Receptor Signaling Research Articles

Endurance exercise attenuates Gαq-RNAi induced hereditary obesity and skeletal muscle dysfunction via improving skeletal muscle Srl/MRCC-I pathway in Drosophila.

G protein alpha q subunit (Gαq) can binds to the G protein-coupled receptor (GPCR) for signaling and is closely related to lipid metabolism. Endurance exercise is an effective means of combating acquired obesity and its complications, but the mechanisms by which endurance exercise modulates hereditary obesity and its complications are unknown. In this study, we achieved knockdown of Gαq in drosophila adipose tissue and skeletal muscle by constructing the Gαq-UAS-RNAi/Ppl-Gal4 and Gαq-UAS-RNAi/Mef2-GAl4 systems. Drosophila were subjected a three-week endurance exercise intervention, and changes in relevant indicators were detected and observed by RT-PCR, ELISA, oil red staining, immunofluorescence staining, and transmission electron microscopy. The results showed that knockdown of Gαq in both adipose tissue and skeletal muscle induced a significant increase in triglycerides accompanied by a decrease in rapid climbing ability, a decrease in Superoxide Dismutase (SOD) activity level, and a decrease in Mitochondrial respiratory chain complexI (MRCC I) content in Drosophila whole body and skeletal muscle, and down-regulated the expression of the G protein alpha q subunit (Gαq), the skeletal muscle myosin heavy chain expression gene (Mhc), mitochondrial biogenesis gene Spargal(the PGC-1alpha homologue in Drosophila). Endurance exercise significantly improved the triglyceride levels in the whole body and skeletal muscle of drosophila with Gαq knockdown in adipose tissue and skeletal muscle, as well as their ability to climb, increased SOD activity level and MRCCI content level, and up-regulated the expression of Gαq, Mhc, and Spargal(Srl). Thus, the present findings suggest that genetic defects in the Gαq gene in adipose and skeletal muscle tissues induce hereditary obesity and skeletal muscle dysfunction, and that endurance exercise attenuates this hereditary obesity and concomitant skeletal muscle dysfunction in drosophila by improving skeletal muscle fiber contractile proteins, mitochondrial function and function, and antioxidant capacity via mediating the Gαq/Mhc, Gαq/Srl/MRCC-I, and Gαq/SOD pathways.

Mechanotransduction governs CD40 function and underlies X-linked hyper-IgM syndrome.

B cell maturation depends on cognate interactions between the T and B cells. Upon interaction with CD40 ligand (CD40L) on T cells, CD40 delivers costimulatory signals alongside B cell antigen receptor (BCR) signaling to regulate affinity maturation and antibody class switch. Mutations affecting CD40-CD40L interactions cause abnormal antibody responses in immunodeficiencies known as X-linked hyper-IgM syndrome (X-HIgM). Here, we study the CD40-mediated mechanotransduction in B cells, which likely occurs during their physical contacts with T cells. We found that CD40 forms catch bond with CD40L that lasts longer at larger forces, both B and T cells exert tension on CD40-CD40L bonds, and force enhances CD40 signaling and antibody class switch. X-HIgM CD40L mutations impair catch bond formation, suppress endogenous tension, and reduce force-enhanced CD40 signaling, leading to deficiencies in antibody class switch. Our findings highlight the role of mechanotransduction in CD40 function and provide insights into the mechanisms underlying X-HIgM syndrome.

Gatekeepers of the brain: Identifying hidden mechanisms of type A GABA receptor signaling and assembly.

Identifying hidden mechanisms of type A GABA receptor signaling and assembly.

Technical challenges of intracellular flow cytometry-based assays as a functional complement to diagnosis of signaling defects of inborn errors of immunity: PI3K pathway as a case of study

BackgroundThe use of next-generation sequencing in inborn errors of immunity (IEI) has considerably increased the identification of novel gene variants, many of which are identified in patients without the described clinical phenotype or with variants of uncertain pathogenic significance in previously described genes. Properly designed functional and cellular assays, many necessarily accomplished by research-based laboratories, reveal the pathogenic consequences of the gene variants and contribute to diagnosis. Activated PI3Kδ syndrome (APDS) is a rare disease that can be divided into APDS1, caused by gain of function (GOF) mutations in PIK3CD gene, and APDS2, with loss of function (LOF) variants in the PIK3R1 gene. Both entities present hyperactivation of the PI3K pathway, which can be analyzed through Akt and S6 phosphorylation status.MethodsOur objective was to perform an accurate, robust, and reproducible functional assay to analyze the phosphorylation status of proteins in the PI3K-Akt-S6 pathway by flow cytometry, to contribute to diagnosis, to monitor treatments, and to establish intra-assay standardization.ResultsWe illustrate the robustness and reproducibility of our experimental procedure in patients with APDS who had high Akt and/or S6 phosphorylation levels at baseline, and after anti-IgM stimulation in B cells. We show the relevance of an appropriate cohort of samples from healthy donors, processed within the same conditions as the suspected samples, in particular the time frame for sample processing once blood is collected.DiscussionWe highlight the importance of B cell stimulation through B cell receptor signaling, which is highly recommended, especially for samples that would be processed more than 24 hours after blood extraction. Also, having a defined experimental procedure is important, including the cytometer setup, which allows cytometer reproducibility for a period of time, enabling the comparison of a sample at different times.

