Elevated mRNA Research Articles

Loss in Pluripotency Markers in Mesenchymal Stem Cells upon Infection with Chlamydia trachomatis.

The intracellular pathogen Chlamydia trachomatis can inflict substantial damage on the host. Notably, Chlamydia infection is acknowledged for its precise modulation of diverse host signaling pathways to ensure cell survival, a phenomenon intricately connected to genetic regulatory changes in host cells. To monitor shifts in gene regulation within Chlamydia-infected cells, we employed mesenchymal stem cells (MSCs) as a naïve, primary cell model. Utilizing biochemical methods and imaging, our study discloses that acute Chlamydia infection in human MSCs leads to the downregulation of transcription factors Oct4, Sox2, and Nanog, suggesting a loss of pluripotency markers. Conversely, pluripotency markers in MSCs were sustained through treatment with conditioned medium from infected MSCs. Additionally, there is an augmentation in alkaline phosphatase activity, along with elevated Sox9 and CD44 mRNA expression levels observed during acute infection. A comprehensive screening for specific cell markers using touchdown PCR indicates an upregulation of mRNA for the early chondrogenesis gene Sox9 and a decrease in mRNA for the MSC marker vimentin. Real-time PCR quantification further corroborates alterations in gene expression, encompassing increased Sox9 and CD44 mRNA levels, alongside heightened alkaline phosphatase activity. In summary, the infection of MSCs with C. trachomatis induces numerous genetic deregulations, implying a potential trend towards differentiation into chondrocytes. These findings collectively underscore a targeted impact of Chlamydia on the gene regulations of host cells, carrying significant implications for the final fate and differentiation of these cells.

Prognostic significance of LOXL2 enzyme activity in primary myelofibrosis.

Primary myelofibrosis (PMF) is characterized by bone marrow fibrosis, but the underlying molecular mechanisms remain incompletely understood. Here, we investigated the role of lysyl oxidase-like 2 (LOXL2), an enzyme involved in extracellular matrix remodeling, in PMF pathogenesis. Analysis of bone marrow cells from PMF patients revealed significantly elevated LOXL2 mRNA expression compared to healthy controls, which was further validated using 2 independent Gene Expression Omnibus datasets (GSE26049 and GSE12234). Serum LOXL2 protein levels were markedly increased in PMF patients (n = 38) compared to controls (n = 15) (P < .01), accompanied by significantly enhanced enzymatic activity (P < .05). Clinical analysis demonstrated that high LOXL2 enzyme activity correlated with adverse clinical features, including higher risk scores in both International Prognostic Scoring System (P = .029) and Dynamic International Prognostic Scoring System (P = .012) systems, and increased peripheral blood blast percentages (≥1%, P = .019). Notably, elevated LOXL2 enzyme activity emerged as an independent adverse prognostic factor in multivariate analysis. Gene set enrichment analysis revealed LOXL2's association with multiple biological processes, including extracellular matrix remodeling, inflammatory responses, and JAK-STAT signaling pathways. Using CellMiner drug prediction analysis, we identified several FDA-approved drugs, particularly kinase inhibitors, as potential LOXL2-targeting therapeutics. Our findings demonstrate LOXL2's critical role in PMF pathogenesis and suggest its potential as both a prognostic marker and therapeutic target in PMF treatment.

Daidzein Inhibits Non-small Cell Lung Cancer Growth by Pyroptosis.

Non-Small-Cell Lung Cancer (NSCLC) represents the leading cause of cancer deaths in the world. We previously found that daidzein, one of the key bioactivators in soy isoflavone, can inhibit NSCLC cell proliferation and migration, while the molecular mechanisms of daidzein in NSCLC remain unclear. We developed an NSCLC nude mouse model using H1299 cells and treated the mice with daidzein (30 mg/kg/day). Mass spectrometry analysis of tumor tissues from daidzein-treated mice identified 601 differentially expressed proteins (DEPs) compared to the vehicle-treated group. Gene enrichment analysis revealed that these DEPs were primarily associated with immune regulatory functions, including B cell receptor and chemokine pathways, as well as natural killer cell-mediated cytotoxicity. Notably, the NOD-like receptor signaling pathway, which is closely linked to pyroptosis, was significantly enriched. Further analysis of key pyroptosis-related molecules, such as ASC, CASP1, GSDMD, and IL-1β, revealed differential expression in NSCLC versus normal tissues. High levels of ASC and CASP1 were associated with a favorable prognosis in NSCLC, suggesting that they may be critical effectors of daidzein's action. In NSCLC-bearing mice treated with daidzein, RT-qPCR and Western blot analyses showed elevated mRNA and protein levels of ASC, CASP1, and IL-1β but not GSDMD, which was consistent with the proteomic data. In summary, this study demonstrated that daidzein inhibits NSCLC growth by inducing pyroptosis. Key pathway modulators ASC, CASP1, and IL-1β were identified as primary targets of daidzein. These findings offer insights into the molecular mechanisms underlying the anti-NSCLC effects of daidzein and could offer dietary recommendations for managing NSCLC.

