Significant Differences In Gene Expression Research Articles

Antibiotics alter development and gene expression in the model cnidarian Nematostella vectensis.

Antibiotics are commonly used for controlling microbial growth in diseased organisms. However, antibiotic treatments during early developmental stages can have negative impacts on development and physiology that could offset the positive effects of reducing or eliminating pathogens. Similarly, antibiotics can shift the microbial community due to differential effectiveness on resistant and susceptible bacteria. Though antibiotic application does not typically result in mortality of marine invertebrates, little is known about the developmental and transcriptional effects. These sublethal effects could reduce the fitness of the host organism and lead to negative changes after removal of the antibiotics. Here, we quantify the impact of antibiotic treatment on development, gene expression, and the culturable bacterial community of a model cnidarian, Nematostella vectensis. Ampicillin, streptomycin, rifampicin, and neomycin were compared individually at two concentrations, 50 and 200 µg mL-1, and in combination at 50 µg mL-1 each, to assess their impact on N. vectensis. First, we determined the impact antibiotics have on larval development. Next Amplicon 16S rDNA gene sequencing was used to compare the culturable bacteria that persist after antibiotic treatment to determine how these treatments may differentially select against the native microbiome. Lastly, we determined how acute (3-day) and chronic (8-day) antibiotic treatments impact gene expression of adult anemones. Under most exposures, the time of larval settlement extended as the concentration of antibiotics increased and had the longest delay of 3 days in the combination treatment. Culturable bacteria persisted through a majority of exposures where we identified 359 amplicon sequence variants (ASVs). The largest proportion of bacteria belonged to Gammaproteobacteria, and the most common ASVs were identified as Microbacterium and Vibrio. The acute antibiotic exposure resulted in differential expression of genes related to epigenetic mechanisms and neural processes, while constant application resulted in upregulation of chaperones and downregulation of mitochondrial genes when compared to controls. Gene Ontology analyses identified overall depletion of terms related to development and metabolism in both antibiotic treatments. Antibiotics resulted in a significant increase to settlement time of N. vectensis larvae. Culturable bacterial species after antibiotic treatments were taxonomically diverse. Additionally, the transcriptional effects of antibiotics, and after their removal result in significant differences in gene expression that may impact the physiology of the anemone, which may include removal of bacterial signaling on anemone gene expression. Our research suggests that impacts of antibiotics beyond the reduction of bacteria may be important to consider when they are applied to aquatic invertebrates including reef building corals.

PC (16:0/14:0) ameliorates hyperoxia-induced bronchopulmonary dysplasia by upregulating claudin-1 and promoting alveolar type II cell repair

Bronchopulmonary dysplasia (BPD) remains a significant challenge in neonatal care, the pathogenesis of which potentially involves altered lipid metabolism. Given the critical role of lipids in lung development and the injury response, we hypothesized that specific lipid species could serve as therapeutic agents in BPD. This study aimed to investigate the role of the lipid Phosphatidylcholine (PC) (16:0/14:0) in modulating BPD pathology and to elucidate its underlying mechanisms of action. Our approach integrated in vitro and in vivo methodologies to assess the effects of PC (16:0/14:0) on the histopathology, cellular proliferation, apoptosis, and molecular markers in lung tissue. In a hyperoxia-induced BPD rat model, we observed a reduction in alveolar number and an enlargement in alveolar size, which were ameliorated by PC (16:0/14:0) treatment. Correspondingly, in BPD cell models, PC (16:0/14:0) intervention led to increased cell viability, enhanced proliferation, reduced apoptosis, and elevated surfactant protein C (SPC) expression. RNA sequencing revealed significant gene expression differences between BPD and PC (16:0/14:0) treated groups, with a particular focus on Cldn1 (encoding claudin 1), which was significantly enriched in our analysis. Our findings suggest that PC (16:0/14:0) might protect against hyperoxia-induced alveolar type II cell damage by upregulating CLDN1 expression, potentially serving as a novel therapeutic target for BPD. This study not only advances our understanding of the role of lipids in BPD pathogenesis, but also highlights the significance of PC (16:0/14:0) in the prevention and treatment of BPD, offering new avenues for future research and therapeutic development.

Diversity and host interaction of the gut microbiota in specific pathogen-free pigs.

Pigs are widely used as animal models in various studies related to humans. The interaction between the gut microbiota and the host has significant effects on the host's health and disease status. However, although there have been many studies investigating the pig gut microbiota, the findings have been inconsistent due to variations in rearing conditions. Interactions between the gut microbiota and host have not been fully explored in pigs. Specific pathogen-free (SPF) pigs are ideal non-primate large animals to study the interactions between the gut microbiota and the host. In this study, we performed high-throughput sequencing analysis of the gut microbiota and the gut tissue transcriptome of six SPF pigs to provide a systematic understanding of the composition, function, and spatial distribution of gut microbiota in SPF pigs. We identified significant differences in microbial diversity and functionality among different gastrointestinal tract sites. Metagenomics data analysis revealed significant differences in alpha diversity and beta diversity of microbiota in different gastrointestinal sites of SPF pigs. Additionally, transcriptomic data indicated significant differences in gene expression as well as KEGG and GO functional enrichment between the small intestine and large intestine. Furthermore, by combining microbial metagenomics and host transcriptomics analyses, specific correlations were found between gut microbiota and host genes. These included a negative correlation between the TCN1 gene and Prevotella dentalis, possibly related to bacterial metabolic pathways involving vitamin B12, and a positive correlation between the BDH1 gene and Roseburia hominis, possibly because both are involved in fatty acid metabolism. These findings lay the groundwork for further exploration of the co-evolution between the microbiota and the host, specifically in relation to nutrition, metabolism, and immunity. In conclusion, we have elucidated the diversity of the gut microbiota in SPF pigs and conducted a detailed investigation into the interactions between the gut microbiota and host gene expression. These results contribute to our understanding of the intricate dynamics between the gut microbiota and the host, offering important references for advancements in life science research, bioproduct production, and sustainable development in animal husbandry.

