SDEAP: a splice graph based differential transcript expression analysis tool for population data.

Accounting for isoforms is likely key to understanding muscle transcriptomic divergence. Muscles offer classic examples of tissues where changes in isoforms alter function and structure in response to stimuli like increased exercise or novel nutrient regimes. To determine how an essential micronutrient alters muscle isoform production, we examined how vitamin D supplementation influences transcription at multiple hierarchical levels across four Atlantic salmon (Salmo salar) muscle tissues. Specifically, we investigated whether analyses of differential transcript expression (DTE), differential transcript usage (DTU), and alternative splicing (DAS) recovered different responses to vitamin D compared to differential gene expression (DGE) alone. Vitamin D modulates salmon muscle transcription at the level of the gene, transcript, and splice junctions in all four muscle tissues. However, the strongest effects at all levels were found in the heart. There was little overlap among significant genes found at the gene, transcript, and splicing levels and the distribution of isoforms per significant gene varied across DGE, DTE, and DTU, indicating that each method tends to identify unique sets of genes. For example, we found that several myosin light chain kinase isoforms were particularly impacted by vitamin D in the heart, but many of the genes exhibited isoforms that were differentially expressed in opposing directions and were thus masked in DGE analysis. We also identified myofibrillar genes that are impacted by vitamin D at all levels of gene regulation, demonstrating that vitamin D impacts many structural proteins that are directly involved in striated muscle contractions. Vitamin D influences several muscle tissue types across multiple levels of transcriptomic divergence. Further, the limited overlap among significant genes found at the gene, transcript, and splicing levels suggests examining only DGE can mask important genes that show differential effects at other levels. Myofibrillar gene isoforms that directly influence muscle contractions in critical organs like the heart could provide an especially fruitful avenue of additional investigation into the transcriptional impacts of vitamin D. Together, these findings clarify how vitamin D influences muscle differentiation, health, and function.

<h3>Introduction</h3> Mean platelet volume is increased in the setting of acute myocardial infarction (AMI) and is a poor prognostic marker despite antiplatelet use. The megakaryocyte (platelet precursor) that resides in the bone marrow has a mean physiological ploidy of 16n. Megakaryocyte size and DNA content are also shown to be increased in AMI. To investigate if megakaryocytes of higher ploidy are transcriptionally different and therefore have the ability to produce larger and more active platelets we performed RNA sequencing in megakaryocytes cultured with or without dimethylfasudil (diMF), an exogenous peptide that drives megakaryocyte ploidy further than previously documented by non-selective aurora kinase inhibition. <h3>Methods</h3> Human adult blood CD34⁺ derived megakaryocytes from 4 individuals underwent 3 days of culture with or without 5 µM diMF. RNA from untreated and treated cells was sequenced and used for differential gene and transcript expression analysis. <h3>Results</h3> Mean ploidy in the untreated megakaryocytes was 2n while diMF treatment drove mean ploidy to approximately 8n. This was associated with a morphological increase in size and nuclear multilobulation. Differential gene expression analysis revealed that in megakaryocytes that had been driven to high ploidy levels, 33 genes were downregulated while 65 genes underwent significant upregulation when compared with untreated megakaryocytes. Gene ontology analysis demonstrated that the downregulated genes were significantly enriched for transcripts involved in the cell cycle including CDC45, chromatin assembly factor 1β, and MCMs. In contrast, genes that were upregulated in higher ploidy megakaryocytes were significantly enriched for transcripts involved in haemostatic and coagulation pathways including VWF, coagulation factor XIII, thrombin receptor-like 2, PDGFβ, angiopoietin 1, thrombospondin 1 and plasminogen activator inhibitor type 1. Furthermore, glycoprotein IIIa (part of the fibrinogen/VWF receptor) and tubulin β1 (involved in microtubule changes in platelet release) were both significantly upregulated in higher ploidy megakaryocytes. Both of these genes are specific to megakaryocytes and platelets. <h3>Conclusion</h3> This is the first analysis of megakaryocytes driven to high ploidy level <i>in vitro</i> using RNA sequencing. Our results demonstrate that along with the anticipated modulation of cell cycle genes with increasing ploidy, higher ploidy megakaryocytes significantly overexpress genes that are involved in megakaryocyte function, platelet activation and thrombosis. These results therefore support a role for platelet production from megakaryocytes of higher ploidy in thrombotic disease.