METTL3 inhibits microglial pyroptosis in neonatal hypoxia-ischemia encephalopathy by regulating GPR39 expression in an m6A-HuR-dependent manner

BackgroundNeonatal hypoxia–ischemia encephalopathy (HIE) is a significant reason for neonatal mortality and prolonged disability. We have previously revealed that GPR39 activation attenuates neuroinflammation in a neonatal HIE rat model. This study aimed to investigate whether GPR39 affected microglial pyroptosis post-HIE. MethodsA neonatal rat model of HIE and a microglia cell model of oxygen-glucose deprivation (OGD) were established. Neuronal loss and cerebral infarction were assessed by using TTC, H&E staining, and Nissl staining. Pyroptosis was evaluated with western blot, LDH assay kit, ELISA, and flow cytometry. Total m6A level and GPR39 m6A modification were determined using m6A dot blot and MeRIP. The interaction between METTL3/HuR/GSK3β and GPR39 was analyzed by performing molecular interaction experiments. GPR39 mRNA stability was examined with actinomycin D. ResultsThe level of GPR39 was increased in neonatal HIE rats and OGD-treated microglia. Brain injury and neuronal loss were significantly increased in the HIE model when GPR39 was knocked down. GPR39 knockdown aggravated NLRP3 inflammasome-mediated microglial pyroptosis. METTL3 upregulated GPR39 expression in an m6A-dependent manner. METTL3 enhanced the interaction of HuR and GPR39. In OGD-exposed microglia, METTL3 elevated GPR39 expression and mRNA stability, which declined after HuR depletion. METTL3 knockdown promoted microglial pyroptosis, which was reversed by GPR39 agonist. Furthermore, microglial pyroptosis was inhibited by GPR39 upregulation, but the outcome was reverted by GSK3β activator SNP. ConclusionMETTL3 inhibits microglial pyroptosis in neonatal HIE via regulating m6A-HuR dependent stabilization of GPR39, which contributes to therapeutics development for neonatal HIE.

Expression patterns of Galectin-3 and NLRP3 in Chagas reactivation and graft damage in heart transplants.

This study aimed to assess the expression patterns of galectin-3 (Gal-3) and NLRP3 in heart transplant recipients according to the presence of reactivated Trypanosoma cruzi infection or allograft rejection in Chagas and non-Chagas heart transplant recipients. Gal-3 and NLRP3 expression levels were analyzed in endomyocardial biopsies from 31 heart transplant recipients, including 16 patients with chronic Chagas disease (ChD) and 15 without ChD. Samples were evaluated during periods of graft rejection or ChD reactivation, characterized by intense myocardial cellular infiltrate, and after remission of the infiltrate, classified by histopathological severity. The transcriptional levels of genes encoding Gal-3, NLRP3, Asc, caspase-1, and IL-1β were identified using the GEO2T tool across different experimental conditions. Gal-3 expression was lower in the myocardial infiltrate of ChD patients compared to non-ChD patients (p < 0.0001), whereas NLRP3 positivity was higher in ChD patients (p < 0.05). In a murine model of T. cruzi infection, elevated Gal-3 mRNA and NLRP3 inflammasome levels were observed in myocardial interstitial cells (p < 0.05). Peripheral blood mononuclear cells and cells from rodent cardiac allografts showed increased Gal-3 mRNA and NLRP3 levels compared to non-transplanted and rodent cardiac isografts (p < 0.001). Our findings suggest that Gal-3 and NLRP3 may be important biomarkers for differentiating heart transplant recipients with and without ChD regarding the myocardial immunological processes.

MicroRNA-34a-5p regulates agouti-related peptide via krüppel-like factor 4 and is disrupted by bisphenol A in hypothalamic neurons