Cell-cycle machinery is critical in regulating uterine steroid hormone for embryo implantation and development.

Proper embryo implantation is necessary for a successful pregnancy. In this issue of the JCI, Aljubran et al. identified the cell cycle regulatory protein cyclin A2 (CCNA2) as a factor in supporting embryo implantation and embryo development. Endometrial stromal cells showed higher levels of CCNA2 in patients undergoing assisted reproductive technology who had successful pregnancies. CCNA2 expression correlated with stromal cell proliferation and the expression of steroid hormone receptors for estrogen (ESR1, also known as ERα) and progesterone (PGR). Notably, loss of Ccna2 in mouse models resulted in infertility. The uteri of these mice were hypoplastic with reduced estrogen sensitivity, resulting in the disruption of stroma cell decidualization and loss of embryo viability after implantation. These findings demonstrate the importance of stroma cell proliferation in preparing the uterus for embryo implantation. They also identify CCNA2 as a coregulator of steroid hormone receptor signaling and suggest that impaired uterine stroma can underly early pregnancy loss.

Chinese herbal medicine, Tongxieyaofang, alleviates diarrhea via gut microbiota remodeling: evidence from network pharmacology and full-length 16S rRNA gene sequencing

BackgroundTongxieyaofang (TXYF) was a traditional Chinese medicine (TCM) formula for the treatment of diarrhea with liver stagnation and spleen deficiency syndrome, but the potential targets and mechanisms have not been fully clarified. This study aims to explore the potential mechanisms of TXYF in alleviating diarrhea using network pharmacology and full-length 16S rRNA gene sequencing.MethodsNetwork pharmacology was applied to identify bioactive compounds and potential targets involved in the role of TXYF in alleviating diarrhea. Meanwhile, a model of diarrhea with liver stagnation and spleen deficiency syndrome was constructed by intragastric administration of Folium senna extract combined with restraint and tail pinch stress. The effect of TXYF on intestinal mucosal microbiota of diarrhea mice was analyzed by full-length 16S rRNA gene sequencing.ResultsNetwork pharmacology analysis showed that kaempferol, wogonin, naringenin, and nobiletin were compounds associated with the efficacy of TXYF. TXYF may alleviate diarrhea via multiple BPs and pathways, including TNF signaling pathway, IL-17 signaling pathway, and Toll-like receptor signaling pathway, which are involved in TCM-gut microbiota-host interactions. Then, we found that TXYF administration reshaped the diversity and composition of the intestinal mucosal microbial community of diarrhea mice. Lactobacillus, primarily Lactobacillus johnsonii, was enriched by the administration of TXYF. After TXYF administration, the abundance of Lactobacillus, particularly Lactobacillus johnsonii, was enriched.ConclusionOral administration of TXYF may alleviate diarrhea through remodeling intestinal mucosal microbiota. Promoting the colonization of beneficial commensal bacteria in the intestinal mucosa through gut microbiota-host interactions may be a potential mechanism of TXYF in the treatment of diarrhea.

Short-term exposure to low doses of aflatoxin B1 aggravates nonalcoholic steatohepatitis by TLR4-mediated necroptosis.

