Gene Ontology Molecular Function Research Articles

Diverse genetic alteration dysregulates neuropeptide and intracellular signalling in the suprachiasmatic nuclei.

In mammals, intrinsic 24 h or circadian rhythms are primarily generated by the suprachiasmatic nuclei (SCN). Rhythmic daily changes in the transcriptome and proteome of SCN cells are controlled by interlocking transcription-translation feedback loops (TTFLs) of core clock genes and their proteins. SCN cells function as autonomous circadian oscillators, which synchronize through intercellular neuropeptide signalling. Physiological and behavioural rhythms can be severely disrupted by genetic modification of a diverse range of genes and proteins in the SCN. With the advent of next generation sequencing, there is unprecedented information on the molecular profile of the SCN and how it is affected by genetically targeted alteration. However, whether the expression of some genes is more readily affected by genetic alteration of the SCN is unclear. Here, using publicly available datasets from recent RNA-seq assessments of the SCN from genetically altered and control mice, we evaluated whether there are commonalities in transcriptome dysregulation. This was completed for four different phases across the 24 h cycle and was augmented by Gene Ontology Molecular Function (GO:MF) and promoter analysis. Common differentially expressed genes (DEGs) and/or enriched GO:MF terms included signalling molecules, their receptors, and core clock components. Finally, examination of the JASPAR database indicated that E-box and CRE elements in the promoter regions of several commonly dysregulated genes. From this analysis, we identify differential expression of genes coding for molecules involved in SCN intra- and intercellular signalling as a potential cause of abnormal circadian rhythms.

Multiomics Analysis of PCB126's Effect on a Mouse Chronic-Binge Alcohol Feeding Model.

Environmental pollutants, including polychlorinated biphenyls (PCBs) have been implicated in the pathogenesis of liver disease. Our group recently demonstrated that PCB126 promoted steatosis, hepatomegaly, and modulated intermediary metabolism in a rodent model of alcohol-associated liver disease (ALD). To better understand how PCB126 promoted ALD in our previous model, the current study adopts multiple omics approaches to elucidate potential mechanistic hypotheses. Briefly, male C57BL/6J mice were exposed to polychlorinated biphenyl (PCB) 126 or corn oil vehicle prior to ethanol (EtOH) or control diet feeding in the chronic-binge alcohol feeding model. Liver tissues were collected and prepared for mRNA sequencing, phosphoproteomics, and inductively coupled plasma mass spectrometry for metals quantification. Principal component analysis showed that PCB126 uniquely modified the transcriptome in EtOH-fed mice. EtOH feeding alone resulted in differentially expressed genes (DEGs), and PCB126 exposure resulted in more DEGs in the EtOH-fed group (907 DEGs) in comparison with the pair-fed group (503 DEGs). Top 20 significant gene ontology (GO) biological processes included "peptidyl tyrosine modifications," whereas top 25 significantly decreasing GO molecular functions included "metal/ion/zinc binding." Quantitative, label-free phosphoproteomics and western blot analysis revealed no major significant PCB126 effects on total phosphorylated tyrosine residues in EtOH-fed mice. Quantified hepatic essential metal levels were primarily significantly lower in EtOH-fed mice. PCB126-exposed mice had significantly lower magnesium, cobalt, and zinc levels in EtOH-fed mice. Previous work has demonstrated that PCB126 is a modifying factor in metabolic dysfunction-associated steatotic liver disease (MASLD), and our current work suggests that pollutants also modify ALD. PCB126 may, in part, be contributing to the malnutrition aspect of ALD, where metal deficiency is known to contribute and worsen prognosis. https://doi.org/10.1289/EHP14132.

Study on the mechanism of Plantago asiatica L in the treatment of viral pneumonia based on network pharmacology

Objective: Viral pneumonia has caused great harm to human health and is infectious, and if it is not treated in time, the consequences will be immeasurable. Chinese medicine treatment of viral pneumonia is promising in clinical research at present. Here, we explore potential targets and mechanisms of Plantago asiatica L in the treatment of viral pneumonia. Methods: Traditional Chinese medicine (TCM) system and traditional Chinese medicine pharmacology database system pharmacology (TCMSP) (https://tcmspw.com/index.php) were used for screening active ingredients of Plantago asiatica L and targets related to active ingredients. The target proteins obtained from the TCMSP database were searched in the UniProt database (https://www.uniprot.org/) to obtain their corresponding gene names. GeneCards database (https://www. Genecards.org) was used to explore related targets closely associated with viral pneumonia. Subsequently, the Venny 2.1 database (https://bioinfogp.cnb.csic.es/tools/venny/) was applied before building a protein-protein interaction (PPI) network. The PPI network was constructed using the String database (https://string-db.org/), decoding the core anti-viral genes in viral pneumonia. Intersected genes were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) to construct a network of "Disease-drug-active component-key target-KEGG pathway". Results: 9 active components, 96 targets, 402 important disease targets, and 3 common targets of viral pneumonia and plantain were obtained. The top 10 GO biological processes, 10 GO cell components, 10 GO molecular functions and the top 10 KEGG signaling pathways were screened out. Conclusion: Apoptosis regulator Bcl-2; CASP3: Caspase - 3; CASP8: Caspase - 8. Those targets act on viral pneumonia by participating in the biological process and regulating the signaling pathway of Kaposi sarcoma-associated herpes virus infection, which provides a basis for future experimental verification and clinical research.