Obesity is a complex disease marked by increased adiposity and impaired metabolic function. While diet and lifestyle are primary causes, endocrine-disrupting chemicals (EDCs), such as bisphenol A (BPA), significantly contribute to obesity. BPA, found in plastic consumer products, accumulates in the hypothalamus and dysregulates energy homeostasis by disrupting the neuropeptide Y (NPY)/agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) neurons.However, the precise molecular mechanisms of how BPA disrupts neuropeptide expression remains unclear. We hypothesized that microRNAs (miRNAs), which regulate approximately 60% of the human protein-coding genome and are crucial for hypothalamic energy regulation, may mediate the effects of BPA on Agrp. Using the TargetScanMouse 8.0 and DIANA microT bioinformatics tools, we identified miR-501-5p as a potential miRNA that directly regulates Agrp and the miR-34 family as miRNAs that indirectly regulate Agrp through its transcription factor krüppel-like factor 4 (KLF4). We found that in an immortalized NPY/AgRP-expressing cell line, mHypoE-41, miR-501-5p unexpectedly upregulated Agrp, while miR-34a-5p reduced Klf4 and Agrp mRNA levels. Serum starvation reduced miR-34a-5p levels and elevated Agrp mRNA levels, suggesting a potential role in AgRP regulation. Inhibiting the miR-34a-5p interaction with the Klf4 3′UTR using a specific target site blocker prevented the downregulation of both Klf4 and Agrp, suggesting miR-34a-5p alters Agrp mRNA levels via regulation of KLF4. BPA treatment increased Agrp and Klf4 expression while simultaneously decreasing miR-34a-5p levels, indicating miR-34a-5p may play a role in BPA-mediated dysregulation of Agrp. Overall, this study highlights indirect miRNA-based regulation of Agrp, which can also be dysregulated by obesogens, such as BPA.

Pharmacological inhibition of ezrin reduces proliferative and invasive phenotype in acute lymphoblastic leukemia cells

Ezrin (EZR) is an actin-associated protein that is often upregulated in cancers. Here we investigate the role of EZR in acute lymphoblastic leukemia (ALL) and explore the therapeutic potential of a pharmacological EZR inhibitor, NSC305787. ALL patient cohorts exhibit significantly elevated EZR mRNA levels, indicating its association with the malignant phenotype. Notably, EZR expression does not impact survival outcomes or relevant clinical-laboratory characteristics, suggesting a role in disease initiation rather than therapy response. NSC305787 induces a dose-dependent reduction in ALL cell viability, and is more potent than a related EZR inhibitor, NSC668394. NSC305787 has multiple effects on ALL cells, including apoptosis induction, clonal growth reduction, and inhibition of cell cycle progression. Importantly, it diminishes adhesiveness and invasiveness in ALL cells. Proteomics analysis highlights changes in translation, RNA catabolism, and cell cycle regulation, emphasizing the broad impact of EZR inhibition on ALL cell biology. Ex vivo assays with primary cells from acute myeloid leukemia (AML) and ALL patients demonstrate NSC305787's efficacy across a molecularly heterogeneous group, independent of risk stratification or recurrent mutations. Notably, NSC305787 shows heightened potency in ALL cells, suggesting its potential as a targeted therapy. In conclusion, our results report high EZR expression in adult ALL patients and support NSC305787 as a promising targeted therapy for ALL that should be further explored.

Deregulation of Casein Kinase-2 in non-small-cell lung cancer: A subunit-specific analysis

Casein kinase-2 (CK2) is a constitutively active kinase that supports neoplastic properties. Although there is an extensive body of preclinical research on CK2, translational and clinical information remains limited and sparse. In this study, we interrogated clinical multiomics databases to examine CK2 deregulation across various cancers. Specifically, we analyzed the mutational frequency, copy-number alterations (CNA), and mRNA expression of CK2 catalytic (CSNK2A1 and CSNK2A2) and regulatory (CSNK2B) subunits across two major cancer genomic repositories (The Cancer Genome Atlas/International Cancer Genome Consortium). These genomic and transcriptomic analyses were further focused on lung cancer and complemented by in situ assessments of CK2 protein subunits and enzymatic activity in biopsies from non-small-cell lung cancer (NSCLC). Our findings indicate that gene mutations and CNA do not account for the elevated mRNA levels and enzymatic activity of CK2 subunits in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Particularly, the upregulation of CSNK2A1 and CSNK2B mRNA was associated with a worse prognosis and showed a direct correlation with the infiltration of myeloid-derived suppressor cells in LUAD. At the protein levels, all CK2 subunits and enzymatic activity were markedly elevated in LUAD (n = 103) and LUSC (n = 31) biopsies; however, only CSNK2A1 expression correlated positively with tumor size and disease stage. Finally, CSNK2A1 appeared to be more tumor-specific than CSNK2A2, suggesting that targeted therapies against this catalytic subunit or its homotetramer may offer a more favorable therapeutic window in NSCLC.

