Protein-coupled Receptor Activation Research Articles

Exploring the activation mechanism of metabotropic glutamate receptor 2

Homomeric dimerization of metabotropic glutamate receptors (mGlus) is essential for the modulation of their functions and represents a promising avenue for the development of novel therapeutic approaches to address central nervous system diseases. Yet, the scarcity of detailed molecular and energetic data on mGlu2 impedes our in-depth comprehension of their activation process. Here, we employ computational simulation methods to elucidate the activation process and key events associated with the mGlu2, including a detailed analysis of its conformational transitions, the binding of agonists, Gi protein coupling, and the guanosine diphosphate (GDP) release. Our results demonstrate that the activation of mGlu2 is a stepwise process and several energy barriers need to be overcome. Moreover, we also identify the rate-determining step of the mGlu2's transition from the agonist-bound state to its active state. From the perspective of free-energy analysis, we find that the conformational dynamics of mGlu2's subunit follow coupled rather than discrete, independent actions. Asymmetric dimerization is critical for receptor activation. Our calculation results are consistent with the observation of cross-linking and fluorescent-labeled blot experiments, thus illustrating the reliability of our calculations. Besides, we also identify potential key residues in the Gi protein binding position on mGlu2, mGlu2 dimer's TM6-TM6 interface, and Gi α5 helix by the change of energy barriers after mutation. The implications of our findings could lead to a more comprehensive grasp of class C G protein-coupled receptor activation.

Targetable leukaemia dependency on noncanonical PI3Kγ signalling.

Phosphoinositide-3-kinase-γ (PI3Kγ) is implicated as a target to repolarize tumour-associated macrophages and promote antitumour immune responses in solid cancers1-4. However, cancer cell-intrinsic roles of PI3Kγ are unclear. Here, by integrating unbiased genome-wide CRISPR interference screening with functional analyses across acute leukaemias, we define a selective dependency on the PI3Kγ complex in a high-risk subset that includes myeloid, lymphoid and dendritic lineages. This dependency is characterized by innate inflammatory signalling and activation of phosphoinositide 3-kinase regulatory subunit 5 (PIK3R5), which encodes a regulatory subunit of PI3Kγ5 and stabilizes the active enzymatic complex. We identify p21 (RAC1)-activated kinase 1 (PAK1) as a noncanonical substrate of PI3Kγ that mediates this cell-intrinsic dependency and find that dephosphorylation of PAK1 by PI3Kγ inhibition impairs mitochondrial oxidative phosphorylation. Treatment with the selective PI3Kγ inhibitor eganelisib is effective in leukaemias with activated PIK3R5. In addition, the combination of eganelisib and cytarabine prolongs survival over either agent alone, even in patient-derived leukaemia xenografts with low baseline PIK3R5 expression, as residual leukaemia cells after cytarabine treatment have elevated G protein-coupled purinergic receptor activity and PAK1 phosphorylation. Together, our study reveals a targetable dependency on PI3Kγ-PAK1 signalling that is amenable to near-term evaluation in patients with acute leukaemia.

Bioinformatic Identification of Signaling Pathways and Hub Genes in Vascular Dementia.

Vascular dementia (VaD) is a prevalent neurodegenerative disease characterized by cognitive impairment due to cerebrovascular factors, affecting a significant portion of the aging population and highlighting the critical need to understand specific targets and mechanisms for effective prevention and treatment strategies. We aimed to identify pathways and crucial genes involved in the progression of VaD through bioinformatics analysis and subsequently validate these findings. We conducted differential expression analysis, Weighted Gene Co-expression Network Analysis (WGCNA), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and Protein-Protein Interaction (PPI) analysis. We utilized pheochromocytoma 12 (PC12) cells to create an in vitro oxygen-glucose deprivation (OGD) model. We investigated the impact of overexpression and interference of adrenoceptor alpha 1D (ADRA1D) on OGD PC12 cells using TdT-mediated dUTP nick-end labeling (TUNEL), reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot (WB), and Fluo-3-pentaacetoxymethyl ester (Fluo-3 AM) analysis. We found 187 differentially expressed genes (DEGs) in the red module that were strongly associated with VaD and were primarily enriched in vasoconstriction, G protein-coupled amine receptor activity, and neuroactive ligand-receptor interaction, mitogen-activated protein kinase (MAPK) signaling pathway, and cell adhesion. Among these pathways, we identified ADRA1D as a gene shared by vasoconstriction, G protein-coupled amine receptor activity, and neuroactive ligand-receptor interaction. The TUNEL assay revealed a significant decrease in PC12 cell apoptosis with ADRA1D overexpression (p < 0.01) and a significant increase in apoptosis upon silencing ADRA1D (p < 0.01). RT-qPCR and WB analysis revealed elevated ADRA1D expression (p < 0.001) and decreased phospholipase C beta (PLCβ) and inositol 1,4,5-trisphosphate receptor (IP3R) expression (p < 0.05) with ADRA1D overexpression. Moreover, the Fluo-3 AM assessment indicated significantly lower intracellular Ca2+ levels with ADRA1D overexpression (p < 0.001). Conversely, interference with ADRA1D yielded opposite results. Our study provides a new perspective on the pathogenic mechanisms of VaD and potential avenues for therapeutic intervention. The results highlight the role of ADRA1D in modulating cellular responses to OGD and VaD, suggesting its potential as a target for VaD treatment.