Aflatoxin B1 (AFB1), a worldwide mycotoxin found in food and foodstuffs, is a potent hepatotoxin in humans and animals. Non-alcoholic fatty liver disease (NAFLD), a widespread disease, could progress from simple steatosis to non-alcoholic steatohepatitis (NASH), hepatic cirrhosis, and even hepatocellular carcinoma (HCC). To date, little is known concerning the relationship between AFB1 and the progression of NAFLD. The effects of low doses of AFB1 on the development of NASH and their mechanism were investigated in vivo and in vitro. The results in vivo showed that AFB1 at 20 and 40 μg/kg.bw aggravated CDAHFD-induced NASH in mice as demonstrated by increasing the serum and liver lipid accumulation, liver inflammation and injury. The results in vitro showed that AFB1 at 1.0 μM aggravated FFA-induced lipid accumulation, inflammation and cell damage in HepG2 cells. In addition, RNA-seq indicated that necroptosis, Toll like receptor signaling and TNF-α signaling showed a significant change in KEGG pathway enrichment in AFB1 at 40 μg/kg.bw. AFB1 significantly upregulated the mRNA and protein levels of TLR4, RIPK3, p-RIPK3, MLKL and p-MLKL, and increased TUNEL positive cells. Also, immunofluorescence results showed that TUNEL, TLR4, TNF-α had co-localized with RIPK3, respectively. Necroptosis inhibitor (GSK-872) attenuated the aggravating effects of AFB1 on NASH. Knockout of TLR4 inhibited necroptosis and rescued the aggravating effects of AFB1 on NASH. These data indicate that low dose of AFB1 aggravated NASH via TLR4-mediated necroptosis. This suggests that low dose of AFB1 is potentially harmful to animals and humans, as they exacerbate NASH.

FRONTIER-2: A phase 2b, long-term extension, dose-ranging study of oral JNJ-77242113 for the treatment of moderate-to-severe plaque psoriasis.

More patients with moderate-to-severe plaque psoriasis achieved responses with JNJ-77242113, a targeted oral peptide inhibiting interleukin (IL)-23 receptor signaling, versus placebo at Week(W)16 of the phase 2 FRONTIER-1 study. FRONTIER-2, a long-term extension of FRONTIER-1, evaluated JNJ-77242113 through 1 year. FRONTIER-1 participants received JNJ-77242113 at doses from 25mg once daily (QD) to 100mg twice daily (BID) or placebo, through W16. Patients completing FRONTIER-1 could enroll in FRONTIER-2 and continue JNJ-77242113 at the same dose through W52. Those on placebo crossed over to JNJ-77242113 100mg QD for W16-52. Safety follow-up continued through W56. Most (89%) FRONTIER-1 patients continued to FRONTIER-2. Across outcomes, response rates were maintained from W16-52. The highest response rates generally occurred with JNJ-77242113 100mg BID. At W52, 76% of patients achieved up to 75% improvement in Psoriasis Area and Severity Index (PASI75) with 100mg BID; rates of clear or almost clear skin were 64% (PASI90), 74% (Investigator's Global Assessment [IGA]0/1), 40% (PASI100), and 43% (IGA 0). From W16-56, 59% of JNJ-77242113-treated patients had ≥1 adverse events (AEs). Serious AEs, considered unrelated to treatment by investigators, occurred in 4% of patients. The study was limited by the small number of patients in each treatment group and the descriptive nature of the longer-term data. Rates of skin clearance with JNJ-77242113 were durable to 1 year and no safety signals were identified.

The brain and hypertension: how the brain regulates and suffers from blood pressure.

The brain plays several roles in the relationship between blood pressure (BP) and the brain: it acts as the control center for BP regulation, a target organ in hypertension, and a crucial component for cognitive function. This mini-review introduces recent findings on "brain and hypertension" from Hypertension Research and other journals. Activation of the angiotensin II type 1 receptor (AT1R) signaling pathway in the brain causes sympathoexcitation and hypertension. AT1R-associated protein and β-arrestin promote AT1R internalization and suppress AT1R signaling, with brain-specific roles in BP regulation. The brain receives various inputs from the peripheral system, including the heart and kidneys, and controls central sympathetic outflow. The brain mechanism involved in the enhanced cardiac sympathetic afferent reflex and the beneficial effects of renal denervation have been demonstrated. The brain's vulnerability in hypertension includes stroke, with cerebral small vessel disease (SVD) contributing to stroke risk and brain changes. Sex differences and the age of hypertension onset influence these outcomes. High salt intake exacerbates hypertension and stroke risk, with central mechanisms like sympathoexcitation implicated. Hypertension significantly impacts cognitive function, linking to cerebral SVD and cognitive decline. Orthostatic BP regulation abnormalities also emerge as early risk markers for dementia. Improved BP control in hypertensive individuals can significantly reduce the risk of stroke and cognitive decline, as well as cardiovascular disease, enhancing overall brain health and quality of life. Further understanding the brain's role in BP regulation and the pathogenesis of hypertension will facilitate the development of novel hypertension treatments and prevention strategies.