Association of Variants in IL-1RN (rs2234663) and IL-1β (rs1143627, rs16944) and Interleukin-1β Levels with Colorectal Cancer: Experimental Study and In Silico Analysis

Background/Objectives. Colorectal cancer (CRC) is a multifactorial disease where the inflammatory state is crucial. This study analyzes the association of the IL-1RN (rs2234663) and IL-1β (rs1143627, rs16944) variants and IL-1β levels with CRC. Methods. This study included 230 CRC patients and 256 controls. Genotypes were determined by PCR and plasma IL-1β levels by ELISA. RegulomeDB analyzed the variants’ functional impacts, while OncoDB assessed IL-1β and IL-1RN expression’s influence on CRC. Results. The A1A1 genotype and dominant pattern of the rs2234663 variant were risk factors for CRC, whereas the A1A2 genotype showed a protective effect. The TC genotype of the rs1143627 variant and the T allele of rs16944 were associated with increased risk, whereas the C allele had a protective effect. The A1A1 genotype was associated with stage I–II CRC diagnosis, while the A2A2 genotype was associated with stage III–IV and ethanol consumption. The CC genotype of rs1143627 was associated with people younger than 50 years and tobacco use, and the TCCC genotype was related to stage III–IV stages and metastasis and hemorrhoids (p &lt; 0.05). IL-1β levels were not associated with CRC. In silico analysis revealed that the variants are in located in important regions regulatory of genes. Elevated IL-1B and IL-1RN mRNA levels were found in CRC, linked to clinicopathological features of the disease. Conclusions. The analyzed variants are associated with CRC and may influence gene regulation by being located at critical sites of key genetic regulators.

HIPK3 hypomethylation as a potential epigenetic biomarker in rheumatic immune diseases with emphasis on rheumatoid arthritis.

This study aimed to explore the potential of HIPK3 DNA methylation as a biomarker for rheumatoid arthritis (RA) by examining its methylation levels in various rheumatic immune diseases and analyzing its correlation with clinical indicators. We recruited 368 participants, including patients with RA, ankylosing spondylitis (AS), GOUT, psoriatic arthritis (PSA), sjogren's syndrome (SS), systemic lupus erythematosus (SLE), dermatomyositis (DM), and healthy controls (HC), and MethylTargetTM targeted region methylation sequencing technology was employed to analyze the DNA methylation levels of HIPK3 (cg15692052). We analyzed the HIPK3 methylation levels and correlated them with common clinical indicators. Transcriptome data from the GEO dataset (GSE93272) were also analyzed to assess HIPK3 mRNA expression levels in RA patients. Statistical analyses included Spearman correlation, One-Way ANOVA, Kruskal-Wallis tests, and ROC curve analysis. HIPK3 methylation levels were significantly lower in the RA, SLE, and DM groups compared to the HC group (P < 0.05). RA subgroups based on RF and CCP statuses also showed lower HIPK3 methylation levels compared to the HC group (P < 0.05). Analysis of the GEO dataset revealed significantly elevated HIPK3 mRNA expression in RA patients (P < 0.05). A negative correlation was found between HIPK3 methylation levels and ESR (r = -0.14, P = 7.1e-3), CRP (r = -0.25, P = 4.1e-7), RF (r = -0.18, P = 5.2e-4), and DAS28-CRP (r = -0.12, P = 0.04) in the RA group. ROC analysis indicated that HIPK3 methylation levels had high diagnostic value for RA (AUC = 0.742), SLE (AUC = 0.701), and DM (AUC = 0.948), especially in distinguishing seronegative RA (AUC = 0.612, 0.747, 0.836) and differentiating RA from AS (AUC = 0.788). The study suggests that HIPK3 hypomethylation in peripheral blood is associated with RA and correlated with inflammatory markers, such as CRP. HIPK3 methylation levels have potential as a novel biomarker for RA diagnosis and predicting inflammation trends, showing particular promise in differentiating RA from other types of arthritis. Key Points • MethylTargetTM targeted region methylation sequencing technology was employed to analyze the DNA methylation levels of HIPK3 (cg15692052). • HIPK3 methylation levels have potential as a novel biomarker for RA diagnosis and predicting inflammation trends.

Methylglyoxal impairs human dermal fibroblast survival and migration by altering RAGE-hTERT mRNA expression in vitro

Fibroblasts are native residents in dermal layer of human skin which are important for dermal regeneration and essential during cutaneous wound healing by releasing inflammatory markers and actively migrate to close an open wound. Premature skin ageing due to methylglyoxal (MGO) has recently caught the attention considering its potential to accelerate the emergence of skin ageing signs, however previous studies were only focused in primary neonatal dermal fibroblast and NIH3t3 fibroblast cell line. Therefore, thorough investigation is required to study the impact of MGO on primary human dermal fibroblast isolated from adult subject (HDFa). In our experiments, short exposure of MGO was observed to induced significant reductions in cell viability at concentrations of 7.5, 10, 12.5, 15, and 17.5 mM (p < 0.005) after 3 hours of treatment. The cellular death of HDFa at 10, 12.5 and 15 mM of MGO were also marked by increased in intracellular ROS level, indicating the involvement of oxidative stress-induced death in these cells. We also observed enlarge scratch areas of cells exposed with 7.5 and 10 mM MGO compared to control after 26 hours, thereby suggesting a decline in cell migration and viability in this group. We propose the increased ROS as the consequence of AGE-RAGE activation which was marked by significant elevation of RAGE mRNA on cells exposed to 10 mM MGO. Our data also suggest the occurrence of DNA damage events via ROS-induced oxidation or mediated by decline in hTERT mRNA expression.

