Log2 Fold Change Research Articles

Transcriptome profiling and characterization of genes associated with tuberization under high temperature in aeroponics in potato cv. Kufri Anand.

High temperature stress is an important abiotic factor, which affects tuberization and ultimately causes heavy yield reduction in potato. Identification and characterization of genes associated with tuberization under high temperature stress is essential for future management through biotechnology. Two contrasting potato varieties Kufri Anand (profuse tuber-bearing) versus Kufri Frysona (very less/scanty tuber-bearing, control) were cultivated in aeroponics under high temperature stress, and transcriptomes were analyzed. Potato cv. Kufri Anand was found superior over control (Kufri Frysona) for tuber yield and its component traits along with root morphology under aeroponics. Transcriptomes of tuber and leaf tissues were analyzed. Statistically significant (p < 0.05) differentially expressed genes (DEGs) were categorised into up-regulated (> 2 log2 fold change, FC) and down-regulated (< -2 log2 FC) genes. DEGs were annotated by gene ontology and KEGG pathways. A few selected up-regulated genes of both tissues were identified, and phylogeny tree and motif analysis were analysed based on 36 peptide sequences representing 15 selected DEGs in this study. Further, gene expression markers were developed and validated by real time qPCR analysis for the identification of high temperature tolerant genotypes. A few key genes associated in tuberization under high temperature conditions were heat shock proteins (e.g. 18.5kDa class I heat shock protein), sugar metabolism (e.g. glucosyltransferase), transcription factor (e.g. WRKY), and phytohormones (e.g. auxin-induced beta-glucosidase). Our study provides an overview of key genes involved in tuberization under high temperature stress in potato cv. Kufri Anand under aeroponics.

Synthetic and natural rubber associated chemicals drive functional and structural changes as well as adaptations to antibiotics in in vitro marine microbiomes

The leaching of additives from plastics and elastomers (rubbers) has raised concerns due to their potential negative impacts on the environment and the development of antibiotic resistance. In this study, we investigated the effects of chemicals extracted from two types of rubber on microbiomes derived from a benthic sea urchin and two pelagic fish species. Additionally, we examined whether bacterial communities preconditioned with rubber-associated chemicals displayed adaptations to antibiotics. At the highest tested concentrations of chemicals, we observed reduced maximum growth rates and yields, prolonged lag phases, and increased alpha diversity. While the effects on alpha and beta diversity were not always conclusive, several bacterial genera were significantly influenced by chemicals from the two rubber sources. Subsequent exposure of sea urchin microbiomes preconditioned with rubber chemicals to the antibiotic ciprofloxacin resulted in decreased maximum growth rates. This indicates a more sensitive microbiome to ciprofloxacin when preconditioned with rubber chemicals. Although no significant interaction effects between rubber chemicals and ciprofloxacin exposure were observed in bacterial alpha and beta diversity, we observed log-fold changes in two bacterial genera in response to ciprofloxacin exposure. These findings highlight the structural and functional alterations in microbiomes originating from various marine species when exposed to rubber-associated chemicals and underscore the potential risks posed to marine life.

A198 RELATIONSHIP BETWEEN HEPATIC GENE EXPRESSION AND MICROBIOME ACCORDING TO DISEASE SEVERITY IN OBESE PATIENTS WITH NON-ALCOHOLIC FATTY LIVER DISEASE

Abstract Background Non-alcoholic fatty liver disease (NAFLD) ranges from steatosis to inflammation (steatohepatitis; NASH), fibrosis and cirrhosis. Development and progression of NAFLD/NASH is attributed to factors such as obesity and metabolic syndrome as well as intestinal microbiome (IM) but research is limited on the relationship between IM and hepatic gene expression. Aims Our objective was to assess in obese patients;1) the transcriptome signature of NAFLD and fibrosis and; 2) determine the relationship between the identified genes and IM. Methods Biochemical and anthropometric measurements as well fecal samples for IM shotgun metagenomic sequencing were collected in those with obesity undergoing bariatric surgery. Liver histology was assessed using Brunt scoring system and hepatic transcriptome was assessed using RNA-Seq. Differences in biochemical data and IM were determined using Kruskal-Wallis test followed by Wilcoxon ranked sum test. Differentially expressed genes (DEGs) in the liver were determined using DESeq2 package in R and had |log2 foldchange| ≥1 with adjusted p-value ampersand:003C0.05. Spearman correlation coefficients was used to evaluate the association between significant DEGs and significant bacteria and adjusted using Benjamini-Hochberg. Results 93 patients had both transcriptome and IM data. Of those, 27 had normal liver obese (NLO) and 66 had NAFLD (including 31 NASH). In those with NAFLD, 19 had no fibrosis (F0) and 47 had presence of fibrosis (F1-F4). We found 71 significantly different DEGs (e.g. PPP1R3G ) in NASH vs NLO and 12 (e.g. THBS2 and GLI2) in NAFLD with presence of fibrosis vs no fibrosis. Those with NASH generally had higher Escherichia coli, Blautia hansenii and lower Alistipees putredinis. Those with NAFLD and fibrosis had increased E. coli and reduced Eubacterium ventriosum and A. putredinis. In the preliminary analysis of NASH vs NLO, B. hansenii negatively correlated with glucose homeostasis-related PPP1R3G. In those with NAFLD, A. putredinis correlated negatively with THBS2 (i.e. Thrombospondin-2) and, in patients with NAFLD and fibrosis, E. coli correlated positively with GLI2 (related to activation of hepatic stellate cells). Conclusions DEGs were associated with specific bacteria in NASH or fibrosis. Next steps include analyzing the microbial metagenomic pathways as well as metabolites and determine the relationship between the IM, hepatic transcriptome and metabolites according to disease severity. Funding Agencies CIHRAmerican College of Gastroenterology Bridge Award, BBDC Tamarack Graduate Award in Diabetes Research