Abstract B026: Identification of small RNAs that function as multi-target therapeutics and as tools for novel target discovery in prostate cancer

Abstract Introduction: A major contributor to prostate cancer (PCa) mortality is the development of resistance to single agents targeting androgen receptor (AR) signaling. We have developed an unbiased shRNA-based negative selection screen to identify small RNAs (shRNAs and siRNAs) that inhibit PCa cell growth and survival (toxic sRNAs). Computational analyses of the most toxic sRNAs identified revealed that each can simultaneously target multiple known AR-coregulatory mRNAs. AR-coregulators modulate AR transcriptional response and are essential to AR signaling. We have proposed that the identified toxic sRNAs inhibit androgen signaling through seed-mediated targeting of androgen regulatory gene networks, resulting in inhibition of cell growth and viability. The goal of the current studies was to begin to analyze the broad therapeutic potential of these toxic sRNAs for PCa. We focused on analyzing i) their potential to thwart cell growth and/or viability when delivered to preclinical models of PCa, and ii) the targetome of the toxic sRNAs as a mean to uncover novel AR-coregulators that represent a targetable axis within androgen signaling. Methods: i) cationic lipid nanoparticles (LNPs), that efficiently deliver the toxic siRNAs to PCa cell lines, were used to deliver toxic siRNAs to organoids grown from PCa patient derived xenografts (PDX) models. 3D image analysis using the Ominer® software package was used to determine organoid count and size, the numbers of nuclei per organoid, and the fraction of dead cells to discriminate between cytotoxic and cytostatic effects. ii) to identify novel AR-coregulators, toxic siRNA direct targets were identified by sequencing of transcripts captured by biotinylated toxic siRNAs. Directly bound targets, were subjected to pathway enrichment and network analysis followed by identification of hub genes (Cytoscape). The effect of individually silencing potential AR-coregulators on the expression of androgen responsive genes was analyzed using Nanostring. Results and Conclusions: i) The effect of the toxic siRNAs on growth and viability of the PCa PDX-derived organoids will be presented and discussed as compelling preliminary data for therapeutic testing in more complex pre-clinical models (PCa PDXs). ii) Based on bioinformatic and downstream analysis of the direct targets of two toxic siRNAs, we selected 12 potentially novel AR- coregulators, with varied known functions including chromatin modifiers, transcriptional regulators, etc. Silencing of several of these potential AR-coregulators significantly affect expression of numerous AR responsive genes. Future experiments will focus on analyzing their interaction and functional association with the AR (i.e. effect of AR binding to chromatin). In conclusion, we have developed an assay that allowed the identification of toxic sRNAs for PCa cells and demonstrated their potential as therapeutics and as a tool for discovery of potential novel targets for PCa. Citation Format: Joshua M Corbin, Hamilton Young, Stefan Wilhelm, Adam S Asch, Chao Xu, Maria J Ruiz Echevarria. Identification of small RNAs that function as multi-target therapeutics and as tools for novel target discovery in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B026.

Maternal obesity changes the small intestine endocannabinoid system and fecal metabolites of weanling rats associated with reduced intestinal permeability and impaired glucose homeostasis.

The small intestine, including the endocannabinoid system (ECS), regulates the energy homeostasis. If maternal obesity modifies the intestinal ECS of the offspring favoring metabolic disorders throughout life is unexplored. Regardless maternal insults, overaction of the ECS has been related to obesity, mainly via type 1 cannabinoid receptor (CB1) signaling, while type 2 cannabinoid receptor (CB2) signaling and the endocannabinoid-like compounds, such as oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), have been associated with anti-inflammatory effects. We hypothesized that maternal obesity changes the ECS in the small intestine of weanling rat offspring in a sex-specific manner associated with altered fecal metabolites. Female rats received a control diet (C; 9% fat) or an obesogenic diet (OD; 37.2% fat, 11.8% sucrose) 9 weeks before mating, gestation and lactation. Offspring were euthanized at weaning. Maternal obesity increased CB2 protein content and mRNA levels of monocyte chemoattractant protein-1 in the small intestine in male offspring, while decreased fecal content of PEA and OEA in both sexes. Maternal obesity decreased gut permeability, but impaired glycemic homeostasis. Concerning fecal levels of γ-aminobutyric acid, amino acids and hypoxanthine, maternal obesity induced a fecal signature related to inflammatory and glycemic homeostasis impairment and dysbiosis. Maternal obesity induced intestinal inflammation and the signaling of CB2, PEA, and OEA might be part of a counter-regulatory response, contributing to reduced gut permeability, but not enough to avoid overweight and glycemic impairment in the offspring at weaning. Our findings provide molecular insights into the intestinal and fecal biomarkers for metabolic disorders.

