Gene Ontology Biological Process Terms Research Articles

#1169 Class-specific gene expression profiling of glomerulus of lupus nephritis by spatial transcriptomics

Abstract Background and Aims Lupus nephritis exhibit various clinical features and subclassifications. Subclassification-specific alteration of transcriptomic profile of lupus nephritis needs additional study which may provide new insights regarding the subclass-specific pathophysiologic mechanism. Method Spatial transcriptomic profiling was conducted using GeoMx Digital Spatial Profiler on formalin-fixed paraffin-embedded kidney biopsy specimens obtained from control (n = 7), LN class I to V (n = 24). We also profiled native kidney primary glomerulonephritis (e.g. anti-PLA2R-associated MN) as additional disease control group. Subclass-specific transcriptomic alteration of glomerulus was compared between the subclasses and primary glomerulonephritis cases. Gene Ontology (GO) terms for DEGs were annotated using the ToppGene suite. Results We first focused on the glomerular transcriptomic alteration in class IV lupus nephritis, which was the most active form with high degree of proteinuria. We found 104 upregulated DEGs and 166 downregulated DEGs which were specifically different in the class IV lupus nephritis cases but not in other subclasses of the disease. The upregulated GO terms included interleukin 10 receptor activity, T cell activation, leukocyte activation, and vesicle membrane. On the other hand, the downregulated DEGs were mostly annotated with GO terms of double-stranded DNA binding domain. In our analysis regarding class V lupus nephritis, total of 128 DEGs were downregulated in class V LN compared to MN. These genes were also downregulated in class V LN when compared to control samples, while no significant expression differences were observed in other LN classes compared to controls. Eleven of 128 downregulated DEGs were annotated with biological process GO terms related to Golgi vesicle transport. Regarding cellular component GO terms, Golgi apparatus (30/128 DEGs), endoplasmic reticulum (ER) membrane (19/128 DEGs), and ER-Golgi intermediate compartment (6/128 DEGs) were among the annotated GO terms associated with the DEGs. DEGs in annotation included GOLGA3, TMED7, BET1L, and RAB10. Conclusion We profiled the class-specific transcriptomic changes in glomerulus of lupus nephritis. Class IV LN was associated upregulation of interleukin 10-mediated inflammatory pathway and downregulation of double stranded-DNA binding domain. The ER-Golgi network and related molecular pathway may be involved in the distinct pathogenesis and clinical features of class V LN. The findings provide novel view of the class-specific pathophysiologic mechanism of lupus nephritis.

#111 RNAseq of microdissected collecting ducts revealing the early signaling mediating the loss of aquaporin 2 after K+ deprivation

Abstract Background and Aims Potassium (K+) deficiency could cause a reduction in urinary concentrating ability, resulting in nephrogenic diabetes insipidus (NDI), but the detailed mechanism remains unclear. Recently, transcriptomic and proteomic data from acquired NDI models reveal that oxidative stress, apoptosis, and inflammatory signaling are associated with AQP2 loss. We aim to explore the early signaling after K+ deprivation in collecting ducts. Method Immunoblotting of kidney lysate were performed at 0, 12, 24, and 48 hours after K+ deprivation in rats. Serum and urine biochemistry were also recorded. Based on immunoblotting, cortical collecting ducts (CCDs) were microdissected from rats at 6 hrs after K+ deprivation versus time controls. Small samples RNA-Seq was carried out independently in K+ deprivation rats versus controls (n = 4). Results Immunoblotting of bulk kidney showed a decreased in AQP2 protein abundance at 12 hours of K+ deprivation diet, and urine osmolality was significantly decreased at 24 hours, confirming the animal model of K+ deprivation-induced NDI. Small samples RNA-Seq data of CCDs at 6 hrs after K+ deprivation showed Aqp2, Aqp3, and Atp1a1 were significantly downregulated. It also revealed that chemokine transcripts (Ccl20 and Ccl28) were increased significantly. We also carried out analysis of Gene Ontology Biological Process terms that are statistically over-represented in the list of 88 “Increased Transcripts” at 6 hrs of K+ deprivation in CCDs, and many of the terms are related to glutathione metabolic process, cell chemotaxis, cellular response to lipopolysaccharide, consistent with an inflammatory response. Conclusion Our small samples RNA-Seq from microdissected CCDs in rats showed early cellular signaling changes in activation of oxidative stress and inflammatory signaling causing loss of aquaporin-2 in K+ deficiency induced NDI.

Unveiling Gene Interactions in Alzheimer's Disease by Integrating Genetic and Epigenetic Data with a Network-Based Approach.

