Overrepresented Gene Ontology Research Articles

O-098 The transcriptome of endometrial organoids of fertile and subfertile women exposed to seminal plasma of fertile men

Abstract Study question What is the effect of seminal plasma (SP) from fertile males on the transcriptome of endometrial epithelial organoids of fertile and subfertile women? Summary answer Transcriptomic changes in fertile and subfertile endometrial organoids are observed upon treatment with SP, related to hormone- and immune-regulation, reproduction and cellular organisation. What is known already Clinical studies have demonstrated the potential beneficial effect of intrauterine SP (liquid fraction of semen) insemination during assisted reproductive technology (ART), resulting in higher implantation- and pregnancy rates. However, the molecular mechanism by which SP exerts its function remains unclear. SP components such as TGF-β1 or IL-8 have the potential capacity of conditioning the uterus for pregnancy. SP has shown to induce significant transcriptional changes in 2D endometrial epithelial cells. 3D self-organising human endometrial organoids have been established over the last years, mimicking a more in vivo-like physiology, and therefore may better represent the interaction of the endometrium with SP. Study design, size, duration Endometrial tissue biopsies from fertile (n = 5) and subfertile (n = 5; subfertile defined as women not having reached clinical pregnancy after at least 12 months of regular unprotected intercourse) were used to establish endometrial epithelial organoids. Subsequently, SP from fertile males (n = 5) was collected and pooled. Endometrial organoids were cultured in complex media supplemented with 1 µM 17β-estradiol (E2) and incubated with or without (control) 1% SP for 6 h. Participants/materials, setting, methods RNA sequencing was performed using NextSeq600 (Illumina). Significant DEGs upon SP exposure were identified using DESeq2 (Fold Change (FC) of ≥ 1.5 or ≤ -1.5). Eight selected DEGs were validated by qPCR in one cell line. Principal Component Analysis (PCA) was performed to compare endometrium donor correlation. Gene Set Enrichment Analysis (GSEA; v4.3.2) was then used to identify overrepresented pathways and gene ontology (GO) gene sets (P < 0.05; False Discovery Rate (FDR) < 0.25). Main results and the role of chance High heterogeneity between donors was observed, regardless of their fertility phenotype. PCA analysis indicated clustering based on donor rather than SP treatment or phenotype. Moreover, the number of significantly DEGs ranged from 629 to 2332. Treatment with SP resulted in identification of 105 significant DEGs (p < 0.05) among all donors (fertile and subfertile) from which 80 DEGs were upregulated and 25 DEGs were downregulated. Several of these DEGs were also reported to be affected by SP in other 2D epithelial cultures studies. Interesting DEGs include progesterone-associated endometrial protein (PAEP; log2FC 2.0 ± 1.0), interleukin-6 (IL6; log2FC 4.5 ± 1.5), insulin-like growth factor-binding protein 3 (IGFBP3; log2FC 1.1 ± 0.2) and mucin 6 (MUC6; log2FC 3.7 ± 1.6). GSEA analysis identified gene set enrichment of pathways and biological processes related to hormone- and immune regulation, reproduction, and cellular organisation in all endometrial organoids treated with SP. Preliminary RNAseq validation with qPCR showed comparable expression patterns for 8 genes (e.g. PAEP and IL6). Moreover, expression of these genes was uniform when using a distinct SP batch (n = 6; fertile males). No clear differential response between fertile and subfertile organoids upon SP exposure was observed. Limitations, reasons for caution The apical-inside organisation of the organoids (i.e. SP interaction from the basolateral side) should be taken into account, since in vivo interaction will be apically. Moreover, high heterogeneity between donors was observed between and within the fertility phenotypes. Therefore, conclusions based on fertility phenotypes should be drawn with caution. Wider implications of the findings Understanding the effect of SP on the endometrium will shed light on male-female reproductive tract crosstalk, which is omitted during ART. Future research should focus on identifying key regulatory SP molecules and functional studies should include apical SP interaction. Potentially providing therapeutic options for improving success rates in ART. Trial registration number Not applicable

Identification of candidate regulatory genes for intramuscular fatty acid composition in pigs by transcriptome analysis