Gallein, G protein βγ subunits inhibitor, suppresses the TGF-α-induced migration of hepatocellular carcinoma cells via inhibition of the c-Jun N-terminal kinase.

G protein-coupled receptor (GPCR) signaling regulates a wide range of pathophysiological cell functions via G protein α and βγ subunits. Small molecules targeting the subunits of Gα and Gβγ have been developed as cancer therapeutics. We have previously reported that transforming growth factor-α (TGF-α) induces the migration of human hepatocellular carcinoma (HCC) HuH7 cells through the activation of AKT, p38 mitogen-activated protein kinase (MAPK), Rho-kinase and c-Jun N-terminal kinase (JNK). This study aims to determine whether Gβγ subunits regulate the TGF-α-induced migration of HCC HuH7 cells using gallein, a Gβγ subunits inhibitor. The Janus family of tyrosine kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway was also involved in the regulation of the migration. Gallein significantly reduced the TGF-α-induced cell migration. In contrast, fluorescein, a gallein-related compound that has no effect on Gβγ subunits, failed to affect the cell migration. Gallein suppressed the TGF-α-stimulated phosphorylation of JNK without affecting the phosphorylation of epidermal growth factor receptor, AKT, p38 MAPK, target protein of Rho-kinase and STAT3. Conversely, fluorescein did not attenuate the phosphorylation of JNK. These results strongly suggest that Gβγ subunits act as positive regulators in TGF-α-induced migration of HCC cells via the JNK signalling pathway.

Vaccination Against Androgen Receptor Splice Variants to Immunologically Target Prostate Cancer

Background/Objectives: Androgen receptor (AR) expression and signaling are critical for the progression of prostate cancer and have been the therapeutic focus of prostate cancer for over 50 years. While a variety of agents have been developed to target this axis, many of these fail due to the emergent expression of AR RNA splice variants, such as AR-V7, that can signal independently of ligand binding. Other therapies, such as vaccination against prostate-specific antigens, have achieved FDA approvals but have fallen short of being incorporated as standard-of-care therapies for advanced prostate cancer. This may be due to the elevated level of immunosuppression observed in prostate cancer, which remains largely refractory to immune checkpoint blockade. Methods: We developed a vaccine targeting AR-V7, a common isoform associated with treatment resistance, and demonstrated its ability to elicit AR-V7-specific immunity and enable anti-tumor responses against AR-V7+ cancers in subcutaneous tumor models. Results: Our studies also revealed that AR-V7 expression conferred an immune suppressive phenotype that was significant in a non-AR-dependent prostate cancer model. Notably, in this model, we found that vaccination in combination with enzalutamide, an AR antagonist, suppressed these aggressive immune suppressive cancers and resulted in enhanced survival in comparison to control vaccinated and enzalutamide-treated mice. While anti-PD-1 immune checkpoint inhibition (ICI) alone slowed tumor growth, the majority of vaccinated mice that received anti-PD-1 therapy showed complete tumor elimination. Conclusions: Collectively, these results validate the importance of AR signaling in prostate cancer immune suppression and suggest the potential of AR-V7-specific vaccines as therapeutic strategies against prostate cancer, offering significant protective and therapeutic anti-tumor responses, even in the presence of androgen signaling inhibitors.

Abstract B036: Pathway repression in progression on osimertinib therapy in NSCLC via a comprehensive genomic and epigenomic circulating tumor DNA (ctDNA) assay