Alzheimer's Disease (AD) is a complex disease and the leading cause of dementia in older people. We aimed to uncover aspects of AD's pathogenesis that may contribute to drug repurposing efforts by integrating DNA methylation and genetic data. Implementing the network-based tool, a dense module search of genome-wide association studies (dmGWAS), we integrated a large-scale GWAS dataset with DNA methylation data to identify gene network modules associated with AD. Our analysis yielded 286 significant gene network modules. Notably, the foremost module included the BIN1 gene, showing the largest GWAS signal, and the GNAS gene, the most significantly hypermethylated. We conducted Web-based Cell-type-Specific Enrichment Analysis (WebCSEA) on genes within the top 10% of dmGWAS modules, highlighting monocyte as the most significant cell type (p < 5 × 10-12). Functional enrichment analysis revealed Gene Ontology Biological Process terms relevant to AD pathology (adjusted p < 0.05). Additionally, drug target enrichment identified five FDA-approved targets (p-value = 0.03) for further research. In summary, dmGWAS integration of genetic and epigenetic signals unveiled new gene interactions related to AD, offering promising avenues for future studies.

Transcriptomic profiling reveals key early response genes during GDF6-mediated differentiation of human adipose-derived stem cells to nucleus pulposus cells.

Stem cell-based therapies show promise as a means of repairing the degenerate intervertebral disc, with growth factors often used alongside cells to help direct differentiation toward a nucleus pulposus (NP)-like phenotype. We previously demonstrated adipose-derived stem cell (ASC) differentiation with GDF6 as optimal for generating NP-like cells through evaluating end-stage differentiation parameters. Here we conducted a time-resolved transcriptomic characterization of ASCs response to GDF6 stimulation to understand the early drivers of differentiation to NP-like cells. Human ASCs were treated with recombinant human GDF6 for 2, 6, and 12 h. RNA sequencing and detailed bioinformatic analysis were used to assess differential gene expression, gene ontology (GO), and transcription factor involvement during early differentiation. Quantitative polymerase chain reaction (qPCR) was used to validate RNA sequencing findings and inhibitors used to interrogate Smad and Erk signaling pathways, as well as identify primary and secondary response genes. The transcriptomic response of ASCs to GDF6 stimulation was time-resolved and highly structured, with "cell differentiation" "developmental processes," and "response to stimulus" identified as key biological process GO terms. The transcription factor ERG1 was identified as a key early response gene. Temporal cluster analysis of differentiation genes identified positive regulation NP cell differentiation, as well as inhibition of osteogenesis and adipogenesis. A role for Smad and Erk signaling in the regulation of GDF6-induced early gene expression response was observed and both primary and secondary response genes were identified. This study identifies a multifactorial early gene response that contributes to lineage commitment, with the identification of a number of potentially useful early markers of differentiation of ASCs to NP cells. This detailed insight into the molecular processes in response to GDF6 stimulation of ASCs is important for the development of an efficient and efficacious cell-based therapy for intervertebral disc degeneration-associated back pain.

Attenuated retinoic acid signaling is among the early responses in mouse uterus approaching embryo attachment.

The uterus is transiently receptive for embryo implantation. It remains to be understood why the uterus does not reject a semi-allogeneic embryo (to the biological mother) or an allogeneic embryo (to a surrogate) for implantation. To gain insights, we examined uterine early response genes approaching embryo attachment on day 3 post coitum (D3) at 22 hours when blue dye reaction, an indication of embryo attachment, had not manifested in mice. C57BL/6 pseudo-pregnant (control) and pregnant mouse uteri were collected on D3 at 22 hours for microarray analysis. The self-assembling-manifold (SAM) algorithm identified 21,858 unique probesets. Principal component analysis indicated a clear separation between the pseudo-pregnant and pregnant groups. There were 106 upregulated and five downregulated protein-coding genes in the pregnant uterus with fold change (fc) >1.5 and q value <5%. Gene ontology (GO) analysis of the 106 upregulated genes revealed 38 significant GO biological process (GOBP) terms (P <0.05), and 32 (84%) of them were associated with immune responses, with a dominant natural killer (NK) cell activation signature. Among the top eight upregulated protein-coding genes, Cyp26a1 inactivates retinoic acid (RA) while Lrat promotes vitamin A storage, both of which are expected to attenuate RA bioavailability; Atp6v0d2 and Gjb2 play roles in ion transport and transmembrane transport; Gzmb, Gzmc, and Il2rb are involved in immune responses; and Tdo2 is important for kynurenine pathway. Most of these genes or their related pathways have functions in immune regulations. RA signaling has been implicated in immune tolerance and immune homeostasis, and uterine NK cells have been implicated in immunotolerance at the maternal-fetal interface in the placenta. The mechanisms of immune responses approaching embryo attachment remain to be elucidated. The coordinated effects of the early response genes may hold the keys to the question of why the uterus does not reject an implanting embryo.