Single-Cell Analyses Offer Insights into the Different Remodeling Programs of Arteries and Veins.

Arteries and veins develop different types of occlusive diseases and respond differently to injury. The biological reasons for this discrepancy are not well understood, which is a limiting factor for the development of vein-targeted therapies. This study contrasts human peripheral arteries and veins at the single-cell level, with a focus on cell populations with remodeling potential. Upper arm arteries (brachial) and veins (basilic/cephalic) from 30 organ donors were compared using a combination of bulk and single-cell RNA sequencing, proteomics, flow cytometry, and histology. The cellular atlases of six arteries and veins demonstrated a 7.8× higher proportion of contractile smooth muscle cells (SMCs) in arteries and a trend toward more modulated SMCs. In contrast, veins showed a higher abundance of endothelial cells, pericytes, and macrophages, as well as an increasing trend in fibroblasts. Activated fibroblasts had similar proportions in both types of vessels but with significant differences in gene expression. Modulated SMCs and activated fibroblasts were characterized by the upregulation of MYH10, FN1, COL8A1, and ITGA10. Activated fibroblasts also expressed F2R, POSTN, and COMP and were confirmed by F2R/CD90 flow cytometry. Activated fibroblasts from veins were the top producers of collagens among all fibroblast populations from both types of vessels. Venous fibroblasts were also highly angiogenic, proinflammatory, and hyper-responders to reactive oxygen species. Differences in wall structure further explain the significant contribution of fibroblast populations to remodeling in veins. Fibroblasts are almost exclusively located outside the external elastic lamina in arteries, while widely distributed throughout the venous wall. In line with the above, ECM-targeted proteomics confirmed a higher abundance of fibrillar collagens in veins vs. more basement ECM components in arteries. The distinct cellular compositions and transcriptional programs of reparative populations in arteries and veins may explain differences in acute and chronic wall remodeling between vessels. This information may be relevant for the development of antistenotic therapies.

Abstract PO1-28-06: Development of a plasma-based real-time qPCR gene expression assay for targeted screening and diagnosis of early-stage breast cancer

Abstract Background Over 12 million US women undergo follow-up breast imaging and exams annually due to breast cancer suspicions, incurring an $8 billion total cost, of which $2.18 billion is attributed to false-positive breast biopsies. Recent updates published to the Mammography Quality Standards Act (MQSA) now recommend supplemental imaging for women with heterogeneously dense and extremely dense breasts, regardless of suspicious findings. Given sensitivity limitations associated with mammography in dense-breasted women, as low as 30-47%, and non-specificity associated with ultrasound and MRI, these recent recommendations are likely to further strain the healthcare system and place substantial burden on patients and providers. With an estimated 25 million dense-breasted women who regularly screen for breast cancer now considered at-risk, there is a critical need for affordable and highly accurate testing, offering better specificity without sacrificing sensitivity. To address this gap, we developed an affordable and accurate plasma-based gene expression assay using real-time qPCR for targeted screening and diagnosis of early-stage breast cancer. Methods In previous studies, 26 cross-correlated mRNA gene targets were discovered and independently validated for non-invasive breast cancer detection across five independent patient cohorts using microarray gene expression profiling, comprised of peripheral blood mononuclear cells (n=337) and saliva (n=20), demonstrating strong efficacy, reproducibility, and concordance across bio-fluids and assay platforms in all studies. A sixth independent validation cohort comprised of 203 plasma samples was subsequently obtained for real-time qPCR clinical assay development and validation in our CLIA laboratory. The CLIA validation cohort was designed to be representative of different diagnosis status, stage, and ethnicity, and included Caucasian, Black/African American, Hispanic, and Asian women. After normalizing mRNA expression and clustering analysis, the signature was refined to 8 target genes and was assessed in a cohort of 87 plasma samples [Table 1] to verify the assay’s diagnostic performance for stage I breast cancer detection, under locked laboratory protocols. We leveraged machine learning methods derived from XGBoost classification, a supervised-learning algorithm that uses sequentially built shallow decision trees to provide accurate results and avoidance of overfitting. Results The XGBoost model, selecting from the 8 mRNA gene targets and patient age, achieved >99% sensitivity, 89% specificity for stage I breast cancer detection in the held-out test set, with an overall diagnostic accuracy of 94.5%. Normalized data showed significant differences in gene expression between healthy controls and stage I patients, and distinct clustering was observed for the 8 mRNA gene signature, including patient age. Conclusions Our plasma-based real-time qPCR gene expression clinical assay, with machine learning, is positioned as a highly accurate non-invasive tool for targeted screening and diagnosis of early-stage breast cancer. Furthermore, a clinically validated assay in this space addresses the need for more targeted diagnostics, while serving as a promising tool for clinicians as they make critical care decisions across the breast health continuum, accelerating time to earlier and more precise intervention and treatment, mitigating unnecessary healthcare expenditures, and reducing the mental, emotional, and financial burden on patients and their families. Citation Format: Martin Keiser, Elizabeth Cormier-May, Matthew Alderdice, Joy Kavanagh, William Guesdon, Heather Healy, Nathalie Jean-Charles, Jay Harness. Development of a plasma-based real-time qPCR gene expression assay for targeted screening and diagnosis of early-stage breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-28-06.

