Ingenuity Pathway Analysis Analysis Research Articles

Analysis of single-cell transcriptome data from a mouse model implicates protein synthesis dysfunction in schizophrenia.

Schizophrenia is a mental disorder that causes considerable morbidity, whose risk largely results from genetic factors. Setd1a is a gene implicated in schizophrenia. To study the gene expression changes found in heterozygous Setd1a± knockout mice in order to gain useful insight into schizophrenia pathogenesis. We mined a single-cell RNA sequencing (scRNAseq) dataset from the prefrontal cortex (PFC) and striatum of Setd1a± mice and identified cell type-specific differentially expressed genes (DEGs) and differential transcript usage (DTU). DEGs and genes containing DTU found in each cell type were used to identify affected biological pathways using Ingenuity Pathway Analysis (IPA). We identified 273 unique DEGs across all cell types in PFC and 675 unique gene peaks containing DTU. In striatum, we identified 327 unique DEGs across all cell types and 8 unique gene peaks containing DTU. Key IPA findings from the analysis of DEGs found in PFC and striatum implicate processes involved in protein synthesis, mitochondrial function, cell metabolism, and inflammation. IPA analysis of genes containing DTU in PFC points to protein synthesis, as well as cellular activities involving intracellular signaling and neurotransmission. One canonical pathway, 'EIF2 Signaling', which is involved in the regulation of protein synthesis, was detected in PFC DEGs, striatum DEGs, and PFC genes containing DTU, drawing attention to its importance in schizophrenia pathophysiology. Processes involving protein synthesis in general and the 'EIF2 Signaling' pathway in particular could be targets for the development of new research strategies and biomarkers in schizophrenia.

#1933 Urinary proteomics identifying the novel biomarker associated with crescentic glomerulonephritis

Abstract Background and Aims As a severe progressive acute renal injury, crescentic glomerulonephritis requires timely pathology diagnosis and treatment. New non-invasive biomarkers will be crucial when the renal biopsy is not available or unacceptable. Urinary proteomics, specifically data-independent acquisition (DIA) proteomics, might provide some potential indicators. Method Urine proteomics examination was on patients with crescentic glomerulonephritis(n = 15), chronic glomerulonephritis (n = 15), acute kidney injury (AKI) without glomerular crescentic(n = 10), and 15 healthy controls. The crescentic glomerulonephritis group is the patients with extensive glomerular crescents over 50%. We performed LC-MS/MS analysis to identify differentially expressed proteins (DEPs), Ingenuity Pathway Analysis (IPA), and the proteome map for significant pathways and crucial proteins among patients and controls. The signature proteins were validated using enzyme-linked immunosorbent assay (ELISA) analysis. Results A total of 55 population, males occupied 45.45%, with the average age being 53 ± 13 years old. A volcano plot among crescentic glomerulonephritis and control groups is in Fig. 1. IPA analysis showed that neutrophil degranulation and complement cascade were the top two pathways in crescentic glomerulonephritis but not in the healthy and nephritis groups. The activated neddylation pathway in patients with crescentic glomerulonephritis compared with AKI patients. After integrating the DEPs among three groups via the Venn plot, we observed eight DEPs significantly associated with the crescentic glomerulonephritis, among which Coagulation factor V (F5), Phospholipid transfer protein (PLTP), and Alcohol dehydrogenase 1C (ADH1C) were significant proteins. Biomarker analysis showed the area under the curve values for predicting crescentic glomerulonephritis were 0.867, 0.851, and 0.815, respectively (P < 0.001). Conclusion Urine proteomics holds promise in identifying the crescentic glomerulonephritis patient, thus providing a potential value as a noninvasive biomarker.

A Pilot Study on the Proteomics Profile of Serum Exosome-Enriched Extracellular Vesicles from Normal versus Individuals with Obesity-Related Insulin Resistance.

Circulating exosome-enriched extracellular vesicles (EVs) have drawn considerable importance in obesity-related insulin-resistance (IR). We sought to compare the proteomics profile of serum exosomes from normal individuals and those with obesity and IR. We isolated serum exosomes from male subjects with obesity and insulin resistance (Ob-IR, HOMA-IR > 2.0) and lean/overweight insulin-sensitive (Normal (N), HOMA-IR < 2.0) individuals. The differential protein expression between the two groups was detected by a label-free quantitative mass spectrometry analysis followed by GO annotation and ingenuity pathway analysis (IPA). We identified 23 upregulated and 46 downregulated proteins between Ob-IR and N groups. Some of these proteins are involved in altering insulin signaling (VPS13C, TBC1D32, TTR, and ADIPOQ), inflammation (NFκB and CRP), and B-cell proliferation/activation (IGLV4-69, IGKV1D-13, and IGHV4-28). GO analysis revealed that the differentially expressed proteins (DEPs) are mainly involved in regulating immune cell activation and are located in extracellular space. IPA analysis showed that top molecules mediating IR, inflammation and B-cell activation were upregulated in Ob-IR subjects compared to N subjects. Serum exosomal proteins can be used as biomarkers to identify the future risk of diabetes and a therapeutic target to prevent or slow down the progression of diabetes in high-risk individuals.

Impact of miR-29c-3p in the Nucleus Accumbens on Methamphetamine-Induced Behavioral Sensitization and Neuroplasticity-Related Proteins.