Sex‐ and APOE‐Specific Transcriptomic Signatures in Alzheimer’s Disease: An AZ‐ADRC‐Research Education Scholars Team Science Project

AbstractBackgroundAge, female sex and APOEε4 allele are among the top risk factors for developing late‐onset Alzheimer’s disease (LOAD). To enable Precision medicine for AD drug development, it is crucial targeting specific biological pathways driving AD pathology. While individual effects of sex or APOE have been studied, genes and biological pathways commonly or uniquely altered by these two factors are less understood.MethodROSMAP RNA‐Seq data were obtained from AMP‐AD including 369 frontal cortex samples from APOEε3/ε3 and APOEε3/ε4 individuals. Of these, 209 had an AD diagnosis and 160 were controls. Differentially expressed genes (DEGs, pvalue&lt;.05) between LOAD and cognitively normal individuals were identified using a generalized linear regression model. Gene Set Enrichment Analysis (GSEA) was performed using Gene Ontology Biological Processes (GOBP). Redundant GOBP enriched terms were removed computing semantic similarity measures (adjusted pvalue&lt;.05). Comparison analyses were conducted to identify common and unique DEGs and enriched GOBPs across sex‐genotype conditions.ResultAPOEε3/ε3 female brains exhibited the greatest DEG number (n = 11,851) while APOEε3/ε3 males exhibited the least (n = 714). Nine DEGs were shared across all comparisons, which were involved in metabolic processes, synaptic function, myelination, and GPCR signaling. GSEA revealed comparable numbers of LOAD‐enriched pathways in all female groups and male APOEε3/ε4s (n = 144‐155 GOBP terms), whereas APOEε3/ε3 males exhibited the fewest enriched terms (n = 13). Identified GOBP terms included pathways related to metabolism, cellular responses and regulation, and immune response regulation. However, there were no common GOBP terms across all sex‐genotype conditions.ConclusionOur analyses provide evidence of sex‐ and APOE‐ specific transcriptomic signatures and biological processes altered in LOAD brains. The findings suggest significantly greater transcriptomic dysregulation in female APOEε3/ε3 brains despite the similar extent of affected biological pathways as other sex‐APOE conditions. In contrast, a significantly smaller number of affected biological processes were identified in male APOEε3/ε3s despite having comparable transcriptomic dysregulation as the male APOε4 carriers but less than the female groups. These analyses provide rationale to develop risk factor‐specific therapeutics for the prevention and treatment of LOAD by considering the interaction between biological sex and APOE genotype.

Signatures of adaptation in myopia-related genes on the sunlight exposure hypothesis

BackgroundMyopia is a common eye disorder that results from gene-environment interactions. The prevalence of myopia varies across populations, and exposure to bright sunlight may prevent its development. We hypothesize that local adaptation to light environments during human migration played a role in shaping the genetic basis of myopia, and we aim to investigate how the environment influences the genetic basis of myopia.MethodWe utilized the whole-genome variant data of the 1000 Genomes Project for analysis. We searched myopia-associated loci that were under selection in Europeans using population branch statistics and the number of segregating sites by length statistics. The outliers of these statistics were enriched in the Kyoto Encyclopedia of Genes and Genomes pathways and the gene ontology biological process terms in searching for pathways that were under selection. We applied Bayesian inference to estimate the correlation between environmental factors and allele frequencies of the selected loci and performed causal inference of myopia using two-sample Mendelian randomization analysis.ResultsWe detected signatures of adaptation in vision and light perception pathways, supporting our hypothesis of sunlight adaptation. We discovered a strong correlation between latitude and allele frequencies in genes that are under significant selection, and we found pleiotropic effects of pigmentation or circadian rhythm genes on myopia, indicating that sunlight exposure influences the genetic diversity of myopia.ConclusionsMyopia genes involved in light perception showed signs of selection. Local adaptation during human migration shaped the genetic basis of myopia and may have influenced its global prevalence distribution.

Identification of SYT10 as a potential prognostic biomarker in esophageal cancer by comprehensive analysis of a mRNA-pseudogene/lncRNA-miRNA ceRNA network

BackgroundEsophageal carcinoma (ESCA) is often diagnosed at the advanced stages, has a poor survival rate and overall is one of the deadliest cancers world-wide. Recent studies have elaborated the significance of non-coding RNAs such as pseudogenes, long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) in cancer progression. In this study, we constructed a four-component competing endogenous RNA (ceRNA) network in ECSA and suggested an RNA with prognostic potential. Materials and methodsExpression profiles of mRNAs, pseudogenes, lncRNAs and miRNAs were collected from The Cancer Genome Atlas (TCGA) database. A ceRNA network was then constructed based on the differentially-expressed RNAs (DERs). KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology) functional analysis and PPI (Protein-Protein Interaction) network analysis was carried out on differentially-expressed (DE) mRNAs of the ceRNA network. Survival analysis was carried out on a selection of RNAs with the highest degree centrality ranks to discover potential prognostic biomarkers. ResultsA four-component ceRNA network with 529 nodes and 729 edges was constructed. The most significant GO biological process terms included positive regulation of transcription from RNA polymerase II promoter, negative regulation of transcription from RNA polymerase II promoter and positive regulation of transcription, DNA-templated. The analysis of KEGG pathways showed that DE mRNAs were significantly enriched in pathways such as pathways in cancer, PI3K-Akt signaling pathways and MicroRNAs in cancer. Among the RNAs that were found to be associated with survival, Synaptotagmin 10 (SYT10) had the highest hazard ratio and thus, proved to be a potential prognostic biomarker for ESCA. ConclusionOur study presented a four-component ceRNA network for ESCA, and identified RNA candidates that were associated with survival of ECSA. Further experimental evaluations and precise validation studies are needed for their clinical significance and roles in the progression of ESCA.

