Analysis Of Gene Expression Changes Research Articles

The involvement of RIPK4 in TNF-α-stimulated IL-6 and IL-8 production by melanoma cells

PurposeThe receptor-interacting protein kinase (RIPK4) has an oncogenic function in melanoma, regulates NF-κB and Wnt/β-catenin pathways, and is sensitive to the BRAF inhibitors: vemurafenib and dabrafenib which lead to its decreased level. As its role in melanoma remains not fully understood, we examined the effects of its downregulation on the transcriptomic profile of melanoma.MethodsApplying RNA-seq, we revealed global alterations in the transcriptome of WM266.4 cells with RIPK4 silencing. Functional partners of RIPK4 were evaluated using STRING and GeneMANIA databases. Cells with transient knockdown (via siRNA) and stable knockout (via CRISPR/Cas9) of RIPK4 were stimulated with TNF-α. The expression levels of selected proteins were assessed using Western blot, ELISA, and qPCR.ResultsGlobal analysis of gene expression changes indicates a complex role for RIPK4 in regulating adhesion, migration, proliferation, and inflammatory processes in melanoma cells. Our study highlights potential functional partners of RIPK4 such as BIRC3, TNF-α receptors, and MAP2K6. Data from RIPK4 knockout cells suggest a putative role for RIPK4 in modulating TNF-α-induced production of IL-8 and IL-6 through two distinct signaling pathways—BIRC3/NF-κB and p38/MAPK. Furthermore, increased serum TNF-α levels and the correlation of RIPK4 with NF-κB were revealed in melanoma patients.ConclusionThese data reveal a complex role for RIPK4 in regulating the immune signaling network in melanoma cells and suggest that this kinase may represent an alternative target for melanoma-targeted adjuvant therapy.

Obesogenic Diet in Mice Leads to Inflammation and Oxidative Stress in the Mother in Association with Sex-Specific Changes in Fetal Development, Inflammatory Markers and Placental Transcriptome.

Obesity during pregnancy is related to adverse maternal and neonatal outcomes. Factors involved in these outcomes may include increased maternal insulin resistance, inflammation, oxidative stress, and nutrient mishandling. The placenta is the primary determinant of fetal outcomes, and its function can be impacted by maternal obesity. The aim of this study on mice was to determine the effect of obesity on maternal lipid handling, inflammatory and redox state, and placental oxidative stress, inflammatory signaling, and gene expression relative to female and male fetal growth. Female mice were fed control or obesogenic high-fat/high-sugar diet (HFHS) from 9 weeks prior to, and during, pregnancy. On day 18.5 of pregnancy, maternal plasma, and liver, placenta, and fetal serum were collected to examine the immune and redox states. The placental labyrinth zone (Lz) was dissected for RNA-sequencing analysis of gene expression changes. the HFHS diet induced, in the dams, hepatic steatosis, oxidative stress (reduced catalase, elevated protein oxidation) and the activation of pro-inflammatory pathways (p38-MAPK), along with imbalanced circulating cytokine concentrations (increased IL-6 and decreased IL-5 and IL-17A). HFHS fetuses were asymmetrically growth-restricted, showing sex-specific changes in circulating cytokines (GM-CSF, TNF-α, IL-6 and IFN-γ). The morphology of the placenta Lz was modified by an HFHS diet, in association with sex-specific alterations in the expression of genes and proteins implicated in oxidative stress, inflammation, and stress signaling. Placental gene expression changes were comparable to that seen in models of intrauterine inflammation and were related to a transcriptional network involving transcription factors, LYL1 and PLAG1. This study shows that fetal growth restriction with maternal obesity is related to elevated oxidative stress, inflammatory pathways, and sex-specific placental changes. Our data are important, given the marked consequences and the rising rates of obesity worldwide.

The host transcriptional response to superinfection by influenza A virus and Streptococcus pneumoniae.