Abstract PS16-03: Intratumoral dosing of INT230-6 in Early-Stage Breast Cancer Patients Induces Tumor Cell Necrosis and Immunomodulatory Effects: A Phase II Randomized Window-Of-Opportunity Study – the INVINCIBLE Trial

Abstract Background: Larger tumors pose an increased risk for breast cancer (BC) recurrence post-surgery. In addition, most BC outside of the triple negative subtype are considered immunological quiescent and minimally responsive to immunotherapies. One potential method to combat disease recurrence risk and induce immune activation pre-surgery is through a local therapy that could cause cell death to expose tumor antigens, provide adjuvants for anti-tumor immune priming, and thus potentially increase responsiveness to immunotherapies or rates of pathological complete response in combination with chemotherapy. We have conducted a randomized, Phase 2 presurgical Window-Of-Opportunity trial for intratumoral (IT) INT230-6 comprising vinblastine, cisplatin and a diffusion enhancer (SHAO) in patients with early-stage operable BC, the INVINCIBLE trial (NCT04781725). Previous in vivo and clinical studies demonstrated that INT230-6 induces cancer cell death and halts replication while maturing dendritic cells and recruiting T-cells into the tumor. In this trial, IT injections of INT230-6 were conducted to 1) evaluate the safety of regional cytotoxic use on BC, 2) assess the drug’s ability to cause necrosis, 3) assess immune response within the tumor, microenvironment and systemically prior to surgical resection, and 4) understand the genetic pathways involved in immune activity. Methods: Women awaiting surgery for newly diagnosed early-stage intermediate or high-grade T1-T2 invasive BC were recruited to the trial. The study has two parts. Part I was a randomized (2:1) open label trial comparing 1-3 doses of INT230-6 injected weekly versus no treatment prior to surgery to evaluate safety, feasibility, and optimal drug dosing. Part II was a double-blinded randomized (2:1) trial where patients received one IT dose of INT230-6 vs saline injection IT. Results: We successfully recruited 91 patients with age ranges of 40-77 yrs (mean of 60 yrs) with tumor size ranging from 1.1 - 4.8 cm (mean 2.5 cm; SD 0.9 cm). The most common (>10%) AEs were injection site pain, injection site reaction and nausea/vomiting. Approximately 90% of AEs were grade 1. In the study INT230-6 induced necrosis in 64% of subjects (37 out of 58, range 0 to 100%); whereas saline induced partial necrosis in 25% of patients (5 out of 20, range 0 to 10%). The table below shows the results of necrosis for INT230-6 use in the entire study compared to saline injection for all subjects and those with tumors of size T2. A single injection of INT230-6 caused necrosis in various histologies, including invasive lobular carcinoma. Gene expression analysis showed significant differential gene expression between the baseline biopsy and surgical specimens using INT230-6. Pathway analysis identified genes associated with TCR signaling, B cells and T cell activation that were significantly upregulated in the post INT230-6 treatment samples. There was a relative increase in CD4 and CD8 T cells and B and mast cells. Conclusion: Preliminary evidence shows that a single dose of INT230-6 can cause significant intratumoral necrosis compared to saline especially in tumors >2. cm. INT230-6 stimulates an immune response in breast cancers prior to surgery with minimal adverse effects and good tolerability. Given its immune activation properties, INT230-6 shows promising potential in future breast cancer neoadjuvant studies. Table 1: INVINCIBLE Study Tumor Necrosis Results Intratumoral INT230-6 injection compared to IT saline injection Citation Format: Angel Arnaout, Lewis Bender, Megan Hopkins, Vanessa Lopez Ozuna, Linda Liao, Susan Robertson, Vida Talebian, Kianoosh Keyhanian, Arif Awan, Franco Abbate, Ian Walters, Gregory Pond, John Bartlett, Lazlo Radvanyi, Melanie Spears. Intratumoral dosing of INT230-6 in Early-Stage Breast Cancer Patients Induces Tumor Cell Necrosis and Immunomodulatory Effects: A Phase II Randomized Window-Of-Opportunity Study – the INVINCIBLE Trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS16-03.