Polo-like Kinase 4: A Multifaceted Marker Linking Tumor Aggressiveness and Unfavorable Prognosis, and Insights into Therapeutic Strategies.

(1) Background: This study investigated whether polo-like kinase 4 (PLK4) is a suitable therapeutic target or biomarker for lung adenocarcinoma (LUAD). (2) Methods: We acquired LUAD data from The Cancer Genome Atlas (TCGA) database through the UCSC Xena data portal. Gene expression, clinical, survival, and mutation data from multiple samples were analyzed. Gene enrichment analysis, unsupervised clustering of PLK4-related pathways, and differential gene expression analyses were performed. Additionally, correlations, t-tests, survival analyses, and statistical analyses were performed. (3) Results: PLK4 expression was higher in LUAD tissues than in normal tissues and was associated with poor prognosis for both overall and progression-free survival in LUAD. PLK4 was highly correlated with cell-proliferation-related pathways using Gene Ontology (GO) biological process terms. PLK4 expression and pathways that were highly correlated with PLK4 expression levels were upregulated in patients with LUAD with the TP53 mutation. (4) Conclusions: PLK4 expression affects the survival of patients with LUAD and is a potential therapeutic target for LUAD with TP53 mutations.

Metabolic changes and propensity for inflammation, fibrosis, and cancer in livers of mice lacking lysosomal acid lipase

Lysosomal acid lipase (LAL) is the sole lysosomal enzyme responsible for the degradation of cholesteryl esters and triacylglycerols at acidic pH. Impaired LAL activity leads to LAL deficiency (LAL-D), a severe and fatal disease characterized by ectopic lysosomal lipid accumulation. Reduced LAL activity also contributes to the development and progression of non-alcoholic fatty liver disease (NAFLD). To advance our understanding of LAL-related liver pathologies, we performed comprehensive proteomic profiling of livers from mice with systemic genetic loss of LAL (Lal−/−) and from mice with hepatocyte-specific LAL-D (hepLal−/−). Lal−/− mice exhibited drastic proteome alterations, including dysregulation of multiple proteins related to metabolism, inflammation, liver fibrosis, and cancer. Global loss of LAL activity impaired both acidic and neutral lipase activities and resulted in hepatic lipid accumulation, indicating a complete metabolic shift in Lal−/− livers. Hepatic inflammation and immune cell infiltration were evident, with numerous upregulated inflammation-related gene ontology biological process terms. In contrast, both young and mature hepLal−/− mice displayed only minor changes in the liver proteome, suggesting that loss of LAL solely in hepatocytes does not phenocopy metabolic alterations observed in mice globally lacking LAL. These findings provide valuable insights into the mechanisms underlying liver dysfunction in LAL-D and may help in understanding why decreased LAL activity contributes to NAFLD. Our study highlights the importance of LAL in maintaining liver homeostasis and demonstrates the drastic consequences of its global deficiency on the liver proteome and liver function.

Comprehensive Transcriptome Analysis in the Testis of the Silkworm, Bombyx mori.

Spermatogenesis is an important process in reproduction and is conserved across species, but in Bombyx mori, it shows peculiarities, such as the maintenance of spermatogonia by apical cells and fertilization by dimorphic spermatozoa. In this study, we attempted to characterize the genes expressed in the testis of B. mori, focusing on aspects of expression patterns and gene function by transcriptome comparisons between different tissues, internal testis regions, and Drosophila melanogaster. The transcriptome analysis of 12 tissues of B. mori, including those of testis, revealed the widespread gene expression of 20,962 genes and 1705 testis-specific genes. A comparative analysis of the stem region (SR) and differentiated regions (DR) of the testis revealed 4554 and 3980 specific-enriched genes, respectively. In addition, comparisons with D. melanogaster testis transcriptome revealed homologs of 1204 SR and 389 DR specific-enriched genes that were similarly expressed in equivalent regions of Drosophila testis. Moreover, gene ontology (GO) enrichment analysis was performed for SR-specific enriched genes and DR-specific enriched genes, and the GO terms of several biological processes were enriched, confirming previous findings. This study advances our understanding of spermatogenesis in B. mori and provides an important basis for future research, filling a knowledge gap between fly and mammalian studies.

