR-504-3p and hsa-miR-6825-5p, as well as with EBV ncRNAs, including EBER1, EBER2, miR-BART7-3p and miR-BART16. The present study contributes to a better understanding of the expression profiling of ncRNAs and their functions in platelets activated by EBV in vitro, and paves the way to further study on platelet function.

Transcriptome analysis of the spleen of heterophils to lymphocytes ratio-selected chickens revealed their mechanism of differential resistance to Salmonella

Salmonella is one of the most common food-borne pathogens and its resistance in chicken can be improved through genetic selection. The heterophils/lymphocytes (H/L) ratio in the blood reflects the immune system status of chicken. We compared the genome data and spleen transcriptomes between the H/L ratio-selected and non-selected chickens, after Salmonella infection, aiming to identify the key genes participating in the antibacterial activity in the spleen. The results revealed that, the selected population had stronger (P<0.05) liver resistance to Salmonella typhimurium (ST) than the non-selected population. In the selected and non-selected lines, the identified differentiation genes encode proteins involved in biological processes or metabolic pathways that included the TGF-beta signaling pathway, FoxO signaling pathway, and Salmonella infection pathway. The results of the analysis of all identified differentially expressed genes (DEGs) of spleen revealed that the G protein-coupled receptor (GPCR) and insulin-like growth factor (IGF-I) signaling pathways were involved in the Salmonella infection pathway. Integrated analysis of DEGs and FST (fixation index), identified candidate genes involved in Salmonella infection pathway, such as GPR39, NTRK2, and ANXA1. The extensive genomic changes highlight the polygenic genetic of the immune response in these chicken populations. Numerous genes related to the immune performance are differentially expressed in the selected and non-selected lines and the selected lines has a higher resistance to Salmonella.

Abstract 3068: Identify targetable molecular drivers of chemotherapy resistance in gastroesophageal adenocarcinoma

Abstract Introduction: Gastroesophageal adenocarcinoma (GEA) is one of the most lethal malignancies worldwide. Even with the standard-of-care chemotherapy, 40% of the patients are innately resistant and half of the initial responders develop acquired resistance. The molecular mechanisms underlying the development of resistance in GEA remain largely unknown. Methods: 30 GEA patients who received chemotherapy were included in this cohort. Both pre-and post-treatment primary tissues were collected to generate patient-derived organoids (PDOs). 29 PDOs were treated with chemotherapeutic agents and the cell viability was measured. We also evaluated the correlation between ex-vivo drug responses of PDOs and in-patient clinical responses. Data obtained from whole-exome (WES) and RNA sequencing of the primary tumor and their PDOs were analyzed to identify targetable molecules associated with chemo-resistance. Results: After treatment, the cell viability of the PDOs obtained from chemo-resistant patients was significantly higher than that of chemo-sensitive patients. The variant allele frequency of the PDOs recapitulated their parental tumors. WES of one patient tumor revealed an increased copy number of EGFR, and their PDOs showed high sensitivity to an EGFR inhibitor in the subsequent drug screens. Furthermore, 40% of chemo-sensitive patients had mutations in one or more genes belonging to the adhesion G protein-coupled receptors (GPCRs) family. The adenylate cyclase-activating G protein-coupled receptor signaling pathway was found to be significantly activated only in chemo-sensitive patients. Single-cell RNA sequencing of pre-and post-treatment tissue revealed a broadly shifted transcriptome in epithelial cells. Patients with upregulated cell stemness genes including CTNNB1, MYC, ETS2, and KLF4 had disease recurrence 6 months post-surgery, while patients with unaltered or downregulated stemness genes remain disease-free 18 months until today. Conclusion: Our PDOs not only reflected the genetic profile of primary tumors but also recapitulated patient responses to chemotherapy. Genomic analysis revealed that mutations in a few GPCR genes might have a role in increased sensitivity to chemotherapy. In addition, the transcriptomic analysis showed stemness genes were associated with disease recurrence. Our GEA PDO platform allows the identification and the subsequent verification of resistant-conferring targets, providing insight for novel therapeutic combinations. Funding: This project is supported by the Cancer Research Society (CCS), and the Department of Defence (DoD), USA. Citation Format: Mingyang Kong, Swneke Bailey, Veena Sangwan, Lorenzo Ferri. Identify targetable molecular drivers of chemotherapy resistance in gastroesophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3068.

Comparison of Fecundity Traits between Mauremys reevesii and Trachemys scripta based on Gene Family Analysis

Background: Chinese pond turtle (CPT, Mauremys reevesii) is a typical species with temperature-dependent sex determination (TSD). Pond slider turtle (PST, Trachemys scripta) is considered one of the worst invasive species in the world. In order to analyze the adaptive evolution of fecundity traits in CPT and TSD, the rapidly evolving gene families were obtained and functional enrichment analysis was performed. At the same time, CPT and PST species-specific genes were also enriched for their fecundity traits. Methods: Orthofinder software was employed to search homologous genes based on protein sequence. CAFÉ software was used to obtain the expansion and contraction gene families of CPT and PST. Then, GO terms and pathway enrichment analyses were performed using TBtools software and Swissprot database. Result: In CPT, 159 rapid expansion gene families GO enrichment results showed that the reproductive activities related pathways were mainly three significant enrichment process, spermatogenesis, steroid hormone mediated signaling pathway and male gamete generation. 2 rapidly contraction gene families showed that there was no significant enrichment pathway related to reproduction. The pathway of significant enrichment were RNA biosynthetic process, regulation of RNA metabolic process and regulation of cellular metabolic process. The gene families unique to the species were significantly enriched in pathways DNA replication-dependent nucleosome organization, DNA replication-dependent nucleosome assembly and negative regulation of megakaryocyte differentiation. In PST, 15 rapid expansion gene families GO enrichment results showed that the most significant GO terms mainly consisted of biological processes which were involved in endocrine process, regulation of endocrine process and cellular response to steroid hormone stimulus, but spermatogenesis and male gamete generation gene families also expanded. 58 rapidly contraction gene families showed that the pathways of significant enrichment were endocrine process, cellular response to steroid hormone stimulus and regulation of endocrine process. At the same time, estrogen secretion, binding of sperm to zona pellucida and sperm-egg recognition gene family also rapidly contraction. The gene families unique to the species were significantly enriched in pathways adenylate cyclase-activating G protein-coupled receptor signaling pathway, double-strand break repair and regulation of potassium ion transport.

