Expression Of CEBPB Research Articles

Effect of acetaminophen use on GABA, NMDA, and synaptic plasticity-related genes in the immature mouse brain: A preliminary study

Abstract Background/aim: Acetaminophen is frequently used as an analgesic during pregnancy. The purpose of the present study was to evaluate the effects of acetaminophen administered to pregnant mice on the fetal brain, attention, memory, and learning functions in the postnatal period, and genetic mechanisms in these mice. Materials and methods: The study was designed with two different groups. The first group consisted of pregnant mice that were injected with acetaminophen, while the second group was comprised of pregnant mice that were injected with saline. 1st, 2nd, and 3rd days of pregnancy, one of the mice was injected subcutaneously with 100 mg/kg acetaminophen, and the other mouse was injected subcutaneously with 0.9% saline. On the 21st day after birth, five female and five male mice were randomly selected for the experimental and control groups. Behavioral tests were performed on mice at 2 months of age. In addition, changes in the transcript levels of 93 genes were evaluated by Real-Time PCR in the hippocampus. Results: The control group showed more interest in the new object than the acetaminophen group (p=0.002). In the marble burying test, greater burying activity was observed in the control group than in the acetaminophen group (p=0.0345). No significant difference was observed between the control and acetaminophen groups in the social interaction and tail suspension tests. GABRG3, GRM3, PICK1, CEBPB, and EGR4 mRNA expression levels increased in the acetaminophen group (0.0317, 0.0159, 0.0069, 0.0457, 0.015, p value respectively). Conclusions: Prenatal acetaminophen exposure affected both behavioral tests and transcript levels. Therefore, the potential effects of prenatal acetaminophen exposure should be carefully investigated.

GATA2 heterozygosity causes an epigenetic feedback mechanism resulting in myeloid and erythroid dysplasia.

The transcription factor GATA2 has a pivotal role in haematopoiesis. Heterozygous germline GATA2 mutations result in a syndrome characterized by immunodeficiency, bone marrow failure and predispositions to myelodysplastic syndrome (MDS) and acute myeloid leukaemia. Clinical symptoms in these patients are diverse and mechanisms driving GATA2-related phenotypes are largely unknown. To explore the impact of GATA2 haploinsufficiency on haematopoiesis, we generated a zebrafish model carrying a heterozygous mutation of gata2b (gata2b+/-), an orthologue of GATA2. Morphological analysis revealed myeloid and erythroid dysplasia in gata2b+/- kidney marrow. Because Gata2b could affect both transcription and chromatin accessibility during lineage differentiation, this was assessed by single-cell (sc) RNA-seq and single-nucleus (sn) ATAC-seq. Sn-ATAC-seq showed that the co-accessibility between the transcription start site (TSS) and a -3.5-4.1 kb putative enhancer was more robust in gata2b+/- zebrafish HSPCs compared to wild type, increasing gata2b expression and resulting in higher genome-wide Gata2b motif use in HSPCs. As a result of increased accessibility of the gata2b locus, gata2b+/- chromatin was also more accessible during lineage differentiation. scRNA-seq data revealed myeloid differentiation defects, that is, impaired cell cycle progression, reduced expression of cebpa and cebpb and increased signatures of ribosome biogenesis. These data also revealed a differentiation delay in erythroid progenitors, aberrant proliferative signatures and down-regulation of Gata1a, a master regulator of erythropoiesis, which worsened with age. These findings suggest that cell-intrinsic compensatory mechanisms, needed to obtain normal levels of Gata2b in heterozygous HSPCs to maintain their integrity, result in aberrant lineage differentiation, thereby representing a critical step in the predisposition to MDS.

Abstract 2059: Therapeutic Delivery Of MICRORNA-369-3p Promotes Atherosclerosis Regression In Diabetic Mice By Regulating Bone Marrow Myeloid Progenitors

Introduction: Hyperglycemia-induced immunometabolic alterations promote myelopoiesis, increase proinflammatory monocytes, and impair atherosclerosis regression. Identifying critical factors to restore metabolic alterations and promote resolution of inflammation remains an unmet medical need. MicroRNAs (miRs) orchestrate multiple signaling events in macrophages and regulate inflammation. Using miR-seq, we identified miR-369-3p as a novel miR expressed higher in regressing mouse plaques. Hypothesis: Therapeutic administration of miR-369-3p promotes plaque regression of diabetes-associated atherosclerosis in mice. Aim: To identify the therapeutic benefits of miR-369-3p overexpression, and the mechanism by which it promotes plaque regression in diabetic mice. Methods: Ldlr-/ - mice were fed HFSC-diet for 12-weeks to develop atherosclerotic plaques and were switched to chow-diet for 6-weeks to induce regression. During regression, mice received either non-specific control mimic (NS-m) or miR-369-3p mimic (369-m) intravenously twice every week. Immunohistological, morphometric, and flow cytometric analyses were performed to define the changes in plaque, circulation, and bone marrow following 369-m delivery. Results: Therapeutic delivery of 369-m promoted plaque resolution in diabetic mice, evident by reduced plaque area and macrophage content when compared to NS-m injected mice. Circulating proinflammatory cytokines (IL1β, IL6 and TNF-α), total circulating monocytes, and M1 macrophages were decreased in lesions while M2 macrophages were increased in 369-m group. 369-m also increased plaque stability and increased FOXP3+ Tregs in plaques when compared to NS-m group. Bioinformatic analyses showed GPR91 and CEBPB as conserved miR targets. Mechanistically, we found that 369-m decreased bone marrow granulocyte-monocyte progenitor population (GMPs), resulting in less inflammatory monocytes, attributed to reduced CEBPB and GPR91 target expression in GMPs. Conclusion: miR-369-3p overexpression reduced plaque-inflammatory macrophages, bone marrow GMPs, and proinflammatory monocytes by regulating macrophage immunometabolism in diabetes-accelerated atherosclerosis in part by targeting CEBPB and GPR91.

CaSSiDI: novel single-cell “Cluster Similarity Scoring and Distinction Index” reveals critical functions for PirB and context-dependent Cebpb repression

PirB is an inhibitory cell surface receptor particularly prominent on myeloid cells. PirB curtails the phenotypes of activated macrophages during inflammation or tumorigenesis, but its functions in macrophage homeostasis are obscure. To elucidate PirB-related functions in macrophages at steady-state, we generated and compared single-cell RNA-sequencing (scRNAseq) datasets obtained from myeloid cell subsets of wild type (WT) and PirB-deficient knockout (PirB KO) mice. To facilitate this analysis, we developed a novel approach to clustering parameter optimization called “Cluster Similarity Scoring and Distinction Index” (CaSSiDI). We demonstrate that CaSSiDI is an adaptable computational framework that facilitates tandem analysis of two scRNAseq datasets by optimizing clustering parameters. We further show that CaSSiDI offers more advantages than a standard Seurat analysis because it allows direct comparison of two or more independently clustered datasets, thereby alleviating the need for batch-correction while identifying the most similar and different clusters. Using CaSSiDI, we found that PirB is a novel regulator of Cebpb expression that controls the generation of Ly6Clo patrolling monocytes and the expansion properties of peritoneal macrophages. PirB’s effect on Cebpb is tissue-specific since it was not observed in splenic red pulp macrophages (RPMs). However, CaSSiDI revealed a segregation of the WT RPM population into a CD68loIrf8+ “neuronal-primed” subset and an CD68hiFtl1+ “iron-loaded” subset. Our results establish the utility of CaSSiDI for single-cell assay analyses and the determination of optimal clustering parameters. Our application of CaSSiDI in this study has revealed previously unknown roles for PirB in myeloid cell populations. In particular, we have discovered homeostatic functions for PirB that are related to Cebpb expression in distinct macrophage subsets.

Itraconazole inhibits tumor growth via CEBPB-mediated glycolysis in colorectal cancer.

Advanced colorectal cancer (CRC) is characterized by a high recurrence and metastasis rate, which is the primary cause of patient mortality. Unfortunately, effective anti-cancer drugs for CRC are still lacking in clinical practice. We screened FDA-approved drugs by utilizing targeted organoid sequencing data and found that the antifungal drug itraconazole had a potential therapeutic effect on CRC tumors. However, the effect and mechanism of itraconazole on CRC tumors have not been investigated. A cell line-derived xenograft model in tumor-bearing mice was established and single-cell RNA sequencing was performed on tumor samples from four mice with or without itraconazole treatment. The proportion of cell populations and gene expression profiles was significantly different between the two groups. We found that itraconazole could inhibit tumor growth and glycolysis. We revealed that CEBPB was a new target for itraconazole, and that silencing CEBPB could repress CRC glycolysis and tumor growth by inhibiting ENO1 expression. Clinical analysis showed that CEBPB expression was obviously elevated in CRC patients, and was associated with poor survival. In summary, itraconazole treatment remodeled cell composition and gene expression profiles. Itraconazole inhibited cell glycolysis and tumor growth via the CEBPB-ENO1 axis. In this study, we illustrate a new energy metabolism mechanism for itraconazole on tumor growth in CRC that will provide a theoretical basis for CRC targeting/combination therapy.