BackgroundIntramuscular fat (IMF) content and its fatty acid (FA) composition are typically controlled by several genes, each with a small effect. In the current study, to pinpoint candidate genes and putative regulators involved in FA composition, we performed a multivariate integrative analysis between intramuscular FA and transcriptome profiles of porcine longissimus dorsi (LD) muscle. We also carried out a combination of network, regulatory impact factor (RIF), in silico prediction of putative target genes, and functional analyses to better support the biological relevance of our findings.ResultsFor this purpose, we used LD RNA-Seq and intramuscular FA composition profiles of 129 Iberian × Duroc backcrossed pigs. We identified 378 correlated variables (13 FA and 365 genes), including six FA (C20:4n-6, C18:2n-6, C20:3n-6, C18:1n-9, C18:0, and C16:1n-7) that were among the most interconnected variables in the predicted network. The detected FA-correlated genes include genes involved in lipid and/or carbohydrate metabolism or in regulation of IMF deposition (e.g., ADIPOQ, CHUK, CYCS, CYP4B1, DLD, ELOVL6, FBP1, G0S2, GCLC, HMGCR, IDH3A, LEP, LGALS12, LPIN1, PLIN1, PNPLA8, PPP1R1B, SDR16C5, SFRP5, SOD3, SNW1, and TFRC), meat quality (GALNT15, GOT1, MDH1, NEU3, PDHA1, SDHD, and UNC93A), and transport (e.g., EXOC7 and SLC44A2). Functional analysis highlighted 54 over-represented gene ontology terms, including well-known biological processes and pathways that regulate lipid and carbohydrate metabolism. RIF analysis suggested a pivotal role for six transcription factors (CARHSP1, LBX1, MAFA, PAX7, SIX5, and TADA2A) as putative regulators of gene expression and intramuscular FA composition. Based on in silico prediction, we identified putative target genes for these six regulators. Among these, TADA2A and CARHSP1 had extreme RIF scores and present novel regulators in pigs. In addition, the expression of TADA2A correlated (either positively or negatively) with C20:4n-6, C18:2n-6, C20:3n-6, C18:1n-9, and that of CARHSP1 correlated (positively) with the C16:1n-7 lipokine. We also found that these two transcription factors share target genes that are involved in lipid metabolism (e.g., GOT1, PLIN1, and TFRC).ConclusionsThis integrative analysis of muscle transcriptome and intramuscular FA profile revealed valuable information about key candidate genes and potential regulators for FA and lipid metabolism in pigs, among which some transcription factors are proposed to control gene expression and modulate FA composition differences.

IDSL.GOA: gene ontology analysis for interpreting metabolomic datasets

Biological interpretation of metabolomic datasets often ends at a pathway analysis step to find the over-represented metabolic pathways in the list of statistically significant metabolites. However, definitions of biochemical pathways and metabolite coverage vary among different curated databases, leading to missed interpretations. For the lists of genes, transcripts and proteins, Gene Ontology (GO) terms over-presentation analysis has become a standardized approach for biological interpretation. But, GO analysis has not been achieved for metabolomic datasets. We present a new knowledgebase (KB) and the online tool, Gene Ontology Analysis by the Integrated Data Science Laboratory for Metabolomics and Exposomics (IDSL.GOA) to conduct GO over-representation analysis for a metabolite list. The IDSL.GOA KB covers 2393 metabolic GO terms and associated 3144 genes, 1,492 EC annotations, and 2621 metabolites. IDSL.GOA analysis of a case study of older versus young female brain cortex metabolome highlighted 82 GO terms being significantly overrepresented (FDR < 0.05). We showed how IDSL.GOA identified key and relevant GO metabolic processes that were not yet covered in other pathway databases. Overall, we suggest that interpretation of metabolite lists should not be limited to only pathway maps and can also leverage GO terms as well. IDSL.GOA provides a useful tool for this purpose, allowing for a more comprehensive and accurate analysis of metabolite pathway data. IDSL.GOA tool can be accessed at https://goa.idsl.me/.

DIFFERENTIAL SIGNALING EFFECTS OF ESCHERICHIA COLI AND STAPHYLOCOCCUS AUREUS IN HUMAN WHOLE BLOOD INDICATE DISTINCT REGULATION OF THE NRF2 PATHWAY.

Escherichia coli and Staphylococcus aureus are two of the most common bacterial species responsible for sepsis. While it is observed that they have disparate clinical phenotypes, the signaling differences elicited by each bacteria that drive this variance remain unclear. Therefore, we used human whole blood exposed to heat-killed E. coli or S. aureus and measured the transcriptomic signatures. Relative to unstimulated control blood, heat-killed bacteria exposure led to significant dysregulation (upregulated and downregulated) of >5,000 genes for each experimental condition, with a slight increase in gene alterations by S. aureus. While there was significant overlap regarding proinflammatory pathways, Gene Ontology overrepresentation analysis of the most altered genes suggested biological processes like macrophage differentiation and ubiquinone biosynthesis were more unique to heat-killed S. aureus, compared with heat-killed E. coli exposure. Using Ingenuity Pathway Analysis, it was demonstrated that nuclear factor erythroid 2-related factor 2 signaling, a main transcription factor in antioxidant responses, was predominately upregulated in S. aureus exposed blood relative to E. coli. Furthermore, the use of pharmacologics that preferentially targeted the nuclear factor erythroid 2-related factor 2 pathway led to differential cytokine profiles depending on the type of bacterial exposure. These findings reveal significant inflammatory dysregulation between E. coli and S. aureus and provide insight into the targeting of unique pathways to curb bacteria-specific responses.

