DESeq Analysis Research Articles

Transcriptomic and proteomic analysis of resistance to matrine in Oedaleus asiaticus (Orthoptera: Oedipodidae)

In order to determine the specific genes and proteins that are affected by matrine and play a role in regulating metabolism in the locust species Oedaleus asiaticus, we conducted RNA-seq, proteomic sequencing, and bioinformatics analyses on third-instar nymphs. These nymphs were divided into two groups: one group was grown under normal conditions, while the other group was treated with matrine. The purpose of this investigation was to gain insight into the molecular mechanisms underlying matrine resistance. Genes and proteins that exhibited differential expression were identified and subjected to analysis using bioinformatics software. The DESeq analysis revealed a total of 743 transcripts that were differentially expressed (DETs). Among these, 208 transcripts were up-regulated, and 535 were downregulated in ZO/ZCK. The iTRAQ discovered that 34 and 65 proteins were, respectively, up- and down-regulated in ZO/ZCK. Additionally, enrichment studies based on Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed. The KEGG and GO analysis of the DEGs suggested the essential roles of matrine treatment in the regulation of O. asiaticus, especially via the biosynthesis of amino acids, glycolysis/gluconeogenesis, iInsulin signaling pathway and MAPK signaling pathway. The findings demonstrated that matrine exerted control of the growth of O. asiaticus via modulating the synthesis of metabolism and biosynthesis. Increased expression of detoxifying enzymes was observed, which may be related to matrine detoxification. These findings provide a basis for better comprehension of the molecular mechanism behind the regulation of development mediated by matrine in Asian locust hoppers.

Responses of soil physico-chemical properties, structure of the microbial community and crop yields to different fertilization practices in Russia's conventional farming system.

The use of fertilizers affects not only the soil fertility and crop yield, but also significantly changes the taxonomic structure of the soil microbiocenosis. Here, based on stationary field experiment, we studied the influence of organo-mineral fertilizer (ОМF), modified by bacteria Bacillus subtilis, H-13 in comparison with different fertilizer systems (organic, mineral, organo-mineral) on (i) crop yield, (ii) physical and chemical properties, and (iii) alpha and beta diversity of the microbial community Albic Retisol (Loamic, Aric, Cutanic, Differentic, Ochric). The studies were carried out against the background of liming (рНКCl - 5.9) and without it (рНКCl - 5.1). The use of only one cattle farmyard manure was less effective than its co-application with mineral fertilizers in half doses. A similar effect was obtained when applying ОМF. In addition, the use of OMF contributes to a significant increase in the reserves of soil organic carbon in the soil layer 0-20 cm by 18%-32%. Using high-throughput sequencing of the 16S rRNA variable V4 gene sequence libraries, 10.759 taxa from 456 genera were identified, assigned to 34 fila (31 bacterial and 3 archaeotic. Unilateral application of mineral fertilizers leads to a significant decrease in the alpha diversity of the structure of soil microbial communities (OTE (other things equal) and Shannon index). A clear clustering of the microbiota was found in the variants with and without the introduction of сattle farmyard manure. It is revealed that the taxonomic structure of the microbiocenosis is formed under the influence of two main factors: crop rotation culture and applied fertilizers. The type of cultivated crop determines the dynamics of the microbiota at the level of larger taxa, such as domains, and fertilizers affect the structure of the microbial community at a lower taxonomic level (phyla, orders, bloodlines). On the basis of the Deseq analysis, marker taxa were identified, according to the share participation of which it is possible to determine the type of cultivated crop and fertilizers used in the experiment. Understanding the dynamics of taxa association and other influential factors can lead to the creation of universal systems of metagenomic indication, where tracking the dynamics of microbial communities will allow for a comprehensive assessment of the agroecological state of soils and timely decisions to prevent their degradation.

Ketamine Promoted Breast Cancer Invasion and Metastasis Through Up-regulating Wnt, BMP, and EGFR Signaling.

