Receptor Transcripts Research Articles

Identification, characterization, and transcription of serotonin receptors in rainbow trout (Oncorhynchus mykiss) in response to bacterial infection and salinity changes

Serotonergic system is involved in the regulation of physiological functions and behavioral traits including cognition, memory, aggression, stress coping, appetite and immunomodulation. Serotonin exerts its functions via binding distinct serotonin receptors which are classified into 7 groups. Salmonid exhibits expanded functional gene copies due to salmonid-specific whole genome duplication. However, serotonin receptor (htr) repertoire is not fully identified in rainbow trout (Oncorhynchus mykiss). In this study, we identified 39 htr genes, including 14 htr1, 4 htr2, 4 htr2 like, 3 htr3, 4 htr4, 2 htr5, 2 htr6, and 6 htr7 subtypes. We investigated physiological functions of serotonin receptors in response to bacterial pathogens exposure and salinity changes. We showed htr1, htr2, htr4 and htr7 subtypes were associated with immunomodulation in response to Vibrio anguillarum or Aeromonas salmonicida infection. Saltwater (salinity of 15) transfer significantly altered htr1, htr2, htr4, and htr7 subtypes, suggesting trout Htr was associated with osmoregulation. We further showed residues interacted with inverse agonist (methiothepin) and serotonin analogue (5-Carboxamidotryptamine) were conserved between trout and human, suggesting exogenous ligands targeting human HTRs might have a role in aquaculture. This study showed duplicated trout Htrs might be physiologically neofunctionalized and potentially exhibit pleiotropic effects in regulating immunomodulation and osmoregulation.

Computational study of SENP1 in cancer by novel natural compounds and ZINC database screening.

Introduction: Sentrin-specific protease 1 (SENP1) is a protein whose main function is deSUMOylation. SENP1 inhibits apoptosis, and increases angiogenesis, estrogen and androgen receptor transcription and c-Jun transcription factor, proliferation, growth, cell migration, and invasion of cancer. The in vivo and in vitro studies also demonstrated which natural compounds, especially phytochemicals, minerals, and vitamins, prevent cancer. More than 3,000 plant species have been reported in modern medicine. Natural compounds have many anti-cancerous andanti-turmeric properties such as antioxidative, antiangiogenic, antiproliferative, and pro-apoptotic properties. Methods: In this study, we investigated the interaction of some natural compounds with SENP1 to inhibit its activity. We also screened the ZINC database including natural compounds. Molecular docking was performed, and toxicity of compounds was determined; then, molecular dynamics simulation (MDS) and essential dynamics (ED) were performed on natural compounds with higher free binding energies and minimal side effects. By searching in a large library, virtual screening of the ZINC database was performed using LibDock and CDOCKER, and the final top 20 compounds were allowed for docking against SENP1. According to the docking study, the top three leading molecules were selected and further analyzed by MDS and ED. Results: The results suggest that resveratrol (from the selected compounds) and ZINC33916875 (from the ZINC database) could be more promising SENP1 inhibitory ligands. Discussion: Because these compounds can inhibit SENP1 activity, then they can be novel candidates for cancer treatment. However, wet laboratory experiments are needed to validate their efficacy as SENP1 inhibitors.

Decreased frequency of Th22 cells and IL-22 cytokine in kidney transplant patients with active cytomegalovirus infection

BackgroundThe immunity of CD4+ T cell subsets against human cytomegalovirus (HCMV) is considerable due to their essential role in controlling the infection in transplant individuals. Previously explained CD4+ subsets such as T helper (Th) 1 have been proven to have a protective role against HCMV infection, while the role of the recently identified Th22 subset has not been described yet. Here, the frequency changes of Th22 cells and the IL-22 cytokine production were investigated in kidney transplant recipients with and without HCMV infection.MethodsTwenty kidney transplant patients and ten healthy controls were enrolled in this study. Patients were categorized into HCMV + and HCMV- groups based on the HCMV DNA real-time PCR results. After isolating CD4+ T cells from PBMCs, the phenotype (CCR6+CCR4+CCR10+) and cytokine profile (IFN-γ−IL-17−IL-22+) of Th22 cells were analyzed by flow cytometry. The gene expression of Aryl Hydrocarbon Receptor (AHR) transcription factor was analyzed by real-time PCR.ResultsThe phenotype frequency of these cells was lower in recipients with infection than in those without infection and healthy controls (1.88 ± 0.51 vs. 4.31 ± 1.05; P = 0.03 and 4.22 ± 0.72; P = 0.01, respectively). A lower Th22 cytokine profile was observed in patients with infection than in the two other groups (0.18 ± 0.03 vs. 0.20 ± 0.03; P = 0.96 and 0.33 ± 0.05; P = 0.04, respectively). AHR expression was also lower in patients with active infection.ConclusionsOverall, this study for the first time suggests that the reduced levels of Th22 subset and IL-22 cytokine in patients with active HCMV infection might indicate the protective role of these cells against HCMV.