Comparative proteome analysis of different Saccharomyces cerevisiae strains during growth on sucrose and glucose

Both the identity and the amount of a carbon source present in laboratory or industrial cultivation media have major impacts on the growth and physiology of a microbial species. In the case of the yeast Saccharomyces cerevisiae, sucrose is arguably the most important sugar used in industrial biotechnology, whereas glucose is the most common carbon and energy source used in research, with many well-known and described regulatory effects, e.g. glucose repression. Here we compared the label-free proteomes of exponentially growing S. cerevisiae cells in a defined medium containing either sucrose or glucose as the sole carbon source. For this purpose, bioreactor cultivations were employed, and three different strains were investigated, namely: CEN.PK113-7D (a common laboratory strain), UFMG-CM-Y259 (a wild isolate), and JP1 (an industrial bioethanol strain). These strains present different physiologies during growth on sucrose; some of them reach higher specific growth rates on this carbon source, when compared to growth on glucose, whereas others display the opposite behavior. It was not possible to identify proteins that commonly presented either higher or lower levels during growth on sucrose, when compared to growth on glucose, considering the three strains investigated here, except for one protein, named Mnp1—a mitochondrial ribosomal protein of the large subunit, which had higher levels on sucrose than on glucose, for all three strains. Interestingly, following a Gene Ontology overrepresentation and KEGG pathway enrichment analyses, an inverse pattern of enriched biological functions and pathways was observed for the strains CEN.PK113-7D and UFMG-CM-Y259, which is in line with the fact that whereas the CEN.PK113-7D strain grows faster on glucose than on sucrose, the opposite is observed for the UFMG-CM-Y259 strain.

DOP71 TREM1, OSM and a co-expressed transcriptional module are core components of the molecular resistome to anti-cytokine therapy in Ulcerative Colitis

Abstract Background Molecular biomarkers are emerging as promising tools to predict response to advanced therapy in IBD. For instance, mucosal expression of Triggering Receptor Expressed on Myeloid cells-1 (TREM1) and oncostatin M (OSM) are associated with anti-TNF non-response in Ulcerative Colitis (UC). The specificity of such transcriptional features is poorly understood. We investigated whether mucosal expression of TREM1 and OSM influences ustekinumab response in UC. Methods UNIFI is a landmark, phase III, registration trial that evaluated the efficacy of ustekinumab as induction and maintenance therapy in patients with moderate to severe UC. In a subset of patients (n=358), colonic biopsies were sampled at baseline and gene expression profiled by RNA microarray (Affymetrix HT HG-U133+ PM). Normalised expression levels of TREM1 and OSM for responders and non-responders to induction (week 8 mucosal healing) were compared using Mann-Whitney U test on GraphPad Prism 9.2.0. Co-expressed transcripts were determined with Morpheus matrix visualisation software (Broad Institute, USA). Gene set variation analysis was used to calculate gene set enrichment scores (ES) within the expression data. Multivariable models were developed to construct receiver operating characteristic curve (ROC) analyses. Deeper biological insights of TREM1- and OSM-expressing cells were explored using single cell RNA sequencing and Gene Ontology over-representation analysis (R 4.2.2). Results Mucosal expression of TREM1 and OSM in inflamed colonic biopsies correlated highly (r = 0.81, p&amp;lt;0.0001) and TREM1 was the third-highest correlate with OSM across the entire genome. Pathway analysis showed that a 20-gene module (GS1) of transcripts co-expressed with TREM1 and OSM was enriched for immune cell adhesion, migration and differentiation (Fig 1). Expression of TREM1 (median: 6.27 vs 5.55, p&amp;lt;0.001), OSM (5.00 vs 4.72, p=0.002) and GS1 (ES: 0.00221 vs -0.496, p&amp;lt;0.0001) were all significantly increased in ustekinumab non-responders compared to responders (Fig 2). Although key clinical parameters correlated weakly with TREM1 and OSM expression, they improved the performance of multivariate models to predict non-response (Fig 3). TREM1 and OSM were predominantly expressed by the same population of inflammatory monocytes accumulating in inflamed colon tissue (Fig 4). Conclusion Colonic expression of TREM1 and OSM is associated with resistance to both anti-TNF therapy and ustekinumab, supporting the notion that they constitute a core molecular resistome in UC patients treated with anti-cytokine agents. Their predominant co-expression in infiltrating, inflammatory monocytes indicates that treatment resistance is likely underpinned by biological pathways regulated by this cell population.

Bioinformatic Data Mining for Candidate Drugs Affecting Risk of Bisphosphonate-Related Osteonecrosis of the Jaw (BRONJ) in Cancer Patients.