Abstract A008: Identification of EYA2 as a promising biomarker for DICER1-related tumor predisposition

Abstract Background: DICER1-related tumor predisposition (DRTP), also known as DICER1 syndrome, is a genetic condition associated with an increased risk of developing various tumors, including pleuropulmonary blastoma, thyroid gland neoplasia, ovarian tumors, and cystic nephroma. Currently, the diagnosis and surveillance of DRTP primarily relies on genetic testing for DICER1 mutations. There is a pressing need for surrogate diagnostic biomarkers to improve early detection and monitoring. Objective: To identify and validate EYA2 as a potential biomarker for DICER1-related tumor predisposition. Methods: Murine mesenchymal stromal cell lines were developed to model the consequences of DICER1 mutations, including the DICER1E1705K mutation found in DRTP. Both bulk RNA sequencing (RNAseq) and microRNA sequencing (miRNAseq) were employed to identify differentially expressed mRNAs in these cell lines. The RNA and protein levels of Eya2 were measured by qRT-PCR and Western blot, respectively. De-repression of Eya2 mRNA by miRNA was evaluated by dual luciferase assay to link overexpression with the DICER1 defect of DRTP. Then, immunohistochemistry staining of EYA2 was performed on a panel of DRTP tumors. Results: We found that more than 2000 mRNAs were derepressed in mesenchymal stromal cells expressing DICER1E1705K as compared to DICER1WT. Among these, Eya2 emerged as a top candidate. We observed elevated Eya2 mRNA and protein levels in DICER1E1705K -expressing mesenchymal stromal cells. Utilizing a reporter assay, we also observed de-repression of the 3’ UTR of Eya2 upon expression of DICER1E1705K as compared to DICER1WT. Strong nuclear EYA2 staining was observed in a subset of DRTP tumor samples. Conclusion: Elevated EYA2 expression in tumor tissues from DRTP patients supports its potential use in both diagnostic and surveillance contexts. Further studies are warranted to validate EYA2 as a clinical biomarker and explore its utility in improving early detection and monitoring strategies for individuals at risk of DRTP. Citation Format: Mona Wu, Shengmei Zhou, Felix Kommoss, Yemin Wang, James F Amatruda. Identification of EYA2 as a promising biomarker for DICER1-related tumor predisposition [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 A008.

Overexpression of the inwardly rectifying potassium channel Kir4.1 or Kir4.1 Tyr9 Asp in Müller cells exerts neuroprotective effects in an experimental glaucoma model

Downregulation of the inwardly rectifying potassium channel Kir4.1 is a key step for inducing retinal Müller cell activation and interaction with other glial cells, which is involved in retinal ganglion cell apoptosis in glaucoma. Modulation of Kir4.1 expression in Müller cells may therefore be a potential strategy for attenuating retinal ganglion cell damage in glaucoma. In this study, we identified seven predicted phosphorylation sites in Kir4.1 and constructed lentiviral expression systems expressing Kir4.1 mutated at each site to prevent phosphorylation. Following this, we treated Müller glial cells in vitro and in vivo with the mGluR I agonist DHPG to induce Kir4.1 or Kir4.1 Tyr9Asp overexpression. We found that both Kir4.1 and Kir4.1 Tyr9Asp overexpression inhibited activation of Müller glial cells. Subsequently, we established a rat model of chronic ocular hypertension by injecting microbeads into the anterior chamber and overexpressed Kir4.1 or Kir4.1 Tyr9Asp in the eye, and observed similar results in Müller cells in vivo as those seen in vitro. Both Kir4.1 and Kir4.1 Tyr9Asp overexpression inhibited Müller cell activation, regulated the balance of Bax/Bcl-2, and reduced the mRNA and protein levels of pro-inflammatory factors, including interleukin-1β and tumor necrosis factor-α. Furthermore, we investigated the regulatory effects of Kir4.1 and Kir4.1 Tyr9Asp overexpression on the release of pro-inflammatory factors in a co-culture system of Müller glial cells and microglia. In this co-culture system, we observed elevated adenosine triphosphate concentrations in activated Müller cells, increased levels of translocator protein (a marker of microglial activation), and elevated interleukin-1β mRNA and protein levels in microglia induced by activated Müller cells. These changes could be reversed by Kir4.1 and Kir4.1 Tyr9Asp overexpression in Müller cells. Kir4.1 overexpression, but not Kir4.1 Tyr9Asp overexpression, reduced the number of proliferative and migratory microglia induced by activated Müller cells. Collectively, these results suggest that the tyrosine residue at position nine in Kir4.1 may serve as a functional modulation site in the retina in an experimental model of glaucoma. Kir4.1 and Kir4.1 Tyr9Asp overexpression attenuated Müller cell activation, reduced ATP/P2X receptor-mediated interactions between glial cells, inhibited microglial activation, and decreased the synthesis and release of pro-inflammatory factors, consequently ameliorating retinal ganglion cell apoptosis in glaucoma.