Osmotic stress tolerance and transcriptomic response of Ramazzottius varieornatus (Eutardigrada: Ramazzottiidae) following tun formation

Abstract To thrive in harsh environments, tardigrades have evolved the ability to enter the quiescent state of cryptobiosis, often characterized by transition into a so-called ‘tun’. Here, we investigate osmobiosis, a substate of cryptobiosis induced by rising osmolyte concentrations. We follow the behaviour and morphology of Ramazzottius varieornatus during transfer from freshwater conditions into 3 Osmol/kg sucrose solution. The tardigrades easily survive the extreme change in external osmolality. During gradual exposure, they initiate tun formation at 0.2 Osmol/kg, with most specimens fully contracted into a tun at 0.5 Osmol/kg. The first transcriptomic profiling of osmobiotic tuns in comparison to active tardigrades reveals a modest shift, with 16% of the 3322 differentially expressed transcripts having a |log2 fold change| > 1. A gene ontology enrichment analysis shows enrichment within protein homeostasis and neurohormonal signalling, with a ubiquitin-conjugating enzyme and neurotransmitter receptor transcripts being down- and upregulated, respectively. A putative Hsp70 is upregulated, whereas transcripts related to eutardigrade-specific proteins, antioxidant defence and DNA repair show minor fold changes. Among putative membrane transporters, a monocarboxylate and two amino acid transporters are downregulated. Our findings are in agreement with previous studies in Ramazzottius indicating that cryptobiosis and tun formation involve no change or modest change in transcription.

Oat beta-glucan reduces colitis by promoting autophagy flux in intestinal epithelial cells via EPHB6-TFEB axis.

Inflammatory bowel disease (IBD) is a group of chronic inflammatory disorders of the gastrointestinal tract, mainly including Crohn's disease and ulcerative colitis. Epidemiological findings suggest that inadequate dietary fibers intake may be a risk factor for IBD. Oat beta-glucan is a type of fermentable dietary fiber and has been proved to reduce experimental colitis. However, the mechanism remains unclear. The aim of this study was to explore the role and possible mechanism of oat beta-glucan in reducing experimental colitis. We used a dextran sulfate sodium (DSS)-induced mice acute colitis model to explore the potential mechanism of oat beta-glucan in reducing experimental colitis. As a result, oat beta-glucan upregulated the expressions of Erythropoietin-producing hepatocyte receptor B6 (EPHB6) and transcription factor EB (TFEB), promoted autophagy flux and downregulated the expressions of interleukin 1 beta (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) in intestinal epithelial cells (IECs). The role of the EPHB6-TFEB axis was explored using a lipopolysaccharide-induced HT-29 cells inflammation model. The results revealed that EPHB6 regulated the expression of TFEB, and knockdown of EPHB6 decreased the protein level of TFEB. When EPHB6 or TFEB was knocked down, autophagy flux was inhibited, and the anti-inflammatory effect of sodium butyrate, a main metabolite of oat beta-glucan in the gut, was blocked. In summary, our findings demonstrated that oat beta-glucan reduced DSS-induced acute colitis in mice, promoted autophagy flux via EPHB6-TFEB axis and downregulated the expressions of IL-1β, IL-6 and TNF-α in IECs, and this effect may be mediated by butyrate.

P-677 Letrozole inhibits ovulation via reducing responsiveness to luteinizing hormone in in vitro-grown mouse follicles