Mechano-sensitivity of β2-adrenoceptors enhances constitutive activation of cAMP generation that is inhibited by inverse agonists

The concept of agonist-independent signalling that can be attenuated by inverse agonists is a fundamental element of the cubic ternary complex model of G protein-coupled receptor (GPCR) activation. This model shows how a GPCR can exist in two conformational states in the absence of ligands; an inactive R state and an active R* state that differ in their affinities for agonists, inverse agonists, and G-protein alpha subunits. The proportion of R* receptors that exist in the absence of agonists determines the level of constitutive receptor activity. In this study we demonstrate that mechanical stimulation can induce β2-adrenoceptor agonist-independent Gs-mediated cAMP signalling that is sensitive to inhibition by inverse agonists such as ICI-118551 and propranolol. The size of the mechano-sensitive response is dependent on the cell surface receptor expression level in HEK293G cells, is still observed in a ligand-binding deficient D113A mutant β2-adrenoceptor and can be attenuated by site-directed mutagenesis of the extracellular N-glycosylation sites on the N-terminus and second extracellular loop of the β2-adrenoceptor. Similar mechano-sensitive agonist-independent responses are observed in HEK293G cells overexpressing the A2A-adenosine receptor. These data provide new insights into how agonist-independent constitutive receptor activity can be enhanced by mechanical stimulation and regulated by inverse agonists.

Antidepressant and anxiolytic potential of Citrus reticulata Blanco essential oil: a network pharmacology and animal model study.

Citrus reticulata Blanco essential oil (CBEO) has attracted increasing attention as a potential treatment for depression and anxiety in recent years. However, there is limited evidence regarding the active compounds responsible for its therapeutic effects. In addition, substantial amounts of CBEO and prolonged therapy are often required. This study aims to investigate the rapid acting antidepressant and anxiolytic effects of CBEO, identify the underlying composition as well as optimize its dosage and duration. CBEO composition was determined using gas chromatography-mass spectrometry (GC-MS), and the corresponding targets were obtained from the SwissTargetPrediction database. Depression-related targets were collected from DisGeNET, GeneCards, Therapeutic Target Database, and Online Mendelian Inheritance in Man. Subsequently, the overlap between CBEO and depression targets was utilized to build a network diagram depicting the relationship between the active ingredients and targets using Cytoscape software. The STRING database facilitated the construction of a protein-protein interaction network, and the Ma'ayan Laboratory Enrichment tool was employed for Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Wiki pathway analyses. Molecular docking was conducted using AutoDock Vina and Discovery Studio Visualizer. Topological analysis predicted the main antidepressant active ingredients in CBEO. A mixture of these compounds was prepared based on their relative GC-MS ratios. Tail suspension test, elevated plus maze, corticosterone-induced PC12 cells, and lipopolysaccharide (LPS)-induced BV2 cells were used to validate the antidepressant and anxiolytic potential of CBEO and CBEO's main bioactive constituents. CBEO contains 18 components that target 121 proteins. We identified 595 targets associated with depression; among them, 29 targets were located between essential oils and depression. Topological results revealed that linalool, p-cymene, α-terpinene, terpinen-4-ol, and α-terpineol were the major active compounds of CBEO in the management of depression. GO analysis identified G protein-coupled opioid receptor activity, phospholipase C-activating G protein-coupled receptor, and neuron projections that were mostly related to molecular functions, cellular components, and biological processes. Neuroactive ligand-receptor interactions, chemical carcinogenesis, and calcium signaling pathways were the major pathways identified in KEGG analysis. Molecular docking showed that the main bioactive ingredients of CBEO had favorable binding affinities for Protein-Protein Interaction's hub proteins, including OPRM1, PTGS2, ESR1, SLC6A4, DRD2, and NR3C1. These five compounds were then mixed at 0.8:5:0.6:2:1 (w/w) ratio to form a CBEO antidepressant active compound mixture. An acute intranasal treatment of CBEO (25mg/kg) only demonstrated an antidepressant effect, whereas the main bioactive compounds combination (12.5mg/kg) illustrated both antidepressant and anxiolytic effects in mice. Linalool, p-cymene, and terpinene-4-ol exhibited neuroprotective and anti-neuroinflammation in the in vitro study, while these effects were not observed for α-terpinene and α-terpineol. Linalool, p-cymene, α-terpinene, terpinen-4-ol, and α-terpineol cymene might be mainly contributing to CBEO's antidepressant effect by regulating neuroactive ligand-receptor interaction, neuron projection, and receptor signaling pathway. A mixture of these compounds showed rapid antidepressant potential via intranasal administration, which was comparable to that of CBEO. The mixture also exhibited an anxiolytic effect while not seen in CBEO.