Cebpβ/IL1/TNFα Positive Feedback Loop Drives Drug Resistance of BCL2 and MDM2 Inhibitors in Monocytic Leukemia Cells

Introduction:Ventoclax-based combinations have emerged as a new standard of care for patients with acute myeloid leukemia (AML) who are not suitable for intense chemotherapy. Not all patients respond to these treatments, and those who do may relapse. MDM2 inhibitors are promising therapeutics for treating TP53 wild-type tumors, including most de novo AML cases, with numerous compounds currently in pre-clinical and clinical evaluation. However, clinical trials of MDM2 inhibitors have shown modest and variable clinical activity. Functional genomic data showed that monocytic leukemia (FAB M4/M5) is resistant to venetoclax-based therapies and MDM2 inhibitors. Notably, venetoclax and an MDM2 inhibitor, idasanutlin, demonstrated a strong positive correlation in the Beat AML cohort. We hypothesize that upregulation of certain myeloid transcription factors in monocytic leukemia may confer intrinsic resistance to BCL2 and MDM2 inhibitors, while certain environmental cues upregulated in these cells may confer extrinsic drug resistance. Additionally, there may be crosstalk between these intrinsic and extrinsic mechanisms. Methods: We performed differential expression gene (DEG) analysis of transcription factors (TFs) between M4/M5 and M0/M1 samples in the Beat AML and TCGA AML cohort. We overexpressed seven myeloid TFs (SPI1, CEBPB, CEBPD, JUNB, IRF8, KLF4, and MAFB) that are upregulated in M4/M5 and performed competitive drug assays. We treated M4/M5 and M0/M1 samples with venetoclax and idasanutlin, in the presence of a panel of myeloid cytokines and measured cell viabilities. A Luminex assay on monocyte supernatants from M4/M5 and M0/M1 AML samples were conducted to identify dysregulated cytokines. Furthermore, RNAseq DEG and immunoblot analysis were performed on CEBPB-overexpressing cell lines and IL-1-treated AML samples to elucidate the underlying mechanisms. Results: CEBPB overexpression conferred drug resistance to a broad range of BH3 mimetics, venetoclax combinations, and MDM2 inhibitors. RNA-seq and immunoblot analyses demonstrated that CEBPB overexpression downregulated CASP3, CASP6, BCL2, and TP53 pathway targets (CDKN1A, PMAIP1, BBC3, BMF, TP53), while upregulated MCL1, BCL2A1, and the NF-κB/IL-1/TNF pathway at transcription and/or translation levels. Phenotyping analysis showed that CEBPB overexpression drives myelo/monocytic differentiation. In accordance, CEBPB expression in primary AML correlates with drug responses of idasanutlin, venetoclax, and many venetoclax combinations. Additionally, primary monocytic leukemia expresses significantly higher levels of IL-1/TNF family genes, BCL2A1, and reduced CASP3, CASP6, and BCL2. Abnormal monocytes, but not granulocytes, T cells, or blasts from M4/M5 leukemia, extrinsically protect leukemia blasts from venetoclax and MDM2 inhibition by secreting elevated IL-1 and TNFα. IL-1β/TNFα treatment drove myelo/monocytic differentiation and up-regulated inflammatory cytokines, including an autoregulatory loop, as well as a number of cytokine receptors, such as IL1R1, TNFRSF1R, TNFRSF2R, and CSF2RB. Remarkably, IL-1α/IL-1β and TNFα uniquely upregulated CEBPB expression in M4/M5 cells and protected them from apoptosis induced by venetoclax and MDM2 inhibitors. Conversely, TNFα treatment induced augmented extrinsic apoptosis in M0/M1 leukemia cells. Interestingly, treatment with venetoclax and idasanutlin led to a feedback upregulation of CEBPB, IL-1β, and/or TNFα, along with their respective receptors, and promoted myelomonocytic differentiation. IL-1/TNFα antagonists or an IRAK inhibitor alone did not kill leukemia cells, but they showed synergistic cytotoxicity when combined with venetoclax and idasanutlin. Conclusions: In summary, we have described a positive feedback loop between CEBPB, IL-1/TNFα, and monocytic differentiation in monocytic leukemia that contributes to intrinsic and extrinsic drug resistance against BCL2 and MDM2 inhibitors. This crosstalk and the consequent drug resistance are further reinforced by venetoclax/MDM2 inhibition treatment. Combining venetoclax or idasanutlin with IL-1/TNFα antagonists or an IRAK inhibitor can abrogate the feedback loop and induce synergistic cytotoxic effects, offering promising therapeutic strategies to enhance the treatment efficacy of venetoclax and MDM2 inhibitors for monocytic leukemia.

Melatonin alleviates the toxic effect of di(2-ethylhexyl) phthalate on oocyte quality resulting from CEBPB suppression during primordial follicle formation

Presently, the exposure of plasticizers to humans and animals occurs daily, which pose a potential threat to reproductive health. In the present study, a pregnant mouse model exposed to di(2-ethylhexyl) phthalate (DEHP, one of the most common plasticizers) and melatonin was established, and the single-cell transcriptome technology was applied to investigate the effects of melatonin in ovarian cells against DEHP. Results showed that DEHP markedly altered the gene expression pattern of ovarian cells, and severely weakened the histone methylation modification of oocytes. The administration of melatonin recovered the expression of LHX8 and SOHLH1 proteins that essential for primordial follicle formation, and increased the expression of CEBPB, as well as key genes of histone methylation modification (such as Smyd3 and Kdm5a). In addition, the ovarian damage caused by DEHP was also relieved after the overexpression of CEBPB, which suggested melatonin could improve primordial follicle formation progress via enhancing CEBPB expression in mice. Besides, the apoptosis of ovarian cells induced by DEHP also was diminished by melatonin. The study provides evidence of melatonin preventing the damage mediated by plasticizers on the reproductive system in females and CEBPB may serve as a downstream target factor of melatonin in the process.

Protective effect of TNFAIP3 on testosterone production in Leydig cells under an aging inflammatory microenvironment

BackgroundThe aging inflammatory microenvironment surrounding Leydig cells is linked to reduced testosterone levels in males. Tumor necrosis factor alpha-induced protein 3 (TNFAIP3) acts as a critical anti-inflammatory factor in various aging-related diseases. This study aims to investigate the protective effect of TNFAIP3 on testosterone production in Leydig cells under an aging inflammatory microenvironment. MethodsBioinformatics analysis examined TNFAIP3 expression differences in aging rat testes and validated the findings in aging mouse testes. In vitro models of inflammation were established using two Leydig cell lines, with tumor necrosis factor alpha (TNF-α) as the inflammatory factor. Lentiviral transduction was utilized to manipulate TNFAIP3 expression in these cell lines. Transcriptomic sequencing identified differentially expressed genes in TNFAIP3-overexpressing cells. ResultsBioinformatics analysis and validation experiments revealed increased inflammatory signaling and elevated TNFAIP3 expression in aging rat and mouse testes. TNFAIP3 knockdown worsened testosterone synthesis inhibition and apoptosis in cells, while TNFAIP3 overexpression reversed these effects. Transcriptome analysis identified alterations in the P38MAPK pathway following TNFAIP3 overexpression. TNFAIP3 knockdown enhanced TNF-induced P38MAPK signaling, whereas its overexpression attenuated this effect. TNFAIP3 was found to regulate testosterone synthesis by upregulating CEBPB expression. ConclusionsTNFAIP3 exhibits inhibitory effects on apoptosis and promotes testosterone production in Leydig cells. The protective influence of TNFAIP3 on Leydig cells within an inflammatory microenvironment is likely mediated through by inhibiting the P38MAPK pathway and upregulating CEBPB expression.

TET2 is recruited by CREB to promote Cebpb, Cebpa, and Pparg transcription by facilitating hydroxymethylation during adipocyte differentiation

Ten-eleven translocation proteins (TETs) are dioxygenases that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), an important epigenetic mark that regulates gene expression during development and differentiation. Here, we found that the TET2 expression was positively associated with adipogenesis. Further, invitro and invivo experiments showed that TET2 deficiency blocked adipogenesis by inhibiting the expression of the key transcription factors CCAAT/enhancer-binding protein beta (C/EBPβ), C/EBPα and peroxisome proliferator-activated receptor gamma (PPARγ). In addition, TET2 promoted 5hmC on the CpG islands (CGIs) of Cebpb, Cebpa and Pparg at the initial time point of their transcription, which requires the cAMP-responsive element-binding protein (CREB). At last, specific knockout of Tet2 in preadipocytes enabled mice to resist obesity and attenuated the obesity-associated insulin resistance. Together, TET2 is recruited by CREB to promote the expression of Cebpb, Cebpa and Pparg via 5hmC during adipogenesis and may be a potential therapeutic target for obesity and insulin resistance.

Activation of BZW1 by CEBPB in macrophages promotes eIF2α phosphorylation-mediated metabolic reprogramming and endoplasmic reticulum stress in MRL/lpr lupus-prone mice

BackgroundLupus nephritis (LN) is associated with significant mortality and morbidity, while effective therapeutics and biomarkers are limited since the pathogenesis is complex. This study investigated the roles of the CEBPB/BZW1/eIF2α axis in metabolic reprogramming and endoplasmic reticulum stress in LN.MethodThe differentially expressed genes in LN were screened using bioinformatics tools. The expression of CEBPB in the renal tissue of patients with LN and its correlation with the levels of creatinine and urinary protein were analyzed. We used adenoviral vectors to construct LN mice with knockdown CEBPB using MRL/lpr lupus-prone mice and analyzed the physiological and autoimmune indices in mice. Chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP–qPCR) and dual-luciferase reporter assays were conducted to explore the regulation of BZW1 by CEBPB, followed by glycolytic flux analysis, glucose uptake, and enzyme-linked immunosorbent assay (ELISA). Finally, the role of eIF2α phosphorylation by BZW1 in bone marrow-derived macrophages (BMDM) was explored using eIF2α phosphorylation and endoplasmic reticulum stress inhibitors.ResultsCEBPB was significantly increased in renal tissues of patients with LN and positively correlated with creatinine and urine protein levels in patients. Downregulation of CEBPB alleviated the autoimmune response and the development of nephritis in LN mice. Transcriptional activation of BZW1 by CEBPB-mediated glucose metabolic reprogramming in macrophages, and upregulation of BZW1 reversed the mitigating effect of CEBPB knockdown on LN. Regulation of eIF2α phosphorylation levels by BZW1 promoted endoplasmic reticulum stress-amplified inflammatory responses in BMDM.ConclusionTranscriptional activation of BZW1 by CEBPB promoted phosphorylation of eIF2α to promote macrophage glycolysis and endoplasmic reticulum stress in the development of LN.

Prioritization of risk genes in colorectal cancer by integrative analysis of multi-omics data and gene networks.

Genome-wide association studies (GWASs) have identified over 140 colorectal cancer (CRC)-associated loci; however, target genes at the majority of loci and underlying molecular mechanisms are poorly understood. Here, we utilized a Bayesian approach, integrative risk gene selector (iRIGS), to prioritize risk genes at CRC GWAS loci by integrating multi-omics data. As a result, a total of 105 high-confidence risk genes (HRGs) were identified, which exhibited strong gene dependencies for CRC and enrichment in the biological processes implicated in CRC. Among the 105 HRGs, CEBPB, located at the 20q13.13 locus, acted as a transcription factor playing critical roles in cancer. Our subsequent assays indicated the tumor promoter function of CEBPB that facilitated CRC cell proliferation by regulating multiple oncogenic pathways such as MAPK, PI3K-Akt, and Ras signaling. Next, by integrating a fine-mapping analysis and three independent case-control studies in Chinese populations consisting of 8,039 cases and 12,775 controls, we elucidated that rs1810503, a putative functional variant regulating CEBPB, was associated with CRC risk (OR=0.90, 95%CI=0.86-0.93, P=1.07×10-7). The association between rs1810503 and CRC risk was further validated in three additional multi-ancestry populations consisting of 24,254 cases and 58,741 controls. Mechanistically, the rs1810503 A to T allele change weakened the enhancer activity in an allele-specific manner to decrease CEBPB expression via long-range promoter-enhancer interactions, mediated by the transcription factor, REST, and thus decreased CRC risk. In summary, our study provides a genetic resource and a generalizable strategy for CRC etiology investigation, and highlights the biological implications of CEBPB in CRC tumorigenesis, shedding new light on the etiology of CRC.

CRISPR screen identifies CEBPB as contributor to dyskeratosis congenita fibroblast senescence via augmented inflammatory gene response.

Aging is the consequence of intra- and extracellular events that promote cellular senescence. Dyskeratosis congenita (DC) is an example of a premature aging disorder caused by underlying telomere/telomerase-related mutations. Cells from these patients offer an opportunity to study telomere-related aging and senescence. Our previous work has found that telomere shortening stimulates DNA damage responses (DDRs) and increases reactive oxygen species (ROS), thereby promoting entry into senescence. This work also found that telomere elongation via TERT expression, the catalytic component of the telomere-elongating enzyme telomerase, or p53 shRNA could decrease ROS by disrupting this telomere-DDR-ROS pathway. To further characterize this pathway, we performed a CRISPR/Cas9 knockout screen to identify genes that extend life span in DC cells. Of the cellular clones isolated due to increased life span, 34% had a guide RNA (gRNA) targeting CEBPB, while gRNAs targeting WSB1, MED28, and p73 were observed multiple times. CEBPB is a transcription factor associated with activation of proinflammatory response genes suggesting that inflammation may be present in DC cells. The inflammatory response was investigated using RNA sequencing to compare DC and control cells. Expression of inflammatory genes was found to be significantly elevated (P < 0.0001) in addition to a key subset of these inflammation-related genes [IL1B, IL6, IL8, IL12A, CXCL1 (GROa), CXCL2 (GROb), and CXCL5]. which are regulated by CEBPB. Exogenous TERT expression led to downregulation of RNA/protein CEBPB expression and the inflammatory response genes suggesting a telomere length-dependent mechanism to regulate CEBPB. Furthermore, unlike exogenous TERT and p53 shRNA, CEBPB shRNA did not significantly decrease ROS suggesting that CEBPB's contribution in DC cells' senescence is ROS independent. Our findings demonstrate a key role for CEBPB in engaging senescence by mobilizing an inflammatory response within DC cells.

Bifidobacterium infantis-Mediated Herpes Simplex Virus-TK/Ganciclovir Treatment Inhibits Cancer Metastasis in Mouse Model.

Previous studies have found that Bifidobacterium infantis-mediated herpes simplex virus-TK/ganciclovir (BF-TK/GCV) reduces the expression of VEGF and CD146, implying tumor metastasis inhibition. However, the mechanism by which BF-TK/GCV inhibits tumor metastasis is not fully studied. Here, we comprehensively identified and quantified protein expression profiling for the first time in gastric cancer (GC) cells MKN-45 upon BF-TK/GCV treatment using quantitative proteomics. A total of 159 and 72 differential expression proteins (DEPs) were significantly changed in the BF-TK/GCV/BF-TK and BF-TK/GCV/BF/GCV comparative analysis. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis enriched some metastasis-related pathways such as gap junction and cell adhesion molecules pathways. Moreover, the transwell assay proved that BF-TK/GCV inhibited the invasion and migration of tumor cells. Furthermore, immunohistochemistry (IHC) demonstrated that BF-TK/GCV reduced the expression of HIF-1α, mTOR, NF-κB1-p105, VCAM1, MMP13, CXCL12, ATG16, and CEBPB, which were associated with tumor metastasis. In summary, BF-TK/GCV inhibited tumor metastasis, which deepened and expanded the understanding of the antitumor mechanism of BF-TK/GCV.

Heart failure in patients is associated with downregulation of mitochondrial quality control genes.

Mitochondrial dysfunction is one of key factors causing heart failure. We performed a comprehensive analysis of expression of mitochondrial quality control (MQC) genes in heart failure. Myocardial samples were obtained from patients with ischemic and dilated cardiomyopathy in a terminal stage of heart failure and donors without heart disease. Using quantitative real-time PCR, we analysed a total of 45 MQC genes belonging to mitochondrial biogenesis, fusion-fission balance, mitochondrial unfolded protein response (UPRmt), translocase of the inner membrane (TIM) and mitophagy. Protein expression was analysed by ELISA and immunohistochemistry. The following genes were downregulated in ischemic and dilated cardiomyopathy: COX1, NRF1, TFAM, SIRT1, MTOR, MFF, DNM1L, DDIT3, UBL5, HSPA9, HSPE1, YME1L, LONP1, SPG7, HTRA2, OMA1, TIMM23, TIMM17A, TIMM17B, TIMM44, PAM16, TIMM22, TIMM9, TIMM10, PINK1, PARK2, ROTH1, PARL, FUNDC1, BNIP3, BNIP3L, TPCN2, LAMP2, MAP1LC3A and BECN1. Moreover, MT-ATP8, MFN2, EIF2AK4 and ULK1 were downregulated in heart failure from dilated, but not ischemic cardiomyopathy. VDAC1 and JUN were only genes that exhibited significantly different expression between ischemic and dilated cardiomyopathy. Expression of PPARGC1, OPA1, JUN, CEBPB, EIF2A, HSPD1, TIMM50 and TPCN1 was not significantly different between control and any form of heart failure. TOMM20 and COX proteins were downregulated in ICM and DCM. Heart failure in patients with ischemic and dilated cardiomyopathy is associated with downregulation of large number of UPRmt, mitophagy, TIM and fusion-fission balance genes. This indicates multiple defects in MQC and represents one of potential mechanisms underlying mitochondrial dysfunction in patients with heart failure.

The circ_006573/miR-376b-3p Axis Advances Spinal Cord Functional Recovery after Injury by Modulating Vascular Regeneration.

Abnormal expression of non-coding RNAs after spinal cord injury (SCI) is associated with pathophysiological outcomes. We bioinformatically predicted a circRNA-miRNA-mRNA axis in SCI. A total of 4690 mRNAs, 17 miRNAs, and 3928 circRNAs were differentially expressed, with co-expressed RNAs predicted to regulate pathways related to wound healing. Among the most highly differentially expressed circRNAs, circ_006573, but not circ_016395, weakened the viability and migration of rat aortic endothelial cells, and its biological effects were rescued with miR-376b-3p mimics. Furthermore, circ_006573 overexpression induced changes in Cebpb, IL-18, and Plscr1 expression that were reversed by miR-376b-3p. In a rat model, circ_006573 shRNA administration improved the pathological manifestations of SCI and ameliorated motor function. Moreover, the expression of CD31, CD34, and VEGF-A in spinal cord tissues was significantly elevated after circ_006573 shRNA treatment, indicating that circ_006573 may be involved in vascular regeneration and functional recovery after SCI. Thus, the circ_006573-miR-376b-3p axis offers a foundation for understanding pathophysiological mechanisms and predicting strategies for treating SCI.

Combined analysis of single-cell and bulk RNA sequencing reveals the expression patterns of circadian rhythm disruption in the immune microenvironment of Alzheimer's disease.