Efficacy of Danlou tablet on myocardial ischemia/ reperfusion injury assessed by network pharmacology and experimental verification.

To investigate the potential pharmacological mechanism of Danlou tablet (, DLT) with a long-term clinical application in the treatment of myocardial ischemia/reperfusion (I/R) injury through network pharmacology, molecular docking and experimental verification. The main chemical ingredients in DLT were retrieved from the Traditional Chinese Medicine (TCM) System Pharmacology Database, the TCM information database, the bioinformatics analysis tool for molecular mechanism of TCM, and HERB database. Disease targets of I/R were accessed from the databases of Online Mendelian Inheritance in Man, GeneCards, Therapeutic Target Database, and DisGeNET database. The overlaying genes of DLT and I/R were obtained from the Venny online platform. The core targets and protein-protein interaction network were constructed and analyzed via the Search Tool for the Retrieval of Interacting Genes Proteins database and Cytoscape software. Furthermore, Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed by the Metascape platform. Based on the results, the component-target-pathway network was constructed and drafted via the Cytoscape software and the platform of Bioinformatics. Furthermore, we performed molecular docking to predict the binding information between chemical molecules and target proteins. Finally, oxygen-glucose deprivation/recovery (OGD/R)-induced H9c2 cardiomyocytes were used to validate the results of network pharmacology in vitro. A total of 189 active chemical components in DLT and 849 correlative targets of I/R were screened. Of note, 133 overlaying genes found from the Venny online platform were concentrated into 28 core genes. Furthermore, the GO and KEGG pathway enrichment analysis presented that DLT might participate in 42 types of GO molecular functions, 747 types of GO biological processes, 19 types of GO cellular components, and 140 kinds of pathways to treat I/R. In the component-target-pathway network, the indirect relationship between herbs and their possible effective pathways was clarified. Based on the molecular docking, we speculated that Baicalein-prostaglandin G/H synthase 2 (PTGS2) with -3.24 kcal/mol, Luteolin-heat shock protein 90 alpha family class A member 1 (HSP90AA1) with -3.22 kcal/mol, Baicalein-HSP90AA1 with -3.13 kcal/mol, and Quercetin-HSP90AA1 with -3.05 kcal/mol possessed the strongest binding force of less than -3 kcal/mol, sequentially. Experimental verification showed that Quercetin, Luteolin, and Baicalein could increase the relative cell viability of OGD/R-stimulated cardiomyocytes, probably by suppressing PTGS2, and activating HSP90AA1 and estrogen receptor 1 expression. We predicted the potential active compounds as the material basis of DLT that may provide a new approach to elucidate the novel pharmacological mechanism underlying the treatment of cardiac I/R damage.

Unraveling the role of miRNAs as biomarkers in Chagas cardiomyopathy: Insights into molecular pathophysiology.

Chagas cardiomyopathy (ChCM) is a severe form of Chagas disease and a major cause of cardiovascular morbidity and mortality. The dysregulation of the immune response leads to cardiac remodeling and functional disruptions, resulting in life-threatening complications. Conventional diagnostic methods have limitations, and therapeutic response evaluation is challenging. MicroRNAs (miRNAs), important regulators of gene expression, show potential as biomarkers for diagnosis and prognosis. This review aims to summarize experimental findings on miRNA expression in ChCM and explore the potential of these miRNAs as biomarkers of Chagas disease. The search was conducted in the US National Library of Medicine MEDLINE/PubMed public database using the terms "Chagas cardiomyopathy" OR "Chagas disease" AND "microRNA" OR "miRNA" OR "miR." Additionally, bioinformatics analysis was performed to investigate miRNA-target interactions and explore enrichment pathways of gene ontology biological processes and molecular functions. The miR-21, miR-146b, miR-146a, and miR-155 consistently exhibited up-regulation, whereas miR-145 was down-regulated in ChCM. These specific miRNAs have been linked to fibrosis, immune response, and inflammatory processes in heart tissue. Moreover, the findings from various studies indicate that these miRNAs have the potential as biomarkers for the disease and could be targeted in therapeutic strategies for ChCM. In this review, we point out miR-21, miR-146b, miR-146a, miR-155, and miR-145-5p role in the complex mechanisms of ChCM. These miRNAs have been shown as potential biomarkers for precise diagnosis, reliable prognostic evaluation, and effective treatment strategies in the ChCM.

Transcriptomic analysis of the submandibular gland under psychological stress condition.

Psychological stress is associated with changes in salivary flow and composition. However, studies to show the effect of psychological stress on the transcriptome of the salivary gland are limited. This study aims to perform a transcriptomic analysis of the submandibular gland under psychological stress using a chronic restraint stress model of rats. Sprague-Dawley rats were divided into stress groups and control groups. Psychological stress was induced in the stress group rats by enclosing them in a plastic tube for 4 h daily over 6 weeks. RNA sequencing was performed on RNA extracted from the submandibular gland. The differentially expressed genes were identified, and the genes of interest were further validated using qRT-PCR, immunofluorescence, and western blot. A comparison between control and stress groups showed 45 differentially expressed genes. The top five altered genes in RNA sequencing data showed similar gene expression in qRT-PCR validation. The most downregulated gene in the stress group, FosB, was a gene of interest and was further validated for its protein-level expression using immunofluorescence and western blot. The genesets for gene ontology cellular component, molecular function, and KEGG showed that pathways related to ribosome biosynthesis and function were downregulated in the stress group compared to the control. Psychological stress showed transcriptomic alteration in the submandibular gland. The findings may be important in understanding stress-related oral diseases.