Recording physiological history of cells with chemical labeling.

Recordings of the physiological history of cells provide insights into biological processes, yet obtaining such recordings is a challenge. To address this, we introduce a method to record transient cellular events for later analysis. We designed proteins that become labeled in the presence of both a specific cellular activity and a fluorescent substrate. The recording period is set by the presence of the substrate, whereas the cellular activity controls the degree of the labeling. The use of distinguishable substrates enabled the recording of successive periods of activity. We recorded protein-protein interactions, G protein-coupled receptor activation, and increases in intracellular calcium. Recordings of elevated calcium levels allowed selections of cells from heterogeneous populations for transcriptomic analysis and tracking of neuronal activities in flies and zebrafish.

TCHH as a Novel Prognostic Biomarker for Patients with Gastric Cancer by Bioinformatics Analysis.

This study aims to investigate the clinicopathological significance and prognostic value of Trichohyalin (TCHH) in gastric cancer patients through bioinformatics analysis. Data on TCHH expression and clinicopathological information were sourced from The Cancer Genome Atlas (TCGA). The Wilcoxon signed-rank test was used for evaluating the correlation between TCHH mRNA expression levels and clinicopathological features. The predictive significance of TCHH mRNA expression for overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) in patients with gastric cancer was assessed using Cox regression models. Furthermore, measures of immune cell infiltration in gastric cancer were made, and studies of gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were also carried out to investigate the possible roles of TCHH in patients with gastric cancer. Compared to normal tissues, gastric cancer had a considerably higher expression of TCHH mRNA (P < 0.05). Wilcoxon analysis revealed a significant association between TCHH mRNA expression and the pathologic M stage (P = 0.017). High TPMT mRNA levels were also correlated with worse OS, DFS, and PFI in gastric cancer patients (both P < 0.05). TCHH showed significant negative correlations with the levels of NK CD56dim infiltration (r = -0.157, p = 0.002), Th17 cells infiltration (r = -0.235, P < 0.001), and Th2 infiltration (r = -0.195, P < 0.001). Furthermore, enrichment analysis indicated potential involvement in intermediate filament cytoskeleton organization, DNA methylation in gamete generation, cell-cell recognition, and G protein-coupled peptide receptor (GPCRs) activity. The level of TCHH mRNA may serve as a novel prognostic biomarker for gastric cancer patients.

Biased Agonism or “Biaism” for Dummies: A Commentary

That signaling bias is a nth level of complexity in the understanding of G protein-coupled receptor (GPCR) activation is a first fact. That its exhaustive description, including the mode d’emploi of its quantitative measurement, remains a challenge is a second fact. That the use of this concept is promising for the design of drug candidates is a third fact. That the translation of signaling biases observed into in vivo specific effects is well documented is a fourth fact. However, the road to apply those aspects of receptology to a systematic description of a ligand and, a fortiori, of a drug candidate, still necessitates a huge body of studies. In the present commentary, the merits of the molecular description of receptor bias signaling are highlighted and the ligand induced-fit impact on GPCR structure, as well as on the functional repertoire of GPCRs, is discussed. An emphasis is given to the practical aspects during drug design, and, thus, the practical limitations of the current approaches, particularly in the context of as soon as the data are transferred to more integrated/living systems, might be a major limitation.

Seascapes Shaped the Local Adaptation and Population Structure of South China Coast Yellowfin Seabream (Acanthopagrus latus).