Abstract Background: Osimertinib is currently the most common precision therapy for patients with non- small cell lung cancer (NSCLC) with EGFR mutations. While it prolongs progression-free survival, resistance eventually develops in many, leading to disease progression. Somatic NGS has identified resistance mutations in the EGFR pathway and other oncogenes, but these account for only 40-45% of cases, suggesting non-genetic factors, such as epigenetic changes, may be involved. This study investigates the epigenetic landscape at diagnosis and progression on first- line osimertinib, focusing on cases with and without canonical resistance mutations. Methods: We defined two cohorts from GuardantINFORM, a clinico-genomics database, restricting to only patients with a test on the Guardant Infinity platform, a comprehensive blood-based NGS assay, that analyzes DNA sequence variants and performs extensive methylation profiling (∼15Mb) in >20,000 differentially methylated regions (DMRs) assessed for sample-specific methylation patterns. (A) Diagnosis Cohort: Comprised of 34 NSCLC patients with blood samples collected within six days prior to the initiation of first-line osimertinib therapy. (B) Progression Cohort: Comprised of 36 patients who were on first-line osimertinib therapy, with blood samples collected within 60 days of discontinuation. The progression cohort was further stratified based on the presence or absence of known resistance mutations identified through somatic variant analysis by Guardant360. Gene set enrichment analysis was conducted on these methylation profiles using the Reactome database to define pathway-level differences between the cohorts. Results: Pathway enrichment analysis in the diagnosis and progression cohorts found expected biological processes in NSCLC such as extracellular matrix organization, G- protein coupled receptor (GPCR) signaling, and FGFR pathways. Additionally, the progression cohort lacking canonical resistance mutations (n=23) exhibited unique enrichment in two specific pathways: Glucose-dependent Insulinotropic Polypeptide and Physiological Factors. These pathways include genes such as GATA4, which has been implicated in the epithelial- mesenchymal transition (EMT)—a process associated with transforming to a small cell phenotype, a known mechanism of resistance to EGFR inhibition, including osimertinib. Conclusion: This study demonstrates the utility of epigenetic profiling via liquid biopsies in uncovering non-genetic mechanisms underlying osimertinib resistance in advanced NSCLC. Identification of unique epigenetic pathways in progressing patients without canonical resistance mutations highlights the potential for discovering novel biomarkers of resistance. Further investigation into these findings may advance our understanding of resistance mechanisms across various cancer therapies, particularly for patients without identified genomic resistance, and inform clinical decision-making and trial design. Citation Format: Aaron Hardin, Leslie Bucheit, Amar Das, Craig Eagel, Helmy Eltoukhy. Pathway repression in progression on osimertinib therapy in NSCLC via a comprehensive genomic and epigenomic circulating tumor DNA (ctDNA) assay [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B036.

Integration of metabolomics and transcriptomics to reveal anti-immunosuppression mechanism of Lycium barbarum polysaccharide

It is well documented that immunosuppression in chickens increases the risk of secondary infections and immunodeficiencies, resulting in significant financial setbacks for the poultry sector. It is crucial to determine if Lycium barbarum polysaccharide (LBP) can counteract immune suppression in young chickens, considering its known ability to modulate immune responses. The aim of this study was to investigate the antagonistic effect and mechanism of LBP on immunosuppression in chicks. A total of 200 seven-day-old Hyland Brown laying hens were used to develop an immunosuppression model and to investigate the optimal time of use and optimal dosage of LBP. A further 120 seven-day-old Hyland Brown laying hens were used to investigate the mechanism of antagonism of LBP against immunosuppression at the optimal time and dosage. The results demonstrated that LBP significantly elevated body weight, spleen index, and peripheral lymphocyte transformation rate, and ameliorated pathological spleen damage in immunosuppressed chickens. A total of 178 differential genes were significantly upregulated following LBP intervention, with a significant enrichment in immune-related pathways, including the chemokine signalling pathway, the C-type lectin receptor signalling pathway, the B-cell receptor signalling pathway, platelet activation, natural killer cell-mediated cytotoxicity, and Th1 and Th2 cell differentiation. A total of 20 different metabolites were identified by metabolomics, which were mainly involved in vitamin metabolism, lipid metabolism, nucleic acid metabolism and amino acid metabolism. The integrated examination of transcriptomic and metabolomic data revealed that the glycerophospholipid metabolic pathway stands out as the most significant among all metabolic pathways. The results demonstrated that LBP regulate the immune system in a multi-pathway and multi-target way.

Abstract PR012: A novel plasma-based epigenomic assay for comprehensive profiling of cancerous and inflammatory disease states