Background Bisphosphonate-related osteonecrosis of the jaw (BRONJ) leads to significant morbidity. Other coadministered drugs may modulate the risk for BRONJ. The present study aimed to leverage bioinformatic data mining to identify drugs that potentially modulate the risk of BRONJ in cancer. Methods A GEO gene expression dataset of peripheral blood mononuclear cells related to BRONJ in multiple myeloma patients was downloaded, and differentially expressed genes (DEGs) in patients with BRONJ versus those without BRONJ were identified. A protein-protein interaction network of the DEGs was constructed using experimentally validated interactions in the STRING database. Overrepresented Gene Ontology (GO) molecular function terms and KEGG pathways in the network were analysed. Network topology was determined, and ‘hub genes' with degree ≥2 in the network were identified. Known drug targets of the hub genes were mined from the ‘drug gene interaction database' (DGIdb) and labelled as candidate drugs affecting the risk of BRONJ. Results 751 annotated DEGs (log FC ≥ 1.5, p < 0.05) were obtained from the microarray gene expression dataset GSE7116. A PPI network with 633 nodes and 168 edges was constructed. Data mining for drugs interacting with 49 gene nodes was performed. 37 drug interactions were found for 9 of the hub genes including TBP, TAF1, PPP2CA, PRPF31, CASP8, UQCRB, ACTR2, CFLAR, and FAS. Interactions were found for several established and novel anticancer chemotherapeutic, kinase inhibitor, caspase inhibitor, antiangiogenic, and immunomodulatory agents. Aspirin, metformin, atrovastatin, thrombin, androgen and antiandrogen drugs, progesterone, Vitamin D, and Ginsengoside 20(S)-Protopanaxadiol were also documented. Conclusions A bioinformatic data mining strategy identified several anticancer, immunomodulator, and other candidate drugs that may affect the risk of BRONJ in cancer patients.

MicroRNA and MicroRNA-Target Variants Associated with Autism Spectrum Disorder and Related Disorders

Autism spectrum disorder (ASD) is a childhood neurodevelopmental disorder with a complex and heterogeneous genetic etiology. MicroRNA (miRNA), a class of small non-coding RNAs, could regulate ASD risk genes post-transcriptionally and affect broad molecular pathways related to ASD and associated disorders. Using whole-genome sequencing, we analyzed 272 samples in 73 families in the New Jersey Language and Autism Genetics Study (NJLAGS) cohort. Families with at least one ASD patient were recruited and were further assessed for language impairment, reading impairment, and other associated phenotypes. A total of 5104 miRNA variants and 1,181,148 3′ untranslated region (3′ UTR) variants were identified in the dataset. After applying several filtering criteria, including population allele frequency, brain expression, miRNA functional regions, and inheritance patterns, we identified high-confidence variants in five brain-expressed miRNAs (targeting 326 genes) and 3′ UTR miRNA target regions of 152 genes. Some genes, such as SCP2 and UCGC, were identified in multiple families. Using Gene Ontology overrepresentation analysis and protein–protein interaction network analysis, we identified clusters of genes and pathways that are important for neurodevelopment. The miRNAs and miRNA target genes identified in this study are potentially involved in neurodevelopmental disorders and should be considered for further functional studies.

MicroSugar: A database of comprehensive miRNA target prediction framework for sugarcane (Saccharum officinarum L.)

microRNA (miRNA) is a group of small non-coding RNA that plays important role in post-transcription of gene expression. With the studies about miRNA increase in sugarcane, the researchers lack an exhaustive resource to achieve the data. To fill this gap, we developed MicroSugar, a database that supported mRNA and miRNA annotation for sugarcane (http://suc.gene-db.com). MicroSugar is an integrated resource developed for 194,528 genes including 80,746 unigenes from long reads of Pacbio platform and 468 miRNAs from 72 samples. Internode elongation (jointing) is the key biological characteristic for the growth of sugarcane tillers into sugarcane stems. The present study combined the sequencing data from the different stages in internode elongation of stem and tiller. In total, the 14,300 3′ untranslated region (UTR) sequences were extracted from the gene sequences and 3019 mRNAs as target of 327 miRNA were identified by miRanda algorithm and Spearman's Rho of expression levels. To determine the gene functions regulated by these miRNAs, the gene ontology enrichment analysis was performed and it confirmed that the over-represented Gene Ontology (GO) terms were associated with organism formation indicating the growth controlling function by miRNAs in sugarcane. Moreover, MicroSugar is a comprehensive and integrated database with a user-friendly responsive template. By browsing, searching and downloading of the nucleotide sequences, expression and miRNA targets, the user can retrieve information promptly. The database provides a valuable resource to facilitate the understanding of miRNA in sugarcane development and growth which will contribute to the study of sugarcane and other plants.

DIPG-21. DIPG cells alter the permeability of the blood-brain barrier in the brainstem leading to treatment failure.