Background: Differences in ER+ and ER- breast cancer tumor biology are well-documented, but little is known about the field of background benign breast in which those cancers arise. We evaluated the transcriptome of benign breast tissues from women with concurrent ipsilateral ER+ and ER- breast cancers (BC) to characterize coding and non-coding RNA profiles. This pilot study provides insight into the transcriptomic landscapes of benign breast tissues in patients with BC, and the microenvironment of the at-risk benign tissue. Methods: With institutional approval, cryobanked breast tissues from patients with concurrent ipsilateral ER+ BC (benign ER+BC, N=14) or ER- BC (benign ER-BC, N=10), were selected for benign tissues with similar epithelial:stromal ratios and grouped into pre (preM) and post-menopausal (PM) groups. Following RNA sequencing (Illumina TruSeq Stranded mRNA kit &amp; Illumina HiSeq 4000), reads were processed (MAP-RSeq v3.0.0) and aligned (STAR aligner; hg38). Differential expression (DE) analysis (edgeR 2.6.2) identified DE genes from normalized RPKM reads (absolute log2 fold change (FC) &amp;gt; 1 and false discovery rate (FDR) &amp;lt; 0.10), corrected for intra-group biases. Over-representation analysis [Ingenuity pathway analysis (IPA), Ingenuity® Systems] and gene set enrichment analysis [(GSEA), GeneTrail 2.0] identified significantly-enriched pathways. Results: In the PM group, there were 144 DE transcripts between benign ER+BC and benign ER-BC, including coding RNAs (40%), antisense RNAs (35%) and lncRNAs (7%). In contrast, the preM group had no significantly DE genes between benign ER+BC and benign ER-BC. In the PM DE coding gene set, the top DE transcripts in benign ER+BC included many genes implicated in BC development# or ER+ BC progression (*) (e.g. up-regulated: KAAG1*, DNAJB7/HSP40*, TMEM151B, ZBTB32# p &amp;lt; 0.001 ; down-regulated: CPB1*, FOS, TPPP3#, CLEC3B#p &amp;lt; 0.001). Top canonical pathways altered in benign ER+BC included MAPK, PI3K, and acute phase response pathways (p&amp;lt;0.05). GSEA of the entire gene set (N=15,223; ranked in order of 144 DE genes) identified 72 altered pathways (P &amp;lt; 0.005); those with the highest normalization enrichment scores (NES) (&amp;gt; 0.4) functionally grouped as immune function-related (T cell function and antigen presentation). Depleted pathways with NES &amp;gt; 0.4, (N=6) functionally grouped into proteasome-related, fatty acid biosynthesis and mitochondrial energy metabolism. Among the non-coding DE gene set, notably, the entire DE antisense RNA gene set (N=51 transcripts) was up-regulated in benign ER+ BC compared to benign ER-BC (P&amp;lt; 0.001) with a subset (N=11) showing marked up-regulation (&amp;gt; 4 log2FC). Among the DE antisense RNAs, 70% have reported roles in carcinogenesis or BC progression (e.g. KRT7-AS, NAV2-AS2, CCDC144NL-AS1, RP11-66B24.4, HSA-MIR4454). Conclusion: The benign breast transcriptome differs between postmenopausal women with ER+ vs ER- BC, with distinctive coding and non-coding RNA signatures. In postmenopausal women with ER+ BC, benign breast expresses a unique antisense RNA set and is enriched in genes implicated in BC development or progression. These data provide insight into at-risk benign breast and facilitate identification of potential biomarkers of carcinogenesis. Citation Format: Carter JM, Nair AA, Davila JI, Heinzen EP, Hoskin TL, Winham SJ, Radisky DC, Visscher DW, Degnim AC. A unique coding and non-coding benign breast transcriptome in post-menopausal ER+ breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-08-10.

Sex-specific gene expression differences in the prefrontal cortex of major depressive disorder individuals

The confounding effects of high genetic diversity on the determination and interpretation of differential gene expression analysis in the parasitic nematode Haemonchus contortus

BackgroundDifferential expression and feature selection analyses are essential steps for the development of accurate diagnostic/prognostic classifiers of complicated human diseases using transcriptomics data. These steps are particularly challenging due to the curse of dimensionality and the presence of technical and biological noise. A promising strategy for overcoming these challenges is the incorporation of pre-existing transcriptomics data in the identification of differentially expressed (DE) genes. This approach has the potential to improve the quality of selected genes, increase classification performance, and enhance biological interpretability. While a number of methods have been developed that use pre-existing data for differential expression analysis, existing methods do not leverage the identities of experimental conditions to create a robust metric for identifying DE genes.ResultsIn this study, we propose a novel differential expression and feature selection method—GEOlimma—which combines pre-existing microarray data from the Gene Expression Omnibus (GEO) with the widely-applied Limma method for differential expression analysis. We first quantify differential gene expression across 2481 pairwise comparisons from 602 curated GEO Datasets, and we convert differential expression frequencies to DE prior probabilities. Genes with high DE prior probabilities show enrichment in cell growth and death, signal transduction, and cancer-related biological pathways, while genes with low prior probabilities were enriched in sensory system pathways. We then applied GEOlimma to four differential expression comparisons within two human disease datasets and performed differential expression, feature selection, and supervised classification analyses. Our results suggest that use of GEOlimma provides greater experimental power to detect DE genes compared to Limma, due to its increased effective sample size. Furthermore, in a supervised classification analysis using GEOlimma as a feature selection method, we observed similar or better classification performance than Limma given small, noisy subsets of an asthma dataset.ConclusionsOur results demonstrate that GEOlimma is a more effective method for differential gene expression and feature selection analyses compared to the standard Limma method. Due to its focus on gene-level differential expression, GEOlimma also has the potential to be applied to other high-throughput biological datasets.

Introduction: High-risk neuroblastoma (H-NB) is an aggressive tumor of the sympathetic nervous system for which there are few predictors of therapeutic response. We sought to determine the association of global alternative splicing (AS) gene expression patterns with NB aggressiveness and MYCN gene status. Methods: As part of the NCI-sponsored Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative, we profiled 190 primary human NBs using Affymetrix Human Exon arrays. Tumors were obtained from children who participated in Children's Oncology Group trials with informed consent. The high-risk cohort included tumors with (H-NBA n=48) and without MYCN amplification (H-NBN n=111), a key molecular determinant of clinical aggressiveness. Twenty-four samples from low-risk NB lacking MYCN amplification (L-NB) were also profiled as a comparison group. Robust multichip analysis (RMA) was used for summarization of probe selection regions (PSR) and transcript regions. Finding isoforms using RMA (FIRMA) was used to generate PSR isoform scores. After an unsupervised pre-filtering step based on coefficient of variation, ANOVA analyses were conducted comparing H-NBA or H-NBN against the L-NB group for differential transcript and isoform expression. Last, gene set enrichment analyses (GSEA) comparing high-risk to low-risk NBs were conducted using unfiltered transcript data and the Molecular Signature Database's (MsigDB) canonical gene sets. Results: 1914 out of the 22,011 core transcripts passed our strict prefiltering criteria that required a high coefficient of variation. There was a larger number of genes with significant differential expression between H-NBA vs. L-NB than H-NBN vs. L-NB (23.5% versus 4.6%, P&amp;lt;0.001). There was also significantly larger number of PSRs exhibiting differential FIRMA scores between H-NBA vs. L-NB compared to H-NBN vs. L-NB (14.6% versus 2.3% of PSRs; P&amp;lt;0.001). NTRK1, a gene with known alternative splicing pattern among neuroblastomas, was identified in our data to have lower expression in both H-NBA and H-NBN vs. the L-NB subgroup but showed significant evidence of alternative splicing only in the H-NBA group. GSEA analyses also identified more differentially expressed pathways in H-NBA vs. L-NB than in H-NBN versus L-NB neuroblastomas (66 versus 31 pathways; P&amp;lt;0.001). Pathways related to cell cycle and axon guidance were among the top differentially expressed pathways in the H-NBA vs. L-NB comparison. Conclusion: Significant differences in gene expression and pathway expression are identified among H-NBA compared to H-NBN tumors. In addition, we show for the first time a high degree of alternative splicing events in the H-NBA tumors. These findings could aid in understanding the regulatory networks in the two subgroups of high-risk neuroblastomas and suggest possible therapeutic targets for further validation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5249.