Abstract Background: We developed a method for comprehensive epigenomic profiling from 1 ml of patient (pt) plasma, targeting histone modifications and DNA methylation. This approach can be applied to plasma from pts with cancer and inflammatory conditions, allowing us to infer expression levels of diagnostic markers and drug targets, measure activity of therapeutically targetable transcription factors, and detect non-genomic mechanisms of resistance. By examining the transcriptional status of therapeutic gene targets and pathways, our method addresses the need for new approaches in liquid biopsy (LBx) assays, providing clinically actionable information that enhances the understanding of cancer and inflammatory biology and therapeutic response. Methods: We developed an innovative multimodal assay capable of revealing genome-wide transcriptional activity of promoters and enhancers from chromatin, as well as surveying the DNA methylome. Utilizing this assay on plasma samples from pts with 5 non- cancer conditions and 15 different tumor types, we generated comprehensive genome-wide transcriptional profiles across 21 conditions. Results: We identified transcriptional activation in cancer pt plasma of cancer-associated biomarkers (e.g., HER2, DLL3) in a tumor-specific manner and in autoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus/lupus nephritis. Epigenomic profiling simultaneously inferred the expression of multiple pertinent ADC and radio-immune targets (e.g., MET, HER2, HER3, TROP2, B7H4 in breast cancer and PSMA in prostate cancer), and SLFN11, a biomarker for sensitivity to these agents. The multimodal platform assesses transcriptional regulation genome-wide, enabling analysis of select genes as well as related pathways. For example, a multimodal classifier robustly predicted HER2 status across multiple HER2-expressing tumors. HER2 classification of breast cancer pts by epigenomic LBx was concordant with standard tissue-based IHC for 64/72 (89%) breast cancer samples (AUC 0.9). Accurate classification was achieved for 11/14 (79%) gastroesophageal adenocarcinoma and 4/4 (100%) ovarian cancer pts. A multi-modal predictive model trained on FOLH1 plasma epigenomic regulatory signals demonstrated significant accuracy in predicting quantitative readouts from PSMA PET scans (total SUVmean) in metastatic castration resistance prostate cancer pts and was associated with response to pluvicto. Epigenomic signatures also delineated activation of the endocrine signaling pathway in breast cancer and multiple mechanisms of resistance to androgen receptor signaling inhibition in prostate cancer, including reactivation of the androgen receptor, glucocorticoid signaling bypass, and histological transformation to a neuroendocrine subtype. Conclusions: This comprehensive epigenomic LBx assay offers a platform for simultaneous genome-wide transcriptional assessment of disease and therapy-relevant genes and pathways, marking a paradigm shift in our ability to assess the underlying transcriptional states of cancer and inflammatory diseases. Citation Format: Travis Clark, Anthony D'Ippolito, Aparna Gorthi, Jonathon Beagan, Jamey Guess, Amy Donahue, Mandy Greene, Alyssa Lau, Fernando Ramirez, Kristian Cibulskis, Jenna Wurster, Michael Coyne, Mary McGillicuddy, Baovy Tran, Tyrone Tamakloe, Hathairat Sawaengsri, Corrie Painter, Juliann Chmielecki, Geoff Otto, Matthew Eaton, Carl Barrett. A novel plasma-based epigenomic assay for comprehensive profiling of cancerous and inflammatory disease states [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR012.

Host Genetic Variations and Their Implications on HBV and HCV Infection in the Iranian Population: A Comprehensive Systematic Review

Background: Background: Hepatitis virus infections are among the serious emerging health issues. They are the primary causes of cirrhosis and hepatocellular carcinoma. Growing evidence shows a link between certain genomic variations and inflammation including viral infection such as HBV and HCV. Therefore, this study aimed to comprehensively review studies that analyze the effect of host genomic variations on the risk of contracting viral hepatitis in Iranian population. Methods: The study was conducted according to the PRISMA Statement. All Persian and English case-control articles published until the beginning of June 2023 were included in the study. Two authors reviewed the articles independently. The third author reviewed the final results. Pathway analysis and protein interactions were also performed using GO and STRING databases. Results: Seventy relevant studies were retrieved. Fifty-three studies examined the association of SNPs with the risk of HBV infection. In terms of genetic variations, 25 genes and 44 SNPs were identified. Tumor necrosis factor alpha, Interleukin 28B, and Interleukin 10 were the most prevalent considered genes. The most common polymorphisms were in the interleukin family. Moreover, the top five identified molecular functions were cytokine activity, cytokine receptor binding, molecular function regulator, protein binding, and signaling receptor binding. Conclusion: The polymorphisms of genes involved in the production of immune factors, cytokines, interleukins, and their receptors are associated with the risk of HBV and HCV infections in the Iranian population. Moreover, the extracellular and intracellular signaling pathways and the regulating molecules of these processes can be considered as important factors in liability for these viral infections.

Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling.

The discovery of Toll-like receptors (TLRs) represented a significant breakthrough that paved the way for the study of host-pathogen interactions in innate immunity. However, there are still major gaps in understanding TLR function, especially regarding the early dynamics of downstream TLR pathways. Here, we present a label-free optical biosensor-based assay as a method for detecting TLR activation in a native and label-free environment and defining the dynamics of TLR pathway activation. This technology is sufficiently sensitive to detect TLR signaling and readily discriminates between different TLR signaling pathways. We define pharmacological modulators of cell surface and endosomal TLRs and downstream signaling molecules and uncover TLR signaling signatures, including potential biased receptor signaling. These findings highlight that optical biosensor assays complement traditional assays that use a single endpoint and have the potential to facilitate the future design of selective drugs targeting TLRs and their downstream effector cascades.