Construction of chronic glomerulonephritis‑related lncRNA‑mRNA regulatory network and lncRNA‑-miRNA‑mRNA ceRNA network by bioinformatics analysis.

Long non-coding RNAs (lncRNAs) are ncRNA transcripts >200 nucleotides that are important genetic regulators. LncRNAs can directly regulate mRNA through a lncRNA-mRNA regulatory mode and can also regulate mRNA through competitive binding to micro (mi)RNA, which is generally known as the competitive endogenous RNA (ceRNA) network. The present study evaluated the functional roles and regulatory networks of lncRNAs in chronic glomerulonephritis (CGN). The proliferative ability of mouse glomerular mesangial cells (GMCs) induced by different concentrations of lipopolysaccharide (LPS) was assessed using the Cell Counting Kit-8 assay, and RNA sequencing (RNA-seq) was performed to identify differentially expressed lncRNAs in LPS-induced GMCs. Based on the sequencing results, six lncRNAs were selected for validation using reverse transcription-quantitative PCR (RT-qPCR). Furthermore, the lncRNA-mRNA regulatory network and the lncRNA-miRNA-mRNA ceRNA network were constructed to assess the role and mechanism of CGN-related lncRNAs. To elucidate the biological functions of lncRNAs, Gene Ontology (GO) biological process term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on all mRNAs involved in the lncRNA-mRNA regulatory network and in the ceRNA network. A total of 1,532 differentially expressed lncRNAs, including 594 upregulated lncRNAs and 938 downregulated lncRNAs, were identified using RNA-seq. The results of RT-qPCR validation were consistent with RNA-seq results. An lncRNA-mRNA regulatory network, including 236 lncRNAs and 556 mRNAs, and a ceRNA network, including 6 lncRNAs, 18 miRNAs and 419 mRNAs, were successfully constructed. The GO biological process term enrichment and KEGG pathway enrichment analyses demonstrated that those lncRNAs were often related to inflammatory response and substance metabolism. The present study identified key CGN-related lncRNAs in LPS-induced GMCs, and further demonstrated a global view of the lncRNA-mRNA regulatory network and ceRNA network involved in CGN. These results offered novel insights into the roles of lncRNAs in the pathogenesis of CGN and identified potential diagnostic biomarkers.

Aberrant Expression of GABA-Related Genes in the Hippocampus of 3xTg-AD Model Mice from the Early to End Stages of Alzheimer's Disease.

We explored the gene expression levels in the brain of 3xTg-AD model mice to elucidate the molecular pathological changes from the early to end stages of Alzheimer's disease (AD). We re-analyzed our previously published microarray data obtained from the hippocampus of 3xTg-AD model mice at 12 and 52 weeks of age. Functional annotation and network analyses of the up- and downregulated differentially expressed genes (DEGs) in mice aged 12 to 52 weeks were performed. Validation tests for gamma-aminobutyric acid (GABA)-related genes were also performed by quantitative polymerase chain reaction (qPCR). In total, 644 DEGs were upregulated and 624 DEGs were downregulated in the hippocampus of both the 12- and 52-week-old 3xTg-AD mice. In the functional analysis of the upregulated DEGs, 330 gene ontology biological process terms, including immune response, were found, and they interacted with each other in the network analysis. In the functional analysis of the downregulated DEGs, 90 biological process terms, including several terms related to membrane potential and synapse function, were found, and they also interacted with each other in the network analysis. In the qPCR validation test, significant downregulation was seen for Gabrg3 at the ages of 12 (p = 0.02) and 36 (p = 0.005) weeks, Gabbr1 at the age of 52 weeks (p = 0.001), and Gabrr2 at the age of 36 weeks (p = 0.02). Changes in immune response and GABAergic neurotransmission may occur in the brain of 3xTg mice from the early to end stages of AD.

Blood-based transcriptional signatures at hospital admission are associated with cardiac magnetic resonance markers of STEMI prognosis