TMIC-36. EVALUATING PHARMACOLOGICAL INHIBITION OF LSD1 IN MODELS OF DIFFUSE MIDLINE GLIOMA AND EFFECTS ON IMMUNE MICROENVIRONMENT SIGNALING

Abstract BACKGROUND Diffuse midline glioma (DMG) is the leading cause of death for pediatric brain tumor patients. DMG is remarkably immunologically senescent, even in comparison to other “immune-cold” tumors like adult glioblastoma. Lysine specific demethylase-1 (LSD1) is a histone demethylase exerting context-specific mechanisms of gene repression and activation and can be inhibited pharmacologically. We found LSD1 regulates the expression of inflammatory cytokines such as Interleukin 18 (IL18) and immune signaling receptors in DMG. Here, we investigated whether IL18 is transcriptionally regulated by LSD1 in vitro and in vivo using clinically relevant small molecule inhibitors. METHODS LSD1 binding to the IL-18 promoter was evaluated using chromatin immunoprecipitation. Transcript and protein expression using qRT-PCR and western blotting assessed the impact of LSD1 inhibition on IL18-related pathways and immune signaling pathways. LSD1 inhibitors (IMG-7289 or GSKLSD1) were evaluated patient-derived xenograft models. Results. We found elevated LSD1 binding on the IL18 promoter in DIPG VI cells. Subsequent qRT-PCR and western blot analyses demonstrated increased IL18 protein levels following treatment with GSKLSD1 or IMG-7289 in DIPG VI cells, alongside heightened IL18 mRNA expression compared to DMSO-treated cells. Either GSKLSD1 or IMG-7289 treatment in DIPG IV cells also led to significantly elevated IL18 binding protein mRNA levels. Orthotopic xenografts using DIPG IV bearing mice dosed with LSD1 inhibitors over the course of 35 days several weeks showed a decrease in IL-18R, and an increase in IL-18 BP transcript levels alongside a significant increase in Slamf7 transcript levels. Some evidence of tumor inhibition was seen in male but not female mice treated with GSKLSD1. CONCLUSIONS Our investigation revealed IL18 as a direct target of LSD1 specifically in DIPG VI, and gender specific effects of GSKLSD1 treatment in vivo. Future studies will prioritize validating and elucidating the underlying mechanisms the therapeutic potential of LSD1 inhibition in DMG.

DNAR-06. EPITRANSCRIPTOMIC TRNA MODIFICATIONS TARGETING ADENOSINE (A) AT POSITION 37 IN GBM

Abstract GBM stands as the most aggressive brain tumor in adults. The median overall survival is less than 2 years despite conventional treatments. Recent advancements in DNA analysis have delineated the molecular characteristics of gliomas, leading to revisions in the WHO classification based on DNA profiles. However, the role of RNA modifications, epitranscriptomics, remains poorly understood in glioma. In this study, we underscore the significance of tRNA modifications targeting adenosine at position 37 in GBM. Transfer RNA isopentenyl transferase 1 (TRIT1) is identified as an isopentenyl transferase catalyzing the conversion of A into i6A at position 37 of tRNA, a nucleotide adjacent to the anticodon region. Additionally, i6A is further modified to 2-methylthio i6A (ms2i6A) by cyclin-dependent kinase 5 regulatory subunit associated-protein 1 (CDK5RAP1) within mitochondrial tRNA. Analysis of TCGA data reveals elevated TRIT1 mRNA expression levels in GBM compared to non-tumor samples. Furthermore, a low expression group of TRIT1 in gliomas demonstrates improved survival outcomes according to data from the CGGA, implicating TRIT1 in glioma progression. Functional assays involving TRIT1 knockdown in human GBM cell lines result in decreased cell viability, indicating its functional significance. Moreover, our data suggest that this phenotype may be mediated through translational control of selenoprotein W (SEPW1) via tRNA^(Ser)Sec modification. GO analysis of TRIT1 and SEPW1 in TCGA data further supports this proposed mechanism. At position 37 of tRNA, we also found that CDK5RAP1 in mitochondrial tRNAs is indispensable for sustaining glioblastoma initiating cell (GIC)-related traits. CDK5RAP1 maintains the self-renewal capacity, undifferentiated state, and tumorigenic potential of GICs, independent of translational control of mitochondrial proteins. Notably, CDK5RAP1 abrogates the antitumor effect of i6A by converting i6A to ms2i6A and shields GICs from excessive autophagy triggered by i6A. This work underscores the critical role of tRNA modifications in glioma pathogenesis. We will report these findings alongside RNA modification profile data and clinical data from glioma patients.