Abstract Study question Does letrozole reduce responsiveness to luteinizing hormone (LH) in in vitro-grown mouse follicles? Summary answer We found that letrozole reduced responsiveness to LH via reducing of luteinizing hormone/chorionic gonadotropin receptor (Lhcgr) transcription in in vitro. What is known already The continuous dosing of letrozole, the third-generation aromatase inhibitor, is used for the controlled ovarian stimulation (COS) of fertility preservation cycles in women with hormone-sensitive breast cancer to prevent the transient rise of serum estrogen levels. Recently, lower oocyte maturation rates and higher abnormal fertilization rates in COS with letrozole compared to standard COS have been reported. The studies using aromatase knockout mice and estrogen receptor beta knockout mice showed that intra-follicular estrogen is essential for responsiveness to LH of granulosa cells. However, whether letrozole reduces responsiveness to LH of follicles remains unknown. Study design, size, duration We evaluated the effect of letrozole on responsiveness to LH using in vitro mouse follicle culture system. Preantral follicles (150-200 µm) with a small number of stromal cells were isolated from 3 weeks old C57BL/6jjcl mouse ovaries and cultured in the presence of letrozole. We analyzed in vitro ovulation ability, and expression levels of ovulation-related genes to clarify the effect of letrozole on responsiveness to LH of follicles. Participants/materials, setting, methods The follicles were cultured with letrozole (0.01µM, 0.1µM, or 1µM) or vehicle (0.1% DMSO) for 5 days followed by 5 IU/ml hCG and 5 ng/ml hEGF stimulation as ovulation induction. The diameter and survival rate of follicles were assessed. After16 hours after ovulation induction, we assessed the ovulation and measured the transcription of Lhcgr, Ptgs2, Runx1 and Actb (internal control) in the follicles by RT-qPCR. We conducted a collateral experiment in the presence of estradiol. Main results and the role of chance The follicle growth and survival rate did not change by letrozole in vitro. However, in vitro ovulation was reduced by the addition of letrozole in a dose-dependent manner (Cochran-Armitage trend test, P < 0.0001): 41.2% in the 0.01µM letrozole treated group (n = 23), 10.5% in the 0.1µM letrozole treated-group (n = 17), and no ovulation in the 1µM letrozole treated-group (n = 19) compared with 69.6% in the vehicle-group (n = 18). Exogenous estrogen addition restored the ovulation ability of 1µM letrozole-treated follicle to the same levels as the vehicle group. RT-qPCR revealed that 0.1 µM letrozole significantly reduce the transcription levels of Lhcgr (P = 0.0027), Runx1 (P = 0.0124), and Ptgs2 (P = 0.0067) compared with vehicle-treated follicles. Exogenous estrogen addition restored the transcription of these genes to levels observed in vehicle-treated follicles. We also observed a positive correlation between the transcription of Lhcgr and Ptgs2 (R = 0.55, P = 0.0003), Lhcgr and Runx1 (R = 0.71, P < 0.0001), and Ptgs2 and Runx1 (R = 0.77, P < 0.0001). Our results suggested that letrozole-treated follicles impaired ovulation due to dysregulation of Lhcgr transcription and its downstream cascade caused by estrogen deficiency. Limitations, reasons for caution This is an in vitro experiment using a mouse model. Further research is needed to determine whether our findings are applicable to humans. Wider implications of the findings Our findings imply that letrozole may have disadvantages in not only ovulation, but also subsequent reproductive processes affected by the effectiveness of the LH surge, such as oocyte maturation, fertilization, and preimplantation embryo development. When evaluating oocyte cryopreservation for fertility preservation, it may be necessary to consider the COS protocol. Trial registration number Not applicable

Melatonin-related dysfunction in chronic restraint stress triggers sleep disorders in mice.

Stress may trigger sleep disorders and are also risk factors for depression. The study explored the melatonin-related mechanisms of stress-associated sleep disorders on a mouse model of chronic stress by exploring the alteration in sleep architecture, melatonin, and related small molecule levels, transcription and expression of melatonin-related genes as well as proteins. Mice undergoing chronic restraint stress modeling for 28days showed body weight loss and reduced locomotor activity. Sleep fragmentation, circadian rhythm disorders, and insomnia exhibited in CRS-treated mice formed sleep disorders. Tryptophan and 5-hydroxytryptamine levels were increased in the hypothalamus, while melatonin level was decreased. The transcription and expression of melatonin receptors were reduced, and circadian rhythm related genes were altered. Expression of downstream effectors to melatonin receptors was also affected. These results identified sleep disorders in a mice model of chronic stress. The alteration of melatonin-related pathways was shown to trigger sleep disorders.

FOXO-like Gene Is Involved in the Regulation of 20E Pathway through mTOR in Eriocheir sinensis

The Forkhead Box O (FOXO) gene plays a key role in various biological processes, such as growth, metabolism, development, immunity and longevity. Molting is an essential process for crustacean growth, which is mainly regulated by 20-hydroxyecdysone (20E) and molt-inhibiting hormone (MIH). Although the role of FOXO in regulating the immune response of crustaceans is well documented, its involvement in controlling crustacean molting remains unclear. In this study, a FOXO-like gene (designed as EsFOXO-like) was identified in Eriocheir sinensis, and the regulation of the 20E pathway by EsFOXO-like was also investigated. The coding sequence of EsFOXO-like was 852 bp, which consisted of 283 amino acids including a conserved Forkhead (FH) domain. EsFOXO-like shared high similarity with FOXO genes from other crustaceans, and the mRNA expression levels of the EsFOXO-like gene were highest in the hepatopancreas and lowest in the hemocytes. However, transcription and protein expression of the EsFOXO-like gene were found to be up-regulated only during the pre-molt stage in the hepatopancreas, with lower expression levels observed at the post-molt stage. To explore the role of EsFOXO-like in the 20E pathway, EsFOXO-like was firstly inhibited by a specific FOXO inhibitor (AS1842856) and then through an EsFOXO-like dsRNA injection, respectively, and the results showed that the relative expression levels of EsFOXO-like were notably decreased in the hepatopancreas after both the inhibitor and dsRNA treatments. The 20E concentration, the mRNA expression levels of the 20E receptors including the ecdysone receptor (EcR) and the retinoid-X receptor (RXR) and EsmTOR transcription in the AS1842856 group or the EsFOXO-RNAi group were all significantly higher than that in the control group, while the mRNA expression level of EsMIH was significantly decreased after EsFOXO-like inhibition. To further investigate whether the EsFOXO-like acts through mTOR or not, Rapamycin was administered to inhibit mTOR activity in EsFOXO-like inhibited crabs. The results revealed a significant reduction in the concentration of 20E and the expression level of EsMIH in the AS1842856 + Rapamycin group compared to the AS1842856 + DMSO group, accompanied by an increase in EsEcR and EsRXR expression. These findings collectively suggest that EsFOXO-like regulates the 20E pathway through mTOR, which offered valuable insights into the understanding of the molting process in crustaceans.

