Regulation Of Cell Differentiation Research Articles

The heterogeneity of NOTCH1 to tumor immune infiltration in pan-cancer.

NOTCH1 signaling, a vital regulator of cell proliferation and differentiation, is widely involved in the occurrence and development of malignant tumors. Pharmacological regulation of NOTCH1 is promising in tumor immunotherapy, whereas the effective rate of existing therapies remains low. NOTCH1 functions, as a cancer suppressor or a cancer promoter in different cancers, is engaged in the crosstalk between the immune microenvironment and cancer cells, posing a major challenge to immunotherapy. Therefore, a comprehensive view of the overall situation of NOTCH1-associated immune infiltration in pan-cancer should be built. The relation between NOTCH1 and immune infiltration was initially investigated in this paper. In this study, the data originated from the Genotype-Tissue Expression (GTEx) and the Cancer Genome Atlas (TCGA) databases were input into multiple online bioinformatic tools to study the characteristics of NOTCH1 in pan-cancer. We found that there was obvious heterogeneity in the NOTCH1-associated tumor immune infiltration in pan-cancer. In accordance with the heterogeneity, pan-cancer mainly fell into two categories, i.e., cancers that NOTCH1 promoted immune infiltration (termed hot tumors) and NOTCH1 inhibited immune infiltration (termed cold tumors). We further analyzed the changes of immune infiltration in pan-carcinoma species from the perspectives of NOTCH1 expression, mutation, gene function, tumor metastasis and drugs. NOTCH1 expression was significantly up-regulated in cold tumors but down-regulated in hot tumors. The Gene ontology (GO) enrichment analysis of NOTCH1 with the two categories placed stress on angiogenesis and protein dealkylation, respectively. Further, the gene sets of angiogenesis facilitated immune infiltration, whereas the gene sets of protein dealkylation hindered immune infiltration. The tsRNA associated with NOTCH1 is a type of angiogenin that potentially exerts a significant influence on angiogenesis. We have conducted a meticulous analysis of the function of this tsRNA. NOTCH1 was conducive to cancer-associated fibroblasts (CAFs) immune infiltration, while the metastatic process was more dependent on the differentiation and angiogenesis function of NOTCH1. Accordingly, the heterogeneity of NOTCH1 in immune infiltration was extensively analyzed in this study based on the pan-cancer study, which can contribute to the formulation of specific immunotherapy strategies.

M5C related-regulator-mediated methylation modification patterns and prognostic significance in breast cancer

5-Methylcytosine (m5C) is closely associated with cancer. However, the role of m5C in breast cancer(BC)remains unclear. This study combined single-cell RNA sequencing (scRNA-Seq) and transcriptomics datasets to screen m5C regulators associated with BC progression and analyze their clinical values. Firstly, This study elucidates the mechanisms of the m5C landscape and the specific roles of m5C regulators in BC patients. we found that the dysregulation of m5C regulators with m5Cscore play the essential role of the carcinogenesis and progression in epithelial cells and myeloid cells of BC at single cell level. External validation was conducted using an independent scRNA-Seq datasets. Then, three distinct m5C modification patterns were identified by transcriptomics datasets. Based on the m5C differentially expressed regulators, the m5Cscore was constructed, and used to divide patients with BC into high and low m5Cscore groups. Patients with a high m5Cscore had more abundant immune cell infiltration, stronger antitumor immunity, and better prognoses. Finally, Quantitative real-time (PCR) and immunohistochemistry were used for the in vitro experimental validation, which had extensive prognostic value. In this study, we aimed to assess the expression of m5C regulators involved in BC and investigate their correlation with the tumor microenvironment, clinicopathological characteristics, and prognosis of BC. The m5C regulators could be used to effectively assess the cell specific regulation prognosis of patients with BC and develop more effective immunotherapy strategies.