Using multiomics to explore the weight differences between genders in Muscovy ducks

Sexual dimorphism in poultry, especially in Muscovy ducks, is a proven phenomenon characterized by significant differences in body weight, growth patterns, and gene expression between male and female individuals. However, there is a dearth of research on the candidate genes and mechanisms underlying these weight differences. We selected 301 Muscovy ducks and recorded their weekly body weights from birth. We utilized 3 non-linear growth models (Logistic, Bertalanffy, and Gompertz) to fit the growth curve of Muscovy ducks, it was found that the logistic model was the most suitable model for describing the growth curve of Muscovy ducks. The results from the logistic model showed that the inflection point of male Muscovy ducks occurred at a later age, and they had a heavier mature body weight than female Muscovy ducks. At 10 wk of age, we collected Muscovy duck breast muscle tissues for transcriptome sequencing (RNA-seq). To exclude the impact of weight difference, 185 differentially expressed genes (DEGs), such as PPAR, FABP3, PLIN1, and FOXO1, were screened. These DEGs were predominantly enriched in terms related to mitochondria, lipids, and nucleic acids. In addition, the gut microbiota has the ability to influence host physiology through the regulation of multiple processes, including playing a crucial role in host muscle growth and development. We randomly selected male and female Muscovy ducks for 16S rRNA sequencing analysis of their cecal microbiota. The results showed that there were significant differences in the composition of cecal microbiota between male and female Muscovy ducks. At the genus level, the relative abundance of Enterenecus and CAG_269 were lower in males compared to females, while Lawsonibacter, Parabacteroides_B, Streptococcus, UBA2658, Caccousia, and Butyricimonas were higher in males than in females. In summary, this study provides a scientific theoretical basis for revealing the different growth patterns of male and female Muscovy ducks, and offers explanations from both the molecular level and microbiological perspectives.

RT-qPCR based quantitative analysis of ARO and ADH genes in Saccharomyces cerevisiae and Metschnikowia pulcherrima strains growth white grape juice.

Yeast biosynthesizes fusel alcohols in fermentation through amino acid catabolism via the Ehrlich pathway. ARO8 and ARO9 genes are involved in the first step of the Ehrlich pathway, while ADH2 and ADH5 genes are involved in the last step. In this study, we describe RT-qPCR methods to determine the gene expression level of genes (ARO8, ARO9, ADH2, ADH5) found in Saccharomyces cerevisiae (Sc) and Metschnikowia pulcherrima (Mp) strains growth pasteurized white grape juice. We used RNA extraction and cDNA synthesis protocols. The RT-qPCR efficiency of primer pairs was evaluated by generating a standard curve through serial dilution of yeast-derived cDNA. Method performance criteria were determined for each RT-qPCR assay. Then, we evaluated the gene expression levels of the four genes in all samples. RNA extraction and cDNA synthesis from yeast samples demonstrated the method's capability to generate high-yield, high-purity nucleic acids, supporting further RT-qPCR analysis. The highest normalized gene expression levels of ARO8 and ARO9 were observed in SC1, SC4, and SC5 samples. No significant difference in ADH2 gene expression among Mp strains was observed during the examination of ADH2 and ADH5 genes (p < 0.05). We observed no expression of the ADH5 gene in Mp strains except MP6 strain. The expression of ADH2 and ADH5 genes was higher in Sc strains compared to Mp strains. The results suggest that the proposed RT-qPCR methods can measure gene expression of ARO8, ARO9, ADH2, and ADH5 in Sc and Mp strains growing in pasteurized white grape juice.

Gene and Protein Expression of Placental Nutrient-Stress Sensor Proteins in Fetal Growth Restriction

Fetal growth restriction (FGR) and low birth weight increase the risk of non-communicable diseases such as type 2 diabetes and heart failure in adulthood. Placental insufficiency is widely considered a major contributor to FGR. Two crucial placental proteins involved in nutrient and stress sensing—O-linked N-acetylglucosamine transferase (OGT) and mechanistic target of rapamycin (mTOR) kinase—play roles in post-translational protein modification and protein translation, influencing cellular growth and metabolism in response to maternal stress, hypoxia, and nutritional status in the placenta. In our study, we examined the gene and protein profiles of OGT and mTOR in FGR and control placentae, comparing those appropriate for gestational age (AGA), while also considering potential confounding effects of fetal sex and delivery mode. Our findings revealed no significant differences in gene expression, protein levels, or activity of OGT, OGA, mTOR, or their associated markers between female AGA and FGR cesarean placentae, nor between female AGA and male AGA cesarean placentae. Additionally, the mode of delivery in female AGA placentae did not affect the levels or activity of these proteins. Overall, our study did not observe significant differences in nutrient sensor protein expression after stratifying by FGR, sex, and delivery mode. Nevertheless, these unbiased results provide a more comprehensive understanding of the complexities of placental gene expression involving OGT and mTOR.

Effect of SNPs in the Promoter Region on the Expression of Cytochrome P450 2E1 (CYP2E1) in Pig Liver.