Secondary bacterial infections are the most frequent complications during influenza A virus (IAV) pandemic outbreaks, contributing to excessive morbidity and mortality in the human population. Most IAV-related deaths are attributed to Streptococcus pneumoniae (SP) infections, which usually begin within the first week of IAV infection in the respiratory tracts. Here, we focused on longitudinal transcriptional responses during a superinfection model consisting of an SP infection that follows an initial IAV infection, comparing superinfection to an IAV-only infection, an SP-only infection, and control treatments. Our longitudinal data allowed a fine analysis of gene expression changes during superinfection. For instance, we found that superinfected mice exhibited rapid gene expression induction or reduction within the first 12 h after encountering the second pathogen. Cell proliferation and immune response activation processes were upregulated, while endothelial processes, vasculogenesis, and angiogenesis were downregulated, providing promising targets for future therapeutic interventions. We further analyzed the longitudinal transcriptional responses in the context of a previously defined spectrum of the host's resistance state, revealing superinfection-specific reprogramming of resistance states, such as reprogramming of fatty acid metabolism and interferon signaling. The reprogrammed functions are compelling new targets for switching the pathogenic superinfection state into a single-infection state.

KDM4C promotes mouse hippocampal neural stem cell proliferation through modulating ApoE expression.

KDM4C is implicated in the regulation of cell proliferation, differentiation, and maintenance in various stem cell types. However, its function in neural stem cells (NSCs) remains poorly understood. Therefore, this study aims to investigate the role and regulatory mechanism of KDM4C in NSCs. Primary hippocampal NSCs were isolated from neonatal mice, and both invivo and invitro lentivirus-mediated overexpression of KDM4C were induced in these hippocampal NSCs. Staining results revealed a significant increase in BrdU- and Ki-67-positive cells, along with an elevated number of cells in S phases due to KDM4C overexpression. Subsequently, RNA-seq was employed to analyze gene expression changes following KDM4C upregulation. GO enrichment analysis, KEGG analysis, and GSEA highlighted KDM4C-regulated genes associated with development, cell cycle, and neurogenesis. Protein-protein interaction analysis uncovered that ApoE protein interacts with several genes (top 10 upregulated and downregulated) regulated by KDM4C. Notably, knocking down ApoE mitigated the proliferative effect induced by KDM4C overexpression in NSCs. Our study demonstrates that KDM4C overexpression significantly upregulates ApoE expression, ultimately promoting proliferation in mouse hippocampal NSCs. These findings provide valuable insights into the molecular mechanisms governing neurodevelopment, with potential implications for therapeutic strategies in neurological disorders.

Pyromeconic acid-enriched Erigeron annuus water extract as a cosmetic ingredient for itch relief and anti-inflammatory activity

Erigeron annuus (EA), traditionally used to treat disorders such as diabetes and enteritis, contains a variety of chemicals, including caffeic acid, flavonoids, and coumarins, providing antifungal and antioxidative benefits. However, the ingredients of each part of the EA vary widely, and there are few reports on the functionality of water extracts in skin inflammation and barrier protection. We assessed the therapeutic properties of the extract of EA without roots (EEA) and its primary ingredient, pyromeconic acid (PA), focusing on their antihistamine, anti-inflammatory, and antioxidative capabilities using HMC-1(human mast cells) and human keratinocytes (HaCaT cells). Our findings revealed that histamine secretion, which is closely related to itching, was notably reduced in HMC-1 cells following pretreatment with EEA (0.1% and 0.2%) and PA (corresponding concentration, 4.7 of 9.4 µg/mL). Similarly, they led to a marked decrease in the levels of pro-inflammatory cytokines, including IL-1β, IL-8, IL-6, and IFN-γ. Furthermore, EA and PA enhanced antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), reduced malondialdehyde (MDA) production, and showed reactive oxygen species (ROS) scavenging activity in HaCaT cells. Moreover, at the molecular level, elevated levels of the pro-inflammatory cytokines IL-1β, IL-6, TARC, and MDC induced by TNF-α/IFN-γ in HaCaT cells were mitigated by treatment with EEA and PA. We also revealed the protective effects of EEA and PA against SDS-induced skin barrier dysfunction in HaCaT cells by enhancing the expression of barrier-related proteins. Using NanoString technology, a comprehensive analysis of gene expression changes indicated significant modulation of autoimmune and inflammatory genes by EEA and PA. In summary, this study suggests that EEA and the corresponding concentration of PA as an active ingredient have functional cosmetic applications to alleviate itching and improve skin health.

Analysis of gene expression changes during lipid droplet formation in HepG2 human liver cancer cells.

Fatty liver is a condition of excessive triglyceride accumulation in hepatocytes. Additionally, hepatocytes exhibit a high degree of fat droplet accumulation during excessive alcohol consumption and metabolic syndrome. However, the molecular mechanisms involved in fat droplet formation remain unknown. The present study used an in vitro fatty liver formation model of the human liver cancer cell line, HepG2, to comprehensively search for fat droplet formation-related genes, and which exhibit changes in expression during fat droplet formation. Microarray analysis with extracted total RNA determined the genes that are involved in fat droplet formation and their expression was confirmed using quantitative polymerase chain reaction following the culture of the HepG2 cells in culture medium containing 0, 50, 200 and 500 µM of oleic acid for 24 h. The results revealed 142 genes demonstrating increased expression levels by >2.0-fold with oleic acid treatment and 426 genes demonstrating decreased expression levels. Perilipin 2 (PLIN2) was estimated as the gene most closely associated with fatty liver. Lipid droplet formation in the HepG2 cells induced by oleic acid led to the upregulation of PLIN2 in a concentration-dependent manner. On the whole, the findings of the present study indicate the involvement of genes in oleic acid-induced lipid droplet formation in HepG2 cells; PLIN2 in particular may play a crucial role in this process.

Abstract B101: Obesity mediates altered triple-negative breast cancer tumor biology in African American women independent of ancestry

Abstract African American (AA) breast cancer (BC) is associated with a higher incidence of aggressive BC phenotypes at younger age, higher grade of tumor as compared to BCs of age-matched patients of European American (EA). For AA women, there is a higher frequency of the more aggressive TNBC subtype and of the PAM50-molecular basal subtype. AA tumors are enriched for inflammatory and immune pathways, implicating inherited susceptibility associated with African ancestry. Obesity induces a state of low-grade inflammation. AA women tend to be more obese than EA and have elevated a inflammatory markers, including CRP, IL6, and IL1. Since inflammation related to obesity is a reflection of dysfunctional adipocyte biology, we used dataset GSE88837 to perform in silico analysis of gene expression changes in visceral adipose tissue for AAs and EAs. AA adipose tissue is more inflamed than EAs at the same level of obesity (BMI>30). There was a high expression of secreted mediators causing systemic inflammation, including heat shock protein 60 (HSPD1), CXCL1, S100A9, and SPP1. VEGFA, S100A9, and SPP, tumor-promoting angiogenic mediators, were higher in obese AAs than EAs. These data indicate that obesity enhances inflammation in visceral adipose tissue of AAs. We performed in silico analysis of dataset GSE78958, of 400 BC patients annotated for race, BMI, and hormone receptor expression. We analyzed BC patients for differential gene expression, PAM50 subtype, tumor stage and grade, based on race and BMI. AA women have a higher BMI (48.9%) than EA women (32.3%) and a higher percentage of poorly differentiated tumors (51.1%) than EAs (40.6%). AA women with BMI >30, had more basal-like tumors (34.8%) than EA women (19.3%). These analyses confirm that excess adiposity promotes the basal BC phenotype. With BMI >30 as a criterion for obesity, the GSE78958 dataset containing BCs for 96 AA and 200 EA patients was analyzed for differences in gene expression. DEGs showed upregulation of T cell exhaustion marker PTPN22 and LAIR2. The metastasis and proliferation promoting chemokines CXCL1, CCL2, CXC12, CXCR4, and S100A9 were higher in the BCs of obese AAs, than BCs of non-obese AAs. Stemness related genes, STAT1, SOX4, and KLF4, were higher in BCs of obese AAs. CD163-expressing M2 macrophages were higher in BCs of obese AAs. Our group showed that there was a considerable overlap of genes among African and AA cases, but there were some non-overlapping genes indicating that ancestry alone cannot explain the genetic diversity in the tumor phenotype of AA patients. The divergence was largely driven by an upregulated gene signature found among AAs and not seen among African patients. GSE78958 (obesity BC dataset) was mapped for ancestral correlation of altered genes in the AA BC dataset. Ancestry mapping showed an inverse correlation of HSPD1 with ancestry. Obesity upregulated HSPD1 in BCs of AAs as well as in AA adipose tissue and decreased the probability of survival for women of AA ethnicity, suggesting delineation of ancestry-specific and race-specific effects. Citation Format: Deepa Bedi, Rachel Martini, Melissa B. Davis, Clayton Yates. Obesity mediates altered triple-negative breast cancer tumor biology in African American women independent of ancestry [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B101.