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Italian Ministry of Health. Background While mortality following ST-segment elevation myocardial infarction (STEMI) is declining, the number of patients developing heart failure due to STEMI is on the rise. Cardiovascular magnetic resonance (CMR) has emerged as an important imaging modality for assessing parameters of the short-term outcome, which add incremental prognostic value above traditional outcome markers alone in acute reperfused STEMI. Prognostic relevant pathways of leukocyte involvement in STEMI outcome are largely unknown. Aim We sought to identify a set of circulating cellular transcripts measured on hospital admission that predict the short-term outcome of STEMI patients as assessed by CMR markers. Methods Thirty consecutive patients (24 males, 6 females, age 61 ± 10 years) admitted with STEMI at our Centre between 2012 and 2015 were enrolled in this retrospective pilot study. The whole-blood transcriptome was analysed by RNA-sequencing using total RNA isolated from peripheral blood samples drawn on hospital admission. Patients were studied with a 1.5T MR scanner within 1 week after primary PCI, and late gadolinium enhancement (LGE) mass and myocardial salvage index (MSI) were measured. K-means clustering was performed to group the samples according to the distribution of values of each CMR variable (i.e., high, medium, and low levels). We used the generalized negative binomial linear model approach with the edgeR package to perform gene-level differential expression analysis among the 3 groups of each CMR variable. To infer the biological functions of genes associated with CMR parameters, we used Gene Ontology Biological Processes terms for Gene Set Enrichment Analysis. To identify the smallest set of genes that can discriminate between groups of patients, we exploited a class of adaptive heuristic search algorithms using the GARS package. Results We identified specific gene expression patterns at baseline associated with high, medium, or low LGE and MSI values at 1-week follow-up. The most representative processes associated with high values of LGE were suggestive of adaptive immune response mediated by T- and B-cells, while innate immune response pathways were associated with medium-lower LGE values. We observed an association of low MSI values with adaptive immune response processes; high-medium values of MSI were, instead, associated with inflammatory-related functions. As for predictors of CMR surrogate markers of patient’s outcome, we identified a set of 13 genes that classified the three LGE mass groups with an accuracy of 91%. Similarly, we identified 13 specific genes that discriminated the three MSI groups with a 100% accuracy. Conclusions We unveiled through RNA-sequencing data mining a set of informative transcriptional features that predict CMR phenotypes after STEMI. Overall, our results could pave the way for the identification of novel blood-based biomarkers to improve early prognosis and therapeutic decision-making in STEMI.

Abstract A006: Chromatin remodeling as a potential epigenetic mechanism of tolerance to KRAS loss

Abstract KRAS-targeted inhibition yielded promising, yet far from ideal, clinical responses, revealing that cancer cells easily bypass KRAS loss. Therefore, we aim to understand how mutant KRAS cancer cells tolerate so well the loss of a key oncogene to which they were addicted to. KRAS expression was silenced through siRNAs in colorectal cancer (CRC) cell lines that carry KRAS mutations. The proteome characterization was obtained by mass spectrometry and the expression of significantly altered proteins was validated by western blotting. Chromatin states were investigated using electron microscopy. Upon KRAS inhibition, there was a significant reduction in the cell number, accompanied by changes in cell cycle, thus supporting KRAS-dependency. Proteomics analysis revealed that KRAS inhibition-tolerant cells upregulated several proteins associated with the extracellular exosome or nuclear compartments. Molecular function and biological process gene ontology terms revealed an up-regulation of proteins mainly associated with binding activities (RNA, protein, nucleosome, and core promoter binding) as well as gene expression regulation, mRNA splicing and processing, and nucleosome assembly and repositioning. In addition, the proteomics data also revealed an upregulation of proteins associated with active chromatin states. Moreover, KRAS-silenced persister cells also presented alterations in some histone post-translational modifications and in chromatin packing, both suggesting that transcription is impacted. Overall, our results suggest an epigenetic mechanism underlying tolerance to KRAS inhibition that involves chromatin structural changes and transcription alterations, which we are currently pursuing. Citation Format: Flavia Martins, Ana L. Machado, Patricia D. Carvalho, Joana Carvalho, Rune Matthiesen, Vadim Backman, Sergia Velho. Chromatin remodeling as a potential epigenetic mechanism of tolerance to KRAS loss [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A006.

Integrated transcriptomic and proteomic analysis of a cytoplasmic male sterility line and associated maintainer line in soybean.

Heterosis is a critical phenomenon in crop improvement. Cytoplasmic male sterility (CMS) and Restorer gene (Rf) systems are essential components for heterosis-based breeding. However, the molecular mechanism underlying CMS remains largely unclear in soybean. We integrated a morphological investigation with comparative analyses of transcriptomic and proteomic changes in pollen from the CMS line W931A and its maintainer line, W931B, at the uninucleate microspore (UM) and binucleate pollen (BP) stages. Compared to W931B, which had healthy, oval pollen grains, W931A showed shrunken or degraded pollen grains with an irregularly thickened endothelium and decreased starch accumulation. Transcriptomic comparisons revealed a total of 865 differentially expressed genes (DEGs) in W931A over the two stages. These genes were primarily associated with pentose and glucuronate interconversions, sphingolipid metabolism, and glycerolipid metabolism. Proteomic analysis revealed 343 differentially expressed proteins (DEPs), which were mainly involved in carbon metabolism, glycolysis/gluconeogenesis, and nitrogen metabolism. Consistently, Gene Ontology (GO) biological process terms related to pollen development were enriched among DEGs at the UM and BP stages. Notably, four genes with demonstrated roles in pollen development were differentially expressed, including AGAMOUS-LIKE 104, PROTEIN-TYROSINE-PHOSPHATASE 1, and PHOSPHOLIPASE A2. A total of 53 genes and the corresponding proteins were differentially expressed in W931A at both the UM and BP stages, and many of these were pectinesterases, polygalacturonases, peroxidases, and ATPases. The results of this study suggest that pollen development in W931A is likely regulated through suppression of the identified DEGs and DEPs. These findings increase our understanding of the molecular mechanism underlying CMS in soybean, aiding future research into soybean fertility and promoting the efficient use of heterosis for soybean improvement.