Circadian rhythm disruption (CRD) represents a critical contributor to the pathogenesis of Alzheimer's disease (AD). Nonetheless, how CRD functions within the AD immune microenvironment remains to be illustrated. Circadian rhythm score (CRscore) was utilized to quantify the microenvironment status of circadian disruption in a single-cell RNA sequencing dataset derived from AD. Bulk transcriptome datasets from public repository were employed to validate the effectiveness and robustness of CRscore. A machine learning-based integrative model was applied for constructing a characteristic CRD signature, and RT-PCR analysis was employed to validate their expression levels. We depicted the heterogeneity in B cells, CD4+ T cells, and CD8+ T cells based on the CRscore. Furthermore, we discovered that CRD might be strongly linked to the immunological and biological features of AD, as well as the pseudotime trajectories of major immune cell subtypes. Additionally, cell-cell interactions revealed that CRD was critical in the alternation of ligand-receptor pairs. Bulk sequencing analysis indicated that the CRscore was found to be a reliable predictive biomarker in AD patients. The characteristic CRD signature, which included 9 circadian-related genes (CRGs), was an independent risk factor that accurately predicted the onset of AD. Meanwhile, abnormal expression of several characteristic CRGs, including GLRX, MEF2C, PSMA5, NR4A1, SEC61G, RGS1, and CEBPB, was detected in neurons treated with Aβ1-42 oligomer. Our study revealed CRD-based cell subtypes in the AD microenvironment at single-cell level and proposed a robust and promising CRD signature for AD diagnosis. A deeper knowledge of these mechanisms may provide novel possibilities for incorporating "circadian rhythm-based anti-dementia therapies" into the treatment protocols of individualized medicine.

Insights into the Potential Impact of Quetiapine on the Microglial Trajectory and Inflammatory Response in Organotypic Cortical Cultures Derived from Rat Offspring.

Atypical antipsychotics currently constitute the first-line medication for schizophrenia, with quetiapine being one of the most commonly prescribed representatives of the group. Along with its specific affinity for multiple receptors, this compound exerts other biological characteristics, among which anti-inflammatory effects are strongly suggested. Simultaneously, published data indicated that inflammation and microglial activation could be diminished by stimulation of the CD200 receptor (CD200R), which takes place by binding to its ligand (CD200) or soluble CD200 fusion protein (CD200Fc). Therefore, in the present study, we sought to evaluate whether quetiapine could affect certain aspects of microglial activity, including the CD200-CD200R and CX3CL1-CX3CR1 axes, which are involved in the regulation of neuron-microglia interactions, as well as the expression of selected markers of the pro- and anti-inflammatory profile of microglia (Cd40, Il-1β, Il-6, Cebpb, Cd206, Arg1, Il-10 and Tgf-β). Concurrently, we examined the impact of quetiapine and CD200Fc on the IL-6 and IL-10 protein levels. The abovementioned aspects were investigated in organotypic cortical cultures (OCCs) prepared from the offspring of control rats (control OCCs) or those subjected to maternal immune activation (MIA OCCs), which is a widely implemented approach to explore schizophrenia-like disturbances in animals. The experiments were performed under basal conditions and after additional exposure to the bacterial endotoxin lipopolysaccharide (LPS), according to the "two-hit" hypothesis of schizophrenia. The results of our research revealed differences between control and MIA OCCs under basal conditions and in response to treatment with LPS in terms of lactate dehydrogenase and nitric oxide release as well as Cd200r, Il-1β, Il-6 and Cd206 expression. The additional stimulation with the bacterial endotoxin resulted in a notable change in the mRNA levels of pro- and anti-inflammatory microglial markers in both types of OCCs. Quetiapine diminished the influence of LPS on Il-1β, Il-6, Cebpb and Arg1 expression in control OCCs as well as on IL-6 and IL-10 levels in MIA OCCs. Moreover, CD200Fc reduced the impact of the bacterial endotoxin on IL-6 production in MIA OCCs. Thus, our results demonstrated that quetiapine, as well as the stimulation of CD200R by CD200Fc, beneficially affected LPS-induced neuroimmunological changes, including microglia-related activation.

Identification of cancer-related genes FGFR2 and CEBPB in choledochal cyst via RNA sequencing of patient-derived liver organoids.

Choledochal cysts (CC) are congenital bile duct anomalies with 6-30% risk for developing bile duct cancer. However, the molecular mechanisms underlying cancer risk of CC are unknown. We sought to identify the gene expression changes underlying the cancer risk of CC patients. Liver organoids (n = 51) were generated from liver/bile duct biopsies of CC (n = 7; type I) and hepatoblastoma (n = 5; HB: non-tumor & tumor) for RNA sequencing. Bioinformatics analysis was conducted to identify differentially expressed cancer-related genes in CC and controls. We compared CC with non-cancerous and cancerous controls, normal adjacent non-tumor region of hepatoblastoma (HB) liver as non-cancerous control and tumor region as non-CC cancer control (HB-tumor). Reverse transcription real-time quantitative PCR (RT-qPCR) verification and immunohistochemistry of selected genes was conducted in additional CC and HB liver biopsies. HB non-tumor and HB tumor organoids displayed distinct gene expression profiles. Expression profiling separated CC organoids into two clusters, one overlapping with HB non-tumor and the other one with HB tumor organoids. Genes selected based on their log2FoldChange values for RT-qPCR verification in 31 CC and 11 HB non-tumor liver tissues revealed significantly elevated expression of FGFR2 in 7 and CEBPB in 2 CC liver tissues (CC vs HB: 4.082 vs. 0.7671, p<0.01; 2.506 vs. 1.210, p<0.01). Distinctive positive staining in bile ducts were seen in CC, HB tumor and non-tumor liver tissues for FGFR2 and CEBPB. Percentages of CEBPB-immuno-positive or FGFR2-immuno-positive bile duct cells in CC and HB-tumor liver were higher than that in HB non-tumor liver. The study identified dysregulated genes related to cancer pathways in CC patients suggesting cancer risk. The findings suggest that the elevated expression of FGFR2 and CEBPB in liver may contribute to cancer development in CC patients.