Abstract B083: Secondary KRAS mutations lead to acquired resistance to KRASG12D inhibitor in colorectal cancer

Abstract KRAS is the most frequently mutated oncogene, with high prevalence in tumor types with a significant unmet clinical need, such as colorectal cancer (CRC). Activating mutations in the KRAS gene leading to amino acid exchange at position 12 from Glycine to Asparagine, G12D, occur in approximately 12.5% of CRC. The approval of mutant specific KRASG12C inhibitors MRTX849 and AMG510 by the FDA raised the question if KRAS targeted therapy approaches could provide benefit to cancer patients with other mutant alleles. Several KRASG12D inhibitors have recently been described and MRTX1133, a selective KRASG12D inhibitor from Mirati Therapeutics, has now entered clinical trial, recruiting patients with advanced solid tumors harboring a KRAS G12D mutation. Alongside the great hopes placed in KRAS targeting therapy, resistance is likely to occur, as it was observed in patients relapsing in response to KRASG12C inhibitors. We aimed to use this pre-clinical study to predict and understand potential resistance mechanisms that may arise during targeted treatment against KRASG12D in the CRC setting. To this aim, the colorectal cancer cell line GP2d was continuously treated with MRTX1133 to generate resistance and individual outgrowing clones were subsequently profiled in a series of assays to identify potential mechanism(s). These assays included analysis of gene expression changes, mutation calling, as well as qPCR and protein-based assays. Interestingly, we identified two individual secondary on-target mutations in the KRAS gene, which could affect binding of MRTX1133 to the pocket in KRAS. These findings were confirmed in an orthogonal assay, by screening for resistance in Ba/F3 cells transduced with the corresponding KRAS double mutant. Combination assays were then performed in parental and resistant cells to study whether additional vertical or alternative pathway inhibition could address the resistance. Collectively our pre-clinical study identified secondary KRAS mutations in the compound binding pocket as a mechanism of resistance to KRAS G12D inhibition by MRTX1133 in colorectal cancer cells. Citation Format: Simone Lieb, Sabine Jurado, Sergej Nowoshilow, Krzysztof M. Zak, Marco H. Hofmann. Secondary KRAS mutations lead to acquired resistance to KRASG12D inhibitor in colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B083.

Blockade of interleukin-6 trans-signaling prevents mitochondrial dysfunction and cellular senescence in retinal endothelial cells

Interleukin-6 (IL-6) is a multifaceted cytokine implicated in the pathogenesis of diabetic retinopathy (DR). Its activity extends through cis- and trans-signaling (TS) pathways, with cis-signaling limited to specific cell types possessing the membrane-bound IL-6 receptor, while trans-signaling broadly activates various cells without the membrane bound IL-6 receptor, including retinal endothelial cells. In this study, we determined the effects of interleukin-6 trans-signaling on mitochondrial dysfunction and cellular senescence in human retinal endothelial cells (HRECs). HRECs were cultured and treated with IL-6 + soluble IL-6R or Hyper IL-6 to activate trans-signaling, along with sgp130Fc for inhibition. RT-PCR was used to analyze gene expression changes associated with inflammation and senescence. Cellular senescence was assessed using SA β-gal staining. Mitochondrial function was evaluated using Seahorse XFe24 Bioanalyzer. IL-6 trans-signaling induced inflammatory gene expression as indicated by the upregulation of ICAM1, MCP1, and SERPINA3 levels. Additionally, it reduced mitochondrial respiration and oxidative phosphorylation, and these effects were counteracted by sgp130Fc. Moreover, IL-6 trans-signaling led to altered expression of apoptosis-associated genes, including downregulation of FIS1, BCL2, and MCL1, while promoting cellular senescence, a phenomenon mitigated by sgp130Fc. These results not only deepen our understanding of IL-6 in DR but also carry broader implications for age-related diseases and the aging process itself. This study underscores the potential therapeutic value of targeting IL-6 trans-signaling with sgp130Fc as a promising anti-inflammatory approach for DR and potentially other inflammatory conditions. Further in-vivo investigations are warranted to elucidate the function of IL-6 trans-signaling in aging-related pathologies and overall organismal health.