The largemouth bass ( Micropterus salmoides ) is a commercially important freshwater aquaculture species. Understanding the molecular mechanisms of gonadal development is critical for improving broodstock quality and enabling monosex culture. However, transcriptional regulatory mechanisms, particularly alternative splicing (AS) events during gonadal differentiation, remain poorly characterized. In this study, Oxford Nanopore Technology (ONT) was employed to generate high-quality full-length transcriptomes from largemouth bass testis, ovary, and muscle tissues. A total of 37,788 unique transcripts were identified, corresponding to 22,273 genes, including 10,065 novel transcripts. Functional annotation revealed distinct transcriptional profiles between gonadal and muscle tissues, with gonadal transcripts enriched in reproductive pathways such as spermatogenesis and steroid hormone biosynthesis, while muscle transcripts displayed functional divergence in non-reproductive processes. Comparative analysis identified 14,112 differentially expressed genes between testes and ovaries, and after refining with muscle tissue controls, 939 testis-specific and 1,353 ovary-specific genes were confirmed. Notably, AS events in gonadal tissues were characterized, uncovering 581 multi-isoform genes in testes and 787 in ovaries. Key genes including dazl (testis-specific) and hsd17b1 (ovary-specific) exhibited differential transcript usage (DTU) and differential transcript expression (DTE), suggesting their roles in sex-specific functional specialization. These findings establish a comprehensive full-length transcriptomic resource for largemouth bass, demonstrate AS as a critical regulatory mechanism in gonadal differentiation, and provide a robust methodological framework for transcriptome studies in aquatic species.

e17601 Background: The oral antioxidant pterostilbene (PT) (3,5-dimethoxy-40-hydroxystilbene) has demonstrated synergistic antiproliferative effect with megestrol acetate (MA) in endometrial cancer (EC). We conducted a preoperative (window of opportunity) study enrolling endometrial cancer patients scheduled for hysterectomy, who were randomized to preoperative PT+MA vs MA alone. Methods: This prospective, randomized phase II clinical trial (NCT03671811) enrolled patients who were surgical candidates for hysterectomy. Patients were randomized 1:1 to each arm and were treated for 3 weeks with either PT+MA (arm 1) or MA alone (arm 2). Exploratory objectives included histologic response by GOG 211 criteria, and transcriptional changes using whole transcriptome analysis. For the RNA-seq analysis, differential expression analysis used the “limma” R limma package. The differentially expressed genes were selected based on the cutoff values of p-value &lt;= 0.05 and fold change &gt;= 1.5. The gene set enrichment analysis was performed using GSEA software v. 4.3.2 to identify the affected Hallmark and KEGG pathways, using the pre-ranked gene list ranked by the -log10(p-value) with a sign determined by the fold change direction. Results: This prospective, randomized phase II clinical trial (NCT03671811) enrolled patients who were surgical candidates for hysterectomy. Patients were randomized 1:1 to each arm and were treated for 3 weeks with either PT+MA (arm 1) or MA alone (arm 2). Exploratory objectives included histologic response by GOG 211 criteria, and transcriptional changes using whole transcriptome analysis. For the RNA-seq analysis, differential expression analysis used the “limma” R limma package. The differentially expressed genes were selected based on the cutoff values of p-value &lt;= 0.05 and fold change &gt;= 1.5. The gene set enrichment analysis was performed using GSEA software v. 4.3.2 to identify the affected Hallmark and KEGG pathways, using the pre-ranked gene list ranked by the -log10(p-value) with a sign determined by the fold change direction. Conclusions: In endometrial cancer, the oral antioxidant Pterostilbene increases innate immune system pathway expression, while reducing mTOR pathway expression, potentially serving as a useful adjunct to progestin hormonal therapy in EC. Clinical trial information: NCT03671811 .