Boar taint, an unfavorable odor in the meat of intact male pigs, is caused primarily by the accumulation of two compounds: androstenone and skatole. This multifactorial trait is regulated by numerous dietary, management and genetic factors. At the mechanistic level, there are many genes known to be involved in boar taint metabolism. Cytochrome P450 2E1 (CYP2E1) impacts boar taint through the phase I metabolism of skatole. The aim of this study was to identify single-nucleotide polymorphisms (SNPs) within the CYP2E1 gene promoter and explore their relationship with the expression of CYP2E1 mRNA and protein. Sequencing of the promoter region using pools of genomic DNA identified seven promoter region SNPs at -159, -586, -1693, -1806, -2322, -2369 and -2514 bp upstream of the ATG start site. Genomic DNA was obtained from 65 boars from the three major swine breeds: Duroc, Landrace and Yorkshire, and individual animals were genotyped for the identified SNPs. RNA was isolated from liver tissue and quantitative PCR was performed to measure CYP2E1 gene expression, while levels of CYP2E1 protein in liver were measured by Western blotting. Significant within-breed variation in CYP2E1 protein and mRNA expression was observed, indicating significant differences in gene expression among individuals. However, levels of CYP2E1 mRNA and protein were not significantly correlated. Two SNPs within the promoter were significantly associated with CYP2E1 mRNA expression, but not with protein expression. This study provides evidence of additional mutations affecting the gene expression of CYP2E1 and suggests that factors that affect the differences in translation of CYP2E1 mRNA may also be important in affecting skatole metabolism.

Experimental validation and comprehensive analysis of m6A methylation regulators in intervertebral disc degeneration subpopulation classification

Intervertebral disc degeneration (IVDD) is one of the most prevalent causes of chronic low back pain. The role of m6A methylation modification in disc degeneration (IVDD) remains unclear. We investigated immune-related m6A methylation regulators as IVDD biomarkers through comprehensive analysis and experimental validation of m6A methylation regulators in disc degeneration. The training dataset was downloaded from the GEO database and analysed for differentially expressed m6A methylation regulators and immunological features, the differentially regulators were subsequently validated by a rat IVDD model and RT-qPCR. Further screening of key m6A methylation regulators based on machine learning and LASSO regression analysis. Thereafter, a predictive model based on key m6A methylation regulators was constructed for training sets, which was validated by validation set. IVDD patients were then clustered based on the expression of key m6A regulators, and the expression of key m6A regulators and immune infiltrates between clusters was investigated to determine immune markers in IVDD. Finally, we investigated the potential role of the immune marker in IVDD through enrichment analysis, protein-to-protein network analysis, and molecular prediction. By analysising of the training set, we revealed significant differences in gene expression of five methylation regulators including RBM15, YTHDC1, YTHDF3, HNRNPA2B1 and ALKBH5, while finding characteristic immune infiltration of differentially expressed genes, the result was validated by PCR. We then screen the differential m6A regulators in the training set and identified RBM15 and YTHDC1 as key m6A regulators. We then used RBM15 and YTHDC1 to construct a predictive model for IVDD and successfully validated it in the training set. Next, we clustered IVDD patients based on the expression of RBM15 and YTHDC1 and explored the immune infiltration characteristics between clusters as well as the expression of RBM15 and YTHDC1 in the clusters. YTHDC1 was finally identified as an immune biomarker for IVDD. We finally found that YTHDC1 may influence the immune microenvironment of IVDD through ABL1 and TXK. In summary, our results suggest that YTHDC1 is a potential biomarker for the development of IVDD and may provide new insights for the precise prevention and treatment of IVDD.

Chronic intermittent hypoxia elicits distinct transcriptomic responses among neurons and oligodendrocytes within the brainstem of mice.

Chronic intermittent hypoxia (CIH) is a prevalent condition characterized by recurrent episodes of oxygen deprivation, linked to respiratory and neurological disorders. Prolonged CIH is known to have adverse effects, including endothelial dysfunction, chronic inflammation, oxidative stress, and impaired neuronal function. These factors can contribute to serious comorbidities, including metabolic disorders and cardiovascular diseases. To investigate the molecular impact of CIH, we examined male C57BL/6J mice exposed to CIH for 21 days, comparing to normoxic controls. We employed single-nucleus RNA sequencing to comprehensively examine the transcriptomic impact of CIH on key cell classes within the brainstem, specifically excitatory neurons, inhibitory neurons, and oligodendrocytes. These cell classes regulate essential physiological functions, including autonomic tone, cardiovascular control, and respiration. Through analysis of 10,995 nuclei isolated from pontine-medullary tissue, we identified seven major cell classes, further subdivided into 24 clusters. Our findings among these cell classes, revealed significant differential gene expression, underscoring their distinct responses to CIH. Notably, neurons exhibited transcriptional dysregulation of genes associated with synaptic transmission, and structural remodeling. In addition, we find dysregulated genes encoding ion channels, and inflammatory response. Concurrently, oligodendrocytes exhibited dysregulated genes associated with oxidative phosphorylation and oxidative stress. Utilizing CellChat network analysis, we uncovered CIH-dependent altered patterns of diffusible inter-cellular signaling. These insights offer a comprehensive transcriptomic cellular atlas of the pons-medulla and provide a fundamental resource for the analysis of molecular adaptations triggered by CIH.