AEBP1 is a negative regulator of skeletal muscle cell differentiation in oral squamous cell carcinoma

The tumor microenvironment plays a pivotal role in cancer development. We recently reported that in oral squamous cell carcinoma (OSCC), adipocyte enhancer-binding protein 1 (AEBP1) is abundantly expressed in cancer-associated fibroblasts (CAFs), leading to CAF activation and inhibition of CD8 + T cell infiltration. In the present study, we investigated whether AEBP1 contributes to the destruction and atrophy of muscle tissues in OSCC. By analyzing human skeletal muscle myoblasts (HSMMs), we found that AEBP1 is downregulated during muscle cell differentiation. Transcriptome analysis revealed that AEBP1 knockdown significantly upregulates myogenesis-related genes in HSMMs, and qRT-PCR and western blot analyses confirmed the induction of muscle-related genes, including MYOG, in HSMMs after AEBP1 knockdown. Conversely, ectopic expression of AEBP1 strongly suppressed myogenesis-related genes in HSMMs. Notably, indirect co-culture of HSMMs with OSCC cells led to AEBP1 upregulation and robust suppression of muscle-related genes in HSMMs. Treatment with TGF-β1 also upregulated AEBP1 and suppressed expression of muscle-related genes in HSMMs. Our findings suggest that AEBP1 is a negative regulator of skeletal muscle cell differentiation and that OSCC cells inhibit muscle cell differentiation, at least in part, by inducing AEBP1.

Characterization of single-cell cis-regulatory elements informs implications for cell differentiation.

Cis-regulatory elements (CREs) govern the specific patterns and dynamics of gene expression in cells during development, which are the fundamental mechanisms behind cell differentiation. However, the genomic characteristics of single-cell CREs (scCREs) closely linked to cell differentiation during development remain unclear. To explore this, we systematically analyzed ∼250,000 putative scCREs obtained from snATAC-seq analysis of the developing mouse cerebellum. We found that over 80% of these scCREs show pleiotropic effects, being active in two or more cell types. The pleiotropic degrees of proximal and distal scCREs are positively correlated with the density and diversity of transcription factor (TF) binding motifs and GC content. There is a negative correlation between the pleiotropic degrees of scCREs and their distances to the nearest TSSs, and proximal scCREs display higher relevance strengths than distal ones. Furthermore, both proximal and distal scCREs related to cell differentiation exhibit enhanced sequence-level evolutionary conservation, increased density and diversity of TF binding motifs, elevated GC content, and greater distances from their nearest genes. Together, our findings reveal the general genomic characteristics of putative scCREs and provide insights into the genomic and evolutionary mechanisms by which scCREs regulate cell differentiation during development.

GABA in early brain development: A dual role review.

This comprehensive review examines the multifaceted roles of gamma-aminobutyric acid (GABA) in early brain development. GABA, traditionally recognized for its inhibitory functions in the mature brain, also exhibits excitatory effects during early neural development. This article explores the mechanisms behind GABA's dual roles, detailing its impact on the properties of the immature brain, the mechanisms of GABA-mediated excitation, the role of GABA-mediated presynaptic inhibition, the trophic actions of GABA during early development, GABA regulation of neurite growth and GABA-mediated cell differentiation in the immature brain. Emphasizing recent research findings, the review highlights the significance of GABAergic signalling in shaping the developing brain and its potential implications for understanding neurodevelopmental disorders.

The sodium/ascorbic acid co-transporter SVCT2 distributes in a striated membrane-enriched domain at the M-band level in slow-twitch skeletal muscle fibers

BackgroundVitamin C plays key roles in cellular homeostasis, functioning as a potent antioxidant and a positive regulator of cell differentiation. In skeletal muscle, the vitamin C/sodium co-transporter SVCT2 is preferentially expressed in oxidative slow fibers. SVCT2 is up-regulated during the early fusion of primary myoblasts and decreases during initial myotube growth, indicating the relevance of vitamin C uptake via SVCT2 for early skeletal muscle differentiation and fiber-type definition. However, our understanding of SVCT2 expression and function in adult skeletal muscles is still limited.ResultsIn this study, we demonstrate that SVCT2 exhibits an intracellular distribution in chicken slow skeletal muscles, following a highly organized striated pattern. A similar distribution was observed in human muscle samples, chicken cultured myotubes, and isolated mouse myofibers. Immunohistochemical analyses, combined with biochemical cell fractionation experiments, reveal a strong co-localization of SVCT2 with intracellular detergent-soluble membrane fractions at the central sarcomeric M-band, where it co-solubilizes with sarcoplasmic reticulum proteins. Remarkably, electrical stimulation of cultured myofibers induces the redistribution of SVCT2 into a vesicular pattern.ConclusionsOur results provide novel insights into the dynamic roles of SVCT2 in different intracellular compartments in response to functional demands.

Judy Lieberman: Stay curious and excited about science.

Judy Lieberman is a professor of pediatrics and adjunct professor of genetics at Harvard Medical School and an endowed chair in cellular and molecular medicine. Her lab studies cytotoxic T lymphocytes (CTL), key cells in the immune defense against viral infection and cancer, as well as molecular pathways activated by the granzymes, and how RNA interference (RNAi) regulates cell differentiation in health and disease states. We spoke to Judy about advice for early career researchers, how she first become interested in cytotoxic T lymphocytes, and key people who have provided mentorship across her career.

Processing and properties of graphene-reinforced polylactic acid nanocomposites for bioelectronic and tissue regenerative functions

An in-situ polymer-solution-processing approach enables the efficient production of uniform graphene-reinforced polylactic acid (G-PLA) nanocomposites with notable physical and biomedical properties. The approach effectively enhances the interfacial bonding between graphene and PLA by creating graphene dangling bonds and defects during exfoliation. As a result, an 182 % increase in Young's modulus and an 85 % increase in tensile strength can be achieved in G-PLA. Only 0.5 wt% graphene addition can reduce the contact angle of the composite from 75.3 to 70.4 and reduce its oxygen permeability by 23 %. The improved hydrophilicity, hermeticity, and mechanical properties make G-PLA an excellent encapsulation material for implantable bioelectronics. Moreover, the composite surface attributes and cell behaviors at the material-tissue interface are investigated histologically through the culture of stem cells on as-synthesized G-PLA. G-PLA composites can significantly boost cell proliferation and regulate cell differentiation towards vascular endothelium, offering tissue regeneration at the surface of implants to recover the injured tissues. The degradation rate of G-PLA nanocomposite can also be regulated since the graphene slows down the autocatalytic chain splitting induced by the terminal carboxylic acid groups of PLA. Therefore, such G-PLA nanocomposites with physical and biomedical properties regulated by graphene loading enable the development of next-generation implantable electronic systems providing both sensing and tissue engineering functions for complicated applications such as implanted sensors monitoring the healing of fractured bones or intracranial pressure.

Green Synthesis of Silver Nanoparticles with Roasted Green Tea: Applications in Alginate–Gelatin Hydrogels for Bone Regeneration

The incorporation of silver nanoparticles (AgNPs) into alginate–gelatin (Alg-Gel) hydrogels can enhance the properties of these materials for bone regeneration applications, due to the antimicrobial properties of AgNPs and non-cytotoxic concentrations, osteoinductive properties, and regulation of stem cell proliferation and differentiation. Here, the hydrogel formulation included 2% (w/v) sodium alginate, 4 µg/mL AgNPs, and 2.5% (w/v) gelatin. AgNPs were synthesized using a 2% (w/v) aqueous extract of roasted green tea with silver nitrate. The aqueous extract of roasted green tea for AgNP synthesis was characterized using HPLC and UHPLC-ESI-QTOF-MS/MS, and antioxidant capacity was measured in Trolox equivalents (TE) from 4 to 20 nmol/well concentrations. Stem cells from human exfoliated deciduous tooth cells were used for differentiation assays including positive (SHEDs/hydrogel with AgNPs) and negative controls (hydrogel without AgNPs). FTIR was used for hydrogel chemical characterization. Statistical analysis (p < 0.05, ANOVA) confirmed significant findings. Roasted green tea extract contained caffeine (most abundant), (−)-Gallocatechin, gallic acid, and various catechins. XRD analysis revealed FCC structure, TEM showed quasispheroidal AgNPs (19.85 ± 3 nm), and UV–Vis indicated a plasmon surface of 418 nm. This integration of nanotechnology and biomaterials shows promise for addressing bone tissue loss in clinical and surgical settings.

Genome-Wide CRISPR Screen Identifies Genes Involved in Metastasis of Pancreatic Ductal Adenocarcinoma.

Background/Objectives: Early and aggressive metastasis is a major feature of pancreatic ductal adenocarcinoma. Understanding the processes underlying metastasis is crucial for making a difference to disease outcome. Towards these ends, we looked in a comprehensive manner for genes that are metastasis-specific. Methods: A genome-wide CRISPR-Cas9 gene knockout screen with 259,900 single guide RNA constructs was performed on pancreatic cancer cell lines with very high or very low metastatic capacity, respectively. Functional aspects of some of the identified genes were analysed in vitro. The injection of tumour cells with or without a gene knockout into mice was used to confirm the effect on metastasis. Results: The knockout of 590 genes-and, with higher analysis stringency, 67 genes-affected the viability of metastatic cells substantially, while these genes were not vital to non-metastasizing cells. Further evaluations identified different molecular processes related to this observation. One of the genes was MYBL2, encoding for a well-known transcription factor involved in the regulation of cell survival, proliferation, and differentiation in cancer tissues. In our metastasis-focussed study, no novel functional activity was detected for MYBL2, however. Instead, a metastasis-specific transformation of its genetic interaction with FOXM1 was observed. The interaction was synergistic in cells of low metastatic capacity, while there was a strong switch to a buffering mode in metastatic cells. In vivo analyses confirmed the strong effect of MYBL2 on metastasis. Conclusions: The genes found to be critical for the viability of metastatic cells form a basis for further investigations of the processes responsible for triggering and driving metastasis. As shown for MYBL2, unexpected processes of regulating metastasis might also be involved.

Cell-microsphere based Living Microhybrids for Osteogenesis Regulating to Boosting Biomineralization

Abstract Biomineralization-based cell-material living composites ex vivo showed great potential for living materials construction and cell regulation. However, cells in scaffolds with unconnected pores usually induce confined nutrient transfer and cell-cell communications, affecting the transformation of osteoblasts into osteocytes and the mineralization process. Herein, the osteoblast-materials living hybrids were constructed with porous PLLA microspheres using a rational design, in which cell-based living materials presented an improved osteoblast differentiation and mineralization model using rationally designed cell-microsphere composites. The results indicated that the microfluidic-based technique provided an efficient and highly controllable approach for producing on-demand PLLA microspheres with tiny pores (< 5 μm), medium pores (5-15 μm), and large pores (> 15 μm), as well as further drug delivery. Furthermore, the simvastatin (SIM)-loaded porous PLLA microsphere with ε-polylysine (ε-PL) modification was used for osteoblast (MC3T3-E1) implantation, achieving the cell-material living microhybrids, and the results demonstrated the ε-PL surface modification and simvastatin could improve osteoblast behavior regulation, including cell adhesion, proliferation, as well as the antibacterial effects. Both in vitro and in vivo results significantly demonstrated further cell proliferation, differentiation, and cascade mineralization regulation. Then, the qPCR or histological staining of typical markers, including collagen type I (Col I), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 (BMP2), as well as the calcium mineral deposition staining in situ, reconfirmed the transformation of osteoblasts into osteocytes. These achievements revealed a promising boost in osteogenesis towards mineralization at the microtissue level by cell-microsphere integration, suggesting an alternative strategy for materials-based ex vivo tissue construction and cell regulation, further demonstrating excellent application prospects in the field of biomineralization-based tissue regeneration.

The transcription factor foxo3 regulates cardiac remodeling after myocardial infarction

Abstract Background Myofibroblast and myeloid cell differentiation following acute myocardial infarction (MI) are important for myocardial fibrosis but also cause adverse remodeling leading to cardiac dysfunction and heart failure. The transcription factor forkhead box O 3 (Foxo3) inhibits hypertrophic cardiac remodeling. We hypothesized that Foxo3, a key regulator of cell differentiation and immunity might inhibit differentiation of fibroblasts and myoloid cells and therefore cardiac fibrosis after MI. Methods MI was induced in Foxo3-/- and wild-type mice by permanent LAD ligation. Cardiac function was determined by transthoracic echocardiography. Cell differentiation was investigated by single nucleus RNA-sequencing (snRNA-seq). Moreover, transdifferentiation assays ex vivo were performed. Results Foxo3-/- mice showed significantly improved survival post- MI (p<0.01) that was in part due to reduced cardiac ruptures (p<0.05). TTE showed reduced LV function, cardiac output, and global strain 4 days post-MI in both groups of animals. However, LV function was significantly attenuated in Foxo3-/- mice 14 d post-MI while diastolic parameters indicated enhanced wall stiffness. snRNA-seq on day 4 post-MI revealed significantly increased numbers of myofibroblasts (p<0.05) transdifferentiating from Progenitor-like state fibroblasts and epicardial derived fibroblasts in Foxo3-/- mice. Myofibroblasts upregulated genes important for extracellular matrix structure and cell migration. Moreover, an upregulation of pro-fibrotic genes was observed in other fibroblast clusters in Foxo3-/- mice and corroborated by significantly elevated collagen type 1 and 3 as well as periostin protein expression. Mechanistically, fibroblasts from Foxo3a-/- mice exhibited enhanced Smad3 dependent myofibroblast marker expression after TGF-ß stimulation. Cell chat analysis indicated enhanced number of interactions and interaction strength of myeloid cells with fibroblasts in Foxo3-/- animals. In line with these observations, differences in cardiac myeloid cell differentiation characterized by reduced center-log ratios of cardiac resident macrophages (Lyve1hiMHCIIlo and Lyve1loMHCIIhi) and increase in monocyte-derived macrophages (Ccr2hi MΦ and Trem2hi MΦ) on day 4 post-MI that was enhanced in Foxo3-/- mice were determined. Differential gene expression patterns on day 4 in pro-inflammatory macrophages (Ccr2hi MΦ) showed upregulation of genes in the interleukin-12 pathway while reparative macrophages (Trem2hi MΦ) revealed an increased expression of genes for extracellular matrix proteins and ECM-collagen interactions in Foxo3-/- mice. Conclusion Our data identify Foxo3 as a master regulator of cardiac remodeling inhibiting myofibroblast and myeloid cell differentiation after acute MI, and suggesting that deficiency of Foxo3 promotes early scar formation but also contributes to adverse matricellular remodeling resulting in impaired cardiac function and enhanced fibrosis.

A LANA peptide inhibits tumor growth by inducing CHD4 protein cleavage and triggers cell death.

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes a latent infection, and viral genes are poised to be transcribed in the latent chromatin. In the poised chromatins, KSHV latency-associated nuclear antigen (LANA) interacts with cellular chromodomain-helicase-DNA-binding protein 4 (CHD4) and inhibits viral promoter activation. CHD4 is known to regulate cell differentiation by preventing enhancers from activating promoters. Here, we identified a putative CHD4 inhibitor peptide (VGN73) from the LANA sequence corresponding to the LANA-CHD4 interaction surface. The VGN73 interacts with CHD4 at its PHD domain with a dissociation constant (KD) of 14nM. Pre-treatment with VGN73 enhanced monocyte differentiation into macrophages and globally altered the repertoire of activated genes in U937 cells. Furthermore, the introduction of the peptide into the cancer cells induced caspase-mediated CHD4 cleavage, triggered cell death, and inhibited tumor growth in a xenograft mouse model. The VGN73 may facilitate cell differentiation therapy.

An Integrated Multi-Model Framework Utilizing Convolutional Neural Networks Coupled with Feature Extraction for Identification of 4mC Sites in DNA Sequences

N4-methylcytosine (4mC) is a chemical modification that occurs on one of the four nucleotide bases in DNA and plays a vital role in DNA expression, repair, and replication. It also actively participates in the regulation of cell differentiation and gene expression. Consequently, it is important to comprehend the role of 4mC in the epigenetic regulation for revealing the complications of the gene expression and their associated governing cellular operations. However, the inherent resource requirements and time constraints of the experimental procedure, present challenges to the cellular culture process. While data-driven methodologies present promising solutions to mitigate the demand for extensive experimental efforts, their performance relies on the suitability and existence of high-quality data. This study presents a multi-model framework that integrates convolutional neural network (CNN) with the distributed k-mer and embedding feature extraction techniques to enhance the identification of 4mC sites in DNA sequences. The integration of k-mers ensures the effective representation of the local sequence patterns, while the utilization of embedding enables a more holistic encoding by considering the broader context and semantics of the sequence data. Following the initial step, the obtained distributed representation of the DNA sequence seamlessly enters the CNN, triggering a crucial convolution operation wherein a set of adaptable filters systematically convolves across the sequence to detect vital local patterns. The proposed integrated multi-model framework was applied to six publicly available datasets and evaluated against the cutting-edge 4mCPred, 4mCCNN, iDNA4mC, Meta-4mCpred, DeepTorrent, 4mCPred-SVM, and DMKL-HFIS methods. The evaluation was based on accuracy, specificity, sensitivity, and Matthews Correlation Coefficient. The results demonstrated that the proposed multi-model framework outperformed the state-of-the-art methods, as well as one-hot encoding and the hybrid of one-hot & TNC features, in accurately identifying 4mC sites.