Understanding the genetic composition and regional adaptation of marine species under environmental heterogeneity and fishing pressure is crucial for responsible management. In order to understand the genetic diversity and adaptability of yellowfin seabream (Acanthopagrus latus) along southern China coast, this study was conducted a seascape genome analysis on yellowfin seabream from the ecologically diverse coast, spanning over 1600 km. A total of 92 yellowfin seabream individuals from 15 sites were performed whole-genome resequencing, and 4,383,564 high-quality single nucleotide polymorphisms (SNPs) were called. By conducting a genotype-environment association analysis, 29,951 adaptive and 4,328,299 neutral SNPs were identified. The yellowfin seabream exhibited two distinct population structures, despite high gene flow between sites. The seascape genome analysis revealed that genetic structure was influenced by a variety of factors including salinity gradients, habitat distance, and ocean currents. The frequency of allelic variation at the candidate loci changed with the salinity gradient. Annotation of these loci revealed that most of the genes are associated with osmoregulation, such as kcnab2a, kcnk5a, and slc47a1. These genes are significantly enriched in pathways associated with ion transport including G protein-coupled receptor activity, transmembrane signaling receptor activity, and transporter activity. Overall, our findings provide insights into how seascape heterogeneity affects adaptive evolution, while providing important information for regional management in yellowfin seabream populations.

Integrated network pharmacology and comprehensive bioinformatics identifying the mechanisms and molecular targets of Jieyu Anshen Granules for treating comorbidity with Alzheimer's disease and depression

Jieyu Anshen Granules (JY) is a traditional Chinese medicine formula commonly used as an adjuvant treatment for Alzheimer's disease (AD) complicated with depression. However, the specific underlying mechanisms and therapeutic targets of JY remain unclear. We used the TCMSP, TCMID, BATMAN-TCM, and other databases to screen the components and assumed targets of JY. Next, the GEO and DisGeNET databases were used to identify the related targets of both AD and Major Depressive Disorder (MDD). Enrichment analyses of core targets were performed, and the main components and core targets of JY for the comorbidity of AD and MDD were identified via protein–protein interaction (PPI) network construction and machine learning algorithms. Lastly, we verified binding affinity using the AutoDock software and molecular docking was performed. A total of 171 components were identified from JY, and 979 targets were obtained from the screening process. Bioinformatics analysis displayed 397 differentially expressed genes (DEGs) were shared by AD and MDD. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis revealed significant enrichment of genes associated with neurotransmitter receptor activity, G protein-coupled amine receptor activity, as well as signaling pathways such as the cAMP signaling pathway, PI3K-Akt signaling pathway, and cholinergic synapses. Through the PPI network and machine learning, we identified three hub genes (Ataxia telangiectasia-mutated gene [ATM], Colony stimulating factor 1 receptor [CSF1R], EPH receptor B2 [EPHB2]) as potential therapeutic targets. Molecular docking confirmed that the components of JY (Mairin, Hederagenin, 3-Epioleanolic Acid) could effectively bind to multiple key targets. This study revealed the effective components and potential mechanisms associated with JY treatment regarding AD and MDD comorbidities, offering valuable insights into promising therapeutic targets for subsequent studies.

A tool kit of highly selective and sensitive genetically encoded neuropeptide sensors.

Neuropeptides are key signaling molecules in the endocrine and nervous systems that regulate many critical physiological processes. Understanding the functions of neuropeptides in vivo requires the ability to monitor their dynamics with high specificity, sensitivity, and spatiotemporal resolution. However, this has been hindered by the lack of direct, sensitive, and noninvasive tools. We developed a series of GRAB (G protein-coupled receptor activation‒based) sensors for detecting somatostatin (SST), corticotropin-releasing factor (CRF), cholecystokinin (CCK), neuropeptide Y (NPY), neurotensin (NTS), and vasoactive intestinal peptide (VIP). These fluorescent sensors, which enable detection of specific neuropeptide binding at nanomolar concentrations, establish a robust tool kit for studying the release, function, and regulation of neuropeptides under both physiological and pathophysiological conditions.

Exploring the mechanism of action of Qian Lie Xing Fang during the treatment of benign prostatic hyperplasia via network pharmacology and molecular dynamics simulation analyses.