Exploring the regulatory interactions between mutated genes and homeobox genes in the head and neck cancer progression

ObjectiveUnderstanding the regulatory role of homeobox (HOX) and mutated genes in the progression of head and neck cancers is essential, although their interaction remains elusive. This study aims to decipher the critical regulation of mutation driven effects on homeobox genes to enhance our understanding of head and neck cancer progression. MethodsGenomic mutation data from The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma were analyzed using VarScan2 for somatic variant detection. Mutational clustering, driver mutation identification, and cancer signaling pathway analysis were performed using the OncodriveCLUST method. Harmonizome datasets were retrieved to identify critical cancer driver genes affecting HOX genes. The effects of HPV infection on HOX and mutated genes were assessed using the oncoviral database. Altered pathway activity due to the effects of cancer drivers on HOX genes was analyzed with Gene Set Cancer Analysis. Functional enrichment analysis of gene ontology biological processes and molecular functions was conducted using the ClusterProfiler R package. ResultsSignificant alterations in HOX genes were observed in head and neck cancer cohorts with mutated TP53, FAT1, and CDKN2A. HOX genes were identified as functionally downstream targets of TP53, signifying transcriptionally mediated regulation. The interaction between HOX genes and mutated TP53, FAT1, and CDKN2A dysregulated the epithelial-to-mesenchymal transition, cell cycle, and apoptosis pathways in head and neck cancer progression. ConclusionThe interplay between cancer driver genes and HOX genes is pivotal in regulating the oncogenic processes underlying the pathogenesis of head and neck squamous cell carcinoma

Antagonistic Activity of Streptomyces alfalfae 11F against Fusarium Wilt of Watermelon and Transcriptome Analysis Provides Insights into the Synthesis of Phenazine-1-Carboxamide.

Streptomyces alfalfa strain 11F has inhibitory effects on many phytopathogenic fungi and improves the establishment and biomass yield of switchgrass. However, the antagonistic effects of strain 11F on Fusarium wilt of watermelon and its secondary metabolites that contribute to its biocontrol activity are poorly understood. We evaluated the antagonistic and growth-promoting effects of strain 11F and conducted a transcriptome analysis to identify the metabolites contributing to antifungal activity. Strain 11F had marked inhibitory effects on six fungal pathogens. The incidence of Fusarium wilt of watermelon seedlings was decreased by 46.02%, while watermelon seedling growth was promoted, as indicated by plant height (8.7%), fresh weight (23.1%), and dry weight (60.0%). Clean RNA-sequencing data were annotated with 7553 functional genes. The 2582 differentially expressed genes (DEGs) detected in the Control vs. Case 2 comparison were divided into 42 subcategories of the biological process, cellular component, and molecular function Gene Ontology categories. Seven hundred and forty functional genes (55.47% of the DEGs) were assigned to Kyoto Encyclopedia of Genes and Genomes metabolic pathways, reflecting the complexity of the strain 11F metabolic regulatory system. The expression level of the gene phzF, which encodes an enzyme essential for phenazine-1-carboxylic acid (PCA) synthesis, was downregulated 3.7-fold between the 24 h and 48 h fermentation time points, suggesting that strain 11F can produce phenazine compounds. A phenazine compound from 11F was isolated and identified as phenazine-1-carboxamide (PCN), which contributed to the antagonistic activity against Fusarium oxysporum f. sp. niveum. PCA was speculated to be the synthetic precursor of PCN. The downregulation in phzF expression might be associated with the decrease in PCA accumulation and the increase in PCN synthesis in strain 11F from 24 to 48 h. Streptomyces alfalfae 11F protects watermelon seedlings from Fusarium wilt of watermelon and promotes seedling growth. The transcriptome analysis of strain 11F provides insights into the synthesis of PCN, which has antifungal activity against F. oxysporum f. sp. niveum of watermelon.

Exploring Potential Molecular Mechanisms of Drug Response in FLT3-ITD Negative AML Patients Treated with Quizartinib Vs Placebo Plus Standard Chemotherapy in the Quiwi Trial