A182 EXPLORING THE LINK BETWEEN SPECIFIC MICROBIAL STRAINS AND ANXIETY AND DEPRESSION IN PATIENTS WITH ULCERATIVE COLITIS

Abstract Background Anxiety and/or depression (A/D) have been identified as significant co-morbidities of ulcerative colitis (UC), and even considered as extraintestinal manifestations, with emerging evidence to support the role for gut microbial dysbiosis in the natural history for both UC and A/D. However, there is a paucity of knowledge regarding the specific attributes of the gut bacteriome in individuals with UC with concomitant A/D. Aims To explore the connection between the gut bacteriome and A/D in a cohort of patients with UC. Methods This cross-sectional study included 29 participants diagnosed with UC either in remission or with mild to severe disease. To test for depression and anxiety, participants completed the Patient Health Questionnaire-8 (PHQ-8) and General Anxiety Disorder-7 (GAD-7) respectively. Participants provided a stool sample for microbiome analysis and the fecal bacteriome was assessed using metagenomic shotgun sequencing. Results Among the 29 patients, 55% were female (n=16) and 45% were male (n=13), 34.5 % (n=10) had a Partial Mayo Score over 5 indicating moderate-severe disease activity (median 3, IQR 1-6). Mean age was 38.3 years (± 12.2 years). Sixty-nine percent were on 5-ASA, 42.9% on steroids, 31.0% on Anti-TNF, and 20.7% on immunomodulators. Mean FCP was 976.4 μg/g (± 1589.2). Forty-four percent (n=12) screened positive for anxiety with a median GAD-7 score of 5.0 (IQR: 1-8), while 26% screened positive for depression with a median PHQ-8 of 3 (IQR: 3-10). Principal Coordinate Analysis (PCoA) reveals distinct clustering between UC patients with or without A/D (Figure 1). Regarding the microbial analyses, the log fold change of Firmicutes bacterium CAG:424, Holdemanella biformis, Blautia hansenii, Bifidobacterium pullorum, Anaerostipes caccae, and Ruminococcus gnavus exhibited positive associations with both A/D, whereas Roseburia sp. CAG:303, Prevotella copri, and Bacteroides stercoris demonstrated negative associations. Uniquely, Faecalicoccus pleomorphus and Lachnospira pectinoschiza showed negative associations only with the PHQ-8. Conclusions Our research affirms the presence of anxiety and depression in a cohort of UC patients. The severity of A/D measured by the GAD-7 and PHQ-8 were linked to the levels of specific fecal microbes. We introduce several novel species that warrant further examination in UC patients with and without A/D. As this was an exploratory study, the findings need replication in a larger sample size. PCoA results reveal distinct bacterial clustering patterns between individuals with minimal anxiety/depression (absence) versus those with mild/moderate/severe anxiety and depression as measured by GAD-7 and PHQ-8. Funding Agencies None

Protective role of arachidonic acid against diabetic myocardial ischemic injury: a translational study of pigs, rats, and humans

AimPatients with diabetes mellitus have poor prognosis after myocardial ischemic injury. However, the mechanism is unclear and there are no related therapies. We aimed to identify regulators of diabetic myocardial ischemic injury.Methods and resultsMass spectrometry-based, non-targeted metabolomic approach was used to profile coronary sinus blood from diabetic and non-diabetic Bama-mini pigs at 0.5-h post coronary artery ligation. Six metabolites had a |log2 (Fold Change)|> 1.3. Among them, the most changed is arachidonic acid (AA), levels of which were 32 times lower in diabetic pigs than in non-diabetic pigs. The AA-derived products, PGI2 and 6-keto-PGF1α, were also significantly reduced. AA treatment of cultured cardiomyocytes protected against cell death by 30% at 48 h of high glucose and oxygen deprivation, which coincided with increased mitophagic activity (as indicated by increased LC3II/LC3I, decreased p62 and increased parkin & PINK1), improved mitochondrial renewal (upregulation of Drp1 and FIS1), reduced ROS generation and increased ATP production. These cardioprotective effects were abolished by PINK1(a crucial mitophagy protein) knockdown or the autophagy inhibitor 3-Methyladenine. The protective effect of AA was also inhibited by indomethacin and Cay10441, a prostacyclin receptor antagonist. Furthermore, diabetic Sprague Dawley rats were subjected to coronary ligation for 40 min and AA treatment (10 mg/day per animal gavaged) decreased myocardial infarct size, cell apoptosis index, inflammatory cytokines and improved heart function. Scanning electron microscopy showed more intact mitochondria in the border zone of infarcted myocardium in AA treated rats. Lastly, diabetic patients after myocardial infarction had lower plasma levels of AA and 6-keto-PGF1α and reduced cardiac ejection fraction, compared with non-diabetic patients after myocardial infarction. Plasma AA level was inversely correlated with fasting blood glucose.ConclusionsAA protects against diabetic ischemic myocardial damage by promoting mitochondrial autophagy and renewal, which is related to AA derived PGI2 signaling. AA may represent a new strategy to treat diabetic myocardial ischemic injury.