Primary mouse myoblast metabotropic purinoceptor profiles and calcium signalling differ with their muscle origin and are altered in mdx dystrophinopathy

Mortality of Duchenne Muscular Dystrophy (DMD) is a consequence of progressive wasting of skeletal and cardiac muscle, where dystrophinopathy affects not only muscle fibres but also myogenic cells. Elevated activity of P2X7 receptors and increased store-operated calcium entry have been identified in myoblasts from the mdx mouse model of DMD. Moreover, in immortalized mdx myoblasts, increased metabotropic purinergic receptor response was found. Here, to exclude any potential effects of cell immortalization, we investigated the metabotropic response in primary mdx and wild-type myoblasts. Overall, analyses of receptor transcript and protein levels, antagonist sensitivity, and cellular localization in these primary myoblasts confirmed the previous data from immortalised cells. However, we identified significant differences in the pattern of expression and activity of P2Y receptors and the levels of the “calcium signalling toolkit” proteins between mdx and wild-type myoblasts isolated from different muscles. These results not only extend the earlier findings on the phenotypic effects of dystrophinopathy in undifferentiated muscle but, importantly, also reveal that these changes are muscle type-dependent and endure in isolated cells. This muscle-specific cellular impact of DMD may not be limited to the purinergic abnormality in mice and needs to be taken into consideration in human studies.

Abstract IA003: Evolution of AR gene structure during prostate cancer progression

Abstract Castration-resistant prostate cancer (CRPC) emerges when endocrine therapies are no longer effective at suppressing activity of the androgen receptor (AR) transcription factor. Our laboratory has implemented DNA sequencing approaches to identify genomic mechanisms contributing to CRPC in prostate cancer cell lines, patient-derived xenografts (PDXs), and clinical specimens. These approaches have revealed AR as one of the most frequently-rearranged genes in CRPC. However the patterns of AR gene rearrangements observed between patients and even between anatomically distinct metastases within a single patient are remarkably complex and heterogeneous. To address this complexity, we analyzed a cohort of prostate cancer specimens using linked-read DNA-sequencing. This approach found that a subset of clinical CRPC metastases had accumulated complex AR rearrangements in conjunction with very high AR copy number, a phenomenon that also occurred in PDX models grown under selective pressure of castration. Using orthogonal approaches of optical genome mapping and fluorescence in situ hybridization, we found that AR residing on extrachromosomal circular DNA (eccDNA) was an underlying mechanism. This study identifies eccDNA as an engine for accumulation of AR gene copy number and structural complexity during CRPC progression. Citation Format: Scott M Dehm. Evolution of AR gene structure during prostate cancer progression [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr IA003.

Interleukin-6 promotes the dedifferentiation of papillary thyroid cancer cells.

Radioiodine treatment is a fundamental therapy for patients with papillary thyroid cancer (PTC). Sodium/iodide symporter (NIS)-mediated iodine uptake is a prerequisite for the efficacy of radioiodine therapy. Interleukin-6 (IL-6) is a pro-tumor cytokine, but its regulation of NIS expression in PTC has not been elucidated. In this study, we found that IL-6 enhanced the proliferation ability of PTC cells. Moreover, the negative association between IL-6 and NIS expression in thyroid cancer tissues was demonstrated. IL-6 downregulated thyroid-specific genes such as NIS, thyroid peroxidase, and thyroid-stimulating hormone receptor and thyroid-specific transcription factors including thyroid transcription factor-1 (TTF-1) and paired box protein-8 (PAX-8). The inhibitory effects of IL-6 on NIS expression were alleviated by mitogen-activated protein kinase and Janus kinase inhibitors. Depletion of c-Jun or STAT3 also rescued IL-6-induced NIS downregulation, with STAT3 depletion exerting a stronger effect. TTF-1 protein expression was also restored by depleting c-Jun or STAT3. STAT3 depletion, but not c-Jun depletion, alleviated the inhibitory effect of IL-6 on PAX-8 expression. Moreover, the downregulation of NIS by IL-6 was rescued by overexpressing TTF-1 and PAX-8. Tocilizumab, an IL-6 receptor blocker, did not have any cytostatic activity in PTC cells, and it also failed to induce redifferentiation in vitro. However, we found that the drug blocked the inhibitory effect of IL-6 on NIS expression. In summary, IL-6 inhibits NIS transcription in PTC cells by activating mitogen-activated protein kinase and Janus kinase signaling.