Neuropeptide signalling orchestrates T cell differentiation.

The balance between T helper type 1 (TH1) cells and other TH cells is critical for antiviral and anti-tumour responses1-3, but how this balance is achieved remains poorly understood. Here we dissected the dynamic regulation of TH1 cell differentiation during in vitro polarization, and during in vivo differentiation after acute viral infection. We identified regulators modulating T helper cell differentiation using a unique TH1-TH2 cell dichotomous culture system and systematically validated their regulatory functions through multiple in vitro and in vivo CRISPR screens. We found that RAMP3, a component of the receptor for the neuropeptide CGRP (calcitonin gene-related peptide), has a cell-intrinsic role in TH1 cell fate determination. Extracellular CGRP signalling through the receptor RAMP3-CALCRL restricted the differentiation of TH2 cells, but promoted TH1 cell differentiation through the activation of downstream cAMP response element-binding protein (CREB) and activating transcription factor 3 (ATF3). ATF3 promoted TH1 cell differentiation by inducing the expression of Stat1, a key regulator of TH1 cell differentiation. After viral infection, an interaction between CGRP produced by neurons and RAMP3 expressed on T cells enhanced the anti-viral IFNγ-producing TH1 and CD8+ T cell response, and timely control of acute viral infection. Our research identifies a neuroimmune circuit in which neurons participate in T cell fate determination by producing the neuropeptide CGRP during acute viral infection, which acts on RAMP3-expressing T cells to induce an effective anti-viral TH1 cell response.

COL8A2 activation enhances function of corneal endothelial cells through HIPPO signaling/mitochondria pathway

Corneal endothelial cells (CECs) are essential for maintaining corneal transparency and hydration through their barrier and pump functions. The COL8A2 gene encodes a component of the extracellular matrix of the cornea, which is crucial for the normal functioning of these cells. Mutations in COL8A2 are linked to corneal dystrophies, emphasizing the gene's importance in corneal health. The purpose of this research is to explore the effects of COL8A2 activation within CECs, to understand its contribution to cellular behavior and health. COL8A2 CRISPR/dCas9 activation system (aCOL8A2) was used to activate the COL8A2. In rats, wound healing and mitochondrial function were assessed after COL8A2 activation. As a result, aCOL8A2 promoted wound healing of rat corneal endothelium by increasing mitochondrial membrane potential. In cultured human CECs, proteomic analysis was performed to screen and identify the differential protein profiles between control and aCOL8A2 cells. Western blot was used to validate the differential proteins from both cells. Mitochondrial function and intracellular distribution were assessed by measuring ATP production and mitochondrial membrane potential. In cultured human CECs, aCOL8A2 increased COL8A2 and phospho-YAP levels. Transendothelial electrical resistance (TEER) was increased and actin cytoskeleton was attenuated by aCOL8A2. Gene ontology analysis revealed that the proteins were mainly involved in the regulation of folate biosynthesis, ECM-receptor interaction, cell differentiation, NADP activity and cytoskeleton. ATP production was increased, mitochondrial membrane potential was polarized and mitochondrial distribution was widespread in the aCOL8A2 group. In conclusion, aCOL8A2 induces a regulatory cascade affecting mitochondrial positioning and efficiency, mediated by alterations in the cytoskeletal architecture and the YAP signaling pathway. This sequence of events serves to bolster the functional capacities of corneal endothelial cells, including their pump and barrier functions, essential for corneal health and transparency.