Analysis of total RNA as a potential biomarker of developmental neurotoxicity in silico.

A vast number of neurodegenerative disorders arise from neurotoxicity. In neurotoxicity, more than 250 RNA molecules are up and downregulated. The manuscript investigates the exposure of chlorpyrifos organophosphate pesticide (COP) effect on total RNA in murine brain tissue in 4 genotypes for in silico neurodegeneration development. The GSE58103 dataset from the Gene Expression Omnibus (GEO) database applies for data preprocessing, normalization, and quality control. Differential expression analysis (DEG) uses the limma package in R. Study compared expression profiles from murine fetal brain tissues across four genotypes: PON-1 knockout (KO), tgHuPON1Q192 (Q-tg), tgHuPON1R192 (R-tg), and wild-type (WT). We analyze 60 samples, 15 samples per genotype, to identify DEGs. The significance criteria are adjusted p-value <.05 and a |log2 fold change| > 1. The study identifies microRNA485 as the potential biomarker of COP toxicity using the GSE58103 dataset. Significant differences exist for microRNA485 between KO and WT groups by differential expression analysis. Moreover, graphical analysis shows sample relationships among genotype groups. MicroRNA485 represents a promising biomarker for developmental COP neurotoxicity by utilizing in silico analysis in scientific practice.

Identification of differentially expressed genes associated with ferroptosis in Crohn's disease.

Ferroptosis-related genes may play a critical regulatory role in the pathogenesis of Crohn's disease (CD). The purpose of the present study was to identify genes expressed in CD that are associated with ferroptosis, and to provide guidance in the diagnosis and therapy of CD. CD mRNA expression data were initially gathered from the Gene Expression Omnibus (GEO) database. GSE75214 and GSE102133 datasets were selected as the major targets and were analyzed for differentially expressed genes (DEGs). Subsequently, R software was used to analyze the common genes among the DEGs between CD and ferroptosis-related genes. Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genome pathway analysis were conducted to identify related pathways and functions. Protein-protein interaction (PPI) analysis was performed to identify target genes. The DSigDB website was used to predict potential target drugs for hub genes. Reverse transcription-quantitative (RT-q) PCR was employed to detect the expression of these ferroptosis-related genes in clinical samples obtained from healthy controls and patients with CD. According to the two GEO datasets, 13 ferroptosis DEGs (11 upregulated genes and two downregulated genes) were identified in CD with thresholds of P<0.05 and |log2 fold change|>1, and were selected for further analysis. PPI analysis indicated the mutual effects among these genes and filtered out five hub genes. The top 10 potential targeted drugs were selected. The qPCR results showed that the expression levels of three genes, namely, IL-6, prostaglandin-endoperoxide synthase 2 (PTGS2) and dual oxidase 2 (DUOX2), were different between CD samples and healthy samples. This result was consistent with the results obtained from the bioinformatics analysis. In conclusion, bioinformatics analysis identified a total of 13 ferroptosis-associated genes in CD. Further verification by qPCR showed that IL-6, PTGS2 and DUOX2 may affect the process of CD by regulating ferroptosis. These findings might provide new biomarkers, diagnostic and therapeutic markers for CD.

Identification and verification of anoikis-related gene markers to predict the prognosis of patients with bladder cancer and assist in the diagnosis and treatment of bladder cancer.

The recurrence and mortality rates of bladder cancer are extremely high, and its diagnosis and treatment are global concerns. The mechanism of anoikis is closely related to tumor metastasis. First, we obtained all the data needed for this study from a public database through a formal operational process. The data were then analyzed by bioinformatics technology. Through the limma package, we screened and obtained 313 anoikis-related genes [false discovery rate (FDR) <0.05, |log fold change (FC) | >0.585]. Then, through univariate independent prognostic analysis, we further screened 146 genes (P<0.05) related to the prognosis of bladder cancer from 313 differential genes. These 146 prognostically relevant differential genes were used for least absolute shrinkage and selection operator (LASSO) regression for further screening to obtain model-related genes and output model formulas. Through the nomogram, we can calculate the survival rate of patients more accurately. The accuracy of the nomogram was also confirmed by calibration curves, independent prognostic analysis, receiver operating characteristic (ROC) curves, decision curve analysis (DCA) curves. We then analysed the sensitivity of immunotherapy in bladder cancer patients with different risk scores via Tumor Immune Dysfunction and Exclusion (TIDE). Through bioinformatics technology and public databases, a prognostic model including 9 anoikis-related genes (KLF12, INHBB, CASP6, TGFBR3, FASN, TPM1, OGT, RAC3, ID4) was obtained. Integrating risk scores with clinical information, we obtained a nomogram that can accurately predict patient survival. By querying the immunohistochemical results of the Human Protein Atlas database, two of the nine model-related genes (FASN, RAC3) have the value of further research and are expected to become new biomarkers to assist the diagnosis and treatment of bladder cancer. Through immune-related analysis, we found that patients in the low-risk group appeared to be more suitable for immunotherapy, while drug sensitivity analysis showed that bladder cancer patients in the high-risk group were more sensitive to common chemotherapy drugs. In this study, a prognostic model that can accurately predict the prognosis of patients with bladder cancer was constructed. FASN and RAC3 are expected to become a new biomarker for the diagnosis and treatment of bladder cancer.

AIM high: Epigenetic modulation and immune stimulation in bladder cancer.