Skeletal muscle regeneration failure in ischemic-damaged limbs is associated with pro-inflammatory macrophages and premature differentiation of satellite cells

BackgroundChronic limb-threatening ischemia (CLTI), a severe manifestation of peripheral arterial disease (PAD), is associated with a 1-year limb amputation rate of approximately 15–20% and substantial mortality. A key feature of CLTI is the compromised regenerative ability of skeletal muscle; however, the mechanisms responsible for this impairment are not yet fully understood. In this study, we aim to delineate pathological changes at both the cellular and transcriptomic levels, as well as in cell–cell signaling pathways, associated with compromised muscle regeneration in limb ischemia in both human tissue samples and murine models of CLTI.MethodsWe performed single-cell transcriptome analysis of ischemic and non-ischemic muscle from the same CLTI patients and from a murine model of CLTI. In both datasets, we analyzed gene expression changes in macrophage and muscle satellite cell (MuSC) populations as well as differential cell–cell signaling interactions and differentiation trajectories.ResultsSingle-cell transcriptomic profiling and immunofluorescence analysis of CLTI patient skeletal muscle demonstrated that ischemic-damaged tissue displays a pro-inflammatory macrophage signature. Comparable results were observed in a murine CLTI model. Moreover, integrated analyses of both human and murine datasets revealed premature differentiation of MuSCs to be a key feature of failed muscle regeneration in the ischemic limb. Furthermore, in silico inferences of intercellular communication and in vitro assays highlight the importance of macrophage-MuSC signaling in ischemia induced muscle injuries.ConclusionsCollectively, our research provides the first single-cell transcriptome atlases of skeletal muscle from CLTI patients and a murine CLTI model, emphasizing the crucial role of macrophages and inflammation in regulating muscle regeneration in CLTI through interactions with MuSCs.

Bone marrow stromal cells in Modic type 1 changes promote neurite outgrowth.

The pain in patients with Modic type 1 changes (MC1) is often due to vertebral body endplate pain, which is linked to abnormal neurite outgrowth in the vertebral body and adjacent endplate. The aim of this study was to understand the role of MC1 bone marrow stromal cells (BMSCs) in neurite outgrowth. BMSCs can produce neurotrophic factors, which have been shown to be pro-fibrotic in MC1, and expand in the perivascular space where sensory vertebral nerves are located. The study involved the exploration of the BMSC transcriptome in MC1, co-culture of MC1 BMSCs with the neuroblastoma cell line SH-SY5Y, analysis of supernatant cytokines, and analysis of gene expression changes in co-cultured SH-SY5Y. Transcriptomic analysis revealed upregulated brain-derived neurotrophic factor (BDNF) signaling-related pathways. Co-cultures of MC1 BMSCs with SH-SY5Y cells resulted in increased neurite sprouting compared to co-cultures with control BMSCs. The concentration of BDNF and other cytokines supporting neuron growth was increased in MC1 vs. control BMSC co-culture supernatants. Taken together, these findings show that MC1 BMSCs provide strong pro-neurotrophic cues to nearby neurons and could be a relevant disease-modifying treatment target.

Gene profiles and mutations in the development of cataracts in the ICR rat model of hereditary cataracts

Cataracts are opacifications of the lens that cause loss of visual acuity and ultimately of eyesight. Age-related cataract develops in most elderly people, but the mechanisms of cataract onset are incompletely understood. The Ihara Cataract Rat (ICR) is an animal model of hereditary cataracts showing cortical opacity that commonly develops prematurely. We identified putative mechanisms of cataract onset in the ICR rat model by measuring gene expression changes before and after cortical cataract development and conducting point mutation analysis. Genes differentially expressed between 4-week-old animals without cortical cataracts and 8–10-week-old animals with cortical cataracts were selected from microarray analysis. Three connections were identified by STRING analysis: (i) Epithelial-Mesenchymal Transition (EMT), including Col1a2, and Pik3r1. (ii) Lens homeostasis, including Aqp5, and Cpm. (iii) Lipid metabolism, including Scd1, Srebf1, and Pnpla3. Subsequently, mutation points were selected by comparing ICR rats with 12 different rats that do not develop cataracts. The apolipoprotein Apoc3 was mutated in ICR rats. Analyses of gene expression changes and point and mutations suggested that abnormalities in EMT or lipid metabolism could contribute to cataract development in ICR rats.