Dual SIFamide receptors in Ixodes salivary glands

Salivary glands are vital to tick feeding success and also play a crucial role in tick-borne pathogen transmission. In previous studies of Ixodes scapularis salivary glands, we demonstrated that saliva-producing type II and III acini are innervated by neuropeptidergic axons which release different classes of neuropeptides via their terminals (Šimo et al., 2009b, 2013). Among these, the neuropeptide SIFamide—along with its cognate receptor—were postulated to control the basally located acinar valve via basal epithelial and myoepithelial cells (Vancová et al., 2019). Here, we functionally characterized a second SIFamide receptor (SIFa_R2) from the I. scapularis genome and proved that it senses a low nanomolar level of its corresponding ligand. Insect SIFamide paralogs, SMYamides, also activated the receptor but less effectively compared to SIFamide. Bioinformatic and molecular dynamic analyses suggested that I. scapularis SIFamide receptors are class A GPCRs where the peptide amidated carboxy-terminus is oriented within the receptor binding cavity. The receptor was found to be expressed in Ixodes ricinus salivary glands, synganglia, midguts, trachea, and ovaries, but not in Malpighian tubules. Investigation of the temporal expression patterns suggests that the receptor transcript is highly expressed in unfed I. ricinus female salivary glands and then decreases during feeding. In synganglia, a significant transcript increase was detected in replete ticks. In salivary gland acini, an antibody targeting the SIFa_R2 recognized basal epithelial cells, myoepithelial cells, and basal granular cells in close proximity to the SIFamide-releasing axon terminals. Immunoreactivity was also detected in specific neurons distributed throughout various I. ricinus synganglion locations. The current findings, alongside previous reports from our group, indicate that the neuropeptide SIFamide acts via two different receptors that regulate distinct or common cell types in the basal region of type II and III acini in I. ricinus salivary glands. Our study investigates the peptidergic regulation of the I. ricinus salivary gland in detail, emphasizing the complexity of this system.

Immunovirological and environmental screening reveals actionable risk factors for fatal COVID-19 during post-vaccination nursing home outbreaks

Coronavirus Disease 2019 (COVID-19) vaccination has resulted in excellent protection against fatal disease, including in older adults. However, risk factors for post-vaccination fatal COVID-19 are largely unknown. We comprehensively studied three large nursing home outbreaks (20–35% fatal cases among residents) by combining severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) aerosol monitoring, whole-genome phylogenetic analysis and immunovirological profiling of nasal mucosa by digital nCounter transcriptomics. Phylogenetic investigations indicated that each outbreak stemmed from a single introduction event, although with different variants (Delta, Gamma and Mu). SARS-CoV-2 was detected in aerosol samples up to 52 d after the initial infection. Combining demographic, immune and viral parameters, the best predictive models for mortality comprised IFNB1 or age, viral ORF7a and ACE2 receptor transcripts. Comparison with published pre-vaccine fatal COVID-19 transcriptomic and genomic signatures uncovered a unique IRF3 low/IRF7 high immune signature in post-vaccine fatal COVID-19 outbreaks. A multi-layered strategy, including environmental sampling, immunomonitoring and early antiviral therapy, should be considered to prevent post-vaccination COVID-19 mortality in nursing homes.

Post-inflammatory administration of N-acetylcysteine reduces inflammation and alters receptor levels in a cellular model of Parkinson's disease.