648 Background: Immune-checkpoint inhibitors (ICI) have emerged as valuable treatment options for patients with bladder cancer, particularly those who are ineligible for other therapies or have experienced disease progression on treatment. Response rates are only 20-30%, highlighting the need for biomarkers to predict treatment response and strategies to overcome resistance. Epigenetic modulators including the DNA methyltransferase (DNMT) inhibitor 5-azacitidine (AZA) and histone deacetylase inhibitors (HDACi) may represent co-targeting therapies in combination with ICI to enhance antitumor immunity. AZA has been demonstrated to increase the expression of a panel of immunomodulatory genes termed the “AZA Immune gene panel (AIM)” in breast, colorectal, and ovarian cancer cell lines. We hypothesized that bladder cancer with low AIM expression can be therapeutically converted by epigenetic modulation to “AIM-high” for future combination with ICI. Methods: Five human bladder cancer cell lines were selected: SCaBER, SW780, T24, RT4 and one developed at our institution (CoCaB1). The Cancer Cell Line Encyclopedia (CCLE) was used to screen the four included lines for intrinsic AIM expression, which were predicted to be AIM-high (n=1) and AIM-low (n=3). A cell viability assay was performed to screen for AZA and HDACi toxicity. Cells were treated with drug or vehicle for 72 hours and harvested at day 6, 14, and 21. RNA was extracted for RNA sequencing (RNA-seq) analyses. For analysis of AIM gene expression in clinical specimens, RNA-seq data was obtained from primary (n=6) and metastatic (n=83) bladder cancer specimens for 20 patients participating in the University of Washington Bladder Cancer Rapid Autopsy Program. Results: RNA-seq analyses of 5 cell lines demonstrated increased expression (&gt;log 2-fold change, p&lt;0.05) of a subset of AIM genes involved in inflammation, interferon, cytokine/chemokine signaling, and cancer testis antigens (CTA, a family of immunogenic proteins) with AZA treatment. Co-treatment with HDACi showed additional upregulation of AIM genes. Gene set enrichment analyses uncovered enrichment in T-cell pathway activation. In metastatic bladder cancer tissues, interferon, cytokine/chemokine, inflammation, and CTA AIM gene set categories were upregulated in a subset of patients. AIM gene enrichment displayed a patient-dependent pattern and was consistent across metastatic sites within a patient. Conclusions: Epigenetic priming therapy increases the expression of immunomodulatory and T-cell related genes in bladder cancer cell lines with low baseline AIM expression. Additional correlative studies in the rapid autopsy series will further determine clinical- and treatment-related factors contributing to intrinsic immune-related gene expression. Building on this paradigm, future in vivo and clinical studies could lead to novel combination therapies for patients with bladder cancer.

Regulation of Follicular Development in Chickens: WIF1 Modulates Granulosa Cell Proliferation and Progesterone Synthesis via Wnt/β-Catenin Signaling Pathway.

Proliferation, apoptosis, and steroid hormone secretion by granulosa cells (GCs) and theca cells (TCs) are essential for maintaining the fate of chicken follicles. Our previous study showed that the Wnt inhibitor factor 1 (WIF1) plays a role in follicle selection. However, the significance of WIF1 in GC- and TC-associated follicular development was not explicitly investigated. This study found that WIF1 expression was strongly downregulated during follicle selection (p < 0.05) and was significantly higher in GCs than in TCs (p < 0.05). WIF1 inhibits proliferation and promotes apoptosis in GCs. Additionally, it promotes progesterone secretion in prehierarchal GCs (pre-GCs, 1.16 ± 0.05 ng/mg vs. 1.58 ng/mg ± 0.12, p < 0.05) and hierarchal GCs (hie-GCs, 395.00 ng/mg ± 34.73 vs. 527.77 ng/mg ± 27.19, p < 0.05) with the participation of the follicle-stimulating hormone (FSH). WIF1 affected canonical Wnt pathways and phosphorylated β-catenin expression in GCs. Furthermore, 604 upregulated differentially expressed genes (DEGs) and 360 downregulated DEGs in WIF1-overexpressed GCs were found through RNA-seq analysis (criteria: |log2⁡(FoldChange)| > 1 and p_adj < 0.05). Cytokine-cytokine receptor interaction and the steroid hormone biosynthesis pathway were identified. In addition, the transcript of estrogen receptor 2 (ESR2) increased significantly (log2⁡(FoldChange) = 1.27, p_adj < 0.05). Furthermore, we found that WIF1 regulated progesterone synthesis by upregulating ESR2 expression in GCs. Additionally, WIF1 suppressed proliferation and promoted apoptosis in TCs. Taken together, these results reveal that WIF1 stimulates follicle development by promoting GC differentiation and progesterone synthesis, which provides an insight into the molecular mechanism of follicle selection and egg-laying performance in poultry.

Co-culturing with Streptococcus anginosus alters Staphylococcus aureus transcriptome when exposed to tonsillar cells.