Introduction Wild-type FLT3 AML presents significant therapeutic challenges due to limited treatment options. The QUIWI trial investigated quizartinib (Quiz) combined with standard chemotherapy, showing promising results. To understand drug response and refractoriness, we conducted a correlative analysis, exploring differential expression patterns among distinct patient groups. We reasoned that the molecular fingerprints of good responders to Quiz plus chemotherapy would be different with respect to patients who respond only to standard chemotherapy, helping to elucidate critical molecular mechanisms associated with Quiz response. Our study provides a basis for personalized therapeutic strategies in FLT3-ITD negative AML. Methods We performed RNA sequencing (RNAseq) analysis on a subset of patients from the clinical trial. RNA was extracted using standard methods, followed by assessment of nucleic acid integrity (TapeStation) and quantification (Qubit). Total mRNA sequencing was performed using polyA RNAseq with TruSeq technology. A total of 206 adequate samples from bone marrow and peripheral blood were sequenced, out of which 55 cases were FLT3-ITD and were discarded for this analysis. For this analysis, only samples with a minimum of 20% blasts were considered. The sequences were aligned to the GRCh37 reference genome using the Hisat algorithm. Differential expression was assessed with the DESeq2 algorithm, using Blast proportion to adjust for disparities among the samples. P-values were adjusted for multiple testing using the FDR method. OS was defined as time from start of screening to death. Gene ontology and pathways analysis were performed using the WebGestalt portal. Results In this study, we compared the gene expression profiles of Quiz versus placebo-treated patients categorized as good responders, defined arbitrarily as those who continue alive and have a minimum follow-up of 6 months after diagnosis. Out of 82 eligible patients, we identified 100 (q-value < 0.05) and 172 (q-value < 0.1) differentially expressed genes between the Quiz and Placebo groups. Notably, 132 genes were overexpressed in the Quiz group, while 40 genes showed overexpression in the placebo group. Moreover, we observed a highly significant overexpression of ribosomal genes in the Quiz group (17 overlaps, KEGG pathways q-value < 2.2 x 10 -16), and a significant overexpression of genes related to the attenuation of the heat shock protein transcriptional response, which is involved in protein folding, in the placebo group (Reactome q-value, 0.008; gene ids: HSPA1A, HSPA1B and DNAJB1). Additionally, the Quiz group exhibited a significant overexpression of transmembrane receptor protein tyrosine phosphatase-related genes (Gene Ontology Molecular Function q-value, 0.02; gene ids: PTPRB, PTPRG and PTPRU). Conclusion This correlative analysis of the Quiwi trial revealed critical molecular mechanisms associated with Quiz response. The overexpression of ribosomal and transmembrane receptor protein tyrosine phosphatase-related genes in good responders within the Quiz group, along with the overexpression of the heat shock pathway genes in the placebo group, aligns with existing knowledge about the FLT3 pathway biology and the molecular determinants to tyrosine-kinase inhibitors. These findings offer potential biomarkers for personalized therapeutic approaches to improve clinical outcomes in this challenging AML population.

Functional enrichment analysis of mutated genes in children with hyperthyroidism.

Hyperthyroidism in Chinese children is relatively high and has been increasing in recent years, which has a significant impact on their healthy development. Hyperthyroidism is a polygenic disorder that presents greater challenges in terms of prediction and treatment than monogenic diseases. This study aims to elucidate the associated functions and gene sets of mutated genes in children with hyperthyroidism in terms of the gene ontology through GO enrichment analysis and in terms of biological signaling pathways through KEGG enrichment analysis, thereby enhancing our understanding of the expected effects of multiple mutated genes on hyperthyroidism in children. Whole-exome sequencing was performed on the DNA samples of children with hyperthyroidism. Screening for pathogenic genes related to hyperthyroidism in affected children was performed using the publicly available disease databases Malacards, MutationView, and Clinvar, and the functions and influences of the identified pathogenic genes were analyzed using statistical analysis and the gene enrichment approach. Through GO enrichment analysis, it was found that the most significant gene ontology enrichment was the function "hormone activity" in terms of gene ontology molecular function. The corresponding mutated genes set that has common effects on hyperthyroidism in children included TG, CALCA, POMC, CGA, PTH, GHRL, FBN1, TRH, PRL, LEP, ADIPOQ, INS, GH1. The second most significant gene ontology enrichment was the function "response to peptide hormone" in terms of biological process. The corresponding mutated genes set that has common effects on hyperthyroidism in children included LRP6, TSC2, KANK1, COL1A1, CDKN1B, POMC, STAT1, MEN1, APC, GHRL, TSHR, GJB2, FBN1, GPT, LEP, ADIPOQ, INS, GH1. Through KEGG enrichment analysis, it was found that the most significant biological signaling pathway enrichment was the pathway "Thyroid hormone signaling pathway" function. The corresponding mutated genes set that has common effects on hyperthyroidism in children included NOTCH3, MYH7, TSC2, STAT1, MED13L, MAP2K2, SLCO1C1, SLC16A2, and THRB. The second most significant biological signaling pathway enrichment was the pathway "Hypertrophic cardiomyopathy" in terms of biological process. The corresponding mutated genes set that has common effects on hyperthyroidism in children included IGF1, CACNA1S, MYH7, IL6, TTN, CACNB2, LAMA2, and DMD. The mutated genes in children with hyperthyroidism were closely linked to function involved in "hormone activity" and "response to peptide hormone" in terms of the biological signaling pathway, and to the functional pathways involved in "Thyroid hormone signaling pathway" and "Hypertrophic cardiomyopathy" in terms of the biological signaling pathway.

Explore the mechanism of ursolic acid acting on atherosclerosis through network pharmacological and bioinformatics methods.