Parkinson's disease (PD) is a complex, multifactorial neurodegenerative disease with a prevalence of 1% over the age of 55. Neuropathological hallmarks of PD include the loss of dopaminergic neurons in the substantia nigra pars compacta and the accumulation of Lewy bodies that contain a variety of proteins and lipids including alpha-synuclein (α-syn). Although the formation of α-syn occurs intracellularly, it can also be found in the extracellular space where it can be taken up by neighboring cells. Toll-like receptor 2 (TLR2) is an immune system receptor that has been shown to recognize extracellular α-syn and modulate its uptake by other cells. Lymphocyte-activation gene 3 (LAG3), an immune checkpoint receptor, has also been proposed to play a role in extracellular α-syn internalization; however, a recent study has disputed this role. Internalized α-syn can trigger expression and secretion of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-2, and IL-6 and induce neuroinflammation, apoptosis, and mitophagy that results in cellular death. In this study, we tested if N-acetylcysteine (NAC), an anti-inflammatory and anti-carcinogenic drug, can circumvent the detrimental effects of neuroinflammation and induce an anti-inflammatory response by modulating transcription and expression of TLR2 and LAG3 receptors. Cells overexpressing wild-type α-syn were treated with TNF-α to induce inflammation followed by NAC to inhibit the deleterious effects of TNF-α-induced inflammation and apoptosis. SNCA gene transcription and α-syn protein expression were validated by q-PCR and Western blot (WB), respectively. Cell viability was measured, and apoptosis was evaluated by WB and terminal deoxynucleotidyl transferase nick end labeling methods. Alterations in LAG3 and TLR2 receptor levels were evaluated by immunofluorescent labeling, WB, and q-PCR. TNF-α not only increased inflammation but also increased endogenous and overexpressed α-syn levels. NAC treatment decreased expression of TLR2 and increased transcription of LAG3 receptor and diminished inflammation-mediated toxicity and cell death. Here, we demonstrate that NAC can reduce neuroinflammation that occurs as a result of alpha-synuclein overexpression, via a TLR2-associated pathway, making it a promising candidate for therapeutic intervention. Further studies are needed to elucidate molecular mechanisms and pathways related to neuroinflammation in PD and to develop possible new therapeutic approaches to slow the clinical progression of PD.

Exogenous Chemicals Impact Virus Receptor Gene Transcription: Insights from Deep Learning.

Despite the fact that coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been disrupting human life and health worldwide since the outbreak in late 2019, the impact of exogenous substance exposure on the viral infection remains unclear. It is well-known that, during viral infection, organism receptors play a significant role in mediating the entry of viruses to enter host cells. A major receptor of SARS-CoV-2 is the angiotensin-converting enzyme 2 (ACE2). This study proposes a deep learning model based on the graph convolutional network (GCN) that enables, for the first time, the prediction of exogenous substances that affect the transcriptional expression of the ACE2 gene. It outperforms other machine learning models, achieving an area under receiver operating characteristic curve (AUROC) of 0.712 and 0.703 on the validation and internal test set, respectively. In addition, quantitative polymerase chain reaction (qPCR) experiments provided additional supporting evidence for indoor air pollutants identified by the GCN model. More broadly, the proposed methodology can be applied to predict the effect of environmental chemicals on the gene transcription of other virus receptors as well. In contrast to typical deep learning models that are of black box nature, we further highlight the interpretability of the proposed GCN model and how it facilitates deeper understanding of gene change at the structural level.

Canonical and truncated transglutaminase-2 regulate mucin-1 expression and androgen independency in prostate cancer cell lines

Androgen independency is associated with poor prostate cancer (PCa) survival. Here we report that silencing of transglutaminase-2 (TG2) expression by CRISPR-Cas9 is associated with upregulation of androgen receptor (AR) transcription in PCa cell lines. Knockout of TG2 reversed the migratory potential and anchorage independency of PC3 and DU145 cells and revealed a reduced level of mucin-1 (MUC1) RNA transcript through unbiased multi-omics profiling, which was restored by selective add-back of the truncated TG2 isoform (TGM2_v2). Silencing of AR resulted into increased MUC1 in TG2KO PC3 cells showing that TG2 affects transcriptional regulation of MUC1 via repressing AR expression. Treatment of PC3 WT cell line with TG2 inhibitor ZDON led to a significant increase in AR expression and decrease in MUC1. ZDON also blocked the formation of MUC1-multimers labelled with TG amine-donor substrates in reducing conditions, revealing for the first time a role for TG2, which we show to be externalised via extracellular vesicles, in MUC1 stabilisation via calcium-dependent transamidation. A specific antibody towards TGM2_v2 revealed its restricted nuclear location compared to the canonical long form of TG2 (TGM2_v1), which is predominantly cytosolic, suggesting that this form contributes to the previously suggested TG2-mediated NF-κB activation and AR transcriptional repression. As TGM2_v2 transcription was increased in biopsies of early-stage prostate adenocarcinoma (PRAD) patients compared to subjects presenting inflammatory prostatitis, and total TG2 protein expression significantly increased in PRAD versus normal tissue, the role of TG2 and its truncated form as a prostate malignancy marker is suggested. In conclusion, this investigation has provided the first unbiased discovery of a novel pathway mediated by TG2 via MUC1, which is shown to contribute to androgen insensitivity and malignancy of PCa cells and be upregulated in PCa biopsies, with potential relevance to cancer immune evasion.