BackgroundDifferential gene expression analysis is a prominent technique for the analysis of biomolecular data to identify genetic features associated with phenotypes. Limma—for microarray data –, and edgeR and DESeq2—for RNA-Seq data–, are the most widely used tools for differential gene expression analysis of bulk transcriptomic data.ResultsWe present the differential expression features of InMoose, a Python implementation of R tools limma, edgeR, and DESeq2. We experimentally show that InMoose stands as a drop-in replacement for those tools, with nearly identical results. This ensures reproducibility when interfacing both languages in bioinformatic pipelines. InMoose is an open source software released under the GPL3 license, available at www.github.com/epigenelabs/inmoose and https://inmoose.readthedocs.io.ConclusionsWe present a new Python implementation of state-of-the-art tools limma, edgeR, and DESeq2, to perform differential gene expression analysis of bulk transcriptomic data. This new implementation exhibits results nearly identical to the original tools, improving interoperability and reproducibility between Python and R bioinformatics pipelines.

Transcription changes in cells taken from bronchoalveolar fluid of COVID-19 patients indicate severe disruption of coagulation and fibrinolytic pathways in the lung and suggest similar processes in other organs.

The subgenome Saccharum spontaneum contributes to sugar accumulation in sugarcane as revealed by full-length transcriptomic analysis

The genus Peromyscus represents a rapidly diverged clade of Cricetid rodents that contains multiple cryptic species and has a propensity for morphologic conservation across its members. The unresolved relationships in previously proposed phylogenies reflect a suspected rapid adaptive radiation. To identify functional groups of genes that may be important in reproductive isolation in a reoccurring fashion across the Peromyscus phylogeny, liver and testis transcriptomes from four species (P. attwateri, P. boylii, P. leucopus, and P. maniculatus) were generated and differential expression (DE) tests were conducted. Taxa were selected to represent members diverged from a common ancestor: P. attwateri + P. boylii (clade A), and P. leucopus + P. maniculatus (clade B). Comparison of clades (A vs. B) suggested that 252 transcripts had significant DE in the liver data set, whereas significant DE was identified for 657 transcripts in the testis data set. Further, 45 genes had DE isoforms in the 657 testis transcripts and most of these functioned in major reproductive roles such as acrosome assembly, spermatogenesis, and cell cycle processes (meiosis). DE transcripts in the liver mapped to more broad gene ontology terms (metabolic processes, catabolic processes, response to chemical, and regulatory processes), and DE transcripts in the testis mapped to gene ontology terms associated with reproductive processes, such as meiosis, sperm motility, acrosome assembly, and sperm–egg fusion. These results suggest that a suite of genes that conduct similar functions in the testes may be responsible for the adaptive radiation events and potential reoccurring speciation of Peromyscus in terms of reproduction through varying expression levels.