Improved understanding of Staphylococcus aureus throat colonization in the presence of other co-existing microbes is important for mapping S. aureus adaptation to the human throat, and recurrence of infection. Here, we explore the responses triggered by the encounter between two common throat bacteria, S. aureus and Streptococcus anginosus, to identify genes in S. aureus that are important for colonization in the presence of human tonsillar epithelial cells and S. anginosus, and further compare this transcriptome with the genes expressed in S. aureus as only bacterium. We performed an in vitro co-culture experiment followed by RNA sequencing to identify interaction-induced transcriptional alterations and differentially expressed genes (DEGs), followed by gene enrichment analysis. A total of 332 and 279 significantly differentially expressed genes with p-value < 0.05 and log2 FoldChange (log2FC) ≥ |2| were identified in S. aureus after 1 h and 3 h co-culturing, respectively. Alterations in expression of various S. aureus survival factors were observed when co-cultured with S. anginosus and tonsillar cells. The serine-aspartate repeat-containing protein D (sdrD) involved in adhesion, was for example highly upregulated in S. aureus during co-culturing with S. anginosus compared to S. aureus grown in the absence of S. anginosus, especially at 3 h. Several virulence genes encoding secreted proteins were also highly upregulated only when S. aureus was co-cultured with S. anginosus and tonsillar cells, and iron does not appear to be a limiting factor in this environment. These findings may be useful for the development of interventions against S. aureus throat colonization and could be further investigated to decipher the roles of the identified genes in the host immune response in context of a throat commensal landscape.

P060 Increased metabolic activity of faecal Sutterella Wadsworthensis at IBD onset is associated with an increased risk of escalation to biologic therapies - Results from the Birimingham Inception Cohort Study

Abstract Background Gut microbiome disruption is an established characteristic of IBD. The lack of treatment naïve longitudinal microbiome data makes it challenging to separate the influence of potential confounders. We present detailed stool metagenomics analysis from a treatment naïve cohort of IBD patients seen in our rapid-access IBD inception clinic. Methods Stool collected in DNA Genotek OM-200 kits was collected pre-diagnosis, alongside a faecal calprotectin (FCP). Microbial DNA was extracted. Libraries were prepared using NEBNext Ultra II FS DNA Library Prep Kit, NEBNext Multiplex Oligos and sequenced using an Illumina NextSeq550. Data processing was undertaken using the HUMAnN 3.0 workflow. After quality control, a median of 3.6 million reads per sample were obtained. Abundance was mapped to the ChocoPhlAn database and functional pathways derived from MetaCyc. Diversity analysis was undertaken in Vegan, with MaAsLin2 for taxa differences. A False discovery Rate (FDR) &amp;lt;0.2 was considered relevant, &amp;lt;0.05 highly significant. Results A total of 45 CD and 33 UC inception patients were included. There was no significant difference in alpha diversity (Shannon; UC median 2.58[IQR 0.8], CD 2.69[1.04] p=0.36) or differential clustering for beta diversity. Shannon alpha diversity significantly associated with FCP across IBD (n=74, Spearman’s Rho [SR] -0.33 p=0.004) and endoscopic severity in both IBD subtypes (UCEIS n=30 SR -0.37 p=0.047, SESCD n=44 SR -0.35 p=0.016). Across IBD, patients going on to require biologic therapy had a significantly lower baseline Shannon (BioYes 2.26[0.98], BioNo 2.77[0.88] p=0.02, split by IBD subtype in Figure 1). Across IBD, increased FCP associated with depletion of two strains of Faecalibacterium Prausnitzii (SGB15318, log2 fold change[L2FC] -1.2 FDR 0.09; SGB15342 L2FC -1.1 FDR 0.15) alongside Roseburia Intestinalis (SGB4951 L2FC -0.7 FDR 0.09). R.Intestinalis also negatively correlated with Harvey Bradshaw Index (L2FC -0.93 FDR 0.02), whilst F.Prausnitzii (SGB15342) did with UCEIS (L2FC -2.3 FDR 0.05). 82 metabolic pathways correlated with UCEIS (FDR &amp;lt;0.2, 8 &amp;lt;0.05), with 89 for SESCD (6 &amp;lt;0.05). Increased activity of Sutterella Wadsworthensis associated positively with UCEIS (5 pathways). On initial comparison across IBD, no significant signal was seen for future biologic utilisation. However, if analyses were corrected for baseline inflammation (derived by FCP), increased activity of S.Wadsworthensis was again seen (Table 1). Conclusion Reduced alpha diversity at IBD onset associated with increased disease severity and future utilisation of biologic treatment. Increased activity of S.Wadsworthensis associated with an increased risk of future biologic requirement above that explained by mucosal inflammation alone.

Comparative Gene Signature of Nociceptors Innervating Mouse Molar Teeth, Cranial Meninges, and Cornea.

The trigeminal ganglion (TG) collects afferent sensory information from various tissues. Recent large-scale RNA sequencing of neurons of the TG and dorsal root ganglion has revealed a variety of functionally distinct neuronal subpopulations, but organ-specific information is lacking. To link transcriptomic and tissue-specific information, we labeled small-diameter neurons of 3 specific subpopulations of the TG by local application of lipophilic carbocyanine dyes to their innervation site in the dental pulp, cornea, and meninges (dura mater). We then collected mRNA-sequencing data from fluorescent neurons. Differentially expressed genes (DEGs) were analyzed and subjected to downstream gene set enrichment analysis (GSEA), and ion channel profiling was performed. A total of 10,903 genes were mapped to the mouse genome (>500 reads). DEG analysis revealed 18 and 81 genes with differential expression (log 2 fold change > 2, Padj < .05) in primary afferent neurons innervating the dental pulp (dental primary afferent neurons [DPAN]) compared to those innervating the meninges (meningeal primary afferent neurons [MPAN]) and the cornea (corneal primary afferent neurons [CPAN]). We found 250 and 292 genes differentially expressed in MPAN as compared to DPAN and to CPAN, and 21 and 12 in CPAN as compared to DPAN and MPAN. Scn2b had the highest log 2 fold change when comparing DPAN versus MPAN and Mmp12 was the most prominent DEG when comparing DPAN versus CPAN and, CPAN versus MPAN. GSEA revealed genes of the immune and mitochondrial oxidative phosphorylation system for the DPAN versus MPAN comparison, cilium- and ribosome-related genes for the CPAN versus DPAN comparison, and respirasome, immune cell- and ribosome-related gene sets for the CPAN versus MPAN comparison. DEG analysis for ion channels revealed no significant differences between the neurons set except for the sodium voltage-gated channel beta subunit 2, Scn2b . However, in each tissue a few ion channels turned up with robust number of reads. In DPAN, these were Cacna1b , Trpv2 , Cnga4 , Hcn1 , and Hcn3 , in CPAN Trpa1 , Trpv1 , Cacna1a , and Kcnk13 and in MPAN Trpv2 and Scn11a . Our study uncovers previously unknown differences in gene expression between sensory neuron subpopulations from the dental pulp, cornea, and dura mater and provides the basis for functional studies, including the investigation of ion channel function and their suitability as targets for tissue-specific analgesia.