Single-cell landscape dissecting the transcription and heterogeneity of innate lymphoid cells in ischemic heart.

Until now, few articles have revealed the potential roles of innate lymphoid cells (ILCs) in cardiovascular diseases. However, the infiltration of ILC subsets in ischemic myocardium, the roles of ILC subsets in myocardial infarction (MI) and myocardial ischemia-reperfusion injury (MIRI) and the related cellular and molecular mechanisms have not been described with a sufficient level of detail. In the current study, 8-week-old male C57BL/6J mice were divided into three groups: MI, MIRI and sham group. Single-cell sequencing technology was used to perform dimensionality reduction clustering of ILC to analyze the ILC subset landscape at a single-cell resolution, and finally flow cytometry was used to confirm the existence of the new ILC subsets in different disease groups. Five ILC subsets were found, including ILC1, ILC2a, ILC2b, ILCdc and ILCt. It is worth noting that ILCdc, ILC2b and ILCt were identified as new ILC subclusters in the heart. The cellular landscapes of ILCs were revealed and signal pathways were predicted. Furthermore, pseudotime trajectory analysis exhibited different ILC statuses and traced related gene expression in normal and ischemic conditions. In addition, we established a ligand-receptor-transcription factor-target gene regulatory network to disclose cell communications among ILC clusters. Moreover, we further revealed the transcriptional features of the ILCdc and ILC2a subsets. Finally, the existence of ILCdc was confirmed by flow cytometry. Collectively, by characterizing the spectrums of ILC subclusters, our results provide a new blueprint for understanding ILC subclusters' roles in myocardial ischemia diseases and further potential treatment targets.

Abstract 584: Myeloid Kruppel-like Factor 14 Mediates Sexual Dimorphism Of Abdominal Aortic Aneurysm Via Estrogen Receptor β

Rationale: Abdominal aortic aneurysm (AAA) is an irreversible degenerative disease of the abdominal aorta. Monocytes/macrophages are important mediators of inflammation and proteolysis during AAA development. In both human and rodent models, AAA has strong sexual dimorphism with a 4-6-fold higher prevalence in males than females, largely attributed to the estrogen/estrogen receptor signaling pathway. Krüppel-Like Factor 14 (KLF14) mediates anti-inflammatory response during disease pathogenesis and shows sexual heterogeneity in human genetic studies. However, whether and how KLF14 regulates sexual dimorphism during AAA progression has largely been unknown. Objective: Herein, we generated a novel insight into the anti-inflammatory effect and sexual dimorphic regulation of KLF14 during AAA pathogenesis. Methods and Results: We induced AAA model in myeloid cell-specific Klf14 knockout mice in ApoE -deficient background ( Klf14 LysM ApoE -/- ) and their littermate control mice ( Klf14 fl/fl ApoE -/- ). Klf14 deficiency in macrophages completely abrogated the sexual dimorphism observed in control mice by showing higher AAA incidence with more macrophage infiltration and more severe elastin degradation in female mice. Mechanistically, KLF14 upregulates the transcription of estrogen receptors, especially ESR2 , in macrophages. Perhexiline, a KLF14 inducer, induces Esr2 expression dependent on Klf14 and exerts an anti-inflammatory effect partially dependent on Esr2 in macrophages. Furthermore, Klf14 or Esr2 deficiency in macrophages abolished the protective effect of exogenous estradiol (E 2 ) treatment in male control mice. Conclusions: These studies provide evidence that Klf14 deficiency within myeloid lineage cells accelerated AAA development in female mice, resulting in the loss of sexual dimorphism in AAA pathogenesis. KLF14 showed protective effects, likely via transcriptionally regulating ESR2 expression and suppressing inflammation.

Early life stress in mice leads to impaired colonic corticosterone production and prolonged inflammation following induction of colitis