A chromosome-level genome assembly of the soybean pod borer: insights into larval transcriptional response to transgenic soybean expressing the pesticidal Cry1Ac protein

BackgroundGenetically modified (GM) crop plants with transgenic expression of Bacillus thuringiensis (Bt) pesticidal proteins are used to manage feeding damage by pest insects. The durability of this technology is threatened by the selection for resistance in pest populations. The molecular mechanism(s) involved in insect physiological response or evolution of resistance to Bt is not fully understood.ResultsTo investigate the response of a susceptible target insect to Bt, the soybean pod borer, Leguminivora glycinivorella (Lepidoptera: Tortricidae), was exposed to soybean, Glycine max, expressing Cry1Ac pesticidal protein or the non-transgenic parental cultivar. Assessment of larval changes in gene expression was facilitated by a third-generation sequenced and scaffolded chromosome-level assembly of the L. glycinivorella genome (657.4 Mb; 27 autosomes + Z chromosome), and subsequent structural annotation of 18,197 RefSeq gene models encoding 23,735 putative mRNA transcripts. Exposure of L. glycinivorella larvae to transgenic Cry1Ac G. max resulted in prediction of significant differential gene expression for 204 gene models (64 up- and 140 down-regulated) and differential splicing among isoforms for 10 genes compared to unexposed cohorts. Differentially expressed genes (DEGs) included putative peritrophic membrane constituents, orthologs of Bt receptor-encoding genes previously linked or associated with Bt resistance, and those involved in stress responses. Putative functional Gene Ontology (GO) annotations assigned to DEGs were significantly enriched for 36 categories at GO level 2, respectively. Most significantly enriched cellular component (CC), biological process (BP), and molecular function (MF) categories corresponded to vacuolar and microbody, transport and metabolic processes, and binding and reductase activities. The DEGs in enriched GO categories were biased for those that were down-regulated (≥ 0.783), with only MF categories GTPase and iron binding activities were bias for up-regulation genes.ConclusionsThis study provides insights into pathways and processes involved larval response to Bt intoxication, which may inform future unbiased investigations into mechanisms of resistance that show no evidence of alteration in midgut receptors.

Abstract LB227: Accelerated intra-clonal transcriptomic diversification of hematopoietic stem cells during aging fuels leukemia evolution

Abstract Cancers are diseases of aging. Many cellular processes characteristic of cancer, e.g., epigenetic dysregulation, are similarly observed during aging. Acute myeloid leukemia (AML) is emblematic of this interplay. As a blood cancer stemming from hematopoietic stem and progenitors (HSPC), its median age of diagnosis is 65. AML is linked to age-related clonal hematopoiesis (CH), a condition resulting from the expansion of HSPC, marked by mutations in leukemia-associated genes. We aim to clarify the intrinsic role of aging in HSPC as it causes CH and leukemic transformation. Hypothesis: aging amplifies the heritable phenotypic variations in hematopoietic stem cells (HSC), enhancing epigenetic heterogeneity. This acts as a substrate for natural selection, driving leukemic transformation. Results: We found a significant increase in epigenetic and transcriptomic heterogeneity in long-term HSC (LT-HSC) from old mice compared to their younger counterparts via single-cell RNA-seq (scRNA-seq)s. This process occurs in an aged bone marrow with higher inflammation and altered HSPC support. Of relevance, we previously show that the rise of epigenetic and transcriptomic heterogeneity in HSC with Tet2 loss and Flt3 ITD precedes leukemic transformation. We further employed CellTag Indexing and scRNA-seq to track the fate of specific HSC subclones. We tracked HSPC cell culture (in vitro) after two-weeks and two-months following transplantation into recipient mice of matching ages (in vivo). Cells of the same clone manifest higher transcriptomic similarities than those from different clones, validating CellTag in mapping clonal evolution. We computed the progeny output activity (Ai) by the ratio of clone i abundance in the committed progenitors to its frequency in the HSC compartment. We found that young mice exhibit significantly higher variability and a higher mean of progeny output activity Ai, indicating the efficiency in self-renewal and cell differentiation. While both young and old mice have low-output (higher stemness) clones, only young mice had significant differential expression of key genes in low-output clones compared to high-output clones, e.g., Dpp4, Eps8, and Col5a1. Depite lower progeny output Ai variation, LT-HSCs from older mice exhibited greater clonal transcriptome diversification both in vitro and in vivo , evidenced by intra-clonal transcriptomic correlation. This pattern also existed in ST-HSC and MPP from older mice. Genes with higher expression variability in older mice highlighted pathways pivotal for HSPC fitness, e.g., ribosome synthesis (RS), inflammation response mechanisms (JAK-STAT), hematopoietic cell lineage, AML and cancer pathways (GSEA). The RS pathway were remarkably enriched in the megakaryocytic-erythroid progenitors, directly before differentiation where substantial protein translation is required. Conclusion: our results suggest that the synergy of aging and somatic mutations in epigenetic regulators may enhance cell-to-cell variations, offering additional heritable phenotypic variations. These, in turn, may facilitate clonal hematopoiesis and leukemic transformation. Future work will delineate specific epigenetic and transcriptomic configurations for mutant HSPC clonal expansion. This study provides a cohesive framework, linking the principles governing aging to cancer. Citation Format: Lamis Naddaf, Marco De Dominici, Xiaowen Chen, Travis Roeder, Hideyuki Oguro, Shilpita Karmakar, Eric Pietras, James DeGregori, Sheng Li. Accelerated intra-clonal transcriptomic diversification of hematopoietic stem cells during aging fuels leukemia evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB227.