Methamphetamine (METH) abuse inflicts both physical and psychological harm. While our previous research has established the regulatory role of miR-29c-3p in behavior sensitization, the underlying mechanisms and target genes remain incompletely understood. In this study, we employed the isobaric tags for relative and absolute quantitation (iTRAQ) technique in conjunction with Ingenuity pathway analysis (IPA) to probe the putative molecular mechanisms of METH sensitization through miR-29c-3p inhibition. Through a microinjection of AAV-anti-miR-29c-3p into the nucleus accumbens (NAc) of mice, we observed the attenuation of METH-induced locomotor effects. Subsequent iTRAQ analysis identified 70 differentially expressed proteins (DEPs), with 22 up-regulated potential target proteins identified through miR-29c-3p target gene prediction and IPA analysis. Our focus extended to the number of neuronal branches, the excitatory synapse count, and locomotion-related pathways. Notably, GPR37, NPC1, and IREB2 emerged as potential target molecules for miR-29c-3p regulation, suggesting their involvement in the modulation of METH sensitization. Quantitative PCR confirmed the METH-induced aberrant expression of Gpr37, Npc1, and Ireb2 in the NAc of mice. Specifically, the over-expression of miR-29c-3p led to a significant reduction in the mRNA level of Gpr37, while the inhibition of miR-29c-3p resulted in a significant increase in the mRNA level of Gpr37, consistent with the regulatory principle of miRNAs modulating target gene expression. This suggests that miR-29c-3p potentially influences METH sensitization through its regulation of neuroplasticity. Our research indicates that miR-29c-3p plays a crucial role in regulating METH-induced sensitization, and it identified the potential molecular of miR-29c-3p in regulating METH-induced sensitization.

TNIK regulation of interferon signaling and endothelial cell response to virus infection.

Traf2 and Nck-interacting kinase (TNIK) is known for its regulatory role in various processes within cancer cells. However, its role within endothelial cells (ECs) has remained relatively unexplored. Leveraging RNA-seq data and Ingenuity Pathway Analysis (IPA), we probed the potential impact of TNIK depletion on ECs. Examination of RNA-seq data uncovered more than 450 Differentially Expressed Genes (DEGs) in TNIK-depleted ECs, displaying a fold change exceeding 2 with a false discovery rate (FDR) below 0.05. IPA analysis unveiled that TNIK depletion leads to the inhibition of the interferon (IFN) pathway [-log (p-value) >11], downregulation of IFN-related genes, and inhibition of Hypercytokinemia/Hyperchemokinemia [-log (p-value) >8]. The validation process encompassed qRT-PCR to evaluate mRNA expression of crucial IFN-related genes, immunoblotting to gauge STAT1 and STAT2 protein levels, and ELISA for the quantification of IFN and cytokine secretion in siTNIK-depleted ECs. These assessments consistently revealed substantial reductions upon TNIK depletion. When transducing HUVECs with replication incompetent E1-E4 deleted adenovirus expressing green fluorescent protein (Ad-GFP), it was demonstrated that TNIK depletion did not affect the uptake of Ad-GFP. Nonetheless, TNIK depletion induced cytopathic effects (CPE) in ECs transduced with wild-type human adenovirus serotype 5 (Ad-WT). Our findings suggest that TNIK plays a crucial role in regulating the EC response to virus infections through modulation of the IFN pathway.

Changes in the Proteome of the Circle of Willis during Aging Reveal Signatures of Vascular Disease.

Approximately 70% of all strokes occur in patients over 65 years old, and stroke increases the risk of developing dementia. The circle of Willis (CoW), the ring of arteries at the base of the brain, links the intracerebral arteries to one another to maintain adequate cerebral perfusion. The CoW proteome is affected in cerebrovascular and neurodegenerative diseases, but changes related to aging have not been described. Here, we report on a quantitative proteomics analysis comparing the CoW from five young (2-3-month-old) and five aged male (18-20-month-old) mice using gene ontology (GO) enrichment, ingenuity pathway analysis (IPA), and iPathwayGuide tools. This revealed 242 proteins that were significantly dysregulated with aging, among which 189 were upregulated and 53 downregulated. GO enrichment-based analysis identified blood coagulation as the top biological function that changed with age and integrin binding and extracellular matrix constituents as the top molecular functions. Consistent with these findings, iPathwayGuide-based impact analysis revealed associations between aging and the complement and coagulation, platelet activation, ECM-receptor interaction, and metabolic process pathways. Furthermore, IPA analysis revealed the enrichment of 97 canonical pathways that contribute to inflammatory responses, as well as 59 inflammation-associated upstream regulators including 39 transcription factors and 20 cytokines. Thus, aging-associated changes in the CoW proteome in male mice demonstrate increases in metabolic, thrombotic, and inflammatory processes.

EPIGENETIC DIFFERENCE BETWEEN INSOMNIA GROUP AND NONINSOMNIA GROUP IN PARTICIPANTS WITH KNEE PAIN

Abstract Chronic knee pain is a common condition that often co-occurs with insomnia. Epigenetic modifications, such as DNA methylation, may play a role in the sleep-pain relationship. In this study, we aimed to identify differentially methylated CpGs/regions and enriched genomic pathways associated with insomnia in participants with knee pain (KP). We recruited 140 cognitively healthy middle to older-aged adults with self-reported KP and extracted DNA from peripheral blood. MethylationEPIC arrays were used for methylation data quantification, R package minfi was used for data preprocessing and quality control, and Ingenuity Pathway Analysis (IPA) was used to explore functional implications of differential methylation in pathways related to insomnia. Annotated genes within ±5kb of the putative differentially methylated regions (DMRs, p&amp;lt; 0.05) were subjected to the IPA analysis. There were no significant differences in sex or race between the two groups (p&amp;gt;0.05). We observed an over-representation of younger participants in the insomnia group (p=0.005) and also from the University of Florida (p=0.008). At raw p &amp;lt; 0.05 cutoff, we identified a total of 20,006 CpG probes, including 12,674 hypermethylated, and 7,332 hypomethylated CpG probes. Pathway analysis using IPA identified multiple immune-related pathways enriched with insomnia-related differentially methylated regions (DMRs), including the antigen presentation pathway (p=3.39E-08). Verapamil was identified as the most significant upstream regulator (p=8.8E-07). Our findings suggest the importance of epigenetic regulation of the immune system in the sleep-pain relationship and highlight the need for further research to understand the epigenetic contributions to sleep disorders in individuals with chronic pain.

Identifying Inflammatory Pathways That Promote RBC Alloimmunization in Patients with Sickle Cell Disease

Introduction: During red blood cell (RBC) transfusion, exposure to unmatched non-ABO antigens can lead to production of alloantibodies that promote potentially fatal hemolytic events. Patients with Sickle Cell Disease (SCD) have the highest incidence of RBC alloimmunization. Prior studies indicate that an altered immuno-regulatory and/or pro-inflammatory state may determine the risk of RBC alloimmunization. For example, one study reported that patients with SCD have inflammatory cytokine gene polymorphisms that could be associated with diverse disease presentations and alloimmunization. We previously reported that inflammatory type 1 interferons (IFNα/β) produced by innate immune cells are required for RBC alloimmunization in mice, while others have suggested that NFkB-induced cytokines play a role in murine transfusion models. However, the inflammatory factors that regulate RBC alloimmunization in patients with SCD are poorly understood. Alloimmunized patients with SCD also have elevated levels of anti-RBC autoantibodies, which facilitate erythrophagocytosis. Given the role of erythrophagocytosis by spleen macrophages in processing RBC antigens, we hypothesized that erythrophagocytosis of SCD RBCs may promote inflammatory pathways that regulate alloimmune responses. Methods: RBCs from control subjects (AA), and reticulocytes and mature RBCs from patients with SCD, were opsonized or not with anti-RBC antibodies (anti-RhD) and co-cultured for 3 hrs with human macrophages (hMac) differentiated from monocytes of healthy subjects. Phagocytosis of labeled RBCs was analyzed by flow cytometry. Bulk mRNA sequencing of co-cultured macrophages was performed to identify differential gene expression. Ingenuity Pathway Analysis (IPA) software was used to identify relevant pathways and upstream regulators of differentially expressed genes. To validate sequencing results, cytokine levels from co-culture supernatants were measured by multiplex cytokine assays. Results: Flow cytometric analysis revealed that opsonization was required for erythrophagocytosis in an FcR dependent manner. RNA-seq Differential Expression analysis of macrophages co-cultured with AA opsonized RBCs vs. non-opsonized RBCs identified 166 significantly upregulated and 24 downregulated genes (p&amp;lt;0.0001). IPA analysis revealed that the top 12 canonical pathways were related to inflammation, including TNF, IL-17, NF-kappa-B and NOD-like receptor signaling pathways. TNF signaling was the top predicted upstream regulator pathway with a fold enrichment score of 10.3 (16 genes) (p&amp;lt;0.0001). Co-cultures of mature RBCs from patients with SCD showed comparable results. When comparing macrophages from co-cultures of opsonized reticulocytes vs. non-opsonized reticulocytes from patients with SCD, 174 genes were upregulated and 31 were downregulated (p&amp;lt;0.0001). Pathway analysis also revealed unique signatures for opsonized reticulocytes from patients with SCD. The top 12 canonical pathways included TNF, NF-kappa-B and NOD-like receptor signaling pathways (Figure 1). The top predicted upstream regulators were TNF and the NF-kappa B signaling pathway with a fold enrichment score of 12.3 (22 genes) (p&amp;lt;0.0001) and 10.9 (18 genes), respectively (Figure 1). Finally, to validate the sequencing results, cytokines levels from supernatants of hMac co-cultures with the RBCs from the patients with SCD and control subjects were measured. Erythrophagocytosis of opsonized reticulocytes and AA RBCs, compared to non-opsonized co-cultures, led to production of NFκB-mediated cytokines, including TNF. Our results show that erythrophagocytosis of SCD RBCs promote NFκB-mediated TNF production. These results warrant further investigation of the role of NFκB-mediated inflammation in RBC alloimmunization in patients with SCD.

Transcriptomic Analyses Indicate That Pre-Leukemic B Cells from People with Monoclonal B Lymphocytosis Are Surprisingly Much Less Active Than Normal B Cells from People with Monoclonal B Lymphocytosis and from Healthy Donors

Introduction. Chronic lymphocytic leukemia (CLL) follows a stepwise developmental process, beginning in a specific group of normal B lymphocytes that undergo clonal expansion. These clonal expansions, known as monoclonal B lymphocytosis (MBL), are considered to be essential precursors of CLL based on immunologic, genetic, and epidemiologic studies. Since MBL represents the overabundance of a single CD5 + B cell, we tested how different the transcriptomes of MBL cells were from normal B cells (NBC) in people with MBL (NBC-MBL) and NBC in healthy donors (NBC-HD), expecting to identify activations pathways in the MBL cells that would explain clonal expansion. Methods. We collected and sorted specific cell populations from 13 patients with monoclonal B lymphocytosis (MBL): CD5 +CD19 +CD20 dimIgL-restricted MBL cells and CD5 -CD19 +CD20 + NBC. Additionally, we obtained CD5 -CD19 +CD20 + NBC from 13 healthy donors (NBC-HD). RNA was extracted and sequenced using SMART-Seq v4 Ultra Low Input and HiSeq platform. DESeq2 was used to analyze RNA-seq data. To identify genes with differential expression between these populations, the transcriptomes of MBL cells were compared to NBC from HD, employing a cutoff of P&amp;lt;0.05 and Log2fc ratio &amp;gt;± 1.5. We also used paired sample t-tests to compare 13 MBL cells to NBC from the same MBL patients. Significantly differentially expressed genes (DEG) resulting from these analyses were then subjected to Ingenuity Pathway Analysis (IPA). Results. When comparing the gene profiles of 13 MBL B cells to 13 NBC-HD cells, we identified 2695 DEG. Next, we compared gene profiles between MBL B cells and NBC-MBL cells obtained from 13 MBL patients, identifying 4821 genes. Using IPA, down-regulated B Cell Receptor Signaling, Cytokine Signaling, and Tumor Microenvironment Pathway were identified in both analyses. Upon overlapping the genes from these two analyses, we found that 803 genes passed the cutoff in both analyses. IPA analysis showed that 14 signaling pathways, including IL-33, IL-17, and HIF1a, were significantly downregulated in MBL cells. Moreover, the negative signaling pathway (Role of p14/p19ARF in Tumor Suppression) was upregulated in MBL cells. PDCD1 expression was significant higher in MBL (Log2Fc 6.8, P&amp;lt;0.0001) and 11 PDCD1 target genes were significantly regulated in MBL cells. IL-6 (Log2Fc -5.4, P&amp;lt;0.0001) expression was significantly lower in MBL and 47 IL-6 target genes were significantly regulated in MBL cells. Surprisingly, genes related to cell activation, proliferation and movement were largely downregulated in MBL cells. There were 2052 genes unique to the analysis MBL vs NBC-HD, IPA analysis showed several T-cell related signaling pathways e.g., T cell Exhaustion Signaling Pathway and T Lymphocyte Apoptosis were upregulated. In the 4018 genes unique in the analysis MBL vs NB-MBL. IPA results showed all the signaling pathways were downregulated in MBL. Discussion. Surprisingly, our transcriptomic analyses indicate that MBL cells are much less active than NBC from people with MBL and from HD. Specifically, we found that many cytokine signaling pathways and genes related with cell activation, proliferation and movement are downregulated in MBL cells. Also, PDCD1 is highly expressed in MBL cells, and PDCD1 target genes are also significantly regulated in MBL cells. This is consistent with the immunosuppressed features of MBL cells since interaction between PD-1 on B cells and its ligands in the microenvironment negatively influence the survival, proliferation, and immune evasion of CLL and MBL cells. Collectively, although functional studies are needed, these transcriptomic data strongly suggest that MBL cells are not in an activated state and are less active than even the NBC from the same person with MBL.

Validation of vaginal microbiome proxies forin vitroexperiments that biomimic Lactobacillus-dominant vaginal cultures.

The vaginal microbiome includes diverse microbiota dominated by Lactobacillus [L.] spp. that protect against infections, modulate inflammation, and regulate vaginal homeostasis. Because it is challenging to incorporate vaginal microbiota into in vitro models, including organ-on-a-chip systems, we assessed microbial metabolites as reliable proxies in addition to traditional vaginal epithelial cultures (VECs). Human immortalized VECs cultured on transwells with an air-liquid interface generated stratified cell layers colonized by transplanted healthy microbiomes (L. jensenii- or L. crispatus-dominant) or a community representing bacterial vaginosis (BV). After 48-h, a qPCR array confirmed the expected donor community profiles. Pooled apical and basal supernatants were subjected to metabolomic analysis (untargeted mass spectrometry) followed by ingenuity pathways analysis (IPA). To determine the bacterial metabolites' ability to recreate the vaginal microenvironment in vitro, pooled bacteria-free metabolites were added to traditional VEC cultures. Cell morphology, viability, and cytokine production were assessed. IPA analysis of metabolites from colonized samples contained fatty acids, nucleic acids, and sugar acids that were associated with signaling networks that contribute to secondary metabolism, anti-fungal, and anti-inflammatory functions indicative of a healthy vaginal microbiome compared to sterile VEC transwell metabolites. Pooled metabolites did not affect cell morphology or induce cell death (∼5.5%) of VEC cultures (n=3) after 72-h. However, metabolites created an anti-inflammatory milieu by increasing IL-10 production (p=.06, T-test) and significantly suppressing pro-inflammatory IL-6 (p=.0001), IL-8 (p=.009), and TNFα (p=.0007) compared to naïve VEC cultures. BV VEC conditioned-medium did not affect cell morphology nor viability; however, it induced a pro-inflammatory environment by elevating levels of IL-6 (p=.023), IL-8 (p=.031), and TNFα (p=.021) when compared to L.-dominate microbiome-conditioned medium. VEC transwells provide a suitable ex vivo system to support the production of bacterial metabolites consistent with the vaginal milieu allowing subsequent in vitro studies with enhanced accuracy and utility.

Developing A Baseline Metabolomic Signature Associated with COVID-19 Severity: Insights from Prospective Trials Encompassing 13 U.S. Centers.

Metabolic disease is a significant risk factor for severe COVID-19 infection, but the contributing pathways are not yet fully elucidated. Using data from two randomized controlled trials across 13 U.S. academic centers, our goal was to characterize metabolic features that predict severe COVID-19 and define a novel baseline metabolomic signature. Individuals (n = 133) were dichotomized as having mild or moderate/severe COVID-19 disease based on the WHO ordinal scale. Blood samples were analyzed using the Biocrates platform, providing 630 targeted metabolites for analysis. Resampling techniques and machine learning models were used to determine metabolomic features associated with severe disease. Ingenuity Pathway Analysis (IPA) was used for functional enrichment analysis. To aid in clinical decision making, we created baseline metabolomics signatures of low-correlated molecules. Multivariable logistic regression models were fit to associate these signatures with severe disease on training data. A three-metabolite signature, lysophosphatidylcholine a C17:0, dihydroceramide (d18:0/24:1), and triacylglyceride (20:4_36:4), resulted in the best discrimination performance with an average test AUROC of 0.978 and F1 score of 0.942. Pathways related to amino acids were significantly enriched from the IPA analyses, and the mitogen-activated protein kinase kinase 5 (MAP2K5) was differentially activated between groups. In conclusion, metabolites related to lipid metabolism efficiently discriminated between mild vs. moderate/severe disease. SDMA and GABA demonstrated the potential to discriminate between these two groups as well. The mitogen-activated protein kinase kinase 5 (MAP2K5) regulator is differentially activated between groups, suggesting further investigation as a potential therapeutic pathway.

MicroRNAs Signature Panel Identifies Heavy Drinkers with Alcohol-Associated Cirrhosis from Heavy Drinkers without Liver Injury.

Background: Alcohol-associated liver disease (ALD) is the most common disorder of prolonged drinking. Mechanisms underlying cirrhosis in such patients remain unclear. MicroRNAs play regulatory role in several diseases, are affected by alcohol and may be important players in alcohol use disorders, such as cirrhosis. Methods: We investigated serum samples from heavy chronic alcohol users (80 g/day (male) and 50 g/day (female) for ≥10 years) that were available from our previously reported GenomALC study. A subset of GenomALC drinkers with liver cirrhosis (cases, n = 24) and those without significant liver disease (drinking controls, n = 23) were included. Global microRNA profiling was performed using high-throughput real-time quantitative PCR to identify the microRNA signatures associated with cirrhosis. Ingenuity Pathway Analysis (IPA) software was utilized to identify target mRNAs of significantly altered microRNAs, and molecular pathways were analysed. Identified microRNAs were analysed for correlation with traditional liver disease biomarkers and risk gene variants previously reported from GenomALC genome-wide association study. Results: The expression of 21 microRNAs was significantly downregulated in cases compared to drinking controls (p < 0.05, ∆∆Ct > 1.5-fold). Seven microRNAs (miR-16, miR-19a, miR-27a, miR-29b, miR-101, miR-130a, and miR-191) had a highly significant correlation (p < 0.001) with INR, bilirubin and MELD score. Three microRNAs (miR-27a, miR-130a and miR-191) significantly predicted cases with AUC-ROC 0.8, 0.78 and 0.85, respectively (p < 0.020); however, INR performed best (0.97, p < 0.001). A different set of six microRNAs (miR-19a, miR-26a, miR-101, miR-151-3p, miR-221, and miR-301) showed positive correlation (ranging from 0.32 to 0.51, p < 0.05) with rs10433937:HSD17B13 gene variant, associated with the risk of cirrhosis. IPA analysis revealed mRNA targets of the significantly altered microRNAs associated with cell death/necrosis, fibrosis and increased steatosis, particularly triglyceride metabolism. Conclusions: MicroRNA signatures in drinkers distinguished those with liver cirrhosis from drinkers without liver disease. We identified mRNA targets in liver functions that were enriched for disease pathogenesis pathways.

SAT632 The Functional Role And Regulatory Mechanism Of FTO M6A RNA Demethylase In Human Uterine Leiomyosarcoma

Abstract Disclosure: Q. Yang: None. A. Al-Hendy: None. Introduction: Uterine leiomyosarcoma (uLMS) is the most frequent subtype of uterine sarcoma that presents a poor prognosis, high rates of recurrence, and metastasis. However, the origin and molecular mechanism underlying driving its clinical and biological behavior remain to be determined. Recently, we and others have revealed the role of microRNAs, DNA methylation, and histone modifications contributing to the pathogenesis of uLMS. However, the connectivity between reversible RNA m6A methylation and uLMS remains unclear. In this study, we assessed the role and mechanism of the m6A demethylase fat mass- and obesity-associated protein (FTO) in the pathogenesis of uLMS. Materials and Methods: We performed immunohistochemistry (IHC) and immunoblot analysis to measure the m6A erasers FTO and ALKBH5 protein levels in LMS and adjacent myometrium tissues (n=8) and cell lines. We performed LMS cell proliferation and cell cycle phase distribution analysis. RNA-seq was performed to determine the molecular mechanism underlying m6A eraser inhibition in LMS cells (n=4 for each group). The R package limma+voom was used for this analysis. Hallmark pathway analysis was used to identify enriched pathways in response to FTO inhibition. Real-time PCR was performed to validate the differentially expressed genes. Ingenuity pathway analysis (IPA) was performed to determine the upstream regulators targeting cell cycle-related genes. We used a cutoff −1.5 &amp;gt; fold-change &amp;gt; 1.5 and an adjusted p-value of 0.05. In addition, data were analyzed by a two-tailed unpaired Student’s t-test between any two groups. Results: IHC analysis revealed that m6A demethylases FTO and ALKBH5 are aberrantly upregulated in uLMS tissues compared to adjacent myometrium (p&amp;lt;0.01, H-score). Furthermore, inhibition of FTO with its small, potent inhibitor (Dac51) decreased the uLMS proliferation dose-dependently via cell cycle arrest at G1. Notably, RNA-seq analysis revealed that inhibition of FTO with Dac51 exhibited a significant decrease in the expression of multiple CDK members and a significant increase in the expression of CDKN1A. Furthermore, IPA analysis revealed that transcription factors, including SP1 and E2F members are the upstream regulators targeting CDK members in response to Dac51 treatment. Moreover, Dac51 treatment altered the expression of several epigenetic regulators, including Sirt1 and 2, HDAC10 and SUV39H1, and rewiring of several critical pathways, including inflammation response, G2M checkpoint, and C-Myc signaling, among others, leading to suppression of uLMS phenotype. Conclusion: Our findings uncover the anti-uLMS function of a potent FTO inhibitor via reprogramming the oncogenic epitranscriptome. Therefore, targeted inhibition of FTO may provide an additional regulatory layer for a promising and novel strategy for treating patients with this aggressive uterine cancer. Presentation: Saturday, June 17, 2023

Unravelling the Link between Psychological Distress and Liver Disease: Insights from an Anxiety-like Rat Model and Metabolomics Analysis.

Psychological distress is associated with an increase in liver disease mortality. This association highlights the close relationship between psychological and physical health. The underlying mechanism of this association needs to be elucidated. In this study, a rat model of anxiety was developed via compound stress. Changes in the HPA axis and inflammatory factors in the brains of the rats were evaluated for behavioral tests and liver function, respectively. The liver metabolic profiles of the rats were characterized through liquid chromatography-mass spectrometry (LC-MS). Differential metabolites were screened based on the conditions of p < 0.05 and VIP > 1. A pathway enrichment analysis was performed on the metabolomics data using the Ingenuity Pathway Analysis (IPA). Immunofluorescence (IF), immunohistochemistry (IHC), and Western blotting assays were performed to examine the expression of the screened target epidermal growth factor receptor (EGFR) and to elucidate the pathway associated with the mechanism. The results showed the impairment of liver function among the rats in an anxiety-like state. Additionally, 61 differential metabolites in the control and anxiety groups were screened using metabolomics (p < 0.05, VIP > 1). The results of the IPA analysis showed that the key target was EGFR. We also found that an anxiety-like state in rats may cause liver injury through the EFGR/PI3K/AKT/NF-κB pathway, which can lead to the production of inflammatory factors in the liver. Our results revealed a mechanism by which anxiety-like behavior leads to liver damage in rats. The findings of this study provided new insights into the deleterious effects of psychological problems on physical health.

Comprehensive analysis of m6A modifications in oral squamous cell carcinoma by MeRIP sequencing.

N6-methyladenosine (m6A) modifications are the most abundant internal modifications of mRNA and have a significant role in various cancers; however, the m6A methylome profile of oral squamous cell carcinoma (OSCC) in the mRNA-wide remains unknown. In this study, we examined the relationship between m6A and OSCC. Four pairs of OSCC and adjacent normal tissues were compared by Methylated RNA immunoprecipitation sequencing (MeRIP-seq). Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Ingenuity Pathway Analysis (IPA) analyses were used to further analyze the MeRIP-seq data. A total of 2,348 different m6A peaks were identified in the OSCC group, including 85 m6A upregulated peaks and 2,263 m6A downregulated peaks. Differentially methylated m6A binding sites were enriched in the coding sequence in proximity to the stop codon of both groups. KEGG analysis revealed genes with upregulated m6A-modified sites in the OSCC group, which were prominently associated with the forkhead box O (FOXO) signaling pathway. Genes containing downregulated m6A-modified sites were significantly correlated with the PI3K/Akt signaling pathway, spliceosome, protein processing in the endoplasmic reticulum, and endocytosis. IPA analysis indicated that several genes with differential methylation peaks form networks with m6A regulators. Overall, this study established the mRNA-wide m6A map for human OSCC and indicated the potential links between OSCC and N6-methyladenosine modification.

Characterization of monoclonal gammopathy of undetermined significance progression to multiple myeloma through meta-analysis of GEO data

The etiology of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) is still obscure as are the processes that enable the progression of MGUS to MM. Understanding the unique vs. shared transcriptomes can potentially elucidate why individuals develop one or the other. Furthermore, highlighting key pathways and genes involved in the pathogenesis of MM or the development of MGUS to MM may allow the discovery of novel drug targets and therapies.We employed STARGEO platform to perform three separate meta-analysis to compare MGUS and MM transcriptomes. For these analyses we tagged (1) 101 MGUS patient plasma cells from bone marrow samples and 64 plasma cells from healthy controls (2) 383 MM patient CD138+ cells from bone marrow and the 101 MGUS samples in the first analysis as controls (3) 517 MM patient peripheral blood samples and 97 peripheral blood samples from healthy controls. We then utilized Ingenuity Pathway Analysis (IPA) to analyze the unique genomic signatures within and across these samples.Our study identified genes that may have unique roles in MGUS (GADD45RA and COMMD3), but also newly identified signaling pathways (EIF2, JAK/STAT, and MYC) and gene activity (NRG3, RBFOX2, and PARP15) in MGUS that have previously been shown to be involved in MM suggesting a spectrum of molecular overlap. On the other hand, genes such as DUSP4, RN14, LAMP5, differentially upregulated in MM, may be seen as tipping the scales from benignity to malignancy and could serve as drug targets or novel biomarkers for risk of progression. Furthermore, our analysis of MM identified newly associated gene/pathway activity such as inhibition of Wnt-signaling and defective B cell development. Finally, IPA analysis, suggests the multifactorial, oncogenic qualities of IFNγ signaling in MM may be a unifying pathway for these diverse mechanisms and prompts the need for further studies.

903-P: Correlation of Gene Expression and Cellular Pathways in Insulin-Expressing Mouse Liver Cells Transduced with an Integrating Adeno-associated Viral Vector (AAV)

Gene therapy is being considered as a treatment/cure for type 1 diabetes (T1D). Previously we used a clinically-applicable AAV to deliver furin cleavable insulin (INS-FUR) to the livers of diabetic non-obese diabetic mice. The traditional AAV8 that allows predominantly episomal expression, and the hybrid AAV8/piggyBac that results in transgene integration into the host genome were used. Euglycaemia was achieved in mice (n=4) that received AAV8/piggyBac-INS-FUR, but not in mice that received AAV8-INS-FUR. In the current study global transcriptomic profiling was carried out, by total RNA sequencing, to compare the key pathways modulated in hepatocytes that reversed diabetes with those that did not. Using Qiagen CLC Workbench, significant genes were identified based on fold change expression cut offs of, and a false discovery rate p-value of &amp;lt;0.05. Selected genes were mapped onto the various cellular pathways using Qiagen Ingenuity Pathway Analysis (IPA) software to determine trends in pathway activation/ inactivation related to the altered gene expression. IPA analysis showed that the canonical pathways identified between the treatments remained largely the same, however their downstream biological processes differed. Key canonical pathways identified include pathways related to insulin signalling, glucose and lipid metabolism. Key pathway players identified as significantly altered in the comparative treatments included insulin, major histocompatibility complex class I, A, glucose-6-phosphatase catalytic subunit 1 and insulin like growth factor binding protein 1. The profile of AAV8/piggyBac-INS-FUR transduced mouse livers resembled that of a normal liver compared to livers transduced with AAV8-INS-FUR. This study confirms the correlation of transcriptomic data mapping and has identified differences in the cellular effects of both vector systems used to deliver INS-FUR for T1D treatment. Disclosure S.Joshua: None. N.T.Nassif: None. A.M.Simpson: None. Funding National Health and Medical Research Council of Australia (1086256)

Dysregulated sphingolipid metabolism contributes to NASH-HCC disease progression

Background: Nonalcoholic fatty liver disease (NAFLD) is the fastest-rising cause of end-stage liver disease for liver transplantation. The underlying mechanisms of NAFLD disease progression from nonalcoholic steatohepatitis (NASH) to hepatocellular carcinoma (HCC) remain unclear. It has been well-accepted that inflammation and dysregulation of sphingolipid metabolism are two important contributors to NASH progression. Recent advances in gene profiling technologies have significantly improved the current understanding of NASH-HCC pathogenesis. However, the key pathway changes underlying liver fibrosis and dysregulation of sphingolipid metabolism haven’t been fully elucidated and are the focus of this study. Methods: DIMOND NASH mouse model (C57BL/6J and 129S1/SvlmJ mixed background) was used in this study. Male mice (21 to 24-week-old) were fed with WDSW (Western diet, Harlan, TD88137, and a high fructose-glucose solution, 23.1g/L D-fructose plus 18.9g/L D-glucose) or standard chow diet ad libitum for six months or one year. At the end of the study, liver tissues were harvested for total RNA isolation using Trizol. Gene profiles were analyzed using the NanoString nCounter® Gene Expression Fibrosis V2 panel with sphingolipid metabolism pathways. The differentially expressed genes (DEGs) between groups were determined using Rosalind. QIAGEN Ingenuity Pathway Analysis (IPA) was used to analyze the DEGs. Human HCC patient data were downloaded from The Cancer Genome Atlas Program (TCGA). The sphingolipid profiles in the serum and liver were determined using LC-MS/MS. Results: All the mice developed NASH and HCC after feeding with WDSW for 6 months and 1 year, respectively. Bioinformatic analysis identified 191 DEGs in the NASH group and 250 DEGs in HCC group compared to the corresponding controls. The IPA analysis revealed that the major pathways related to inflammation and fibrosis were upregulated in both NASH and HCC mice. The expression levels of key genes related to ceramide synthesis and metabolism [serine palmitoyltransferase long chain base subunit 2 (Sptlc2), ceramide synthase 6 (Cers6), N-acylsphingosine amidohydrolase 2 (Asah2)] and sphingosine-1-phosphate receptor 2 (S1PR2) were significantly upregulated in NASH mice. In HCC group, Cers5, Cers6, sphingomyelin synthase 1 (Sgms1), Asah1, and S1PR3 were significantly increased. The expression levels of sphingosine kinase 2 and S1P lyase 1 were decreased both in NASH and HCC. Analysis of the TCGA data set showed that CERS5, CERS6, SPTLC2, ASAH1 and SGMS1 were highly expressed in HCC compared to healthy controls. The LC-MS/MS data also showed significant changes in sphingolipid profiles in serum and liver NASH and HCC. Conclusion: Our study suggests that dysregulated sphingolipid metabolism is associated with NASH and HCC disease progression. Further validation and research are needed for the potential application of sphingolipid profiles as diagnostic or prognostic markers in the clinic. This study was supported by VA Merit Award I01BX004033; Research Career Scientist Award (IK6BX004477); VA ShEEP grants (1IS1BX005517-01 and 1 IS1 BX004777-01) National Institutes of Health Grant R01 DK104893, R01DK-057543 and R21 AA026629-01. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of SGLT2 Inhibition on the transcriptomic and proteomic profile of non-diabetic Dahl SS Rats

Sodium-Glucose Cotransporter-2 (SGLT2) inhibitors are new, highly effective drugs for treating type 2 diabetes mellitus. SGLT2 inhibitors also substantially reduced the risk of hospitalization for heart failure. Previous studies in rodents and humans have shown that SGLT2 inhibitors lower blood pressure levels, cardiovascular risk events, and kidney diseases in subjects with and without diabetes mellitus. However, the mechanisms behind those effects are still unknown. Our previous study showed that SGLT2 inhibition blunted the development of salt-sensitive (SS) hypertension and caused glucosuria and natriuresis in Dahl SS rats without affecting the expression and activity of Na + channels and changes in the renin-angiotensin-aldosterone system. Here, we aim to investigate the effect of SGLT2 treatment on non-diabetic SS rats using multi-omics analysis, which may provide insights into the underlying mechanisms. 8-week-old male SS rats were treated with or without SGLT2 inhibitor (dapagliflozin, 2 mg/kg/day in drinking water) for 3 weeks. Kidney cortex and medulla tissue were collected for RNA-Seq analysis. Proteomics analysis was performed on kidney cortex tissue and plasma. The data were further processed by Ingenuity Pathway Analysis (IPA).RNA-Seq analysis indicated that despite SLGT2 mainly being expressed in the kidney cortex, dapagliflozin treatment altered the expression of more genes in the medulla (855 in the cortex vs 2244 differentially expressed genes (DEGs) in the medulla). Dapagliflozin treatment also caused significant changes in multiple genes related to inflammation and hypertension, with IPA analysis predicting the downregulation of both functions. In hypertension network Adrb3, Retn, Foxn4 were the most upregulated genes, and Ptgs1, Nos1, Oxtr – downregulated. In addition, disease and biological function enrichment analysis revealed that the altered genes were mainly related to the following functions: lipid metabolism, small molecule biochemistry, amino acid metabolism, cell cycle and morphology, and molecular transport. Interestingly, lipid metabolism was predicted to be downregulated in the cortex but upregulated in the medulla.Proteomic analysis identified 450 differentially expressed proteins (DEPs) in the kidney cortex and 22 in plasma. Disease and biological function enrichment analysis on proteomics data revealed mostly the same biological functions as RNA-Seq data. In addition, canonical pathways analysis based on RNA-Seq and proteomics data revealed that endothelin-1 and oxytocin signaling pathways were the most downregulated pathways in the kidney cortex and medulla under the dapagliflozin treatment. Urea cycle and glucocorticoid receptor signaling were the most changed canonical pathway in proteomic data. In conclusion, our results indicated that inhibition of SGLT2 by dapagliflozin altered various genes and proteins in the kidney of Dahl SS rats, which may provide insights for future mechanistic studies. This research was supported by National Institutes of Health grants R35 HL135749 (to AS). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Tear Fluid as a Biomarker for Parkinson’s Disease: Downregulation of DNA Repair Genes/Pathways via RNA-Seq Analysis (S37.007)

<h3>Objective:</h3> We performed an RNA-Seq analysis of tears from 16 Parkinson’s Disease (PD) patients and 16 healthy, age-matched healthy controls (HC) to identify genes and pathways that may serve as biomarkers of PD. <h3>Background:</h3> We previously demonstrated that PD tears have significantly higher levels of oligomeric a-synuclein than HC tears. Analyses of hundreds of subjects demonstrated this biomarker predicted PD diagnosis, irrespective of disease duration (AUROC=0.76). To improve this model, we performed RNA-Seq on PD and HC tears. Pilot studies of brain and tears revealed ~74% of genes expressed in aged cortex were expressed in tears. <h3>Design/Methods:</h3> Schirmer’s Tear Strips were frozen at −80°C. RNA was extracted and evaluated for quantity and integrity (RIN). RNA-Seq libraries were prepared with the NEBNext Ultra II RNA Library Prep Kit and sequenced on an Illumina NovaSeq sequencer. FASTQ files were aligned with Tophat and transcripts were counted and compared with Cufflinks. Pathway analysis was performed using the Qiagen Ingenuity Pathway Analysis (IPA) software and unsupervised, hierarchical clustering of significantly affected pathways was tested with RStudio. <h3>Results:</h3> RNA-Seq analysis showed more genes were downregulated in PD tears (N=898) vs. upregulated (N=238). IPA analysis of downregulated genes revealed DNA repair pathways were significantly affected, and reflected the downregulation of 60 genes related to DNA excision repair, recombination, and damage in PD tears. These 60 genes included <i>HNRNPC</i>, <i>TARDBP</i>, <i>USP47</i>, <i>EZH2</i>, <i>FANCC.</i> Unsupervised hierarchical clustering of these 60 genes’ transcript levels (FPKMs) separated PD and HC with 83.9% accuracy (sensitivity 93.3% and specificity 75%). <h3>Conclusions:</h3> RNA-Seq may provide a useful strategy for PD diagnosis using tears, a non-invasive biofluid. Preliminary data suggests increased diagnostic accuracy compared to our tear investigations of oligomeric a-synuclein levels alone. Downregulation of DNA repair genes/pathways suggests that alterations observed in PD brains and dopaminergic neurons are also observed in tears <b>Disclosure:</b> Dr. Lew has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Supernus. Dr. Lew has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for kyowa. Dr. Lew has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for neurocrine. Dr. Lew has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Acorda. Dr. Lew has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Acadia. Dr. Lew has received personal compensation in the range of $500-$4,999 for serving as a Consultant for RegenXBio. Dr. Lew has received personal compensation in the range of $10,000-$49,999 for serving on a Speakers Bureau for Neurocrine. Dr. Lew has received personal compensation in the range of $5,000-$9,999 for serving on a Speakers Bureau for Kyowa. Dr. Lew has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for acorda. The institution of Dr. Lew has received research support from MJFF. Dr. Lew has received research support from NIH. Miss Omidsalar has nothing to disclose. Miss Singh Kakan has nothing to disclose. Mr. Gerke has nothing to disclose. Ms. Tanveer has nothing to disclose. Dr. Feigenbaum has nothing to disclose. Dr. Tamadonfar has nothing to disclose. Sarah Hamm-Alvarez has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Oyster Point. The institution of Sarah Hamm-Alvarez has received research support from Oyster Point. Dr. Hjelm has nothing to disclose.