Diffuse Intrinsic Pontine Glioma (DIPG) is an aggressive paediatric high-grade glioma with no effective therapies. The blood-brain barrier (BBB) presents a significant obstacle for delivery of therapeutics into the brain, especially in the brainstem. This study aims to investigate the effect of DIPG cells on the BBB in the brainstem. We hypothesized that the location of DIPG may result in a less permeable BBB than other brain regions. We compared two independent orthotopic models with the same DIPG cells injected in the cortical region or brainstem. We found that treatment with mTOR inhibitor, temsirolimus, significantly extended survival for cortical tumors compared with the same tumor in the brainstem (p=0.0097). Immunohistochemical analysis showed significant reduction of mTOR target, P-p70S6K, in the cortical region compared to brainstem in treated animals, with pharmacokinetic analysis confirming significantly higher temsirolimus levels in the cortical region. These findings suggest that cortical tumors respond better than brainstem tumors, and that a less permeable BBB in brainstem tumors contributes to treatment failure. To understand whether DIPG cells affect the BBB in the brainstem, single-cell RNA sequencing experiments were conducted on vasculature isolated from DIPG and matrigel-injected mice. Gene-ontology overrepresentation analyses identified downregulation in the P38MAPK pathway in endothelial cells from DIPG-injected mice, suggesting the potential for therapeutic manipulation with cytokines. Treatment with SNGR-TNFα, a derivative of an agent successfully used in improving drug penetration in CNS lymphoma patients, in an in vitro BBB/DIPG model significantly reduced transendothelial resistance, and further exploration into the effects on the BBB in vivo is currently being undertaken. Our studies indicate that the intact BBB in the brainstem in DIPG is a major reason for treatment failure, and DIPG cells directly influence the vasculature and response to treatment. This may lead to a novel DIPG treatment strategy and for other brain tumors.

Use Of Weighted Gene Coexpression Network Analysis To Identify Connectivity Between Gut And Brain Gene Expression

BackgroundAlterations in the gut brain axis are being recognized as a pathogenic factor for an increasing number of diseases, including neurodegenerative diseases such as Parkinson Disease (PD). The extent to which gene expression profiling in the gut and the brain is co‐regulated is not well understood. Weighted gene coexpression network analysis (WGCNA) uses unbiased hierarchical clustering to reduce gene expression profiling data to modules of highly correlated genes.HypothesisGut‐brain connectivity will be strongest for modules related to physiologic mechanisms with a systemic component, such as immune reactivity.MethodsWGNCA networks were constructed for distal colon and striatum RNA seq data from mice overexpressing human wild type alpha synuclein (ASO, n=18) and wild type (WT, n=16) mice at 1 and 3 months. Mice with matched colon and striatum data (n=10/6 ASO/WT) were included in this analysis. Linear regression identified associations between colon and striatum modules. For this analysis, we controlled for both age and genotype as genotype‐ and age‐associated modules could be correlated due to these covariates alone. Colon‐striatum intermodular connectivity was visualized in Cytoscape. Overrepresented gene ontology (GO) terms in WGCNA modules were determined using the hypergeometric function in the GOstats package in R. Enrichment for gene signatures from single cell sequencing data was determined using the hypergeometric test against cell type signatures from the Panglao and Cellmarker databases.ResultsSelected associations are shown in Figure 1. There are strong correlations between colon and striatum modules that are enriched for terms related to the immune response. Modules most clearly related to the immune response are highlighted in yellow, but several other modules are also closely related to the immune response. For example, there is an association between the striatum module enriched for endothelial cells of the blood brain barrier, and the colon module enriched for goblet cells which produce the mucus layer of the epithelium, a key component of the innate immune system.ConclusionsThrough analysis of matched colon and striatum samples, we can see correlations between gene expression in the colon and striatum. Most modules in this gut‐brain network have some relevance to the immune system which is not unexpected. These results demonstrate the feasibility of “profiling” the gut‐brain axis. A larger sample size would permit evaluation of disease‐related changes in this profile.

Gene Expression Profile Changes in the Stimulated Rat Brain Cortex After Repetitive Transcranial Magnetic Stimulation.

Repetitive transcranial magnetic stimulation (rTMS) is gaining popularity as a research tool in neuroscience; however, little is known about its molecular mechanisms of action. The present study aimed to investigate the rTMS-induced transcriptomic changes; we performed microarray messenger RNA, micro RNA, and integrated analyses to explore these molecular events. Eight adult male Sprague-Dawley rats were subjected to a single session of unilateral rTMS at 1 Hz (n = 4) or sham (n = 4). The left hemisphere was stimulated for 20 minutes. To evaluate the cumulative effect of rTMS, eight additional rats were assigned to the 1-Hz (n = 4) or sham (n = 4) rTMS groups. The left hemisphere was stimulated for 5 consecutive days using the same protocol. Microarray analysis revealed differentially expressed genes in the rat cortex after rTMS treatment. The overrepresented gene ontology categories included the positive regulation of axon extension, axonogenesis, intracellular transport, and synaptic plasticity after repeated sessions of rTMS. A single session of rTMS primarily induced changes in the early genes, and several miRNAs were significantly related to the mRNAs. Future studies are required to validate the functional significance of selected genes and refine the therapeutic use of rTMS.