GIRK3 deletion facilitates kappa opioid signaling in chondrocytes, delays vascularization and promotes bone lengthening in mice.

Long bones are formed and repaired through the process of endochondral ossification. Activation of G protein-coupled receptor (GPCR) signaling pathways is crucial for skeletal development and long bone growth. G protein-gated inwardly-rectifying K+ (GIRK) channel genes are key functional components and effectors of GPCR signaling pathways in excitable cells of the heart and brain, but their roles in non-excitable cells that directly contribute to endochondral bone formation have not been studied. In this study, we analyzed skeletal phenotypes of Girk2-/-, Girk3-/- and Girk2/3-/- mice. Bones from 12-week-old Girk2-/- mice were normal in length, but femurs and tibiae from Girk3-/- and Girk2/3-/- mice were longer than age-matched controls at 12-weeks-old. Epiphyseal chondrocytes from 5-day-old Girk3-/- mice expressed higher levels of genes involved in collagen chain trimerization and collagen fibril assembly, lower levels of genes encoding VEGF receptors, and produced larger micromasses than wildtype chondrocytes in vitro. Girk3-/- chondrocytes were also more responsive to the kappa opioid receptor (KOR) ligand dynorphin, as evidenced by greater pCREB expression, greater cAMP and GAG production, and upregulation of Col2a1 and Sox9 transcripts. Imaging studies showed that Kdr (Vegfr2) and endomucin expression was dramatically reduced in bones from young Girk3-/- mice, supporting a role for delayed vasculogenesis and extended postnatal endochondral bone growth. Together these data indicate that GIRK3 controls several processes involved in bone lengthening.

Near-infrared photoimmunotherapy induced tumor cell death enhances tumor dendritic cell migration.

Near-infrared photoimmunotherapy (NIR-PIT) selectively kills tumor cells to which the photo-absorber dye IR700DX-conjugated antibodies are bound and induces a systemic anti-tumor immune response. NIR-PIT induces immunogenic cell death (ICD), releases damage-associated molecular patterns (DAMPs) molecules from dying tumor cells, and activates dendritic cells (DCs). However, it is unclear whether NIR-PIT affects migration of tumor-infiltrating (Ti)-DCs to draining lymph nodes (dLNs), where a systemic anti-tumor response is induced. Here, we utilized in vivo photolabeling of Ti-DCs in tumors in photoconvertible protein KikumeGreen-Red (KikGR) mice to show that NIR-PIT enhanced migration of Ti-DCs including cDC1s, cDC2s, and CD326+ DCs to dLNs. This effect was abolished by blocking adenosine triphosphate (ATP), one of the DAMPs molecules, as well as by inhibition of Gαi signaling by pertussis toxin. Thus, ICD induction by NIR-PIT stimulates Ti-DC migration to dLNs via ATP-P2X7 receptor and Gαi protein-coupled receptor signaling pathways and may augment tumor antigen presentation to induce anti-tumor T cells in dLNs.

Bioinformatics study of the potential therapeutic effects of ginsenoside Rf in reversing nonalcoholic fatty liver disease

ObjectiveGinsenoside Rf, a tetracyclic triterpenoid only present in Panax ginseng, has been proven to relieve lipid metabolism and inflammatory reactions, which can be a potential treatment for nonalcoholic fatty liver disease (NAFLD). Therefore, this study aimed to reveal the underlying mechanisms of ginsenoside Rf in the treatment of early-stage NAFLD (NAFL) by using a bioinformatics method and biological experiments. MethodsTarget genes associated with NAFL were screened from the Gene Expression Omnibus (GEO) database, a database repository of high-throughput gene expression data and hybridization arrays, chips, and microarrays. Subsequently, gene set enrichment analysis was performed by using Gene Ontology enrichment analysis tool. Then, the binding capacity between ginsenoside Rf and NAFL-related targets was evaluated by molecular docking. Finally, the FFA-induced HepG2 cell model treated with ginsenoside Rf was adopted to verify the effect of ginsenoside Rf and the related mechanisms. ResultsThere were 41 common differentially expressed genes in the GEO dataset. Gene Ontology and Reactome pathway enrichment analysis of the differentially expressed genes showed that many pathways could be related to the pathogenesis of NAFL, including those participating in the cytokine-mediated signaling pathway, G protein-coupled receptor signaling pathway, and response to lipopolysaccharide. Finally, the qRT–PCR analysis results indicated that ginsenoside Rf therapy could ameliorate the transcription of ANXA2, BAZ1A, DNMT3L and MMP9. ConclusionOur research discovered the relevant mechanisms and basic pharmacological effects of ginsenoside Rf in the treatment of NAFL. These results might facilitate the development of ginsenoside Rf as an alternative medication for NAFL.