CWH43 Is a Novel Tumor Suppressor Gene with Negative Regulation of TTK in Colorectal Cancer.

Colorectal cancer (CRC) ranks among the most prevalent forms of cancer globally, and its late-stage survival outcomes are less than optimal. A more nuanced understanding of the underlying mechanisms behind CRC's development is crucial for enhancing patient survival rates. Existing research suggests that the expression of Cell Wall Biogenesis 43 C-Terminal Homolog (CWH43) is reduced in CRC. However, the specific role that CWH43 plays in cancer progression remains ambiguous. Our research seeks to elucidate the influence of CWH43 on CRC's biological behavior and to shed light on its potential as a therapeutic target in CRC management. Utilizing publicly available databases, we examined the expression levels of CWH43 in CRC tissue samples and their adjacent non-cancerous tissues. Our findings indicated lower levels of both mRNA and protein expressions of CWH43 in cancerous tissues. Moreover, we found that a decrease in CWH43 expression correlates with poorer prognoses for CRC patients. In vitro experiments demonstrated that the suppression of CWH43 led to increased cell proliferation, migration, and invasiveness, while its overexpression had inhibitory effects. Further evidence from xenograft models showed enhanced tumor growth upon CWH43 silencing. Leveraging data from The Cancer Genome Atlas (TCGA), our Gene Set Enrichment Analysis (GSEA) indicated a positive relationship between low CWH43 expression and the activation of the epithelial-mesenchymal Transition (EMT) pathway. We conducted RNA sequencing to analyze gene expression changes under both silenced and overexpressed CWH43 conditions. By identifying core genes and executing KEGG pathway analysis, we discovered that CWH43 appears to have regulatory influence over the TTK-mediated cell cycle. Importantly, inhibition of TTK counteracted the tumor-promoting effects caused by CWH43 downregulation. Our findings propose that the decreased expression of CWH43 amplifies TTK-mediated cell cycle activities, thus encouraging tumor growth. This newly identified mechanism offers promising avenues for targeted CRC treatment strategies.

Bioinformatics analysis to identify potential biomarkers for the pulmonary artery hypertension associated with the basement membrane.

Pulmonary arterial hypertension (PAH) is a rapidly progressing cardiopulmonary disease. It is characterized by increased pulmonary artery pressure and vascular resistance. The most notable histopathological characteristic is vascular remodeling. The changes in the basement membrane (BM) are believed to be related to vascular remodeling. It is crucial to identify potential biomarkers associated with the BM in PAH, to guide its treatment. The microarray datasets GSE117261 and GSE113439 were downloaded from the Gene Expression Omnibus. Two data sets were examined to identify genes associated with the BM by analyzing gene expression changes. Next, we analyzed the relevant genes in the Kyoto Encyclopedia of Genes and Genomes using Gene Ontology and Disease Ontology annotationand conducted pathway enrichment analysis. We conducted a protein-protein interaction network analysis on the genes related to BMs and used the cell cytoHubba plug-in to identify the hub genes. Furthermore, we conducted an immune infiltration analysis and implemented a histogram model. Finally, we predicted and analyzed potential therapeutic drugs for PAH and set up a miRNA network of genetic markers. Six candidate genes related to BMs, namely Integrin Subunit Alpha V, Integrin Subunit Alpha 4, ITGA2, ITGA9, Thrombospondin 1, and Collagen Type IV Alpha 3 Chain, were identified as potential modulators of the immune process in PAH. Furthermore, ginsenoside Rh1 was found to significantly impact drug targeting based on its interactions with the six BM-related genes identified earlier. A novel biomarker related to the BM, which plays a crucial role in the development of PAH, has been identified.

Regional Gene Expression in the Retina, Optic Nerve Head, and Optic Nerve of Mice with Optic Nerve Crush and Experimental Glaucoma.