Overexpression of microRNAs miR-25-3p, miR-185-5p and miR-132-3p in Late Onset Fetal Growth Restriction, Validation of Results and Study of the Biochemical Pathways Involved.

In a prospective study, 48 fetuses were evaluated with Doppler ultrasound after 34 weeks and classified, according to the cerebroplacental ratio (CPR) and estimated fetal weight (EFW), into fetuses with normal growth and fetuses with late-onset fetal growth restriction (LO-FGR). Overexpression of miRNAs from neonatal cord blood belonging to LO-FGR fetuses, was validated by real-time PCR. In addition, functional characterization of overexpressed miRNAs was performed by analyzing overrepresented pathways, gene ontologies, and prioritization of synergistically working miRNAs. Three miRNAs: miR-25-3p, miR-185-5p and miR-132-3p, were significantly overexpressed in cord blood of LO-FGR fetuses. Pathway and gene ontology analysis revealed over-representation of certain molecular pathways associated with cardiac development and neuron death. In addition, prioritization of synergistically working miRNAs highlighted the importance of miR-185-5p and miR-25-3p in cholesterol efflux and starvation responses associated with LO-FGR phenotypes. Evaluation of miR-25-3p; miR-132-3p and miR-185-5p might serve as molecular biomarkers for the diagnosis and management of LO-FGR; improving the understanding of its influence on adult disease.

Coexpression of Gene Transcripts with Monoamine Oxidase A Quantified by Human In Vivo Positron Emission Tomography.

The monoamine oxidase A (MAO-A) is integral to monoamine metabolism and is thus relevant to the pathophysiology of various neuropsychiatric disorders; however, associated gene-enzyme relations are not well understood. This study aimed to unveil genes coexpressed with MAO-A. Therefore, 18 179 mRNA expression maps (based on the Allen Human Brain Atlas) were correlated with the cerebral distribution volume (VT) of MAO-A assessed in 36 healthy subjects (mean age ± standard deviation: 32.9 ± 8.8 years, 18 female) using [11C]harmine positron emission tomography scans. Coexpression analysis was based on Spearman's ρ, over-representation tests on Fisher's exact test with false discovery rate (FDR) correction. The analysis revealed 35 genes in cortex (including B-cell translocation gene family, member 3, implicated in neuroinflammation) and 247 genes in subcortex (including kallikrein-related peptidase 10, implicated in Alzheimer's disease). Significantly over-represented Gene Ontology terms included "neuron development", "neuron differentiation", and "cell-cell signaling" as well as "axon" and "neuron projection". In vivo MAO-A enzyme distribution and MAOA expression did not correlate in cortical areas (ρ = 0.08) while correlation was found in subcortical areas (ρ = 0.52), suggesting influences of region-specific post-transcriptional and -translational modifications. The herein reported information could contribute to guide future genetic studies, deepen the understanding of associated pathomechanisms and assist in the pursuit of novel therapeutic targets.

Integrated mRNA-miRNA transcriptome analysis of bladder biopsies from patients with bladder pain syndrome identifies signaling alterations contributing to the disease pathogenesis

BackgroundInterstitial cystitis, or bladder pain syndrome (IC/BPS), is a chronic bladder disorder characterized by lower abdominal pain associated with the urinary bladder and accompanied by urinary frequency and urgency in the absence of identifiable causes. IC/PBS can be separated into the classic Hunner’s ulcerative type and the more prevalent non-ulcerative disease. Our aim was to unravel the biological processes and dysregulated cell signaling pathways leading to the bladder remodeling in non-ulcerative bladder pain syndrome (BPS) by studying the gene expression changes in the patients’ biopsies.MethodsWe performed paired microRNA (miRNA) and mRNA expression profiling in the bladder biopsies of BPS patients with non-Hunner interstitial cystitis phenotype, using comprehensive Next-generation sequencing (NGS) and studied the activated pathways and altered biological processes based on the global gene expression changes. Paired mRNA-miRNA transcriptome analysis delineated the regulatory role of the dysregulated miRNAs by identifying their targets in the disease-induced pathways.ResultsEIF2 Signaling and Regulation of eIF4 and p70S6K Signaling, activated in response to cellular stress, were among the most significantly regulated processes during BPS. Leukotriene Biosynthesis nociceptive pathway, important in inflammatory diseases and neuropathic pain, was also significantly activated. The biological processes identified using Gene Ontology over-representation analysis were clustered into six main functional groups: cell cycle regulation, chemotaxis of immune cells, muscle development, muscle contraction, remodeling of extracellular matrix and peripheral nervous system organization and development. Compared to the Hunner’s ulcerative type IC, activation of the immune pathways was modest in non-ulcerative BPS, limited to neutrophil chemotaxis and IFN-γ-mediated signaling. We identified 62 miRNAs, regulated and abundant in BPS and show that they target the mRNAs implicated in eIF2 signalling pathway.ConclusionsThe bladders of non-ulcerative BPS patients recruited in this study had alterations consistent with a strong cell proliferative response and an up-regulation of smooth muscle contractility, while the contribution of inflammatory processes was modest. Pathway analysis of the integrated mRNA-miRNA NGS dataset pinpointed important regulatory miRNAs whose dysregulation might contribute to the pathogenesis. Observed molecular changes in the peripheral nervous system organization and development indicate the potential role of local bladder innervation in the pain perceived in this type of BPS.