Abstract A006: Differential gene expression to delineate racial disparities in the molecular landscape of pancreatic ductal adenocarcinoma

Abstract Introduction: In the US, there are racial disparities in PDAC incidence and mortality, higher among blacks than whites. In addition to socioeconomic status, lifestyles, and age, genetics also contributes to these disparities. Thus, we conducted transcriptomic analyses (RNA-seq) of PDAC samples collected from African American (AA) and Caucasian (CA) patients to identify race/ethnicity-specific gene expression profiles and their related pathways to find determinants that contribute to the aggressive phenotypes of PDACs. This study is relevant to the UAB catchment area, since about 30% of patients with PDAC are AAs. Methodology: Histologically confirmed PDACs (n=40) from AA (9 PDAC and 3 matching normal tissues) and CA (31 PDAC and 5 matching normal tissues) were included in this study. FFPE sections of PDACs and their corresponding normal tissues were macro-dissected for RNA isolation. Whole transcriptomic sequencing was performed with a NextSeq 500/550 platform. Trimmed reads were mapped to a human reference genome (hg38) using HISAT, and gene level read count data were obtained using HTSeq. Differential expression analysis was performed using the DESeq2 bioconductor package. ClusterProfiler/DOSE R packages were used for gene ontology and KEGG pathway enrichment analyses. Genes with log 2-fold change of ≥1 and adjusted P-value &amp;lt;0.05 were considered as differentially expressed. Results: Among the top upregulated genes altered in only AA PDACs, compared to their normal tissues, were RNF144B, TESPA1, KLHL17, H2AX, MDGA1, CDK20, PHLDA3, SLC6A16, PARVG, GATD3, NUDT16, RENBP, RTL8C, C1QTNF1, HLA−DRB5, PARP15, PPP1R16B, RASGRP2, and CHST15. Of note, targeting CHST15, by an RNA oligonucleotide, STNM01 in Phase I/IIa trial on unresectable PDAC patients showed improved overall survival. Additionally, inhibition of KLHL17, an upstream activator of Ras/MAPK, could be a candidate target in PDAC. The KEGG pathways altered in AA PDACs, were glycerophospholipid metabolism; bile secretion; retinol metabolism; regulation of lipolysis in adipocytes; and pantothenate and CoA biosynthesis. In CAs, the top upregulated genes in PDACs, compared to their normal tissues, were PPY, UGT1A10, GPR20, SDR16C5, KLK7, MYBPC1, ITLN1, PADI1, CLCA1, UGT1A9, and SERPINB3.The KEGG pathways altered in CA PDACs, were cellular senescence; AGE−RAGE signaling pathway in diabetic complication; PD−L1 and PD−1 checkpoint pathway; and central carbon metabolism. There were 13 genes differentially modulated in AA PDACs as compared to CA PDACs. The 6 down-regulated were USP17L1, C2CD4D, MMP13, DCUN1D5, and 2 genes without annotations (ENSG00000276345 and ENSG00000280966); the 7 up-regulated genes were SEMA4A, LETM2, HMOX1, OTUB2, MEDAG, VEGFD, and HBA1. Immunohistochemical validation of these markers is in progress. Conclusions: Findings of this study showed distinct gene expression profiles and differentially modulated pathways in AA and CA PDAC patients. These results will aid in identifying aggressive phenotypes and new targets for developing race/ethnicity-based therapeutic interventions. Citation Format: Prachi Bajpai, Ravi Paluri, Sameer Al Diffalha, Darshan S. Chandrashekar, Farrukh Afaq, Ryan Bash, C. Ryan Miller, Sooryanarayana Varambally, Moh’d Khushman, Upender Manne. Differential gene expression to delineate racial disparities in the molecular landscape of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A006.