A major risk factor for glaucomatous optic neuropathy is the level of intraocular pressure (IOP), which can lead to retinal ganglion cell axon injury and cell death. The optic nerve has a rostral unmyelinated portion at the optic nerve head followed by a caudal myelinated region. The unmyelinated region is differentially susceptible to IOP-induced damage in rodent models and human glaucoma. While several studies have analyzed gene expression changes in the mouse optic nerve following optic nerve injury, few were designed to consider the regional gene expression differences that exist between these distinct areas. We performed bulk RNA-sequencing on the retina and separately micro-dissected unmyelinated and myelinated optic nerve regions from naïve C57BL/6 mice, mice after optic nerve crush, and mice with microbead-induced experimental glaucoma (total = 36). Gene expression patterns in the naïve unmyelinated optic nerve showed significant enrichment of the Wnt, Hippo, PI3K-Akt, and transforming growth factor β pathways, as well as extracellular matrix-receptor and cell membrane signaling pathways, compared to the myelinated optic nerve and retina. Gene expression changes induced by both injuries were more extensive in the myelinated optic nerve than the unmyelinated region, and greater after nerve crush than glaucoma. Changes present three and fourteen days after injury largely subsided by six weeks. Gene markers of reactive astrocytes did not consistently differ between injury states. Overall, the transcriptomic phenotype of the mouse unmyelinated optic nerve was significantly different from immediately adjacent tissues, likely dominated by expression in astrocytes, whose junctional complexes are inherently important in responding to IOP elevation.

Improved chilling tolerance in glasshouse-grown potted sweet basil by end-of-production, short-duration supplementary far red light.

Sweet basil is a popular culinary herb used in many cuisines around the world and is widely grown commercially for retail as a live potted plant. However, basil is easily damaged by temperatures below 12 °C meaning plants must be transported from the grower to the retailer in a warm transport chain, adding considerable commercial cost in temperate countries. Improvement of chilling tolerance has been demonstrated in post-harvest crops such as tomato fruits and, indeed, fresh cut basil, by manipulation of the red:far red ratio of light provided to plants throughout the photoperiod and for a significant duration of the growing process in controlled environment chambers. We tested the effectiveness of periodic short-duration end-of-production supplementary far red light treatments designed for use with basil plants grown in a large scale commercial glasshouse for the live potted basil market. Four days of periodic, midday supplementary far red light given at end of production induced robust tolerance to 24 h of 4 °C cold treatment, resulting in greatly reduced visual damage, and reduced physiological markers of chilling injury including electrolyte leakage and reactive oxygen species accumulation. Antioxidant levels were also maintained at higher levels in live potted basil following this cold treatment. RNAseq-based analysis of gene expression changes associated with this response pointed to increased conversion of starch to soluble raffinose family oligosaccharide sugars; increased biosynthesis of anthocyanins and selected amino acids; inactivation of gibberellin signaling; and reduced expression of fatty acid desaturases, all previously associated with increased chilling tolerance in plants. Our findings offer an efficient, non-invasive approach to induce chilling tolerance in potted basil which is suitable for application in a large-scale commercial glasshouse.

A TRANSCRIPTOMIC APPRECIATION OF CHILDHOOD MENINGOCOCCAL AND POLYMICROBIAL SEPSIS FROM A PRO-INFLAMMATORY AND TRAJECTORIAL PERSPECTIVE, A ROLE FOR VASCULAR ENDOTHELIAL GROWTH FACTOR A AND B MODULATION?

This study investigated the temporal dynamics of childhood sepsis by analyzing gene expression changes associated with proinflammatory processes. Five datasets, including four meningococcal sepsis shock (MSS) datasets (two temporal and two longitudinal) and one polymicrobial sepsis dataset, were selected to track temporal changes in gene expression. Hierarchical clustering revealed three temporal phases: early, intermediate, and late, providing a framework for understanding sepsis progression. Principal component analysis supported the identification of gene expression trajectories. Differential gene analysis highlighted consistent upregulation of vascular endothelial growth factor A (VEGF-A) and nuclear factor κB1 (NFKB1), genes involved in inflammation, across the sepsis datasets. NFKB1 gene expression also showed temporal changes in the MSS datasets. In the postmortem dataset comparing MSS cases to controls, VEGF-A was upregulated and VEGF-B downregulated. Renal tissue exhibited higher VEGF-A expression compared with other tissues. Similar VEGF-A upregulation and VEGF-B downregulation patterns were observed in the cross-sectional MSS datasets and the polymicrobial sepsis dataset. Hexagonal plots confirmed VEGF-R (VEGF receptor)-VEGF-R2 signaling pathway enrichment in the MSS cross-sectional studies. The polymicrobial sepsis dataset also showed enrichment of the VEGF pathway in septic shock day 3 and sepsis day 3 samples compared with controls. These findings provide unique insights into the dynamic nature of sepsis from a transcriptomic perspective and suggest potential implications for biomarker development. Future research should focus on larger-scale temporal transcriptomic studies with appropriate control groups and validate the identified gene combination as a potential biomarker panel for sepsis.