Molecular and physiological mechanisms of tea (Camellia sinensis (L.) O. Kuntze) leaf and root in response to nitrogen deficiency

BackgroundAs an economically important crop, tea is strongly nitrogen (N)-dependent. However, the physiological and molecular mechanisms underlying the response of N deficiency in tea are not fully understood. Tea cultivar “Chunlv2” [Camellia sinensis (L.) O. Kuntze] were cultured with a nutrient solution with 0 mM [N-deficiency] or 3 mM (Control) NH4NO3 in 6 L pottery pots containing clean river sands.ResultsN deficiency significantly decreased N content, dry weight, chlorophyll (Chl) content, L-theanine and the activities of N metabolism-related enzymes, but increased the content of total flavonoids and polyphenols in tea leaves. N deficiency delayed the sprouting time of tea buds. By using the RNA-seq technique and subsequent bioinformatics analysis, 3050 up-regulated and 2688 down-regulated differentially expressed genes (DEGs) were isolated in tea leaves in response to N deficiency. However, only 1025 genes were up-regulated and 744 down-regulated in roots. Gene ontology (GO) term enrichment analysis showed that 205 DEGs in tea leaves were enriched in seven GO terms and 152 DEGs in tea roots were enriched in 11 GO items based on P < 0.05. In tea leaves, most GO-enriched DEGs were involved in chlorophyll a/b binding activities, photosynthetic performance, and transport activities. But most of the DEGs in tea roots were involved in the metabolism of carbohydrates and plant hormones with regard to the GO terms of biological processes. N deficiency significantly increased the expression level of phosphate transporter genes, which indicated that N deficiency might impair phosphorus metabolism in tea leaves. Furthermore, some DEGs, such as probable anion transporter 3 and high-affinity nitrate transporter 2.7, might be of great potential in improving the tolerance of N deficiency in tea plants and further study could work on this area in the future.ConclusionsOur results indicated N deficiency inhibited the growth of tea plant, which might be due to altered N metabolism and expression levels of DEGs involved in the photosynthetic performance, transport activity and oxidation–reduction processes.

Comparative Transcriptome Analysis of Tolerant and Sensitive Genotypes of Common Bean (Phaseolus vulgaris L.) in Response to Terminal Drought Stress

We conducted a genome-wide transcriptomic analysis of three drought tolerant and sensitive genotypes of common bean to examine their transcriptional responses to terminal drought stress. We then conducted pairwise comparisons between the root and leaf transcriptomes from the resulting tissue based on combined transcriptomic data from the tolerant and sensitive genotypes. Our transcriptomic data revealed that 491 (6.4%) DEGs (differentially expressed genes) were upregulated in tolerant genotypes, whereas they were downregulated in sensitive genotypes; likewise, 396 (5.1%) DEGs upregulated in sensitive genotypes were downregulated in tolerant genotypes. Several transcription factors, heat shock proteins, and chaperones were identified in the study. Several DEGs in drought DB (data Base) overlapped between genotypes. The GO (gene ontology) terms for biological processes showed upregulation of DEGs in tolerant genotypes for sulfate and drug transmembrane transport when compared to sensitive genotypes. A GO term for cellular components enriched with upregulated DEGs for the apoplast in tolerant genotypes. These results substantiated the temporal pattern of root growth (elongation and initiation of root growth), and ABA-mediated drought response in tolerant genotypes. KEGG (kyoto encyclopedia of genes and genomes) analysis revealed an upregulation of MAPK (mitogen activated protein kinase) signaling pathways and plant hormone signaling pathways in tolerant genotypes. As a result of this study, it will be possible to uncover the molecular mechanisms of drought tolerance in response to terminal drought stress in the field. Further, genome-wide transcriptomic analysis of both tolerant and sensitive genotypes will assist us in identifying potential genes that may contribute to improving drought tolerance in the common bean.