This study aimed to explore the historical research progress on benign prostatic hyperplasia from the perspective of traditional Chinese medicine theory and the treatment of benign prostatic hyperplasia (BPH) with Qian Lie Xing Fang (QLXF) via the warming and tonifying of kidney yang, promotion of blood circulation, and clearing of meridians. First, network pharmacology analysis was used to screen and identify possible pathways for BPH treatment with QLXF. Subsequently, molecular docking analysis helped explore the mechanism of action by which the components of QLXF affected androgen receptor (AR) and type 5 phosphodiesterase inhibitor (PDE-5) levels. Targets for treatment with QLXF were identified from the online Mendelian inheritance in man and DisGeNET databases. BPH-related genes were identified using GeneCards and online Mendelian inheritance in man databases, and their intersection was used to construct a protein-protein interaction network analysis graph. Subsequently, gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses were performed. The semiflexible docking of the ingredients of QLXF acting on the 2 targets was performed via molecular docking and molecular dynamics simulation, to elucidate the mechanism of action by which the active ingredients affect AR and PDE-5 levels further. This enabled us to explore the pattern of interactions between small active ingredient molecules, the target protein, and the stability after binding at the microscopic level. Gene ontology enrichment analysis showed that QLXF affected several processes, such as DNA transcription factor binding, kinase binding, protein homodimerization activity, protein structure domain-specific binding, and protein-coupled amine receptor activity in BPH patients. KEGG results showed that chemical carcinogenic reactive oxidative species and the JAK-STAT, Pl3k-Akt, FoxO, NF-κB, and other pathways were significantly enriched. Conducting molecular docking studies to investigate the interaction of active components from QLXF with AR and PDE-5, it was found that MOL002260 may possess the potential to inhibit PDE-5 activity, while MOL010578 may exhibit the capability to inhibit AR activity. QLXF is closely associated with various biological processes and KEGG signaling pathways related to BPH. The active ingredients of QLXF were investigated for their interactions with AR and PDE-5, with a primary focus on the small molecules MOL002260 and MOL010578.

Identification and characterization of ecdysis-related neuropeptides in the lone star tick Amblyomma americanum.

The lone star tick, Amblyomma americanum, is an important ectoparasite known for transmitting diseases to humans and animals. Ecdysis-related neuropeptides (ERNs) control behaviors crucial for arthropods to shed exoskeletons. However, ERN identification and characterization in A. americanum remain incomplete. We investigated ERNs in A. americanum, assessing their evolutionary relationships, protein properties, and functions. Phylogeny, sequence alignment, and domain structures of ERNs were analyzed. ERN functionality was explored using enrichment analysis, and developmental and tissue-specific ERN expression profiles were examined using qPCR and RNAi experiments. The study shows that ERN catalogs (i.e., eclosion hormone, corazonin, and bursicon) are found in most arachnids, and these ERNs in A. americanum have high evolutionary relatedness with other tick species. Protein modeling analysis indicates that ERNs primarily consist of secondary structures and protein stabilizing forces (i.e., hydrophobic clusters, hydrogen bond networks, and salt bridges). Gene functional analysis shows that ENRs are involved in many ecdysis-related functions, including ecdysis-triggering hormone activity, neuropeptide signaling pathway, and corazonin receptor binding. Bursicon proteins have functions in chitin binding and G protein-coupled receptor activity and strong interactions with leucine-rich repeat-containing G-protein coupled receptor 5. ERNs were expressed in higher levels in newly molted adults and synganglia. RNAi-mediated knockdown of burs α and burs β expression led to a significant decrease in the expression of an antimicrobial peptide, defensin, suggesting they might act in signaling or regulatory pathways that control the expression of immune-related genes. Arthropods are vulnerable immediately after molting because new cuticles are soft and susceptible to injury and pathogen infections. Bursicon homodimers act in prophylactic immunity during this vulnerable period by increasing the synthesis of transcripts encoding antimicrobial peptides to protect them from microbial invasion. Collectively, the expression pattern and characterization of ERNs in this study contribute to a deeper understanding of the physiological processes in A. americanum.

Intracellular cAMP Signaling Pathway via Gs Protein-Coupled Receptor Activation in Rat Primary Cultured Trigeminal Ganglion Cells.

G protein-coupled receptors in trigeminal ganglion (TG) neurons are often associated with sensory mechanisms, including nociception. We have previously reported the expression of P2Y12 receptors, which are Gi protein-coupled receptors, in TG cells. Activating P2Y12 receptors decreased the intracellular free Ca2+ concentration ([Ca2+]i). This indicated that intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels can mediate Ca2+ signaling in TG cells. Here, we report more extensive-expression patterns of Gs protein-coupled receptors in primary cultured TG neurons isolated from 7-day-old newborn Wistar rats and further examine the roles of these receptors in cAMP signaling using the BacMam sensor in these neurons. To identify TG neurons, we also measured [Ca2+]i using fura-2 in TG cells and measured intracellular cAMP levels. TG neurons were positive for Gαs protein-coupled receptors, beta-2 adrenergic (β2), calcitonin gene-related peptide (CGRP), adenosine A2A (A2A), dopamine 1 (D1), prostaglandin I2 (IP), and 5-hydroxytriptamine 4 (5-HT4) receptor. Application of forskolin (FSK), an activator of adenylyl cyclase, transiently increased intracellular cAMP levels in TG neurons. The application of a phosphodiesterase inhibitor augmented the FSK-elicited intracellular cAMP level increase. These increases were significantly suppressed by the application of SQ22536, an adenylyl cyclase inhibitor, in TG neurons. Application of agonists for β2, CGRP, A2A, D1-like, IP, and 5-HT4 receptors increased intracellular cAMP levels. These increases were SQ22536-sensitive. These results suggested that TG neurons express β2, CGRP, A2A, D1, IP, and 5-HT4 receptors, and the activations of these Gαs protein-coupled receptors increase intracellular cAMP levels by activating adenylyl cyclase.

G protein-coupled estrogen receptor activation by bisphenol-A disrupts lipid metabolism and induces ferroptosis in the liver

As a metabolic disruptor, bisphenol A (BPA) has been widely reported to disrupt lipid balance. Moreover, BPA has gained significant attention due to its estrogenic activity. While both ferroptosis and the G-protein-coupled estrogen receptor (GPER) have been implicated in lipid metabolism, their link to BPA-induced lipid accumulation remains unclear. In this study, chickens were randomly assigned to three groups and housed them for 4 weeks: a control group (0 μg/L BPA), a low dose group (50 μg/L BPA) and a high dose group (5000 μg/L BPA) to investigate the underlying mechanism of BPA-induced hepatotoxicity. Our results showed that BPA exposure significantly increased the contents of TG, TC, and LDL-C while decreasing HDL-C levels. We also found that BPA treatment altered the levels of genes involved in fatty acid β-oxidation (ampkα, cpt-1, and ppaα), synthesis (acc, fas, scd-1, and srebp-1) and absorption (lpl and cd36). Moreover, the results showed that the BPA group had higher levels of IL-1β, IL-18 and TNF-α. These results indicated that BPA exposure disrupted lipid metabolism and induced inflammation in the liver. We also demonstrated that BPA caused hepatic ferroptosis by raising iron content and the expression of genes related to lipid peroxidation (lpcat3, acsl4 and alox15), while reducing the expression of antioxidant system-associated genes (gpx4, slc7a11 and slc3a2). Importantly, BPA remarkably activated GPER expression in the liver. Interestingly, inhibition of GPER remarkably ameliorated BPA-induced lipid metabolism disorder, inflammatory response, and ferroptosis, indicating the crucial role of GPER in BPA-induced liver abnormalities. These findings highlight the link between GPER and ferroptosis in BPA-induced hepatotoxicity, providing new insights into the potential hazard of BPA.

G protein-coupled estrogen receptor (GPER) selective agonist G1 attenuates the neurobehavioral, molecular and biochemical alterations induced in a valproic acid rat model of autism

AimsAutism Spectrum Disorder (ASD) is a neurodevelopmental disorder with a rising prevalence in boys rather than girls. G protein-coupled estrogen receptor (GPER) activation by its agonist G1 showed a neuroprotective effect, similar to estradiol. The present study aimed to examine the potential of the selective GPER agonist G1 therapy on the behavioral, histopathological, biochemical, and molecular alterations induced in a valproic acid (VPA)-rat model of autism. Main methodsVPA (500 mg/kg) was intraperitoneally administered to female Wistar rats (on gestational day 12.5) to induce the VPA-rat model of autism. The male offspring were intraperitoneally administered with G1 (10 and 20 μg/kg) for 21 days. After the treatment process, rats performed behavioral assessments. Then, sera and hippocampi were collected for biochemical and histopathological examinations and gene expression analysis. Key findingsGPER agonist G1 attenuated behavioral deficits, including hyperactivity, declined spatial memory and social preferences, anxiety, and repetitive behavior in VPA rats. G1 improved neurotransmission and reduced oxidative stress and histological alteration in the hippocampus. G1 reduced serum free T levels and interleukin-1β and up-regulated GPER, RORα, and aromatase gene expression levels in the hippocampus. SignificanceThe present study suggests that activation of GPER by its selective agonist G1 altered the derangements induced in a VPA-rat model of autism. G1 normalized free T levels via up-regulation of hippocampal RORα and aromatase gene expression. G1 provoked estradiol neuroprotective functions via up-regulation of hippocampal GPER expression. The G1 treatment and GPER activation provide a promising therapeutic approach to counteract the autistic-like symptoms.