9246 Mineralocorticoid Receptor Regulation of the Molecular Clock in Cardiomyocytes Is Modulated by Time of Day

Abstract Disclosure: M. Kanki: None. S. Heanue: None. P.J. Fuller: None. T.J. Cole: None. C.D. Clyne: None. M.J. Young: None. The onset of cardiovascular disease events peaks in the early morning, which coincides with the diurnal surge in aldosterone and cortisol. Aldosterone-induced mineralocorticoid receptor (MR) activation in cardiomyocytes has established roles in cardiac pump function and electrophysiology and inappropriate MR activation promotes cardiovascular pathophysiology in experimental models of hypertension and heart failure and in the clinic. Recently, we identified a novel role for MR in regulating the circadian clock signalling network in cardiac cells and showed that time of day modulates MR activation in the heart. Whether time-of-day-dependent MR regulation of circadian clocks in cardiomyocytes modulates temporal expression of key functional pathways is unknown. Therefore, we sought to identify cardiac responsiveness to MR stimulation at lights on (7AM) versus lights off (7PM) in ≥8-week old male wildtype (WT) and cardiomyocyte-MR knockout (MyoMRKO) mice. RNA-seq analyses revealed that aldosterone (50 ug/kg) administered at 7PM but not at 7AM significantly enriched transcription of genes associated with 52 biological processes including “Circadian rhythm”, “Reg. of blood pressure”, “Ion transport” and “Reg. of the force of heart contraction” in WT hearts (p<0.05 versus 7PM vehicle-injected WT mice). These outcomes were absent in hearts isolated from MyoMRKO mice administered aldosterone at 7AM or 7PM. Analysis by RT-qPCR revealed that aldosterone versus vehicle administered at 7AM increased expression of circadian Period (Per) and Thyrotroph embryonic factor (Tef) genes in WT hearts, which was lost in MyoMRKO mice. Whole transcriptome analysis also detected significant enrichment in “Cell-cell signalling”, “Positive regulation of cell differentiation”, “Positive regulation of glucose metabolic process”, “Regulation of sodium ion transport”, and “Inflammatory response" processes between 7AM and 7PM in WT hearts, which was also lost in MyoMRKO mice. Our data show that MR transcription of genes associated with circadian rhythms and ion transport is temporally regulated, and demonstrates specificity for cardiomyocyte-MR regulation of circadian clocks. Taken together, we highlight that the role for cardiomyocyte-MR regulation of cardiac electrophysiology, heart rate variability, and metabolic processes is time-of-day-dependent. Understanding temporal coordination of MR activation in physiology and pathophysiology may inform new strategies for optimising MR-directed therapies in patients with heart disease. Presentation: 6/2/2024

8639 Clinical characterization and germline DNA methylation analysis of a large cohort of patients with paraganglioma syndrome type 4

Abstract Disclosure: R. Halperin: None. R. Horowitz: None. A. Jabarin: None. A. Tirosh: None. Background: Patients harboring germline pathogenic variant (PV) in the SDHB gene (Paraganglioma syndrome type 4, PPGL4) have a higher risk of developing paragangliomas and pheochromocytomas. PPGL4 is considered a higher-risk syndrome for aggressive and metastatic abdominal-thoracic paragangliomas compared with other paraganglioma syndromes. The penetrance in PPGL4 reaches 65% by the age of 80 years. Aims: To assess possible germline DNA epigenetic alterations in patients harboring the SDHB PV. Materials and Methods: A large cohort of patients with PPGL4 was characterized clinically in our clinic and genetically via germline DNA sequencing for the familial variant (SDHB c.640C>T p.Q214Ter, n=144) and whole genome methylation analysis compared 19 patients vs. 129 healthy controls. The control group consisted of four SDHB PV-negative family members and an additional 125 samples retrieved from a public database (GSE153712). Analysis was performed on Rstudio, using the ChAMP suite for normalization, imputation, differentially methylated probes (DMP), and regions (DMR) analyses. Pathway analysis and visualization were executed using the clusterprofiler and enrichplot packages. Promoter regions were defined by the transcription start site (TSS1500). Results: The cohort includes three kindreds, and each presented independently with an index patient: Index patient in kindred A, a 12-year-old boy, with thoracic paraspinal and testicular paragangliomas, non-secretory, requiring several surgeries due to spinal compression with subsequent improvement of neurological deficit. Index patient in kindred B, a 26-year-old woman with abdominal paraganglioma, metastatic to the lungs and spine. Index patient in kindred C, a 41-year-old man with a locally aggressive abdominal paraganglioma, metastatic to the skull and spine. A total of 114 (44.2% females) patients underwent genetic evaluation (45/50, 16/28, and 25/36 in kindreds A, B and C, respectively). Of those, 50 (58.1%) harbored the familial SDHB PV (68.9%, 56.2%, and 40.0%, respectively). Twenty-six patients underwent at least partial clinical evaluation: Eight had paraganglioma (4/8 had metastatic disease), three had pheochromocytoma, and two patients had neck masses that are currently being evaluated. Pathway analysis revealed enrichment of the germline DNA alterations to the regulation of cell differentiation and development, metabolic processes, and cellular component organization (p<0.03 for all pathways). Conclusions: We report one of the largest cohorts of patients with SDHB PV, showing typical low penetrance and aggressive phenotype. Genomic analysis shows distinct germline DNA methylation in SDHB PV-positive compared with the control group. Future studies may reveal an association between specific epigenetic alterations and penetrance/phenotypic patterns in this rare hereditary syndrome. Presentation: 6/2/2024