In Silico Target Prediction of Overexpressed microRNAs from LPS-Challenged Zebrafish (Danio rerio) Treated with the Novel Anti-Inflammatory Peptide TnP.

miRNAs regulate gene expression post-transcriptionally in various processes, e.g., immunity, development, and diseases. Since their experimental analysis is complex, in silico target prediction is important for directing investigations. TnP is a candidate peptide for anti-inflammatory therapy, first discovered in the venom of Thalassophryne nattereri, which led to miRNAs overexpression in LPS-inflamed zebrafish post-treatment. This work aimed to predict miR-21, miR-122, miR-731, and miR-26 targets using overlapped results of DIANA microT-CDS and TargetScanFish software. This study described 513 miRNAs targets using highly specific thresholds. Using Gene Ontology over-representation analysis, we identified their main roles in regulating gene expression, neurogenesis, DNA-binding, transcription regulation, immune system process, and inflammatory response. miRNAs act in post-transcriptional regulation, but we revealed that their targets are strongly related to expression regulation at the transcriptional level, e.g., transcription factors proteins. A few predicted genes participated concomitantly in many biological processes and molecular functions, such as foxo3a, rbpjb, rxrbb, tyrobp, hes6, zic5, smad1, e2f7, and npas4a. Others were particularly involved in innate immunity regulation: il17a/f2, pik3r3b, and nlrc6. Together, these findings not only provide new insights into the miRNAs mode of action but also raise hope for TnP therapy and may direct future experimental investigations.

DNA methylation profile of liver of mice conceived by invitro fertilization.

Offspring generated by invitro fertilization (IVF) are believed to be healthy but display a possible predisposition to chronic diseases, like hypertension and glucose intolerance. Since epigenetic changes are believed to underlie such phenotype, this study aimed at describing global DNA methylation changes in the liver of adult mice generated by natural mating (FB group) or by IVF. Embryos were generated by IVF or natural mating. At 30 weeks of age, mice were sacrificed. The liver was removed, and global DNA methylation was assessed using whole-genome bisulfite sequencing (WGBS). Genomic Regions for Enrichment Analysis Tool (GREAT) and G:Profilerβ were used to identify differentially methylated regions (DMRs) and for functional enrichment analysis. Overrepresented gene ontology terms were summarized with REVIGO, while canonical pathways (CPs) were identified with Ingenuity® Pathway Analysis. Overall, 2692 DMRs (4.91%) were different between the groups. The majority of DMRs (84.92%) were hypomethylated in the IVF group. Surprisingly, only 0.16% of CpG islands were differentially methylated and only a few DMRs were located on known gene promoters (n = 283) or enhancers (n = 190). Notably, the long-interspersed element (LINE), short-interspersed element (SINE), and long terminal repeat (LTR1) transposable elements showed reduced methylation (P < 0.05) in IVF livers. Cellular metabolic process, hepatic fibrosis, and insulin receptor signaling were some of the principal biological processes and CPs modified by IVF. In summary, IVF modifies the DNA methylation signature in the adult liver, resulting in hypomethylation of genes involved in metabolism and gene transcription regulation. These findings may shed light on the mechanisms underlying the developmental origin of health and disease.