GTADC: A Graph-Based Method for Inferring Cell Spatial Distribution in Cancer Tissues.

The heterogeneity of tumors poses a challenge for understanding cell interactions and constructing complex ecosystems within cancer tissues. Current research strategies integrate spatial transcriptomics (ST) and single-cell sequencing (scRNA-seq) data to thoroughly analyze this intricate system. However, traditional deep learning methods using scRNA-seq data tend to filter differentially expressed genes through statistical methods. In the context of cancer tissues, where cancer cells exhibit significant differences in gene expression compared to normal cells, this heterogeneity renders traditional analysis methods incapable of accurately capturing differences between cell types. Therefore, we propose a graph-based deep learning method, GTADC, which utilizes Silhouette scores to precisely capture genes with significant expression differences within each cell type, enhancing the accuracy of gene selection. Compared to traditional methods, GTADC not only considers the expression similarity of genes within their respective clusters but also comprehensively leverages information from the overall clustering structure. The introduction of graph structure effectively captures spatial relationships and topological structures between the two types of data, enabling GTADC to more accurately and comprehensively resolve the spatial composition of different cell types within tissues. This refinement allows GTADC to intricately reconstruct the cellular spatial composition, offering a precise solution for inferring cell spatial composition. This method allows for early detection of potential cancer cell regions within tissues, assessing their quantity and spatial information in cell populations. We aim to achieve a preliminary estimation of cancer occurrence and development, contributing to a deeper understanding of early-stage cancer and providing potential support for early cancer diagnosis.

298 Differential Gene Expression Underlying Glioma Related Epilepsy

INTRODUCTION: Seizures are a common sequalae for patients suffering from gliomas. There are several factors, such as molecular characteristics, known to influence the initiation of seizures that may ultimately drive tumor growth. However, different levels of gene expression associated with seizures for gliomas remain unclear. Here, we review current literature and analyze RNA sequencing of gliomas to further probe these differences. METHODS: Utilizing total RNA sequencing obtained from The Cancer Genome Atlas - Lower Grade Glioma to identify and characterize differential expression in patients that do and do not experience seizures. Utilizing QIAGEN Ingenuity Pathways Analysis (IPA), we identified canonical and functional pathways to further characterize differential expression. RESULTS: From 289 patients with gliomas, 83 (28.7%) had available information regarding seizures and other variables of interest. Of these, 50 (60.2%) were allocated to the seizure group and the remaining 33 (39.8%) to the non-seizure group. There were no statistically significant differences in age (p = 0.4661), sex (p = -0.1338), race/ethnicity (p = 0.7331), molecular subtype (0.8656), tumor grade (p = 0.2260), or anatomical location. When comparing the level of RNA expression from these tumors between the seizure and non-seizure groups, we identified 52 genes that were significantly differentially regulated. Critical canonical pathways were identified, most significantly RHOGDI and Semaphorin Neuronal Repulsive signaling.Finally, in the functional analysis, tumors that promoted seizures had gene sets that were significantly increased involving neuronal differentiation and synaptogenesis. CONCLUSIONS: In the setting of gliomas, differences in tumor gene expression exist between individuals with seizures and without seizures despite similarities in patient demographics and other tumor characteristics. There was significant difference in gene expression that were most associated with neuron development and synaptogenesis, ultimately suggesting a mechanistic role of a tumor-neuron synapse in seizure initiation and potentially tumor growth.

Molecular Profiling to Understand Treatment Resistance and Response in Glioblastoma

INTRODUCTION: Patients with glioblastoma experience a wide variation in response to standard treatment, with nearly 30% experiencing tumour progression during treatment, and nearly 6% surviving more than 5 years. To date, there are few non-invasive clinical biomarkers to predict response to firstline treatment. Chemical exchange saturation transfer (CEST) MRI may have the potential to fill this gap. CEST MRI is sensitive to treatment-induced changes and changes in tumor metabolism1. Our team has obtained CEST data for patients before, during and after standard chemoradiation treatment, and found that CEST provides markers of early response and can identify early, standard and late progressors before treatment initiation.2 METHODS: Patients (n = 104) with primary, IDH wild-type glioblastoma were imaged with CEST-MRI at multiple time points throughout standard chemoradiation treatment. DNA and RNA were co-extracted from matched normal and tumour pairs and processed for whole genome sequencing and gene expression analysis using Nanostring. Clinical variables such as age, extent of resection, sex, ECOG status and MGMT promoter methylated were also collected. A survival analysis was conducted using the Kaplan-Meier method with log-rank tests. Univariate and multi-variate hazard ratios for clinical variables were calculated by fitting Cox Proportional Hazards Models. RESULTS: Early progressors reported a median progression-free survival (PFS) of 142 days compared to 296.5 in standard and 832 days in late progressors (p &lt; 0.0001). Early progressors also harbored distinct and statistically significant differences in gene expression and genomic alterations, namely in DNA damage repair, glucose transport and arginine metabolism pathways. A gene signature was prognostic of PFS and overall survival in a Cox proportional hazards model. CONCLUSIONS: This data may serve as a radiogenomic biomarker to assess treatment response within early phases of treatment and allow for a personalized treatment plan.