RNA sequencing to characterize pathways in EGFR-mutated non-small cell lung cancer.

e21019 Background: The purpose of our study is to explore patterns in gene expression for patients with EGFR-mutated non-small cell lung cancer (NSCLC). Methods: 287 patients with EGFR-mutated lung cancer were identified using the Tempus Lens database. Raw RNA sequence expression data was requested from TEMPUS XT RNA analysis. Sequences were processed through the EdgeR analysis in the R programming environment. Differential abundance analysis was done to further filter out genes and Benjamini-Hochberg correction selected genes with a corrected p-value of &lt; 0.05 for further pathway analysis. Pathway analysis was performed using GSEA (Gene Set Enrichment Analysis). Enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and GSEA Molecular Signatures Database (MsigDB) was performed for overexpressed and under-expressed genes in patients. Results: Pathway analysis run against the KEGG reference database and GSEA MSigDB helped to illustrate genes shared in activated and suppressed pathways in our patient cohort. For example, using the KEGG reference database, enriched pathways meeting p value significance threshold of &lt; 0.005 included the nicotine addiction, olfactory transduction, and cortisol synthesis and secretion pathways, amongst others. Suppressed pathways using the KEGG reference database meeting p value significance threshold of &lt; 0.005 included fat digestion and absorption, vitamin digestion and absorption, PPAR signaling pathway, and drug metabolism by cytochrome P450 and other enzymes. Using the GSEA MSigDB, enriched pathways included G protein-coupled receptor activity, sensory perception, synaptic signaling and trans-synaptic signaling, meanwhile suppressed pathways included cytolysis by host of symbiont cells, negative regulation of very-low-density lipoprotein particle remodeling, and oxidoreductase activity. Conclusions: Patterns in gene and pathway expression were observed in our cohort of patients with EGFR-mutated NSCLC. Analyzing RNA sequencing pathways showed differential up and down regulation of multiple pathways. For example, suppression of the fat and vitamin digestion and absorption pathways may shed light on processes involved in cancer cachexia and metabolism. Though further work needs to be done, RNA sequencing results for patients with EGFR-mutated lung cancer may lead to information on cancer development and additional therapeutic targets.

A network pharmacological-based study of the mechanism of Liuwei Dihuang pill in the treatment of chronic kidney disease.

Chronic kidney disease (CKD) is a progressive disease that poses a huge economic burden to society. Liuwei Dihuanng pill is an effective treatment for chronic kidney disease, but its treatment mechanism is unclear. The rapid development of network pharmacology has provided new strategies for studying Chinese medicine. The traditional Chinese medicine systems pharmacology database and analysis platform was used to obtain the bioactive components and targets of Liuwei Dihuanng pill. The sources for the CKD-related targets were then obtained from the Genecards, OMIM, TTD, and DisGeNET databases. R was used to identify the intersecting genes for Liuwei Dihuang pill and CKD-related targets. Analysis of protein-protein interactions (PPI) was performed using STRING, and PPI networks and drug-component-target networks were constructed using Cytoscape software. Kyoto encyclopedia of genes and genomes pathway and gene ontology enrichment analyses were performed using R. Finally, molecular docking was performed to determine the binding activity between bioactive components and the targets. After screening and data de-duplication of 74 active components, 209 drug targets, and 14,794 disease targets, a total of 204 drug-disease targets were acquired. Subsequently, a drug-component-target network and PPI network were established. The primary components of Liuwei Dihuang pill included quercetin, stigmasterol, kaempferol, beta-sitosterol, tetrahydroalstonine, kadsurenone, hederagenin, hancinone C, diosgenin, and sitosterol. In addition, JUN, AKT1, TP53, RELA, MAPK1, FOS, TNF, IL6, ESR1, and RXRA were identified as the main targets. Gene ontology function enrichment analysis revealed that these targets were involved in reactive oxygen species metabolic processes, responses to metal ions and to chemical stimuli, G protein-coupled amine receptor activity, and nuclear factor receptor activity. Kyoto encyclopedia of genes and genomes enrichment analysis showed that these targets were involved in the AGE-RAGE signaling pathway, IL-17 signaling pathway, TNF signaling pathway, and so on. Molecular docking results indicated good binding activity between the core targets and core components. The potential mechanism of Liuwei Dihuanng pill in the treatment of CKD was preliminarily discussed in this study, providing a theoretical basis and evidence for further experimental research.