Transcriptome profile of the anterior pituitary gland in pigs during maternal recognition of pregnancy

The activity of the pituitary gland determines the success of female reproduction. The knowledge regarding the reproductive-status-related changes in the transcriptome of the porcine pituitary is limited. This study aimed to determine and compare the transcriptome profile of the pituitary gland collected from pigs during maternal recognition of pregnancy, i.e. on days 12–13 of pregnancy and during the respective days of the estrous cycle. Analysis indicated 482 differentially expressed genes (DEGs) with an FC > 1.5 (P < 0.05) in the pituitary of pregnant vs. estrous-cyclic pigs. Among them, 68 were up-regulated and 414 were down-regulated. The evaluated DEGs were annotated into 39 gene ontology (GO) biological process terms, 13 GO cellular component terms, and 10 GO molecular function terms. Among the evaluated DEGs were selected genes coding for proteins potentially involved in the regulation of early pregnancy in pigs and used for gene interaction analysis and validation of microarray results. An analysis of the relationships among DEGs in pituitaries collected during maternal recognition of pregnancy showed that some of them are connected with, for example, TGFβ signaling pathway, PRL synthesis, adipocytokines pathway and immune response during maternal recognition of pregnancy. These findings expand the knowledge regarding the molecular mechanisms appearing in the porcine pituitary during the maternal recognition period of pregnancy.

Proximity Proteomics Identifies Network of Lipid Metabolism Regulators in JAK2 V617F Interactome

Background: Janus kinase 2 (JAK2) is a non-receptor tyrosine kinase that activates transcriptional programs of cell survival and proliferation, through signal transducers and activators of transcription (STAT) family of transcription factors, in response to stimulation by growth factors and cytokines. Upregulation of JAK/STAT signaling is common in myeloproliferative neoplasms (MPNs), which are also clinically associated with accelerated catabolism (weight loss, exhaustion). JAK2 inhibitors can improve these symptoms and improve quality of life for patients with MPNs, but they are unable to eradicate disease-initiating malignant hematopoietic stem cells, indicating that our understanding of the molecular pathophysiology of MPNs is incomplete and that new therapeutically targetable mechanisms need to be uncovered to improve therapy. Objective: To better understand signaling patterns implicating JAK2 V617F in malignant transformation, we interrogated the JAK2 WT and JAK2 V617F cellular interactomes using proximity dependent labeling with biotin (PDL) and mass spectrometry. Methods: Retroviral (MSCV-IRES-GFP) vectors expressing JAK2 WT or JAK2 V617F fused with biotin ligase (TurboID, PMID: 30125270) were established and used to transduce murine myeloid FDC-P1 cells. FDC-P1 cell lines stably expressing TurboID alone, TurboID-JAK2 WT, or TurboID-JAK2 V617F were incubated with 50 μM biotin for 3 hours. Cells were lysed and biotinylated proteins were pulled down using streptavidin conjugated beads. On-bead trypsin digestion followed by mass spectrometry were performed to determine a pool of biotinylated proteins representing a network of proximal interactors of TurboID, TurboID-JAK2 WT, or TurboID-JAK2 V617F. Significantly enriched proximal interacting proteins identified in TurboID-JAK2 V617F or TurboID-JAK2 WT pulldowns were designated as having 1.5-fold more enrichment (FDR ≤ 0.05) over TurboID, and showing 1.5-fold difference in enrichment (FDR ≤ 0.05) between TurboID-JAK2 V617F and TurboID-JAK2 WT. Results: 441 proteins were identified to be significantly enriched in TurboID-JAK2 WT over TurboID control and significantly enriched over TurboID-JAK2 V617F. 330 proteins were significantly enriched in TurboID-JAK2 V617F over TurboID control and significantly enriched over TurboID-JAK2 WT. Bioinformatic analyses of proteins more enriched by TurboID-JAK2 WT or TurboID-JAK2 V617F both identified hematopoietic system as a significant tissue, and nucleoside triphosphatase activity as a significant gene ontology (GO) molecular function. Top 10 GO biological process terms enriched exclusively in TurboID-JAK2 WT included "Positive regulation of establishment of protein localization to telomere", and "Glucose catabolic process" (Panel 1). GO biological process terms exclusively enriched in TurboID-JAK2 V617F included "Cellular response to interleukin-3", "Fatty acid beta-oxidation", and "Mitochondrial gene expression" (Panel 1). See table in Panel 1 for list of proteins assigned to significantly enriched GO biological process terms. Conclusions: Our data indicate that JAK2 V617F is associated with changes in cellular energy metabolism, characterized by increased association with proteins involved in fatty acid beta-oxidation, and decreased association with proteins involved in glucose metabolism. These data are consistent with the report of Skoda group showing that expression of JAK2 V617F resulted in systemic metabolic changes including hypoglycemia, adipose tissue atrophy, and early mortality (PMID: 31511238). Our study identifies new potential JAK2 V617F interactors involved in regulation of fatty-acid oxidation and mitochondrion linked processes. Further studies are warranted to determine druggability of the newly identified proximal interactors to revert JAK2 V617F-dependent pathologies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal