Ingenuity Pathway Analysis Software Research Articles

Pathway analysis of peripheral blood CD8+ T cell transcriptome shows differential regulation of sphingolipid signaling in multiple sclerosis and glioblastoma.

Multiple sclerosis (MS) and glioblastoma (GBM) are CNS diseases in whose development and progression immune privilege is intimately important, but in a relatively opposite manner. Maintenance and strengthening of immune privilege have been shown to be an important mechanism in glioblastoma immune evasion, while the breakdown of immune privilege leads to MS initiation and exacerbation. We hypothesize that molecular signaling pathways can be oppositely regulated in peripheral blood CD8+ T cells of MS and glioblastoma patients at a transcriptional level. We analyzed publicly available data of the peripheral blood CD8+ T cell MS vs. control (MSvsCTRL) and GBM vs. control (GBMvsCTRL) differentially expressed gene (DEG) contrasts with Qiagen's Ingenuity pathway analysis software (IPA). We have identified sphingolipid signaling pathway which was significantly downregulated in the GBMvsCTRL and upregulated in the MSvsCTRL. As the pathway is important for the CD8+ T lymphocytes CNS infiltration, this result is in line with our previously stated hypothesis. Comparing publicly available lists of differentially expressed serum exosomal miRNAs from MSvsCTRL and GBMvsCTRL contrasts, we have identified that hsa-miR-182-5p has the greatest potential effect on sphingolipid signaling regarding the number of regulated DEGs in the GBMvsCTRL contrast, while not being able to find any relevant potential sphingolipid signaling target transcripts in the MSvsCTRL contrast. We conclude that the sphingolipid signaling pathway is a top oppositely regulated pathway in peripheral blood CD8+ T cells from GBM and MS, and might be crucial for the differences in CNS immune privilege maintenance of investigated diseases, but further experimental research is necessary.

Genome‑wide association study and polygenic risk scores predict psoriasis and its shared phenotypes in Taiwan.

Psoriasis is a chronic inflammatory dermatological disease, and there is a lack of understanding of the genetic factors involved in psoriasis in Taiwan. To establish associations between genetic variations and psoriasis, a genome‑wide association study was performed in a cohort of 2,248 individuals with psoriasis and 67,440 individuals without psoriasis. Using the ingenuity pathway analysis software, biological networks were constructed. Human leukocyte antigen (HLA) diplotypes and haplotypes were analyzed using Attribute Bagging (HIBAG)‑R software and chi‑square analysis. The present study aimed to assess the potential risks associated with psoriasis using a polygenic risk score (PRS) analysis. The genetic association between single nucleotide polymorphisms (SNPs) in psoriasis and various human diseases was assessed by phenome‑wide association study. METAL software was used to analyze datasets from China Medical University Hospital (CMUH) and BioBank Japan (BBJ). The results of the present study revealed 8,585 SNPs with a significance threshold of P<5x10‑8, located within 153 genes strongly associated with the psoriasis phenotype, particularly on chromosomes 5 and 6. This specific genomic region has been identified by analyzing the biological networks associated with numerous pathways, including immune responses and inflammatory signaling. HLA genotype analysis indicated a strong association between HLA‑A*02:07 and HLA‑C*06:02 in a Taiwanese population. Based on our PRS analysis, the risk of psoriasis associated with the SNPs identified in the present study was quantified. These SNPs are associated with various dermatological, circulatory, endocrine, metabolic, musculoskeletal, hematopoietic and infectious diseases. The meta‑analysis results indicated successful replication of a study conducted on psoriasis in the BBJ. Several genetic loci are significantly associated with susceptibility to psoriasis in Taiwanese individuals. The present study contributes to our understanding of the genetic determinants that play a role in susceptibility to psoriasis. Furthermore, it provides valuable insights into the underlying etiology of psoriasis in the Taiwanese community.

Multi-Functional Role of RECK in Metabolic Dysfunction-Associated Steatotic Liver Disease Development: A Mass Spectrometry-Proteomic Approach

Metabolic dysfunction-associated steatotic liver disease (MASLD, previously referred to as nonalcoholic fatty liver disease) is characterized by increased fat deposition and inflammation in the liver as well as dysregulation of cellular processes that affect the extracellular matrix (ECM). We previously observed that the expression of Reversion Inducing Cysteine Rich Protein with Kazal motifs (RECK), a key ECM regulatory protein, negatively correlates with MASLD-associated fibrosis and inflammation (metabolic dysfunction-associated steatohepatitis, MASH). However, mechanisms governing this relationship remain unclear. Therefore, we hypothesized that overexpression of RECK is hepatoprotective in the setting of MASH by altering several ECM-linked pathways, as analyzed by proteomics. Accordingly, we generated hepatocyte-specific Reck-overexpressing transgenic mice. Male hepatocyte-specific Reck-overexpressing transgenic mice and littermate controls were fed a Western diet high in fat, carbohydrate, and cholesterol for 24 weeks (n=6/group). Whole liver tissue was submitted for mass spectrometry-proteomic analysis. We identified 370 differentially expressed proteins out of a total of 4693 proteins using a q-value cutoff of ≤0.10; of which 90 proteins were significantly upregulated while 280 significantly downregulated. This included an upregulation of Cyp2b (adjusted p≤0.01) and downregulation of Taok1, C/EBPβ, and Vitronectin (all adjusted p≤0.01), all of which have been shown to be important in liver health and metabolism in the context of MASH. Predictive pathway analysis using Ingenuity Pathway Analysis software (Qiagen) identified several significantly altered pathways, including those involved in ECM regulator pathways (integrin signaling, z-score=6.0; mTOR signaling, z-score=5.3; leukocyte extravasation signaling, z-score=5.1), cell-signaling (chemokine signaling, z-score=5.4; CXCR4 signaling chemokine signaling, z-score=5.0), and inflammatory responses (leukocyte extravasation signaling, z-score=5.1; CXCR4 signaling, z-score=5.0). Interestingly, pathway analysis based on significantly altered proteins predicted the downregulation of critical components of hepatic inflammation and fibrosis, including TGF-β, IL-1β, and TNFα. These novel results reveal the regulatory role of RECK in MASH progression, specifically governing the components of ECM remodeling and inflammation. Our goal is to develop RECK inducers and identify additional therapeutic targets in MASH development and progression. This work was funded by the NIH award R01DK130243. This is the full abstract presented at the American Physiology Summit 2024 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.

Resistance-Exercise-Induced Circulating Factors on Damaged Skeletal Muscle Tissue Proteome in Young Untrained Men

Recovery from muscle damage is tightly orchestrated by multiple physiological processes that can be altered by circulating factors (e.g., cytokines, growth factors) and intracellular signaling pathways (e.g., autophagy). Our previous studies have demonstrated that resistance exercise (RE)-induced circulating factors alter the expression of intramuscular cytokine mRNA and autophagic and myogenic markers in exercise-induced skeletal muscle damage (EIMD). However, the global effects of these circulating factors on damaged muscle are largely understudied. Purpose: To identify global changes in the proteome and functional pathways in skeletal muscle when EIMD is followed by an upper body RE-induced circulating milieu. Hypothesis: RE-induced circulating factors alter protein expression profile in damaged muscle. Methods: Vastus lateralis samples from 3 men included in the parent study were utilized for this report. Untrained men (age: 23 ± 5yr; height: 181.6 ± 5.9cm; weight: 86.0 ± 15.8kg) completed 2 identical bouts of maximal unilateral eccentric knee extensions for 8 sets of 10 repetitions, which were immediately followed by either heavy upper body RE to induce changes in circulating factors (EX) or 20 min seated rest (CON). Muscle samples from the damaged leg at 24h were used to compare between conditions. The total number of proteins was measured using LC-MS/MS. Bioinformatic analyses were performed via Qiagen Ingenuity Pathway Analysis software. For identifying enriched biological processes and functions, proteins had to have at least 1.0-fold differential expression with an FDR-adjusted significance level &lt; 0.05. Z scores were calculated to predict changes in biological functions. Results: A total of 900 proteins were identified. For EX, 79 proteins were downregulated, and 15 were upregulated compared to CON at 24h. Pathway analyses demonstrated that for EX, there was decreased activation in acute phase response signaling, liver X receptor/retinoid X receptor activation, nitric oxide production and reactive oxygen species in macrophages, and neutrophil extracellular trap signaling pathways compared to CON. In addition, for EX, there was an increase in macrophage IL-12 signaling and production than CON. Conclusion: RE-induced changes in circulating factors modulated pathways associated with the immune response and cytokine signaling in untrained men. Given that immune response is critical for recovery from muscle damage, these results highlight the potential role of RE-induced circulating factors (e.g., hormones, growth factors, etc.) which could play a role in mediating muscle recovery. This is the full abstract presented at the American Physiology Summit 2024 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.