Shatavari supplementation in postmenopausal women alters the skeletal muscle proteome and pathways involved in training adaptation

PurposeShatavari is an understudied, widely available herbal supplement. It contains steroidal saponins and phytoestrogens. We previously showed that six weeks of shatavari supplementation improved handgrip strength and increased markers of myosin contractile function. Mechanistic insights into shatavari’s actions are limited. Therefore, we performed proteomics on vastus lateralis (VL) samples that remained from our original study.MethodsIn a randomised double-blind trial, women (68.5 ± 6 years) ingested either placebo or shatavari (equivalent to 26,500 mg/d fresh weight) for six weeks. Tandem mass tag global proteomic analysis of VL samples was conducted (N = 7 shatavari, N = 5 placebo). Data were normalized to total peptides and scaled using a reference sample. Data were filtered using a 5% FDR. For each protein, the pre to post supplementation difference was expressed as log2 fold change. Welch’s t tests with Benjamini–Hochberg corrections were performed for each protein. Pathway enrichment (PADOG, CAMERA) was interrogated in Reactome (v85).ResultsNo individual protein was significantly different between supplementation conditions. Both PADOG and CAMERA indicated that pathways related to (1) Integrin/MAPK signalling, (2) metabolism/insulin secretion; (3) cell proliferation/senescence/DNA repair/cell death; (4) haemostasis/platelets/fibrin; (5) signal transduction; (6) neutrophil degranulation and (7) chemical synapse function were significantly upregulated. CAMERA indicated pathways related to translation/amino acid metabolism, viral infection, and muscle contraction were downregulated.ConclusionOur analyses indicate that shatavari may support muscle adaptation responses to exercise. These data provide useful signposts for future investigation of shatavari’s utility in conserving and enhancing musculoskeletal function in older age. Trial registrationNCT05025917 30/08/21, retrospectively registered.

Maternal PM2.5 exposure is associated with preterm birth and gestational diabetes mellitus, and mitochondrial OXPHOS dysfunction in cord blood

Maternal exposure to fine particulate matter (PM2.5) is associated with adverse pregnancy and neonatal health outcomes. To explore the mechanism, we performed mRNA sequencing of neonatal cord blood. From an ongoing prospective cohort, Air Pollution on Pregnancy Outcome (APPO) study, 454 pregnant women from six centers between January 2021 and June 2022 were recruited. Individual PM2.5 exposure was calculated using a time-weighted average model. In the APPO study, age-matched cord blood samples from the High PM2.5 (˃15 ug/m3; n = 10) and Low PM2.5 (≤ 15 ug/m3; n = 30) groups were randomly selected for mRNA sequencing. After selecting genes with differential expression in the two groups (p-value < 0.05 and log2 fold change > 1.5), pathway enrichment analysis was performed, and the mitochondrial pathway was analyzed using MitoCarta3.0. The risk of preterm birth (PTB) increased with every 5 µg/m3 increase of PM2.5 in the second trimester (odds ratio 1.391, p = 0.019) after adjusting for confounding variables. The risk of gestational diabetes mellitus (GDM) increased in the second (odds ratio 1.238, p = 0.041) and third trimester (odds ratio 1.290, p = 0.029), and entire pregnancy (odds ratio 1.295, p = 0.029). The mRNA-sequencing of cord blood showed that genes related to mitochondrial activity (FAM210B, KRT1, FOXO4, TRIM58, and FBXO7) and PTB-related genes (ADIPOR1, YBX1, OPTN, NFkB1, HBG2) were upregulated in the High PM2.5 group. In addition, exposure to high PM2.5 affected mitochondrial oxidative phosphorylation (OXPHOS) and proteins in the electron transport chain, a subunit of OXPHOS. These results suggest that exposure to high PM2.5 during pregnancy may increase the risk of PTB and GDM, and dysregulate PTB-related genes. Alterations in mitochondrial OXPHOS by high PM2.5 exposure may occur not only in preterm infants but also in normal newborns. Further studies with larger sample sizes are required.

Astragaloside IV Alleviates Acute Liver Failure Induced by D-GalN/LPS by Upregulating Autophagy and Reducing Inflammation

Abstract Acute liver failure (ALF) is a life-threatening condition that manifests in an extremely serious manner and progresses rapidly. The following study investigated the protective effect of astragaloside IV (AS-IV), a traditional Chinese drug, on ALF, and its underlying mechanisms, focusing on autophagy and inflammation regulation. Mice were randomly divided into a saline group, a D-galactosamine and lipopolysaccharide (D-GalN/LPS) group and an AS-IV group. Biochemical analysis, immunohistochemistry, cytometric bead array, high-throughput quantitative PCR, flow cytometry and Western analysis were used to assess inflammation and liver damage 5 hours after D-GalN/LPS exposure. Astragaloside IV treatment reduced mortality by alleviating D-GalN/LPS–induced hepatic damage and decreasing inflammation (decreasing Ly6c+ monocyte levels, reducing inflammatory cytokines and increasing anti-inflammatory factors) as well as upregulating autophagy. Furthermore, PCR array was used to detect expression of autophagy-related genes, which demonstrated a Log2 fold change in gene expression between the AS-IV and D-GalN/LPS groups ranging from 1.19 to −3.53, with Tnfsf10 showing the largest alteration between the two groups. These data suggest that AS-IV may alleviate ALF by upregulating autophagy and reducing inflammation, and it may therefore be an interesting drug for alleviating ALF.

Identification of M2 Macrophage-Related Key Genes in Advanced Atherosclerotic Plaques by Network-Based Analysis.