Epitranscriptomic regulation of cardiac fibrosis via YTHDF1-dependent PIEZO2 mRNA m6A modification.

Mechanosensitive ion channels play a key role in heart development, physiology, and disease. However, little is known about the molecular mechanisms of the mechanosensitive nonselective cationic channel Piezo family in cardiac fibrosis. Mice were treated with ISO/Ang-II/TAC to induce cardiac fibrosis. AAV9 carrying POSTN promoter-driven small hairpin RNA targeting YTHDF1, and Piezo2 were administered to ISO mice to investigate their roles in cardiac fibrosis. RNA-seq, single-cell sequencing, and histological and biochemical analyses were performed to determine the mechanism by which YTHDF1 regulates Piezo2 expression in cardiac fibrosis. Piezo2 was reconstituted in YTHDF1-deficient cardiac fibroblasts and mouse hearts to study its effects on cardiac fibroblast autophagy and fibrosis. Piezo2 but not Piezo1 expression increased in experimental cardiac fibrosis and TGF-β1-induced cardiac fibroblasts. Fibroblast-specific Piezo2 deficiency ameliorated fibroblast activation and autophagy and inhibited cardiac fibrosis. Mechanistically, Piezo2 upregulation was associated with elevated m6A mRNA levels. Site-specific m6A modifications at peak_26355 were crucial for regulating the binding of YTHDF1 to Piezo2 mRNA and inducing Piezo2 translation. Notably, Piezo2 epitranscriptomic repression ameliorated experimental cardiac fibrosis. We demonstrated a novel epitranscriptomic mechanism through which YTHDF1 recognizes Piezo2 and controls cardiac fibroblast autophagy and fibrosis through m6A-dependent modulation. Our findings provide new insights for the development of preventive measures for cardiac fibrosis.

Preclinical Evaluation of MK-8189: A Novel Phosphodiesterase 10A Inhibitor for the Treatment of Schizophrenia.

MK-8189 is a novel phosphodiesterase 10A (PDE10A) inhibitor being evaluated in clinical studies for the treatment of schizophrenia. PDE10A is a cyclic nucleotide phosphodiesterase enzyme highly expressed in medium spiny neurons of the striatum. MK-8189 exhibits sub-nanomolar potency on the PDE10A enzyme and has excellent pharmaceutical properties. Oral administration of MK-8189 significantly increased cGMP and pGluR1 in rat striatal tissues. Activation of the dopamine D1 direct and D2 indirect pathways was demonstrated by detecting significant elevation of mRNA encoding substance P (Sub P) and enkephalin (ENK) after MK-8189 administration. The PDE10A tracer [3H]MK-8193 was used determine the PDE10A enzyme occupancy (EO) required for efficacy in behavioral models. In the rat conditioned avoidance responding assay, MK-8189 significantly decreased avoidance behavior at PDE10A EO greater than ~48%. MK-8189 significantly reversed an MK-801-induced deficit in pre-pulse inhibition at PDE10A EO of ~47% and higher. Target engagement of MK-8189 in rhesus monkeys was examined with [11C]MK-8193 in PET studies and plasma concentrations of 127nM MK-8189 yielded ~50% EO in the striatum. The impact of MK-8189 on cognitive symptoms was evaluated using the objective retrieval task in rhesus monkeys. MK-8189 significantly attenuated a ketamine-induced deficit in object retrieval performance at exposure that yielded ~29% PDE10A EO. These findings demonstrate the robust impact of MK-8189 on striatal signaling and efficacy in preclinical models of symptoms associated with schizophrenia. Data from these studies were used to establish the relationship between preclinical efficacy, plasma exposures, and PDE10A EO to guide dose selection of MK-8189 in clinical studies. Significance Statement We describe the primary pharmacology of MK-8189 a PDE10A inhibitor under evaluation for the treatment of schizophrenia. We report efficacy in preclinical models that have been used to characterize other PDE10A inhibitors and atypical antipsychotics. The PDE10A occupancy achieved by MK-8189 in behavioral studies was used to support dose selection in clinical trials. This work provides evidence to support exploration of higher levels of PDE10A occupancy in clinical trials to determine if this translates to improved efficacy in patients.

Molecular and structural analyses of voltage-dependent anion channel 2 and its anti-apoptotic function in stress and pollutant resistance in Pacific abalone

This study aimed to identify voltage-dependent anion channel 2 (Hdh-VDAC2) and determine its functional role in response to acute thermal stress, H2O2-induced stress, heavy metal toxicity, bacterial and viral infections, and during metamorphosis. Structural analysis confirmed that Hdh-VDAC2 is a pore-forming β-barrel protein. Molecular docking further confirmed the protein-protein interactions of Hdh-VDAC2 with Hdh-BAX, Hdh-caspase 3, and Hdh-BCL2. In the Hdh-VDAC2-inhibited hemocytes (HCY), apoptotic genes (Hdh-caspase-3 and Hdh-BAX) exhibited elevated mRNA expression, while the anti-apoptotic gene (Hdh-BCL2) was downregulated. Further, fluorescent techniques confirmed excessive reactive oxygen species (ROS) production, lower cell viability, elevated caspase 3 activity, and increased DNA fragmentation in Hdh-VDAC2-inhibited HCY, indicating an anti-apoptotic role of Hdh-VDAC2 in Pacific abalone. Transcriptomic analysis revealed differential expression patterns, with upregulation in the digestive gland (DG) and downregulation in the gill (GIL) and HCY when comparing heat-tolerant (HT) versus heat-sensitive (HS) abalone groups. Additionally, both cold and heat stresses induced Hdh-VDAC2 expression. Other environmental factors including H2O2, cadmium, bacteria, and viruses, were also shown to induce Hdh-VDAC2 mRNA expression in the GIL and DG of Pacific abalone. During metamorphosis, the blastula (BLS) stage exhibited higher Hdh-VDAC2 mRNA expression. These findings suggest that Hdh-VDAC2 plays a crucial anti-apoptotic role and may be a biomarker for summer mortality in Pacific abalone.

Tensile stress induced osteogenesis of periodontal ligament cells via Piezo1 mediated TAZ-Cbfα1 signaling

ObjectiveCyclic tensile stress (CTS) is known to induce osteogenesis of periodontal ligament cells (PDLCs), in which the molecular mechanism remains to be elucidated.This study aimed to investigate the role of the mechanosensitive calcium channel Piezo1 in the osteogenesis of PDLCs under tensile strain, as well as the signal regulation of the TAZ-Cbfα1 pathway in this process. DesignPDLCs were isolated from periodontal ligament tissues and subjected to CTS. Alizarin red staining (ARS) and alkaline phosphatase (ALP) assay were used to detect the osteogenesis of PDLCs. RT-qPCR and Western blot were used to detect the transcripts and protein expression levels of Piezo1, Transcriptional co-activator with PDZ binding motif (TAZ), and Core-binding factor α1 (Cbfα1) respectively. Immunofluorescence staining was used to detect the nuclear aggregation of TAZ. Small interfering RNA (siRNA) targeting Piezo1 (Piezo1-siRNA) was adopted to inhibit Piezo1 mRNA expression. ResultsThe results showed that the osteogenic differentiation capacity of PDLCs was significantly enhanced under CTS, along with elevated mRNA and protein expression levels of Piezo1, TAZ, and Cbfα1. Moreover, the ALP activity and the formation of calcium nodules by ARS staining were significantly increased. In addition, Piezo1 siRNA infection significantly inhibited the CTS-induced TAZ-Cbfα1 pathway and the osteogenesis of PDLCs, suggesting the regulatory role of Piezo1. ConclusionsWe provided evidence that the application of CTS encourages the osteogenic differentiation of PDLCs, which could be mediated by the Piezo1 targeted TAZ-Cbfα1 signaling.

Sex differences in the expression of metabolic and pro-inflammatory markers in the initial post-COVID-19 phase

Abstract Cardiovascular complications are a common manifestation of post-acute COVID-19 infection. Post-COVID-19 syndrome is often associated with the female sex and exacerbated inflammation; however, the underlying molecular mechanisms of this sex difference remain poorly understood. In the present study, we aimed to investigate several metabolic, mitochondrial, autophagy, oxidative stress, and inflammatory markers in peripheral blood mononuclear cells (PBMCs) and plasma isolated from older (&amp;gt;75) male and female patients at the initial phase (1-2 weeks) after COVID-19 pneumonia. Age-matched non-infected women and men were used as a control group. We found a significantly higher plasma TNF-α in post-COVID-19 women, but not in men, compared to the control group. Similarly, a significant elevation of IL-1β and IL-18 mRNA in PBMCs from post-COVID-19 women, but not men, was observed. These effects were associated with marked activation of AMPK (defined as pAMPK/AMPK ratio), suggesting potential metabolic stress in female post-COVID-19 PBMCs. Further analysis revealed increased expression of mitochondrial genes, which may be due to AMPK activation as a compensatory reaction to metabolic stress. Analysis of autophagy or oxidative stress (serum 8-OHdG) markers did not reveal any differences between the groups investigated. We conclude that women in the initial phase of post-COVID-19 have elevated expression of inflammatory markers in plasma and in immune cells, which is associated with metabolic stress. This may contribute to the cardiovascular complications in female post-COVID-19 patients.