Abstract 5895: Analysis of microbiome in gastric MALT lymphoma

Abstract Gastric MALT (mucosa-associated lymphoid tissue) lymphoma is found at a rate of 50% or more among the types of MALT lymphoma. Majority of patients with gastric MALT lymphoma are infected with Helicobacter pylori and also have clinical improvement with eradication therapy. However, it is unclear how H. pylori cause gastric MALT lymphoma. To explore it, we prospectively collected 13 patients who are newly diagnosed with gastric MALT lymphoma with H. pylori. We gathered samples from normal regions and lesions during endoscopy for diagnosis. Samples of lesion were harvested again after eradication of H. pylori. we sequenced 10 of WES (whole exome sequencing) and 13 of WTS (whole transcriptome sequencing) with these samples. With sequencing data, we aligned the sequenced reads to human reference genome (hg19) using BWA and called the variants with Mutect2. We aligned the reads and calculated TPM (Transcripts Per Million) from WTS with STAR and RSEM. We performed DEG (Differentially Expressed Genes) and GSEA (Gene Set Enrichment Analysis) for three comparison groups which are divided into normal, lesion, and after eradication. Gene fusions were detected using STAR-Fusion and Arriba with WTS. To detect potential microbiome from the samples, we used Kraken2, a taxonomic classification tool. We found that MUC6 was mutated from all samples and some samples have mutated MUC3A, KMT2C, ZNF595, CDC27, etc., and two have BIRC3-MALT1 fusion. From DEG and GSEA analysis, G protein-coupled receptor activity was activated in the group of lesion samples and suppressed in normal samples and eradication samples. Lymphocyte activation and immune response were also activated in the cancerous lesion. Kraken2 classified the reads which were not aligned to the human reference genome as reads from microbiota including H. pylori. The reads of H. pylori were more abundant in the lesion group than the other groups. According to the result of differentially expressed species analysis between the groups of lesion and eradication, the following species were also more abundant in the lesion group; Veillonella dispar, Helicobacter hepaticus, Helicobacter winghamensis, etc. We analyzed genetic alterations and differentially expressed genes and also classified non-human reads as microbiome with sequencing data from gastric MALT lymphoma. Our results suggest how H.pylori infection is related to carcinogenesis of gastric MALT lymphoma. Citation Format: SeungJoo Yoo, Dong-Yeop Shin, Ja Min Byun, Junshik Hong, Hongseok Yun, Youngil Koh, Sung-Soo Yoon. Analysis of microbiome in gastric MALT lymphoma [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 5895.

Integrated UPLC-MS, network pharmacology, and intestinal flora analysis to determine the treatment effect of Qiangzhi decoction on comorbid Tourette syndrome and RRTI.

Tourette syndrome (TS) is a complex neurodevelopmental disorder characterized by vocal and motor tics. Recurrent respiratory tract infection (RRTI), a commonly occurring disease in childhood, correlates with recurrent and severe course of tic symptoms. Qiangzhi decoction (QZD) is a traditional Chinese medicine that can alleviate TS symptoms while reducing the recurrence of RRTI. However, the mechanism of QZD on TS and RRTI remains unclear. This study aimed to determine the treatment effect of QZD on comorbid TS and RRTI by integrating ultrahigh-performance liquid chromatography mass spectrometry (UPLC-MS), network pharmacology, and intestinal flora analysis. The components of QZD were first identified by UPLC-quadrupole (Q)-orbitrap-MS/MS. The mechanism of QZD on comorbid RRTI and TS was investigated by a series of network pharmacological methods, including target prediction and bioinformatics analysis. Finally, a comorbid TS and RRTI rat model was established by intraperitoneal injection of 3,3-iminodipropionitrile (IDPN), cyclophosphamide (CTX), and lipopolysaccharide (LPS). Alteration of gut microbiota in the alleviation of TS and RRTI by QZD was investigated via intestinal flora analysis. The results of UPLC-Q-orbitrap-MS/MS showed that QZD had 96 types of chemical components. The network pharmacology results demonstrated that targets of QZD involved in the treatment of TS and RRTI involved 1045 biological processes (BPs), 109 cellular components (CCs), and 133 molecular functions (MFs), including synaptic and transsynaptic signaling, chemical synaptic transmission, neurotransmitter receptor activity, G protein-coupled amine receptor activity, and serotonin receptor activity, among others. Firmicutes, Bacteroidetes, Coprococcus, and Lachnospiraceae played crucial roles in gut microbiota of a QZD-treated comorbid TS and RRTI model. Our results revealed QZD provided a multicomponent, multitarget, and multipathway synergistic treatment of comorbid TS and RRTI.