Atherosclerotic plaque accounts for major adverse cardiovascular events because of its vulnerability. The classically activated macrophage (M1) and alternatively activated macrophage (M2) are implicated in the progression and regression of plaque, respectively. However, the therapeutic targets related to M2 macrophages still remain largely elusive. In this study, cell-type identification by estimating relative subsets of RNA transcripts and weighted gene coexpression network analysis algorithms were used to establish a weighted gene coexpression network for identifying M2 macrophage-related hub genes using GSE43292 data set. The results showed that genes were classified into 7 modules, with the blue module (Cor = 0.67, P = 3e-05) being the one that was most related to M2 macrophage infiltration in advanced plaques, and then 99 hub genes were identified from blue module. Meanwhile, 1289 differentially expressed genes were produced in GSE43292 data set. Subsequently, the intersection genes of hub genes and differentially expressed genes, including AKTIP , ASPN , FAM26E , RAB23 , PLS3 , and PLSCR4 , were obtained by Venn diagrams and named as key genes. Further validation using data sets GSE100927 and GSE41571 showed that 6 key genes all downregulated in advanced and vulnerable plaques compared with early and stable plaque samples (|Log2 (fold change)| > 0.5, P < 0.05 or 0.001), respectively. Receiver operator characteristic curve analysis indicated that the 6 key genes might have potential diagnostic value. The validation of key genes in the model in vitro and in vivo also demonstrated decreased mRNA expressions of AKTIP , ASPN , FAM26E , RAB23 , PLS3 , and PLSCR4 ( P < 0.05 or 0.001). Collectively, we identified AKTIP, ASPN, FAM26E, RAB23, PLS3, and PLSCR4 as M2 macrophage-related key genes during atherosclerotic progression, proposing potential intervention targets for advanced atherosclerotic plaques.

Aging or chronic stress impairs working memory and modulates GABA and glutamate gene expression in prelimbic cortex.

The glucocorticoid (GC) hypothesis posits that effects of stress and dysregulated hypothalamic-pituitary-adrenal axis activity accumulate over the lifespan and contribute to impairment of neural function and cognition in advanced aging. The validity of the GC hypothesis is bolstered by a wealth of studies that investigate aging of the hippocampus and decline of associated mnemonic functions. The prefrontal cortex (PFC) mediates working memory which also decreases with age. While the PFC is susceptible to stress and GCs, few studies have formally assessed the application of the GC hypothesis to PFC aging and working memory. Using parallel behavioral and molecular approaches, we compared the effects of normal aging versus chronic variable stress (CVS) on working memory and expression of genes that encode for effectors of glutamate and GABA signaling in male F344 rats. Using an operant delayed match-to-sample test of PFC-dependent working memory, we determined that normal aging and CVS each significantly impaired mnemonic accuracy and reduced the total number of completed trials. We then determined that normal aging increased expression of Slc6a11, which encodes for GAT-3 GABA transporter expressed by astrocytes, in the prelimbic (PrL) subregion of the PFC. CVS increased PrL expression of genes associated with glutamatergic synapses: Grin2b that encodes the GluN2B subunit of NMDA receptor, Grm4 that encodes for metabotropic glutamate receptor 4 (mGluR4), and Plcb1 that encodes for phospholipase C beta 1, an intracellular signaling enzyme that transduces signaling of Group I mGluRs. Beyond the identification of specific genes that were differentially expressed between the PrL in normal aging or CVS, examination of Log2 fold-changes for all expressed glutamate and GABA genes revealed a positive association between molecular phenotypes of aging and CVS in the PrL but no association in the infralimbic subregion. Consistent with predictions of the GC hypothesis, PFC-dependent working memory and PrL glutamate/GABA gene expression demonstrate comparable sensitivity to aging and chronic stress. However, changes in expression of specific genes affiliated with regulation of extracellular GABA in normal aging vs. genes encoding for effectors of glutamatergic signaling during CVS suggest the presence of unique manifestations of imbalanced inhibitory and excitatory signaling in the PFC.

Identification of new diagnostic targets for hepatitis B virus‐induced liver fibrosis

AbstractBackgroundLiver fibrosis is a transitional stage from hepatitis to cirrhosis, and hepatitis B virus (HBV) is the most common cause of liver disease. Transcriptome sequencing technology and bioinformatics analysis are increasingly being used to screen diagnostic targets for liver fibrosis.MethodsThe GSE171294 dataset of HBV‐induced liver fibrosis tissue and normal tissue was obtained from the Gene Expression Omnibus (GEO) public database and used to screen for differentially expressed mRNAs using R software. mRNAs with |log fold change| &gt;1 and p &lt; 0.05 were considered to be differentially expressed. A heat map was drawn to visualize the expression patterns of the differentially expressed mRNAs. To screen for candidate target mRNAs, the differentially expressed mRNAs were annotated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis. Finally, a protein–protein interaction (PPI) network was constructed to analyze the relationships between the differentially expressed mRNAs.ResultsA total of 243 differentially expressed mRNAs were identified (p &lt; 0.05); 129 were up‐regulated and 114 were down‐regulated. The up‐regulated and down‐regulated mRNAs were significantly enriched in 16 and 8 KEGG pathways, respectively. The enriched KEGG pathways included Salmonella infection, Protein processing in the endoplasmic reticulum, IL‐17 signaling pathway, and Aldosterone synthesis and secretion. The enriched GO terms were related mainly to cell proliferation, apoptosis, endoplasmic reticulum complex assembly, and myosin synthesis. The PPI network contained 161 nodes and 120 pairs of interactions. The top 10 key nodes were CAV1, CD4, NR3C1, PDIA3, EZR, IRF4, SOX9, HSP90AB1, CD40, and SEC13.ConclusionsBioinformatics analysis of the transcriptome sequencing data in the GSE171294 dataset identified CD4, NR3C1, and EZR and other genes at key nodes as new targets for the treatment of liver fibrosis caused by HBV. These results provide new insights for HBV‐induced liver fibrosis research and clinical treatment.