GENES MEDIATING CELL GROWTH IN ALDOSTERONE-PRODUCING ADENOMAS

Objective: Aldosterone-producing adenomas (APA) are a major cause of primary aldosteronism. Somatic mutations explain the excess aldosterone production in the majority of patients with APA with mutations in KCNJ5 encoding a potassium channel the most prevalent in most reported populations. Mechanisms driving cell proliferation are largely undefined. Design and method: Quantitative transcriptome analysis using RNA-seq was used to identify differentially expressed genes between macro-APAs (n = 9, diameter &gt;= 30 mm) and micro-APAs (n = 12, diameter &lt;= 10 mm). Validation of RNA-seq data by TaqMan real-time PCR was performed for 14 genes in a broader cohort of APAs (NMD, no mutation detected, n = 28; KCNJ5-mutated, n = 43). Results: Hierarchical cluster analysis of the 500 genes with largest coefficient of variation indicated sample clustering based on genotype (KCNJ5 or NMD) and APA diameter. Differential expression of 155 and 348 genes was found between micro- and macro-APAs with KCNJ5 mutations and NMD, respectively. Among the top 5 overrepresented Gene Ontology terms (biological process), cell death was exclusively enriched in NMD micro- vs NMD macro-APAs. The expression levels of 10 genes were validated with a potential function related to cell growth. Expression of BEX1 (a reported tumour suppressor) was 2.8-fold down regulated in macro-APAs relative to micro-APAs (P &lt; 0.001), and a linear negative correlation of BEX1 expression with APA diameter was observed in NMD APAs (r = -0.501, P = 0.007). Genes involved in beta-catenin signalling, SFRP2, DKK1 and TSPAN12 were 5.9-fold (P &lt; 0.001), 1.8-fold (P = 0.007) and 8.3-fold (P &lt; 0.0001) down regulated, respectively, in macro-APAs compared with micro-APAs. BAI1 and CCL21 were also down regulated in macro-APAs relative to micro-APAs (3.3-fold, P = 0.026; 2.6-fold, P = 0.145, respectively). Additionally, the expression of TMPRSS3 (r = 0.727, P &lt; 0.001), BMP4 (r = 0.389, P = 0.041) and FBXL21 (r = 0.761, P &lt; 0.001) showed a linear positive correlation with adenoma diameter in NMD subgroup. Conclusions: APA display distinct transcriptome profiles according to adenoma diameter which may help identify genes involved in APA tumour cell growth.

Transcriptome Analyses of Myometrium from Fibroid Patients Reveals Phenotypic Differences Compared to Non-Diseased Myometrium.

Uterine fibroid tissues are often compared to their matched myometrium in an effort to understand their pathophysiology, but it is not clear whether the myometria of uterine fibroid patients represent truly non-disease control tissues. We analyzed the transcriptomes of myometrial samples from non-fibroid patients (M) and compared them with fibroid (F) and matched myometrial (MF) samples to determine whether there is a phenotypic difference between fibroid and non-fibroid myometria. Multidimensional scaling plots revealed that M samples clustered separately from both MF and F samples. A total of 1169 differentially expressed genes (DEGs) (false discovery rate < 0.05) were observed in the MF comparison with M. Overrepresented Gene Ontology terms showed a high concordance of upregulated gene sets in MF compared to M, particularly extracellular matrix and structure organization. Gene set enrichment analyses showed that the leading-edge genes from the TGFβ signaling and inflammatory response gene sets were significantly enriched in MF. Overall comparison of the three tissues by three-dimensional principal component analyses showed that M, MF, and F samples clustered separately from each other and that a total of 732 DEGs from F vs. M were not found in the F vs. MF, which are likely understudied in the pathogenesis of uterine fibroids and could be key genes for future investigation. These results suggest that the transcriptome of fibroid-associated myometrium is different from that of non-diseased myometrium and that fibroid studies should consider using both matched myometrium and non-diseased myometrium as controls.

RNAseq Reveals Differential Gene Expression Contributing to Phytophthora nicotianae Adaptation to Partial Resistance in Tobacco

Phytophthora nicotianae is a devastating oomycete plant pathogen with a wide host range. On tobacco, it causes black shank, a disease that can result in severe economic losses. Deployment of host resistance is one of the most effective means of controlling tobacco black shank, but adaptation to complete and partial resistance by P. nicotianae can limit the long-term effectiveness of the resistance. The molecular basis of adaptation to partial resistance is largely unknown. RNAseq was performed on two isolates of P. nicotianae (adapted to either the susceptible tobacco genotype Hicks or the partially resistant genotype K 326 Wz/Wz) to identify differentially expressed genes (DEGs) during their pathogenic interactions with K 326 Wz/Wz and Hicks. Approximately 69% of the up-regulated DEGs were associated with pathogenicity in the K 326 Wz/Wz-adapted isolate when sampled following infection of its adapted host K 326 Wz/Wz. Thirty-one percent of the up-regulated DEGs were associated with pathogenicity in the Hicks-adapted isolate on K 326 Wz/Wz. A broad spectrum of over-represented gene ontology (GO) terms were assigned to down-regulated genes in the Hicks-adapted isolate. In the host, a series of GO terms involved in nuclear biosynthesis processes were assigned to the down-regulated genes in K 326 Wz/Wz inoculated with K 326 Wz/Wz-adapted isolate. This study enhances our understanding of the molecular mechanisms of P. nicotianae adaptation to partial resistance in tobacco by elucidating how the pathogen recruits pathogenicity-associated genes that impact host biological activities.