Spaceflight effects on human vascular smooth muscle cell phenotype and function

The cardiovascular system is strongly impacted by the hazards of spaceflight. Astronauts spending steadily increasing lengths of time in microgravity are subject to cardiovascular deconditioning resulting in loss of vascular tone, reduced total blood volume, and diminished cardiac output. Appreciating the mechanisms by which the cells of the vasculature are altered during spaceflight will be integral to understanding and combating these deleterious effects as the human presence in space advances. In this study, we performed RNA-Seq analysis coupled with review by QIAGEN Ingenuity Pathway Analysis software on human aortic smooth muscle cells (HASMCs) cultured for 3 days in microgravity and aboard the International Space Station to assess the transcriptomic changes that occur during spaceflight. The results of our RNA-Seq analysis show that SMCs undergo a wide range of transcriptional alteration while in space, significantly affecting 4422 genes. SMCs largely down-regulate markers of the contractile, synthetic, and osteogenic phenotypes including smooth muscle alpha actin (αSMA), matrix metalloproteinases (MMPs), and bone morphogenic proteins (BMPs). Additionally, components of several cellular signaling pathways were strongly impacted including the STAT3, NFκB, PI3K/AKT, HIF1α, and Endothelin pathways. This study highlights the significant changes in transcriptional behavior SMCs exhibit during spaceflight and puts these changes in context to better understand vascular function in space.

Abstract 7097: Integrated proteomics and metabolomics reveals new insight into the synergistic interaction of valproic acid plus simvastatin in prostate cancer xenograft model associated with down-modulation of YAP and TAZ signaling

Abstract Background: Despite advancements in therapeutic approaches, including taxane-based chemotherapy and androgen receptor targeting agents, metastatic castration-resistant prostate cancer (mCRPC) remains an incurable disease, underlying the need of novel strategies that can target the complexities of this disease and bypass the development of drug-resistance mechanisms. We previously demonstrated the synergistic antitumor interaction of the antiepileptic with histone deacetylase inhibitory activity valproic acid (VPA), and the lipid-lowering drug simvastatin (SIM). This combination sensitizes mCRPC cells to docetaxel treatment and revert docetaxel-resistance, both in vitro and in vivo models, by targeting cancer stem cells compartment via mevalonate pathway/YAP axis modulation. Methods: Proteomic and metabolomic/lipidomic analysis on tumor samples were performed by LC-MS/MS-based shotgun proteomics and by 1H-NMR approach, respectively. Progenesis QI for proteomics v. 4.2 was used as label-free quantification platform. Functional annotation analysis were performed by using the DAVID v6.8 (Database for Annotation, Visualization and Integrated Discovery); g:Profiler WikiPathways databases; Reactome version 66; Ingenuity Pathway Analysis software. Partial least squares-discriminant analysis (PLS-DA) and Loading plot were performed by Metabo Analyst v5.0 tool. Results: Tumor samples derived from 22Rv1 mCRPC cells xenografted mice, treated or not with VPA/SIM combination were characterized by a combined proteomic and metabolomic/lipidomic approach. In deep bioinformatics analysis confirmed a specific impact of VPA/SIM on Hippo pathway, with a clear down regulation of YAP and its targets (CTGF, CycD1 ANKH1) in treated vs untreated tumors, functionally related with modulation of cancer-related extracellular matrix remodelling and metabolic reprogramming. Treatment also downregulated TEAD transcription factor mediating YAP-induced expression, along with the upregulation of 14-3-3, responsible for YAP cytoplasmic retention and degradation, paralleled by the up-regulation of YAP up-stream negative regulators such as LATS1 and AMPK. VPA/SIM also modulated intermediates of TCA, urea cycles and glycolysis and decreased cholesterol and fatty acids. Conclusion: In summary, this study provides further insights into the molecular mechanism of the VPA/SIM antitumor effect suggesting the potential of repurposing these two generic and safe drugs in combination with standard antitumor therapies also in other tumors besides mCRCP. Citation Format: Federica Iannelli, Rita Lombardi, Susan Costantini, Maria Serena Roca, Laura Addi, Francesca Bruzzese, Elena Di Gennaro, Alfredo Budillon, Biagio Pucci. Integrated proteomics and metabolomics reveals new insight into the synergistic interaction of valproic acid plus simvastatin in prostate cancer xenograft model associated with down-modulation of YAP and TAZ signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7097.

Abstract 3406: Transcriptomic analysis reveals differentially expressed pathways in colon tumors of cancer patients with vs without obesity: Results from the ColoCare Study

Abstract Despite the rise of global obesity rates and obesity’s implication in increased colon cancer risk, the understanding of mechanisms underlying these associations and opportunities for interception are limited. This work leveraged transcriptomic data from colon tumors to identify pathways and biologically relevant functions that characterize the tumors among obese vs nonobese individuals. Fresh-frozen colon tumor samples were collected from 155 patients who underwent surgery without neoadjuvant treatment as part of the ColoCare Study at Huntsman Cancer Institute, Utah, and Heidelberg University Hospital, Germany. RNA was sequenced using the NovaSeq X. The DESeq2 R package identified differentially expressed genes stratified by body mass index (BMI) at diagnosis (&amp;gt;30 kg/m2 vs &amp;lt;30 kg/m2) adjusting for age, sex, study site, and tumor stage. Genes with padj &amp;lt; 0.2 were entered into Ingenuity Pathway Analysis (IPA) software for pathway and expression analyses. Significant pathways were identified using Fischer’s Exact Test as the probability of molecules’ association with canonical pathways due to chance. The analysis was also repeated testing a median BMI categorization of &amp;gt;27.7 kg/m2 vs &amp;lt;27.7 kg/m2 using padj &amp;lt; 0.1 for IPA entry. Patients were 49% female, 63±14 years old, 47% stage III with a mean baseline BMI of 28.8±6.0 kg/m2. In colon tumors of those with &amp;gt;BMI 30 kg/m2, 213 genes were differentially expressed compared to BMI &amp;lt;30 kg/m2. IPA identified five upregulated pathways (neurovascular coupling signaling, adrenergic receptor signaling, activation of N-methyl-D-aspartate receptors and postsynaptic events, response to elevated platelet cytosolic Ca2+, docosahexaenoic acid signaling) and one downregulated pathway (WNT/B-catenin signaling) in patients with vs without obesity (p &amp;lt;0.05). The top predicted functional pathways, based on activation z-scores, identified in expression analysis included significant activation in chromosomal instability and aberration, size of body, phosphorylation of L-tyrosine, protein kinase cascade, and cell death and survival categories among colon tumors in obese patients. Using a median BMI categorization, 458 genes and 35 pathways were differentially expressed in those with BMI &amp;gt; 27.7 kg/m2 vs &amp;lt; 27.7 kg/m2. Both BMI models had good agreement with 5 pathways consistently significantly (z-score &amp;gt; |2|) up- or down-regulated, including predicted downregulation of organismal death and increased body size and phosphorylation of L-tyrosine in the higher BMI group. Colon tumors from patients with a higher BMI had pathways associated with more aggressive tumors, such as increased chromosomal instability and decreased cell death. Our ongoing work investigates the role of the adipose-tissue tumor microenvironment among patients, and the potential for interception using parallel mouse models. Citation Format: Victoria M. Bandera, Tengda Lin, Caroline Himbert, Elaine M. Glenny, Aik Choon Tan, Jennifer Ose, Christy Warby, Olena Aksonova, Alessandro Carpanese, Chris Stubben, David Nix, Kenneth Boucher, Peter Schirmacher, Ildiko Strehli, Jolanta Jedrzkiewicz, Lyen C. Huang, Jessica N. Cohan, Alexander Brobeil, Martin A. Schneider, Christoph Kahlert, Erin M. Siegel, Adetunji T. Toriola, David Shibata, Christopher I. Li, Jane C. Figueiredo, Biljana Gigic, Jatin Roper, Stephen Hursting, Cornelia M. Ulrich. Transcriptomic analysis reveals differentially expressed pathways in colon tumors of cancer patients with vs without obesity: Results from the ColoCare Study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3406.

Platycodi Radix Extract Prevents Hepatic Steatosis by Enhancing Bile Acid Synthesis in a High-Fat Diet-Induced Fatty Liver Mouse Model.

We aimed to identify the mechanism underlying the preventive effects of non-alcoholic fatty liver disease (NAFLD) through Platycodi Radix consumption using liver proteomic and bioinformatic analysis. C57BL/6J mice were categorized into three groups: those receiving a standard chow diet (NCD), those on a high-fat diet (HFD), and those on an HFD supplemented with 5% Platycodi Radix extract (PRE). After a 12-week period, PRE-fed mice exhibited a noteworthy prevention of hepatic steatosis. Protein identification and quantification in liver samples were conducted using LC-MS/MS. The identified proteins were analyzed through Ingenuity Pathway Analysis software, revealing a decrease in proteins associated with FXR/RXR activation and a concurrent increase in cholesterol biosynthesis proteins in the PRE-treated mouse liver. Subsequent network analysis predicted enhanced bile acid synthesis from these proteins. Indeed, the quantity of bile acids, which was reduced in HFD conditions, increased in the PRE group, accompanied by an elevation in the expression of synthesis-related proteins. Our findings suggest that the beneficial effects of PRE in preventing hepatic steatosis may be mediated, at least in part, through the modulation of FXR/RXR activation, cholesterol biosynthesis, and bile acid synthesis pathways.

Whole-exome sequencing reveals genetic variants that may play a role in neurocytomas.

Neurocytomas (NCs) are rare intracranial tumors that can often be surgically resected. However, disease course is unpredictable in many patients and medical therapies are lacking. We have used whole exome sequencing to explore the molecular etiology for neurocytoma and assist in target identification to develop novel therapeutic interventions. We used whole exome sequencing (WES) to compare the molecular landscape of 21 primary & recurrent NCs to five normal cerebellar control samples. WES data was analyzed using the Qiagen Clinical Insight program, variants of interest (VOI) were interrogated using ConSurf, ScoreCons, & Ingenuity Pathway Analysis Software to predict their potential functional effects, and Copy number variations (CNVs) in the genes of interest were analyzed by Genewiz (Azenta Life Sciences). Of 40 VOI involving thirty-six genes, 7 were pathogenic, 17 likely-pathogenic, and 16 of uncertain-significance. Of seven pathogenic NC associated variants, Glucosylceramidase beta 1 [GBA1 c.703T > C (p.S235P)] was mutated in 5/21 (24%), Coagulation factor VIII [F8 c.3637dupA (p.I1213fs*28)] in 4/21 (19%), Phenylalanine hydroxylase [PAH c.975C > A (p.Y325*)] in 3/21 (14%), and Fanconi anemia complementation group C [FANCC c.1162G > T (p.G388*)], Chromodomain helicase DNA binding protein 7 [CHD7 c.2839C > T (p.R947*)], Myosin VIIA [MYO7A c.940G > T (p.E314*)] and Dynein axonemal heavy chain 11 [DNAH11 c.3544C > T (p.R1182*)] in 2/21 (9.5%) NCs respectively. CNVs were noted in 85% of these latter 7 genes. Interestingly, a Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 2 [CTDSP2 c.472G > A (p.E158K)] of uncertain significance was also found in > 70% of NC cases. The variants of interest we identified in the NCs regulate a variety of neurological processes including cilia motility, cell metabolism, immune responses, and DNA damage repair and provide novel insights into the molecular pathogenesis of these extremely rare tumors.

Grade 2, 3 and Dedifferentiated Chondrosarcomas: A Comparative Study of Isocitrate Dehydrogenase-Mutant and Wild-Type Tumors with Implications for Prognosis and Therapy.

Grade 2 and 3 and dedifferentiated chondrosarcomas (CS) are frequently associated with isocitrate dehydrogenase (IDH) mutations and often exhibit a poor clinical outcome. Treatment is limited mainly to surgery. Defining IDH status (wild type (WT) and mutant) and the associated transcriptome may prove useful in determining other therapeutic options in these neoplasms. Formalin-fixed paraffin-embedded material from 69 primary and recurrent grade 2, 3 and dedifferentiated CS was obtained. DNA sequencing for IDH1 and IDH2 mutations (n = 47) and RNA sequencing via Nextseq 2000 (n = 14) were performed. Differentially expressed genes (DEGs) were identified and used to predict aberrant biological pathways with Ingenuity Pathway Analysis (IPA) software (Qiagen). Gene Set Enrichment Analyses (GSEA) using subsets C3, C5 and C7 were performed. Differentially expressed genes were validated by immunohistochemistry. Outcome analysis was performed using the Wilcoxon test. A set of 69 CS (28 females, 41 males), average age 65, distributed among femur, pelvis, humerus, and chest wall were identified from available clinical material. After further selection based on available IDH status, we evaluated 15 IDH WT and 32 IDH mutant tumors as part of this dataset. Out of 15 IDH WT tumors, 7 involved the chest wall/scapula, while 1 of 32 mutants arose in the scapula. There were far more genes overexpressed in IDH WT tumors compared to IDH mutant tumors. Furthermore, IDH WT and IDH mutant tumors were transcriptomically distinct in the IPA and GSEA, with IDH mutant tumors showing increased activity in methylation pathways and endochondral ossification, while IDH WT tumors showed more activity in normal matrix development pathways. Validation immunohistochemistry demonstrated expression of WT1 and AR in IDH WT tumors, but not in IDH mutants. SATB2 was expressed in IDH mutant tumors and not in WT tumors. Outcome analysis revealed differences in overall survival between mutant and WT tumors (p = 0.04), dedifferentiated mutant and higher-grade (2, 3) mutant tumors (p = 0.03), and dedifferentiated mutant and higher-grade (2, 3) WT tumors (p = 0.03). The longest survival times were observed in patients with higher-grade WT tumors, while patients with dedifferentiated mutant tumors showed the lowest survival. Generally, patients with IDH WT tumors displayed longer survival in both the higher-grade and dedifferentiated groups. Grade 2, 3 and dedifferentiated chondrosarcomas are further characterized by IDH status, which in turn informs transcriptomic phenotype and overall survival. The transcriptome is distinct depending on IDH status, and implies different treatment targets.

Protective effects of Jing-Si-herbal-tea in inflammatory cytokines-induced cell injury on normal human lung fibroblast via multiomic platform analysis.

The protective effects and related mechanisms of Jing-Si herbal tea (JSHT) were investigated in cellular damage mediated by pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor-α, on normal human lung fibroblast by multiomic platform analysis. The in silico high-throughput target was analyzed using pharmacophore models by BIOVIA Discovery Studio 2022 with ingenuity pathway analysis software. To assess cell viability, the study utilized the MTT assay technique. In addition, the IncuCyte S3 ZOOM System was implemented for the continuous monitoring of cell confluence of JSHT-treated cytokine-injured HEL 299 cells. Cytokine concentrations were determined using a Quantibody Human Inflammation Array. Gene expression and signaling pathways were determined using next-generation sequencing. In silico high-throughput target analysis of JSHT revealed ingenuity in canonical pathways and their networks. Glucocorticoid receptor signaling is a potential signaling of JSHT. The results revealed protective effects against the inflammatory cytokines on JSHT-treated HEL 299 cells. Transcriptome and network analyses revealed that induction of helper T lymphocytes, TNFSF12, NFKB1-mediated relaxin signaling, and G-protein coupled receptor signaling play important roles in immune regulatory on JSHT-treated cytokine-injured HEL 299 cells. The findings from our research indicate that JSHT holds promise as a therapeutic agent, potentially offering advantageous outcomes in treating virus infections through various mechanisms. Furthermore, the primary bioactive components in JSHT justify extended research in antiviral drug development, especially in the context of addressing coronavirus.

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.

The Neurotoxic Effect of Environmental Temperature Variation in Adult Zebrafish (Danio rerio).

Neurotoxicity consists of the altered functionality of the nervous system caused by exposure to chemical agents or altered chemical-physical parameters. The neurotoxic effect can be evaluated from the molecular to the behavioural level. The zebrafish Danio rerio is a model organism used in many research fields, including ecotoxicology and neurotoxicology. Recent studies by our research group have demonstrated that the exposure of adult zebrafish to low (18 °C) or high (34 °C) temperatures alters their brain proteome and fish behaviour compared to control (26 °C). These results showed that thermal variation alters the functionality of the nervous system, suggesting a temperature-induced neurotoxic effect. To demonstrate that temperature variation can be counted among the factors that generate neurotoxicity, eight different protein datasets, previously published by our research group, were subjected to new analyses using an integrated proteomic approach by means of the Ingenuity Pathway Analysis (IPA) software (Release December 2022). The datasets consist of brain proteome analyses of wild type adult zebrafish kept at three different temperatures (18 °C, 26 °C, and 34 °C) for 4 days (acute) or 21 days (chronic treatment), and of BDNF+/- and BDNF-/- zebrafish kept at 26 °C or 34 °C for 21 days. The results (a) demonstrate that thermal alterations generate an effect that can be defined as neurotoxic (p value ≤ 0.05, activation Z score ≤ -2 or ≥2), (b) identify 16 proteins that can be used as hallmarks of the neurotoxic processes common to all the treatments applied and (c) provide three protein panels (p value ≤ 0.05) related to 18 °C, 34 °C, and BDNF depletion that can be linked to anxiety-like or boldness behaviour upon these treatments.

198 Differential Gene Expression and Metabolic Pathway Comparisons of Liver and Duodenum from Suckling Piglets Given One or Two Iron Dextran Injections

Abstract Six female littermate pairs were used in an experiment to evaluate the mRNA expression in tissues from piglets given one or two 1 mL injections of iron dextran (200 mg Fe/mL, Uniferon, Pharmacosmos Inc. Watchung, NJ). All pigs in the litter were administered the first 1 mL injection &amp;lt; 24 hours after birth and on d 7, pigs were paired by body weight (BW; 1.72 ± 0.13 kg) and one pig from each pair was randomly selected as control (CON) and the other received a second injection (+Fe). At weaning on d 22, each piglet was anesthetized, and samples of liver and duodenum were taken from the anesthetized piglets and placed in DNA/RNA Shield (Zymo, Irvine, CA) for preservation until mRNA extraction. RNA-Seq libraries were sequenced to a depth of at least 30 million read pairs (150 bp paired-end sequencing) per sample. Differential Gene Expression data were analyzed with GeneSpring (Agilent, Santa Clara, CA) with a fold-change (FC) of |1.2| P &amp;lt; 0.05 (Table 1). Pathway analysis was conducted with Ingenuity Pathway Analysis (IPA QIAGEN, Redwood City, CA) software with Z-score cutoff of P &amp;lt; 0.05. In the duodenum 435 genes were significantly changed with a FC ≥|1.2| P &amp;lt; 0.05. Most notably, Claudin 1 and Claudin 2 were inversely affected by the second dose of iron. Claudin 1 (CLDN1, FC = 4.48, P = 0.0423) is responsible for cell-to-cell adhesion in the epithelial cell sheets and serves as a barrier to prevent water and solutes from passing through the paracellular space. Claudin 2 (CLDN2, FC = -1.41, P = 0.0097) was down regulated because it is expressed in cation leaky epithelia. In the liver 362 genes were expressed with a FC ≥|1.2| P &amp;lt; 0.05. The gene most affected by a second dose of 200 mg Fe was HAMP (Hepcidin Antimicrobial Peptide) with an FC of 40.8. HAMP is a liver-produced hormone that is the main circulating regulator of Fe absorption and distribution across tissues. It also controls the major flows of Fe into plasma by promoting endocytosis and degradation of ferroportin (SLC4A1). This leads to the retention of Fe in Fe-exporting cells and decreased flow of Fe into plasma. Metabolic pathway changes in the duodenum and liver provide evidence for the improved feed conversion and growth rates in pigs given two iron injections pre-weaning. In the duodenum, there is a down regulation of gluconeogenesis (P &amp;lt; 0.05). Concurrently, there is a decrease in the production of urea in the liver (P &amp;lt; 0.05). These observations show that there is less need for gluconeogenesis, thus less urea production from deaminated amino acids. The genomic and pathway analyses provided empirical evidence linking gene expression with phenotypic observations of piglet health and growth improvements.

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.

FRI135 Differential Expression Of Circulating MicroRNAs In Primary Aldosteronism: Potential Biomarkers For Improved Diagnosis

Abstract Disclosure: S. Lamprou: None. S.M. MacKenzie: None. J.D. McClure: None. S. Robertson: None. A. Riddell: None. J.C. van Kralingen: None. A. Gimenez-Roqueplo: None. P. Reel: None. S. Reel: None. G. Assié: None. A. Laurence: None. F. Beuschlein: None. G. Rossi: None. J. Deinum: None. M. Reincke: None. G. Eisenhofer: None. E. Jefferson: None. M. Zennaro: None. E. Davies: None. Primary Aldosteronism (PA) is the most common endocrine hypertension subtype, caused by autonomous aldosterone secretion. While readily treatable, accurate diagnosis of PA – particularly its differentiation from primary hypertension (PHT) – is a complex and protracted process that can entail delays in effective treatment and lead to more severe comorbidities. Given the established role of microRNAs (miRNA) in regulating aldosterone biosynthesis, we hypothesize that circulating miRNAs are potentially viable biomarkers for PA that could inform more accurate and rapid diagnosis. To detect changes in circulating miRNA profiles specific to PA, we analyzed plasma levels of 172 selected microRNAs in a retrospectively-generated cohort of PHT (n=111) and PA patients (n=109). The dataset consisted of ΔCt values for each miRNA (normalized to multiple endogenous miRNAs), as determined by real time RT-qPCR assay. Two-sample t-tests were used to assess whether the means of the two disease groups are equal, corrected using False Discovery Rate (FDR; significance level q &amp;lt; 0.05). Ingenuity Pathway Analysis software (QIAGEN) was employed to analyze the miRNAs bioinformatically and identify mRNA targets involved in blood pressure regulation. Pathway analysis was performed to discover associations between PA and differentially-expressed miRNAs. We identified 83 circulating microRNAs that showed significant differential expression: 27 were upregulated and 56 downregulated in PA relative to PHT. The upregulated miRNA demonstrating the greatest fold change was hsa-miR-451a (FC:1.8, q=0.001), while the most downregulated was hsa-miR-409-3p (FC:0.5, q=0.001). Bioinformatics analysis predicted several dysregulated miRNAs to target mRNAs in PA-related pathways. For example, the most upregulated miRNA, hsa-miR-451a, is predicted to target mRNAs in biochemical pathways relevant to PA pathophysiology, including SGK1 and MAP3K1. SGK1 contributes to the signaling cascade induced by the mineralocorticoid receptor-aldosterone complex that stimulates Na+ reabsorption in the kidney, while MAP3K1 participates in the Renin-Angiotensin system, a key regulator of aldosterone secretion, as part of a signaling cascade that leads to the activation of the AP1 complex in the nucleus. In addition to hsa-miR-451a, other differentially-expressed miRNAs were also revealed to target multiple mRNAs with direct and indirect connections to PA. In summary, analysis has identified miRNAs that are significantly differentially expressed in the plasma of PA patients compared to PHT patients, indicating promise for a biomarker-based approach to PA diagnosis utilizing distinctive circulating microRNA signatures. Further analysis is now being conducted to validate our findings in an independent population of hypertensive patients. Presentation: Friday, June 16, 2023

The endometrial transcriptome transition preceding receptivity to embryo implantation in mice

BackgroundReceptivity of the uterus is essential for embryo implantation and progression of mammalian pregnancy. Acquisition of receptivity involves major molecular and cellular changes in the endometrial lining of the uterus from a non-receptive state at ovulation, to a receptive state several days later. The precise molecular mechanisms underlying this transition and their upstream regulators remain to be fully characterized. Here, we aimed to generate a comprehensive profile of the endometrial transcriptome in the peri-ovulatory and peri-implantation states, to define the genes and gene pathways that are different between these states, and to identify new candidate upstream regulators of this transition, in the mouse.ResultsHigh throughput RNA-sequencing was utilized to identify genes and pathways expressed in the endometrium of female C57Bl/6 mice at estrus and on day 3.5 post-coitum (pc) after mating with BALB/c males (n = 3–4 biological replicates). Compared to the endometrium at estrus, 388 genes were considered differentially expressed in the endometrium on day 3.5 post-coitum. The transcriptional changes indicated substantial modulation of uterine immune and vascular systems during the pre-implantation phase, with the functional terms Angiogenesis, Chemotaxis, and Lymphangiogenesis predominating. Ingenuity Pathway Analysis software predicted the activation of several upstream regulators previously shown to be involved in the transition to receptivity including various cytokines, ovarian steroid hormones, prostaglandin E2, and vascular endothelial growth factor A. Our analysis also revealed four candidate upstream regulators that have not previously been implicated in the acquisition of uterine receptivity, with growth differentiation factor 2, lysine acetyltransferase 6 A, and N-6 adenine-specific DNA methyltransferase 1 predicted to be activated, and peptidylprolyl isomerase F predicted to be inhibited.ConclusionsThis study confirms that the transcriptome of a receptive uterus is vastly different to the non-receptive uterus and identifies several genes, regulatory pathways, and upstream drivers not previously associated with implantation. The findings will inform further research to investigate the molecular mechanisms of uterine receptivity.

Characterizing the urinary proteome of prematurity-associated lung disease in school-aged children

IntroductionAlthough different phenotypes of lung disease after preterm birth have recently been described, the underlying mechanisms associated with each phenotype are poorly understood. We, therefore, compared the urinary proteome for different spirometry phenotypes in preterm-born children with preterm- and term-born controls.MethodsPreterm and term-born children aged 7–12 years, from the Respiratory Health Outcomes in Neonates (RHiNO) cohort, underwent spirometry and urine collection. Urine was analysed by Nano-LC Mass-Spectrometry with Tandem-Mass Tag labelling. The preterm-born children were classified into phenotypes of prematurity-associated preserved ratio impaired spirometry (pPRISm, FEV1 < lower limit of normal (LLN), FEV1/FVC ≥ LLN), prematurity-associated obstructive lung disease (POLD, FEV1 < LLN, FEV1/FVC < LLN) and preterm controls (FEV1 ≥ LLN,). Biological relationships between significantly altered protein abundances were analysed using Ingenuity Pathways Analysis software, and receiver operator characteristic curves were calculated.ResultsUrine was analysed from 160 preterm-born children and 44 term controls. 27 and 21 were classified into the pPRISm and POLD groups, respectively. A total of 785 proteins were detected. Compared to preterm-born controls, sixteen significantly altered proteins in the pPRISm group were linked to six biological processes related to upregulation of inflammation and T-cell biology. In contrast, four significantly altered proteins in the POLD group were linked with neutrophil accumulation. Four proteins (DNASE1, PGLYRP1, B2M, SERPINA3) in combination had an area under the curve of 0.73 for pPRISm and three combined proteins (S100A8, MMP9 and CTSC) had AUC of 0.76 for POLD.ConclusionsIn this exploratory study, we demonstrate differential associations of the urinary proteome with pPRISm and POLD.Trial registrationEudraCT: 2015-003712-20

Differences in the metabolic profiles of brown and white adipocytes based on secretomics

Brown adipocytes and white adipocytes play important roles in systemic metabolism and energy homeostasis. Recent studies have demonstrated that white adipocytes and brown adipocytes secrete numerous adipokines and thus act as endocrine cells. However, differences in the metabolites secreted from white adipocytes and brown adipocytes have never been reported. In this study, we assessed the metabolites secreted from white adipocytes and brown adipocytes. In total, the levels of 47 metabolites in brown adipocytes were significantly different from those in white adipocytes, with 31 high and 16 low in brown adipocytes as compared with those in white adipocytes. We classified these secreted metabolites as amino acids and peptides, fatty acids, and conjugates, glycerophosphocholines, furanones, and trichloroacetic acids. In addition, we identified the glycerophospholipid metabolism activated in white adipocytes, and these differentially expressed metabolites were associated with the mitogen-activated protein kinase pathway and Janus kinase-signal transducer and activator of transcription signaling pathway according to the Ingenuity Pathway Analysis (IPA) software analysis. This study revealed novel metabolites secreted from brown adipocytes and white adipocytes, and these metabolites from adipocytes may perform specific biological functions based on the type of adipocyte that secretes them, and this forms the material basis of the interaction between adipocytes and other cells.

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)