Early life stress (ELS) is an environmental trigger linked to increased risk of inflammatory bowel disease (IBD). Our goal was to identify mechanisms whereby ELS in mice affects susceptibility to, and/or severity of, intestinal inflammation. We hypothesized that ELS alters the delicate balance of typical homeostatic mediators in the intestinal microenvironment, predisposing ELS-exposed individuals to IBD onset or worsened disease upon a pro-inflammatory triggering event within the gut. We utilized 2 published animal models of ELS. In the first model, newborn mice were separated from the dam daily for 4-8 hours beginning on postnatal day 2, then weaned early on postnatal day 17. Normally reared (NR) control mice were left undisturbed with the dams until standard weaning on postnatal day 21. In the second model, dams were fed the synthetic glucocorticoid – dexamethasone, or vehicle ad libitum in drinking water (3 μg/mL) on post-partum days 1-14. Colitis was induced in 4-week-old mice via intraperitoneal injection of IL-10 receptor blocking antibody (100 μg) every 5 days for 15 days. Five or 15 days later, histological colitis scores and colonic transcripts for Tnf, glucocorticoid receptors, and steroidogenic enzymes were measured. ELS-exposed mice displayed altered mucosa-associated immune cell balances, with an initial deficit in anti-inflammatory IL-10-producing CD4 T cells at 4-wks of age (n≥11, p<0.05), followed by an increase in IFNγ-producing CD4 T cells at 8-wks of age (n≥7, p<0.05). ELS-exposed mice also displayed reduced levels of the glucocorticoid, corticosterone in the plasma (n≥10, p<0.01) as well as locally within the colon (n≥7, p<0.05). Upon colitis induction, NR control animals showed improvements in indices of inflammation following cessation of IL-10 receptor blockade, whereas ELS-exposed animals maintained high levels of intestinal Tnf (n≥7, p<0.01) and histological signs of colitis (n≥7, p<0.05). In colitic animals, prior exposure to ELS was associated with significantly lower expression of genes associated with corticosterone synthesis ( Cyp11a1; n≥5, p<0.05) and responsiveness ( Nr3c1, Nr5a2; n≥5, p<0.01). Finally, ex-vivo TNF stimulation of colonic crypt cells from ELS mice led to increased inhibition of corticosterone synthesis, relative to NR control mice (n≥4, p<0.05). Our study identifies impaired local colonic glucocorticoid production and responsiveness as a potential mechanism whereby ELS predisposes to chronic colitis in susceptible hosts. Crohn's and Colitis Foundation Research Initiatives Award This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sex Differences in Gut Microbiota Fatty Acid Metabolism in Human Salt Sensitivity of Blood Pressure

Salt sensitivity is a trait in which high dietary sodium (Na+) intake causes an increase in blood pressure. We previously demonstrated that in the gut, elevated dietary Na+ causes dysbiosis. The mechanistic interplay between excess dietary Na+-induced alteration in the gut microbiome and sex differences is less understood. The goal of this study was to identify novel metabolites in salt sensitivity and sex differences in response to a high dietary Na+ intake. We hypothesized that excess dietary salt-induced dysbiosis is associated with distinct metabolomic changes in men and women. We performed plasma metabolomics analysis and measured blood pressure of human volunteers who completed a validated 3-day food record to estimate dietary Na+ intake. Based on the recommendations by the American Heart Association, we classified daily Na+ intake of <2.3g as normal salt and high salt for subjects consuming ≥ 2.3g Na+. Spearman correlation was used to assess the relationship between Na+ intake and blood pressure. We also performed RNA sequencing on human (N=11) monocytes treated with high salt in vitro. The relationship between dietary Na+ intake and mean arterial pressure was different in women (r=0.3422, p=0.0022, N=81) and men (r=0.4552, p=0.0010, N=49). Network analysis of human plasma untargeted metabolome revealed fatty acids as top subnetworks differentially changed with salt intake. In subjects consuming a high Na+ diet, levels of linoleate (1.211 ± 0.330 vs. 0.869 ± 0.170; p<0.001); palmitate (1.155 ± 0.292 vs. 0.924 ± 0.233; p= 0.003); arachidonic acid (1.119 ± 0.242 vs. 0.965 ± 0.201; p=0.015); and 12-hydroxyeicosatetraenoic acid (1.329 ± 0.925 vs. 0.902 ± 0.520; p=0.0469) were higher in women (28) than men (25). In contrast, we observed no sex differences in these parameters between men and women consuming a normal salt diet. RNA sequencing analysis revealed that exposure of human monocytes to high salt in vitro upregulated transcription of fatty acid receptors and arachidonic acid-related genes. Specifically, ALOX12 (277.8 ± 13.8 vs. 169.5 ± 16.7, p<0.0001) which encodes for arachidonate 12-lipoxygenase, ALOX5AP (6459 ± 1553 vs. 2598 ± 30, p=0.0310) which encodes for 5-lipoxygenase activating protein, and FFR2 (418.1 ± 97.8 vs. 115.9 ± 29.4, p=0.0078) which encodes for free fatty acids receptor 2 were increased in human monocytes, and this was associated with a pro-inflammatory phenotype. These findings suggest that excess dietary Na+ is associated with fatty acid metabolism dysregulation, and that the arachidonic acid pathway may potentially play a role in sex differences of salt-sensitive hypertension. This work was supported by American Heart Association grants POST9034281 (to JAI) and 15SDG24890015 (to JFF) and NIH grants K01HL130497 (to AK) as well as Venture Pilot Funding (to AK) from Vanderbilt Microbiome Initiative and Vanderbilt Institute for Infection, Immunology, and Inflammation (VI4) and a P&F award (to JFF) from Vanderbilt University Medical Center's Digestive Disease Research Center supported by NIH grant P30DK058404. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.