Study question Can higher concentrations of di-2-ethylhexyl phthalate (DEHP) in follicular fluid (FF) lead to an altered microRNA expression profile? Summary answer The present study indicates the upregulation of miR-203a-3p, miR-150-5p and miR-28-3p in the follicular fluid of women with higher DEHP concentrations (FDR&amp;lt;0.05). What is known already Women are exposed daily to endocrine-disrupting chemicals (EDCs) such as phthalates which can be found in hundreds of personal care products and plastic items. In studies of rodents exposed to certain phthalates, high doses have been shown to change hormone levels, disrupt folliculogenesis and cause birth defects. Recently, we reported a strong inverse association between DEHP metabolites and ovarian sensitivity index (OSI) in females undergoing ART treatment. Several mechanisms have been proposed to explain the association between DEHP and fertility in rodents, however not much is known about the mechanisms in humans. Study design, size, duration FF was collected from 96 women undergoing IVF treatment after controlled ovarian stimulation during ovarian puncture. 48 participants from Estonia were recruited at Nova Vita Clinic AS in Tallinn, Estonia in 2019 and 48 participants from Sweden were recruited at Carl von Linnekliniken in Uppsala, Sweden in 2016. In both cohorts, patients signed a written informed consent form. Both cohorts were matched for age, BMI, parity and the number of previous IVF treatments. Participants/materials, setting, methods DEHP metabolite concentrations in the FF samples were measured in our earlier study by LC-MS/MS. Cell free RNA was extracted from FF samples with Qiagen miRNeasy Micro kit, sequencing libraries were prepared with QIAseq miRNA library kit and sequenced on Illumina NextSeq 550 instrument. Differential miRNA expression analysis was performed with the DESeq2 package. Pathway enrichment analysis for differentially expressed miRNA targets was conducted with the miEAA tool integrated with WikiPathways. Main results and the role of chance In total, 465 miRNAs were observed at least twice in ≥ 24 samples. Differential expression analysis was conducted between DEHP-high and DEHP-low groups in combined cohorts using the cohort as a covariate. 16 miRNAs were found to be differentially expressed between DEHP groups with FDR&amp;lt;0.1. Three miRNAs: miR-203a-3p, miR-150-5p and miR-28-3p, were differentially expressed with FDR&amp;lt;0.05 and were more abundant in women with high DEHP-levels in both cohorts. Of these, miR-203a-3p was negatively correlated with OSI (Pearson R = -0.27, p = 0.0089). Six miRNAs (miR-10b-5p, miR-112-5p, miR-132-5p, miR-203-3p, miR-205-5p and miR-27-3p) were found to have predicted targets in the androgen receptor signalling pathway. Limitations, reasons for caution The miRNAs found in this study need to be validated in a larger set of patients as a cohort-dependent difference in the results was observed. There is a need for further functional studies to investigate miRNAs as possible mediators of the association between DEHP and ovarian sensitivity index. Wider implications of the findings The current study presents evidence that DEHP exposure alters the expression of FF miRNAs which are associated with androgen receptor signalling. These results provide novel evidence of the potential influence of endocrine-disrupting chemicals on female reproduction. Trial registration number NA

The insulin sensitizer drug, rosiglitazone, has been shown to have a protective effect on pancreatic islet cell structure and function in animal models of type 2 diabetes. The identification of new molecular targets associated both with islet cell dysfunction and protection is a crucial research goal. In the present study, a proteomics approach has been used to identify such targets. Obese C57Bl/6J lep/lep mice and lean littermates were given the insulin sensitizer drug BRL49653, rosiglitazone. It normalized the impaired glucose tolerance in lep/lep mice but had no significant effect on glucose tolerance in the lean mice. Pancreatic islet polypeptides were arrayed by a two-dimensional gel electrophoresis system that separated more than 2500 individual spots. Three overexpressed and six underexpressed proteins were significant (p < 0.05) between lep/lep and lean mice, and four were modulated significantly (p < 0.05) by the rosiglitazone treatment of the obese mice. The identity of these differentially expressed proteins was made using mass spectrometric analysis and provided evidence that differential expression of actin-binding proteins may be an important aspect of defective islet function. Rosiglitazone increased carboxypeptidase B expression in both lep/lep and normal mice suggesting that this might be an independent effect of rosiglitazone that contributes to improved insulin processing.