Abstract 4138639: Exploring advanced therapeutic approaches for atherosclerosis: Targeting efferocytosis and EndoMT with a focus on the CD47

Background: The anti-phagocytic molecule CD47 is upregulated in the atherosclerotic plaques of patients with cerebrovascular disease; however, the molecular mechanisms responsible for the development and progression of atherosclerotic lesions have not been fully established. In this study, we postulate how endothelial-specific CD47 promotes endothelial-to-mesenchymal transition (EndoMT) in potentiating atherosclerosis and assess the efficacy of the development of novel therapeutic interventions for cardiovascular disease. Methods: Using single-cell RNA sequencing (scRNA-seq), combined with molecular, cellular, and biochemical analyses, we investigated the role of endothelial CD47 in stimulating EndoMT using knock-out in ApoE-/- atherosclerotic mouse models. Results: ScRNA-seq revealed that CD47 promotes EndoMT, and the loss of endothelial CD47 inhibits EndoMT marker expression as well as transforming growth factor-beta signaling in vitro and atherosclerotic mice, which is associated with smaller lesions in Apoe-/- mouse model. In endothelial cells, CD47 hinders efferocytosis of senescent ECs and represses macrophage’s efferocytotic capacity to propel arterial inflammation and atherogenesis. Mechanistically, the interaction between CD47 and efferocytic receptor MerTK exacerbates inflammation by inhibiting the process of efferocytosis in endothelial cells and macrophages under atherogenic conditions. In response to atherogenic stimuli, endothelial CD47 upregulates the endocytic adaptor protein epsin, causing fibroblast growth factor receptor-1 (FGFR1) signal attenuation that upregulates TGF-β signaling and promoting EndoMT and potentiating atherosclerosis. Conclusion: We conclude that endothelial CD47 potentiates EndoMT during atherogenesis by increasing TGF-β signaling through FGFR1 internalization and degradation and targeted manipulating CD47 expression with precision nanomedicine offering a novel approach for mitigating cardiovascular disease.

Abstract 4120290: Sex-specific Impact of Early to Mid-gestational Testosterone Excess on Sheep Fetal and Adult Offspring Lungs

Using the translationally relevant sheep model of prenatal testosterone (T) excess, we previously reported intrauterine growth retardation (IUGR) in both sexes, adverse sex specific programming of fetal heart and cardiometabolic dysfunction in adult female offspring. Because of the intricate reciprocal relationship between the heart and lungs, we hypothesized that gestational T excess will have detrimental sex-specific effects on offspring lungs beginning as early as fetal life and extending into adulthood. Pregnant ewes were intramuscularly injected with 100 mg of testosterone propionate twice weekly from gestational days (GD) 30-90 (term 147 days) and offspring lungs were harvested at GD120, 30 days after cessation of T treatment; (n=4-7/group) and in adulthood (n=4-6/group) and processed for molecular and histological analyses. Data was analyzed by student T test within each sex and Cohen (d) effect size was calculated (Cohens d>0.8 represents large effect size. Lung collagen was significantly increased in T-male fetuses (TM) vs control males (CM) (p=0.01, d=1.99) that persisted into adulthood (TM vs CM: p=0.01, d=1.83). The effects of gestational T excess was evidenced as an increase in collagen only in the lungs of adult T-female fetuses (TF) relative to control females (CF) (p=0.003, d=2.38), but not during fetal life. Gestational T excess induced significant parallel increases in TUNEL positive nuclei/frame in TM fetuses relative to CM (n=3/group, p=0.01, d=3.35) persisting as a large magnitude increase of TUNEL positive nuclei/frame in adult TM (p=0.12, d=0.96). In TF females, apoptotic changes were not noted in fetal lungs but an increase of TUNEL positive nuclei/frame was evidenced in adult TF (p=0.08, d=1.14). At the molecular level prenatal T excess induced an increase in histone deacetylase 1 (HDAC1) expression in fetal TM vs CM (p=0.04, d=1.50), and decrease in HDAC1 in TF vs CF (p=0.10, d=1.09) suggestive of T-induced epigenetic alteration. Transcriptomic analysis of fetal lungs revealed upregulation of pathways associated with estrogen signaling and neuroactive ligand receptor interaction, and downregulation of pathways associated with protein processing in endoplasmic reticulum and antigen processing&presentation (unadjusted p< 0.05, log FC>1.5) in both sexes. Overall, early to mid-gestational exposure to testosterone adversely reprograms the lungs in sex specific fashion as early as fetal life extending into postnatal adult life.