RNA Sequencing of Pooled Samples Effectively Identifies Differentially Expressed Genes.

Analysis of gene expression changes across the genome provides a powerful, unbiased tool for gaining insight into molecular mechanisms. We have effectively used RNA sequencing to identify differentially expressed genes in long-lived genetic mutants in C. elegans to advance our understanding of the genetic pathways that control longevity. Although RNA sequencing costs have come down, cost remains a barrier to examining multiple strains and time points with a sufficient number of biological replicates. To circumvent this, we have examined the efficacy of identifying differentially expressed genes by sequencing a pooled RNA sample from long-lived isp-1 mitochondrial mutant worms. We found that sequencing a pooled RNA sample could effectively identify genes that were found to be significantly upregulated in the two individually sequenced RNA-seq experiments. Finally, we compared the genes significantly upregulated in the two individually sequenced RNA-seq experiments to two previous microarray experiments to come up with a high-confidence list of modulated genes in long-lived isp-1 mutant worms. Overall, this work demonstrates that RNA sequencing of pooled RNA samples can be used to identify differentially expressed genes.

Specific processing of meiosis-related transcript is linked to final maturation in human oocytes.

Human meiosis in oocytes entails an intricate regulation of the transcriptome to support late oocyte growth and early embryo development, both crucial to reproductive success. Currently, little is known about the co- and post-transcriptional mRNA processing mechanisms regulating the last meiotic phases, which contribute to transcriptome complexity and influence translation rates. We analyzed gene expression changes, splicing and pre-mRNA processing in an RNA sequencing set of 40 human oocytes at different meiotic maturation stages, matured both in vivo and in vitro. We found abundant untranslated region (UTR) processing, mostly at the 3' end, of meiosis-related genes between the germinal vesicle (GV) and metaphase II (MII) stages, supported by the differential expression of spliceosome and pre-mRNA processing related genes. Importantly, we found very few differences among GV oocytes across several durations of IVM, as long as they did not reach MII, suggesting an association of RNA processing and successful meiosis transit. Changes in protein isoforms are minor, although specific and consistent for genes involved in chromosome organization and spindle assembly. In conclusion, we reveal a dynamic transcript remodeling during human female meiosis, and show how pre-mRNA processing, specifically 3'UTR shortening, drives a selective translational regulation of transcripts necessary to reach final meiotic maturation.

Differential Gene Expression Data Analysis of ASD Using Random Forest.

Autism spectrum disorder (ASD) is a developmental disability caused by differences in the brain regions. Analysis of differential expression (DE) of transcriptomic data allows for genome-wide analysis of gene expression changes related to ASD. De-novo mutations may play a vital role in ASD, but the list of genes involved is still far from complete. Differentially expressed genes (DEGs) are treated as candidate biomarkers and a small set of DEGs might be identified as biomarkers using either biological knowledge or data-driven approaches like machine learning and statistical analysis. In this study, we employed a machine learning-based approach to identify the differential gene expression between ASD and Typical Development (TD). The gene expression data of 15 ASD and 15 TD were obtained from the NCBI GEO database. Initially, we extracted the data and used a standard pipeline to pre-process the data. Further, Random Forest (RF) was used to discriminate genes between ASD and TD. We identified the top 10 prominent differential genes and compared them with the statistical test results. Our results show that the proposed RF model yields 5-fold cross-validation accuracy, sensitivity and specificity of 96.67%. Further, we obtained precision and F-measure scores of 97.5% and 96.57%, respectively. Moreover, we found 34 unique DEG chromosomal locations having influential contributions in identifying ASD from TD. We have also identified chr3:113322718-113322659 as the most significant contributing chromosomal location in discriminating ASD and TD. Our machine learning-based method of refining DE analysis is promising for finding biomarkers from gene expression profiles and prioritizing DEGs. Moreover, our study reported top 10 gene signatures for ASD may facilitate the development of reliable diagnosis and prognosis biomarkers for screening ASD.