7763 Mineralocorticoid Receptor Regulation of the Molecular Clock in Cardiomyocytes Is Modulated by Time of Day

Abstract Disclosure: M. Kanki: None. S. Heanue: None. P.J. Fuller: None. T.J. Cole: None. C.D. Clyne: None. M.J. Young: None. The onset of cardiovascular disease events peaks in the early morning, which coincides with the diurnal surge in aldosterone and cortisol. Aldosterone-induced mineralocorticoid receptor (MR) activation in cardiomyocytes has established roles in cardiac pump function and electrophysiology and inappropriate MR activation promotes cardiovascular pathophysiology in experimental models of hypertension and heart failure and in the clinic. Recently, we identified a novel role for MR in regulating the circadian clock signalling network in cardiac cells and showed that time of day modulates MR activation in the heart. Whether time-of-day-dependent MR regulation of circadian clocks in cardiomyocytes modulates temporal expression of key functional pathways is unknown. Therefore, we sought to identify cardiac responsiveness to MR stimulation at lights on (7AM) versus lights off (7PM) in ≥8-week old male wildtype (WT) and cardiomyocyte-MR knockout (MyoMRKO) mice. RNA-seq analyses revealed that aldosterone (50 ug/kg) administered at 7PM but not at 7AM significantly enriched transcription of genes associated with 52 biological processes including “Circadian rhythm”, “Reg. of blood pressure”, “Ion transport” and “Reg. of the force of heart contraction” in WT hearts (p<0.05 versus 7PM vehicle-injected WT mice). These outcomes were absent in hearts isolated from MyoMRKO mice administered aldosterone at 7AM or 7PM. Analysis by RT-qPCR revealed that aldosterone versus vehicle administered at 7AM increased expression of circadian Period (Per) and Thyrotroph embryonic factor (Tef) genes in WT hearts, which was lost in MyoMRKO mice. Whole transcriptome analysis also detected significant enrichment in “Cell-cell signalling”, “Positive regulation of cell differentiation”, “Positive regulation of glucose metabolic process”, “Regulation of sodium ion transport”, and “Inflammatory response" processes between 7AM and 7PM in WT hearts, which was also lost in MyoMRKO mice. Our data show that MR transcription of genes associated with circadian rhythms and ion transport is temporally regulated, and demonstrates specificity for cardiomyocyte-MR regulation of circadian clocks. Taken together, we highlight that the role for cardiomyocyte-MR regulation of cardiac electrophysiology, heart rate variability, and metabolic processes is time-of-day-dependent. Understanding temporal coordination of MR activation in physiology and pathophysiology may inform new strategies for optimising MR-directed therapies in patients with heart disease. Presentation: 6/2/2024

Discovery of reversible and covalent TEAD 1 selective inhibitors MSC-1254 and MSC-5046 based on one scaffold

The Transcriptional Enhanced Associated Domain (TEAD) family of transcription factors are key components of the Hippo signalling family which play a crucial role in the regulation of cell proliferation, differentiation and apoptosis. The identification of inhibitors of the TEAD transcription factors are an attractive strategy for the development of novel anticancer therapies. A HTS campaign identified hit 1, which was optimised using structure-based drug design, to deliver potent TEAD1 selective inhibitors with both a reversible and covalent mode of inhibition. The preference for TEAD1 could be rationalised by steric differences observed in the lower pocket of the palmitoylation-site between subtypes, with TEAD1 having the largest available volume to accommodate substitution in this region