CEBPB/POU2F2 modulates Endothelin1 expression in prehypertensive SHR vascular smooth muscle cells.

The pathogenesis of hypertension is not fully understood; ET1 is involved in developing essential hypertension. ET1 can promote VSMC proliferation or hypertrophy through autocrine and paracrine effects. Proliferating smooth muscle cells in the aorta are "dedifferentiated" cells that cause increased arterial stiffness and remodeling. Male SHRs had higher aortic stiffness than normal control male WKY rats. Male SHR VSMCs expressed high levels of the ET1 gene, but endothelial cells did not. Therefore, it is necessary to understand the molecular mechanism of enhanced ET1 expression in SHR VSMCs. We identified POU2f2 and CEBPB as the main molecules that enhance ET1 expression in male SHR VSMCs. A promoter activity analysis confirmed that the enhancer region of the ET1 promoter in male SHR VSMCs was from -1309 to -1279 bp. POU2f2 and CEBPB exhibited an additive role in the enhancer region of the ET1 promoter. POU2f2 or CEBPB overexpression sufficiently increased ET1 expression, and co-transfection with the CEBPB and POU2f2 expression plasmids had additive effects on the activity of the ET1 promoter and ET1 secretion level of male WKY VSMCs. In addition, knockdown of POU2f2 also revealed that POU2f2 is necessary to enhance ET1 expression in SHR VSMCs. The enhancer region of the ET1 promoter is highly conserved in rats, mice, and humans. POU2f2 and CEBPB mRNA levels were significantly increased in remodeled human VMSCs. In conclusion, the novel regulation of POU2f2 and CEBPB in VSMCs will help us understand the pathogenesis of hypertension and support the development of future treatments for hypertension.

LBSUN152 The Warburg Effect And Complex Thermomics Inform Phenotypic Expression

Abstract Introduction As technology reveals biological complexity, integration of vast clinical and multi-omic data utilizing artificial intelligence promises to clarify confusion. We have utilized challenge tests to inform algorithmic graphs recognizing heat as foundational for understanding phenotypic variation. Thermomics, describing complex thermic influences that are difficult to objectively quantify thereby compromising application to AI, are readily included in practice-derived models. Methods For the past 45 years cases have been evaluated with oral calcium challenge and immersion in water while observing changes in urinary composition. Between 2001 and 2010 respiratory gas exchange following a high carbohydrate meal combined with pulsatile insulin revealed metabolic variance that combined with basic, translational and clinical data of others informed generation of algorithmic graphs. COVID 19 served to clarify the effect of ACE2 disruption by predicting divergent phenotypic expression mediated by calciotropic hormones. Results Extraction of O2 from glucose, a consequence of fatty acid synthesis producing R/Q &amp;gt; 1 is modeled as foundational for metabolic evolution. This Warburg effect, recognized in proliferating malignant cells, was observed at the systemic level in humans and, modeled as mediated by hypoxia inducible factors regulating glucose uptake and suppression of beta-oxidation. Higher order orchestration is governed by a signaling structure including PTH, PTHrP, FGF23, Klotho, prolactin, growth hormone, IGFs, insulin, SGLT2, thyroid, adrenal and vit D while salt inducible kinase contributes to understanding of terrestrial adaptation. Inclusion of thermomics leads to prediction of a favorable effect of sub-cutaneous adipose beiging on CNS function mediated by circulating Klotho as a constraint on FGF23 in amelioration of Long COVID by phentermine that is also effective in hypophosphatasia where the exercise induced beiging, induced by irisin is known to be dependent upon alkaline phosphatase. The post-exertional malaise of Myalgic Encephalomyelitis, where exercise induces elevation in R/Q and reduced work capacity, has informed modeling of the systemic Warburg effect. The models are further validated with mechanistic explanation for recent reports of marked increases of R/Q in human Paget's disease and CEBPb expression dependence mimicking Alzheimer's and limiting life span in mice. Conclusions The value of practice is demonstrated by the ability to model thermomics even when difficult to quantify. The work is validated by explanation of old conundrums, discovery of treatment for unresolved problems, facilitation of case-based discovery and integration of emerging data from basic sciences. The Warburg effect when recognized as foundational to biology, contributes to a General Theory of Metabolism applicable to effective personalization of care. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.