To explore the deep mechanisms of ursolic acid (UA) for treating atherosclerosis based on network pharmacology and bioinformatics. UA target genes were derived from traditional Chinese medicine system pharmacology, BATMAN-TCM, and SwissTargetPrediction databases. Atherosclerosis-related genes were derived from genecards, NCBI genes, and OMIM databases. The protein interaction network was constructed through the STRING database, and the hub network was extracted by using the Cytoscape software MCODE app. The enrichment analysis of gene ontology and Kyoto encyclopedia of genes and genomes was performed by the R software clusterProfiler package, and the expression and prognostic value of the hub genes were verified on the data set. Screen the genes for expression and prognosis conclusions, conduct methylation analysis, and ceRNA construction. UA had 145 targets in the treatment of atherosclerosis. The top 7 gene ontology (biological process, molecular function, and cellular component) and pathways related to atherosclerosis were screened out. It is principally involved in biological processes, including response to lipopolysaccharide and regulation of inflammatory response. The main signaling pathways incorporated the TNF signaling pathway and the AGE-RAGE signaling pathway. Androgen receptor (AR) and interleukin-1 beta gene (IL1B) were further screened as core target genes. Methylation analysis demonstrated that the AR methylation level was elevated in the atherosclerotic group. On the contrary, the IL1B methylation level was lower in the atherosclerotic group. The results of the ceRNA analysis indicated that there were 43 targeted miRNAs in AR and 3 miRNAs in IL1B. We speculate that the target genes of UA regulating atherosclerosis are AR and IL1B. The mechanism may be that UA regulates the expression of target genes by regulating the methylation of target genes.

P-155 MicroRNA-155 (miR-155) expression profile and developmental characteristics of bovine early embryos are altered when cultured in group versus individually

Abstract Study question Culturing bovine preimplantation embryos in group versus individually has an impact on the expression profile of miR-155 and developmental characteristics. Summary answer MiR-155 was significantly greater in its expression when embryos were cultured in groups versus singly. Developmental characteristics of blastocysts were similar regardless of culture condition. What is known already MiRNAs are short non-coding RNAs that play important roles in many biological pathways including embryogenesis and are potent effectors of post-transcriptional gene silencing. It has been estimated that approximately 60% of all proteins-coding genes are regulated by miRNAs. MiR-155 is involved in early events of embryogenesis; however, it has not been characterized in bovine embryos and this knowledge has not yet been clinically translated. Existing literature indicates that group culture yields superior embryo development as embryos can benefit from autocrine signaling and paracrine communication but embryos in groups may be exposed to negative effects of dying or delayed embryos. Study design, size, duration The study consisted of two groups: zygotes cultured to blastocysts in group and those cultured individually. Each sample consists of 3 blastocysts. Droplet digital PCR (dd-PCR) was used to examine the expression of miR-155. Gene Set Enrichment Analysis (GSEA) of miRNAs was carried out to identify functional Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that are enriched for the targets of one or more miRNAs. Participants/materials, setting, methods Oocytes were aspirated from antral follicles of bovine ovaries and matured in vitro. Twenty-four hours post retrieval, matured oocytes were inseminated with sperm from similar bull. Presumptive zygotes were cultured either in groups or individually until blastocyst stage, Blastocyst were collected and purified for RNA. Analysis for KEGG and GO Biological Process, Cellular Component and Molecular Functions were carried out on the identified target genes. Results were plotted according to level of enrichment for miR-155. Main results and the role of chance Five hundred and seventy-five oocytes were retrieved from 120 bovine ovaries. Cleavage rate and blastocyst formation after IVF were compared between those blastocysts cultured in groups versus individually. There was no significant difference among the two groups with regards to cleavage rate (69% vs. 54%) and blastocyst formation (42% vs.35%). ddPCR assessment showed significantly greater MiR-155 expression in those blastocysts cultured in groups (p = 0.002). The highest level of miR-151 expressed was 20 copies/sample. Every sample, regardless of the culture conditions, had detectable miR-155 level. The interaction of predicted miR-155 target clusters based on the GO Biological Processes shows that miR-155 plays a role in DNA binding, transcription factor binding, regulates transcription and affects cell adhesion. KEGG analysis revealed an association between miR-155 to steroidogenesis and controls apoptosis pathways. Limitations, reasons for caution The number of copies expressed for this miRNA was relatively low, which could suggest that miR-155 may not have a functional role at the blastocyst stage as the effectiveness of a miRNA depends on the relative number of binding site and target molecules. Blastocysts were pooled based on morphological assessment. Wider implications of the findings MiRNAs offer a novel mean to assess embryonic fitness. Further validation and more complex study designs are required to implement the profiling of embryonic miRNAs as a diagnostic test for embryo selection. Group culture increased miR-155 expression, possibly reflecting differences in cellular behavior between group cultured embryos and individually ones. Trial registration number Not applicable

Proteomic Profiling of Chemotherapy Responses in FOLFOX-Resistant Colorectal Cancer Cells.

Chemoresistance mechanisms of colorectal cancer remain largely elusive. We aim to compare the difference of chemotherapy responses between FOLFOX-resistant and wild-type colorectal cancer cells by proteomic profiling to suggest novel treatment targets. FOLFOX-resistant colorectal cancer cells DLD1-R and HCT116-R were developed by chronic exposure to progressive FOLFOX doses. Proteomic profiling of FOLFOX-resistant and wild-type cells under FOLFOX exposure were conducted by mass-spectrometry-based protein-analysis technology. Verification of selected KEGG pathways was conducted by Western blot. DLD1-R had significantly higher FOLFOX-chemoresistance (10.81 times) than its wild-type counterpart. A total of 309 and 90 differentially expressed proteins were identified in DLD1-R and HCT116-R, respectively. In terms of gene ontology molecular function, RNA binding and cadherin binding ranked first for DLD1 and HCT116 groups, respectively. For gene set enrichment analysis, ribosome pathway and DNA replication were significantly up-regulated and down-regulated in DLD1-R, respectively. The most significantly up-regulated pathway in HCT116-R was regulation of the actin cytoskeleton. Up-regulations in the ribosome pathway (DLD1-R) and actin cytoskeleton (HCT116-R) were verified by Western blot. There were several significantly altered signaling pathways in FOLFOX-resistant colorectal cancer cells under FOLFOX with notable up-regulations in the ribosomal process and actin cytoskeleton.

Effect of preventive training in cardiac transcriptoma in rats with pulmonary hypertension

Abstract Funding Acknowledgements Type of funding sources: None. Introduction Pulmonary hypertension (PH) is a progressive disease characterized by elevated right ventricular afterload, vascular remodeling, right ventricular hypertrophy and dysfunction, and ultimately heart failure. Physical exercise has been recognized as a treatment for this condition. Although different mechanisms have been proposed to explain PH development, the influence of preventive exercise training interventions onset of pulmonary hypertension in right ventricular hypertrophy is underexplored. Purpose Analyze the effect of preventive training in global gene expression profiling of right ventricular in PH from rats. Methods Twenty-four male Wistar rats (206-220 g) were divided into three four groups (n=8/group): S, sedentary control; SPH, sedentary pulmonary hypertension; and TPH, trained pulmonary hypertension group. The preventive training protocol was performed on a treadmill for 13 weeks, five times/week. Cardiac hypertrophy by PH was experimentally induced by a single intraperitoneal injection (60 mg/kg) of monocrotaline after the protocol training. The S were injected with saline solution. The rats were evaluated 28 days after monocrotaline administration, in the early stage of the PH. Left ventricle weight, right ventricle weight, and atrium weight normalized by body weight were used as indexes of heart hypertrophy. Gene expression profiling was performed using the Rat Gene ST Array platform. To further understand the biological relevance of differential expressed genes, we performed a functional enrichment analysis in the context of the gene ontology (biological process, molecular function, and cellular component) and interaction networks (KEGG, Reactome, Wikipathways and BioPlanet). Results Through the analysis, the HP group had cardiac hypertrophy, and the training mitigated this change p< 0.05. Global gene expression profiling identified 687 differentially expressed genes (S vs. SPH), in the hypertrophy right ventricular during PH, and when compared to preventive training, 91 genes were dysregulated (SPH vs TPH). Gene ontology analysis on expression profiling data revealed alteration on genes that regulate the inflammatory process, muscle growth, metabolic processes, cell death, and ion regulation comparison between SPH vs. TPH The genes that were altered in the hypertrophy phase of PH and modified by the exercise were platelet alpha granule lumen (genes Fgg and Qsox1), NAD + binding (gene Cryl1), positive regulation of cAMP-mediated signaling (gene Cxcl11), positive regulation of macrophage differentiation (Il34 gene), mRNA cleavage (Cstf3 gene), negative regulation of transporter activity (Wwp2 gene), regulation of respiratory system process (Mtg2 gene) p< 0.05. Conclusion Preventive training influences changes in cardiac gene expression profile in the hypertrophy stage of PH. New genes have been identified, and these could be biomarkers and potential therapeutic targets in the early stage of PH.

Abstract 5177: Nilotinib suppresses the myofibroblastic cancer-associated fibroblast phenotype

Abstract Background: Cancer-associated fibroblasts (CAFs) modulate the tumor immune microenvironment and are an exciting target for improving response to immunotherapy. Major roles of CAFs in the immune microenvironment include deposition of extracellular matrix (ECM) to prevent immune cell infiltration, a function associated with myofibroblastic CAFs (myCAFs), and production of cytokines to alter the immune milieu, associated with inflammatory CAFs (iCAFs). Recently we have demonstrated the potential for tyrosine kinase inhibitors (TKIs) to alter CAF phenotypes. Here we investigate the ability of dasatinib and nilotinib to alter the immune regulatory functions of CAFs. Methods: Bulk RNA sequencing comparing the effects of nilotinib and dasatinib on primary-derived patient CAFs was performed. CAFs from two rectal cancer patient tumors were isolated, cultured, and treated for 96 hours with 2.5μM nilotinib, 100nM dasatinib, or control feeding media. RNA isolation, library preparation, sequencing, data processing, and differential expression analysis was done through the University of Wisconsin - Madison Gene Expression Center, Biotechnology Center, and Bioinformatics Resource. Gene set enrichment analyses were done using GSEA 4.2.3 (Broad Institute) and all other analyses done in R. Results: The primary variance in samples is between CAF lines (RC1 and RC2), followed by treatments. RNA expression markers for myCAFs significantly associate with the Gene Ontology molecular function ECM structural constituent (q < 0.001). Nilotinib decreased expression of this gene set in RC1 (q < 0.001), while dasatinib treatment increased these genes in RC2, though not significantly (q = 0.2). Both dasatinib and nilotinib downregulated immune-related hallmark gene sets including IL6/JAK/STAT3 signaling (RC2 dasatinib q < 0.05, q < 0.001 for others) and TNFα signaling through NFκB (RC1 dasatinib q < 0.001, RC2 niltoinib q < 0.05, others q = 0.001,). Additionally, dasatinib induced increased expression of genes involved in myogenesis in both CAF lines (RC1 q = 0.001, RC2 q < 0.001), while nilotinib significantly decreased these genes in RC1 (q = 0.001). MYOCD is a transcription factor involved in myogenesis that regulates important myCAF genes such as ACTA2 and TAGLN. Nilotinib treatment decreased expression of MYOCD (RC1 log2FC -1.3 q < 0.001, RC2 -0.75 q < 0.05) and its target genes in both lines, whereas dasatinib did not significantly alter expression. Conclusions: CAFs derived from different cancers harbor different transcriptional profiles and have different responses to TKIs. Dasatinib treatment led to an increase in expression of ECM genes associated with the myCAF phenotype, while nilotinib decreased this phenotype, potentially by inhibiting MYOCD expression. Further investigation into the mechanisms by which nilotinib treatment decreases expression of ECM genes and whether these trends continue in vivo are warranted. Citation Format: Katherine Anne Johnson, Yousef Gadalla, Cheri A. Pasch, Dustin A. Deming. Nilotinib suppresses the myofibroblastic cancer-associated fibroblast phenotype. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5177.