A novel anoikis-related gene signature can predict the prognosis of hepatocarcinoma patients.

Hepatocellular carcinoma (HCC) is a major health problem with more than 850,000 cases per year worldwide. This cancer is now the third leading cause of cancer-related deaths worldwide, and the number is rising. Cancer cells develop anoikis resistance which is a vital step during cancer progression and metastatic colonization. However, there is not much research that specifically addresses the role of anoikis in HCC, especially in terms of prognosis. This study obtained gene expression data and clinical information from 371 HCC patients through The Cancer Genome Atlas (TCGA) Program and The Gene Expression Omnibus (GEO) databases. A total of 516 anoikis-related genes (ANRGs) were retrieved from GeneCard database and Harmonizome portal. Differential expression analysis identified 219 differentially expressed genes (DEGs), and univariate Cox regression analysis was utilized to select 99 ANRGs associated with the prognosis of HCC patients. A risk scoring model with seven genes was established using the least absolute shrinkage and selection operator (LASSO) regression model, and internal validation of the model was performed. The identified 99 ANRGs are closely associated with the prognosis of HCC patients. The risk scoring model based on seven characteristic genes demonstrates excellent predictive performance, further validated by receiver operating characteristic (ROC) curves and Kaplan-Meier survival curves. The study reveals significant differences in immune cell infiltration, gene expression, and survival status among different risk groups. The prognosis of HCC patients can be predicted using a unique prognostic model built on ANRGs in HCC.

Humic Acid Mitigates Drought Stress in Tomato

Drought stress, one of the most important abiotic stresses, severely limits global crop production. To increase tolerance to this stress, environmentally friendly practices are emphasised. Humic acid, one of the most important natural biostimulants, has positive effects on plant growth and yield. Recently, it has also been reported to play an important role in resistance to various abiotic stresses. However, many physiological and molecular mechanisms by which humic acid confers drought resistance have not been fully elucidated. Therefore, the effects of humic acid application on different morphological and physiological stress indicators and some antioxidative enzyme gene expressions of tomato seedlings under drought stress conditions were investigated in this study. It was found that drought stress decreased shoot fresh/dry weight, root fresh/dry weight, shoot and root length, chlorophyll and relative water content of plants by 67%, 56%, 31%, 38%, 22%, 20%, 15% and 25%, respectively. Humic acid application significantly increased these parameters, while reducing ion leakage, MDA and proline levels. The antioxidant enzyme gene expression of tomato seedlings under drought conditions showed no significant difference in SOD and APX gene expression, whereas CAT gene expression increased and GR gene expression decreased with humic acid application. Our results showed that humic acid application interacted with stress-related antioxidant enzyme gene expression and may be effective in reducing drought stress.

Comparative analysis of gene expression between mice and humans in acetaminophen-induced liver injury by integrating bioinformatics analysis

ObjectiveMice are routinely utilized as animal models of drug-induced liver injury (DILI), however, there are significant differences in the pathogenesis between mice and humans. This study aimed to compare gene expression between humans and mice in acetaminophen (APAP)-induced liver injury (AILI), and investigate the similarities and differences in biological processes between the two species.MethodsA pair of public datasets (GSE218879 and GSE120652) obtained from GEO were analyzed using “Limma” package in R language, and differentially expressed genes (DEGs) were identified, including co-expressed DEGs (co-DEGs) and specific-expressed DEGS (specific-DEGs). Analysis of Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed analyses for specific-DEGs and co-DEGs. The co-DEGs were also used to construct transcription factor (TF)-gene network, gene-miRNA interactions network and protein-protein interaction (PPI) network for analyzing hub genes.ResultsMouse samples contained 1052 up-regulated genes and 1064 down-regulated genes, while human samples contained 1156 up-regulated genes and 1557 down-regulated genes. After taking the intersection between the DEGs, only 154 co-down-regulated and 89 co-up-regulated DEGs were identified, with a proportion of less than 10%. It was suggested that significant differences in gene expression between mice and humans in drug-induced liver injury. Mouse-specific-DEGs predominantly engaged in processes related to apoptosis and endoplasmic reticulum stress, while human-specific-DEGs were concentrated around catabolic process. Analysis of co-regulated genes reveals showed that they were mainly enriched in biosynthetic and metabolism-related processes. Then a PPI network which contains 189 nodes and 380 edges was constructed from the co-DEGs and two modules were obtained by Mcode. We screened out 10 hub genes by three algorithms of Degree, MCC and MNC, including CYP7A1, LSS, SREBF1, FASN, CD44, SPP1, ITGAV, ANXA5, LGALS3 and PDGFRA. Besides, TFs such as FOXC1, HINFP, NFKB1, miRNAs like mir-744-5p, mir-335-5p, mir-149-3p, mir-218-5p, mir-10a-5p may be the key regulatory factors of hub genes.ConclusionsThe DEGs of AILI mice models and those of patients were compared, and common biological processes were identified. The signaling pathways and hub genes in co-expression were identified between mice and humans through a series of bioinformatics analyses, which may be more valuable to reveal molecular mechanisms of AILI.