Transcriptome Dynamics in Triticum aestivum Genotypes Associated with Resistance against the Wheat Dwarf Virus.

Wheat dwarf virus (WDV) is one of the most important pathogens of cereal crops worldwide. To understand the molecular mechanism of resistance, here we investigated the comparative transcriptome of wheat genotypes with different levels of resistance (Svitava and Fengyou 3) and susceptibility (Akteur) to WDV. We found a significantly higher number of differentially expressed transcripts (DETs) in the susceptible genotype than in the resistant one (e.g., Svitava). The number of downregulated transcripts was also higher in the susceptible genotype than in the resistant one (Svitava) and the opposite was true for the upregulated transcripts. Further functional analysis of gene ontology (GO) enrichment identified a total of 114 GO terms for the DETs. Of these, 64 biological processes, 28 cellular components and 22 molecular function GO terms were significantly enriched. A few of these genes appear to have a specific expression pattern related to resistance or susceptibility to WDV infection. Validation of the expression pattern by RT-qPCR showed that glycosyltransferase was significantly downregulated in the susceptible genotype compared to the resistant genotypes after WDV infection, while CYCLIN-T1-3, a regulator of CDK kinases (cyclin-dependent kinase), was upregulated. On the other hand, the expression pattern of the transcription factor (TF) MYB (TraesCS4B02G174600.2; myeloblastosis domain of transcription factor) was downregulated by WDV infection in the resistant genotypes compared to the susceptible genotype, while a large number of TFs belonging to 54 TF families were differentially expressed due to WDV infection. In addition, two transcripts (TraesCS7A02G341400.1 and TraesCS3B02G239900.1) were upregulated with uncharacterised proteins involved in transport and regulation of cell growth, respectively. Altogether, our findings showed a clear gene expression profile associated with resistance or susceptibility of wheat to WDV. In future studies, we will explore the regulatory network within the same experiment context. This knowledge will broaden not only the future for the development of virus-resistant wheat genotypes but also the future of genetic improvement of cereals for resilience and WDV-resistance breeding.

Exploring the active ingredients and potential mechanisms of action of sinomenium acutum in the treatment of rheumatoid arthritis based on systems biology and network pharmacology.

Objective: To investigate and predict the targets and signaling pathways of sinomenium acutum (SA) in the treatment of rheumatoid arthritis (RA) through systems biology and network pharmacology, and to elucidate its possible mechanisms of action. Methods: We screened the active ingredients and corresponding target proteins of SA in Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), Traditional Chinese Medicines Integrated Database (TCMID) and Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine (BATMAN); and obtained the targets of rheumatoid arthritis diseases in a database of gene-disease associations (DisGeNET), Online Mendelian Inheritance in Man (OMIM) database. The two targets were mapped by Venn diagram and the intersection was taken. The intersecting targets were used to construct protein-protein interaction (PPI) network maps in the String database, and Metascape was used for Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Finally, the molecular docking technique was applied to validate and further clarify the core target of SA for the treatment of rheumatoid arthritis. Results: A total of six active ingredients and 217 potential targets were obtained after screening; 2,752 rheumatoid arthritis-related targets and 66 targets common to RA and SA. GO function and KEGG pathway enrichment analysis yielded 751 GO function entries (652 GO biological processes, 59 GO molecular functions and 40 GO cellular components) and 77 KEGG signaling pathways. It mainly involves pathways related to neural activity ligand-receptor interaction pathways, cancer pathways, calcium signaling channels, Th17 cell differentiation and others, which are mainly classified into four categories, including regulation of immunity, anti-inflammation, regulation of cell growth and apoptosis, and signaling. The molecular docking results showed that the binding energy of PTGS2, CASP3, JUN and PPARG to the key components beta-sitosterol, 16-epi-Isositsirikine, Sinomenine and Stepholidine were ≤ -6.5kcal/mol, suggesting the existence of molecular binding sites. Conclusion: SA acts on key targets such as PTGS2, CASP3, JUN, and PPARG to modulate signaling pathways such as neural activity ligand-receptor interaction, cancer, calcium ion, NF-κB, and Th17 cell differentiation to regulate immunity, anti-inflammation, modulation of cell cycle, bone metabolism, and signaling for the treatment of RA. It was also confirmed that the treatment of RA with SA has multi-component, multi-target, multi-pathway and multi-mechanism characteristics.

Genomic Alterations, Gene Expression Profiles and Functional Enrichment of Normal-Karyotype Acute Myeloid Leukaemia Based on Targeted Next-Generation Sequencing

Characterising genomic variants is paramount in understanding the pathogenesis and heterogeneity of normal-karyotype acute myeloid leukaemia (AML-NK). In this study, clinically significant genomic biomarkers were ascertained using targeted DNA sequencing and RNA sequencing on eight AML-NK patients' samples collected at disease presentation and after complete remission. In silico and Sanger sequencing validations were performed to validate variants of interest, and they were followed by the performance of functional and pathway enrichment analyses for overrepresentation analysis of genes with somatic variants. Somatic variants involving 26 genes were identified and classified as follows: 18/42 (42.9%) as pathogenic, 4/42 (9.5%) as likely pathogenic, 4/42 (9.5%) as variants of unknown significance, 7/42 (16.7%) as likely benign and 9/42 (21.4%) as benign. Nine novel somatic variants were discovered, of which three were likely pathogenic, in the CEBPA gene with significant association with its upregulation. Transcription misregulation in cancer tops the affected pathways involving upstream genes (CEBPA and RUNX1) that were deregulated in most patients during disease presentation and were closely related to the most enriched molecular function gene ontology category, DNA-binding transcription activator activity RNA polymerase II-specific (GO:0001228). In summary, this study elucidated putative variants and their gene expression profiles along with functional and pathway enrichment in AML-NK patients.

HMM-based profiling identifies the binding to divalent cations and nucleotides as common denominators of suramin targets

Introduction: Suramin is one of the pharmacopeia’s most promiscuous drugs. Originally developed for African trypanosomiasis, suramin was also used for onchocerciasis and it has been proposed as an anticancer agent, antiviral drug, therapy for arthritis, autism, and antidote for snake bites. Target proteins of suramin have been described from different species. Here we identify the common motifs among these various targets, aiming to explain the promiscuous nature of suramin.Methods: We have searched for suramin target proteins in the literature and in chemical databases. Applying rigorous inclusion criteria, a list of 44 diverse proteins was assembled with experimental evidence for direct interaction with, and inhibition by, suramin. Hidden Markov model-based target profiling was performed by running the full set of Pfam protein family domains against these proteins.Results: Common denominators were identified by mapping the identified Pfam domains to molecular function gene ontology terms. This in silico pipeline identified nucleotide binding, nucleic acid binding, and binding to divalent cations as the most common denominators of the suramin targets.Discussion: Our results suggest that the extraordinary polypharmacology of suramin may be caused by its ability to inhibit the interaction of proteins with nucleotides or nucleic acids and with divalent cations (Mg2+, Ca2+, Zn2+). Suramin is well known to inhibit nucleotide receptors and nucleic acid-binding enzymes. The association with divalent cations is new and might be key towards the design of better, more selective inhibitors.

Novel insights into the somatic proteome of Strongyloides stercoralis infective third-stage larvae

BackgroundStrongyloidiasis is a neglected tropical disease affecting an estimated 600 million people, particularly in resource-limited settings. The infection can persist lifelong due to unusual auto-infective cycle of Strongyloides stercoralis. The lack of a diagnostic gold standard and limited knowledge of the mechanisms underpinning this chronic infection are key issues in disease management. To date, only a few proteomics studies have been conducted to elucidate the molecular mechanisms associated with Strongyloides parasitism or to highlight novel immunological markers, with the result that our knowledge of S. stercoralis proteome remains limited. This study aims at expanding the characterization of S. stercoralis infective larvae (iL3) in order to further explore the mechanisms of parasitism and to highlight possible novel targets for serodiagnosis.MethodsiL3 obtained from an infected subject were analysed by high-throughput tandem mass spectrometry. To achieve a more comprehensive characterization of the iL3 proteome we analysed the experimental dataset using an automatic search strategy combined with manual annotation, which included gene ontology (GO) analysis, InterPro annotation, assessment of the homology with Homo sapiens and other pathogens of clinical importance and B-cell epitope prediction.ResultsOur pipeline identified 430 S. stercoralis proteins, 187 (43%) of which were uncharacterized. Oxidoreductases and peptidases were amongst the most represented protein categories, as highlighted by molecular function GO analyses, while membrane and mitochondrial proteins were the most represented cellular component GO categories. A high proportion of proteins bearing the CAP, SCP or thioredoxin domain or belonging to cysteine-rich secretory, transthyretin-like or peptidase protein families were also identified. Additionally, we highlighted nine proteins displaying low homology with H. sapiens or other related pathogens and bearing amino acid sequences with immunogenic properties.ConclusionsOur comprehensive description and annotation of the S. stercoralis iL3 proteome contribute to expanding the ‘omics characterization of this parasite and provide experimental evidence on the most represented proteins associated with S. stercoralis parasitism, as inferred from genomic and transcriptomic data. Moreover, novel candidate immunogenic proteins to be evaluated as novel serological diagnostic markers are highlighted.Graphical