In this study, we used an orthotropic breast cancer model combined with ketamine addiction and next-generation sequencing (NGS) to comprehensively investigate molecular alterations in ketamine-mediated metastasis. Ketamine is widely used in anesthesia and drug abuse. Our previous study revealed that ketamine promotes the growth of breast cancer cells; however, the detailed molecular mechanism remains unknown. An orthotropic breast cancer model was established by injecting EO771 breast cancer cells into the mammary fat pad of mice intraperitoneally administered ketamine (30 mg/kg, daily) for 68 days. Tumors collected at day 38 were frozen for future analysis, and their metastasis state was checked at day 68. Tumors were grouped and subjected to NGS analysis, followed by differential gene expression analysis (DEseq) and pathway identification. DEseq analysis showed that ketamine up-regulated metastasis-related signaling, and the key genes were BMP5, FZD6, MMP1B, EGFR, WNT5A, BMP7, and DCN. Ketamine addiction up-regulates the expression of genes involved in the Wnt, EGFR, and BMP signaling cascades, which may be associated with breast cancer progression and metastasis.

DOP79 Alternative polygenic forms of Inflammatory Bowel Disease are not captured by current generations of polygenic risk scores

Abstract Background Polygenic risk scores (PRSs) have emerged as a promising tool to ascertain individuals at high risk of developing IBD. However, the sensitivity of current PRSs is limited, and will likely remain so in the immediate future. The default expectation is that patients not captured by PRSs must be enriched for rare variants with strong effects and/or environmental risk factors. We set out to investigate an alternative scenario, namely that combinations of common variants in non-GWAS genes conform population of patients that are missed by large genetic studies for the main forms of the disease. Methods MAGMA was used to identify genes from the International Inflammatory Bowel Disease Genetics Consortium (IIBDGC) GWAS. We selected a sort of monogenic IBD genes1 which were significant for IBD (pval ≈ 10-4) but do not overlap with polygenic IBD genes. These genes were compared using DESeq tool on two transcriptomic databases: RISK (Crohn’s disease (CD) onset vs non-IBD) and PROTECT (ulcerative colitis (UC) onset vs 52 weeks). A second DESeq analysis was performed between patients with high against low expression of these 13 genes. We explored these genes using in-silico predicted expression based on eQTL in 9 different tissues for UK Biobank (UKBB) population, comparing UKBB individuals according to disease and polygenic risk score status. Results The starting point based on MAGMA analysis led to the detection of 13 monogenic IBD genes which may partially participate in polygenic forms of the disease. The transcriptomic analyses, based on stratification according to the levels of these 13 genes, implied that these genes participate in regulatory programmes that are particularly heterogeneous and variable among cases. The detected differentially expressed genes are associated with immune processes and receptors, inflammation transporters, and digestive problems, as well as skin development, humoral response, extracellular matrix, and mitochondrial component. The TWAS-based estimation analyses hint that low-PRS patients are indeed enriched for common variants associated with higher pathogenic levels at the 13 selected genes. Conclusion Although polygenic risk scores may play a valuable role in patient management, our study hints at the presence of patients with common polygenic forms that are not captured by current genetic risk estimators. 1. Chrissy B. et al, 2022. Gastroenterology.

Bioinformatics analysis of miRNA and its associated genes to identify potential biomarkers of oral submucous fibrosis and oral malignancy.

MicroRNAs are a group of non-coding RNA that controls the gene expression. The interaction between miRNA and mRNA is thought to be dynamic. Oral cancer "The cancer of mouth" is quite prevailing in developing countries. miRNA has been found associated with oral cancer targeting tumor growth, cell proliferation, metastasis, invasion. The significant association of miRNA with genes could be used as a remarkable tool for diagnosis as well as prognostic analysis of oral cancer. The aim of the present study is to evaluate common upregulated and downregulated miRNAs in oral submucous fibrosis (OSMF) and oral malignancy (OM) patients that can be used as diagnostic biomarkers, and to find out their interactions with target genes to establish associated networks in cancer pathways. Using miRDeep2 and DESeq analysis, the upregulated and downregulated miRNA in OSMF (Oral Submucous Fibrosis) and OM (Oral Malignancies) samples were compared to GEO (Gene Expression Omnibus) control dataset. There were 50 common downregulated miRNAs and 13 common upregulated miRNAs in OSMF and OM samples. miRNet analysis of common upregulated miRNA and common downregulated miRNA identified 1295 and 5954 genes, respectively connected with cancer pathways. From analysis of Hub genes, HRAS, STAT3, TP53, MYC, PTEN, CTNNB1, CCND1, JUN, VEGFA, KRAS were found associated with downregulated miRNA and VEGFA, TP53, MDM2, PTEN, MYC, ERBB2, CDKN1A, HSP90AA1, CCND1, AKTI were found associated with upregulated miRNA. The gene enrichment analysis of these hub genes were associated with cell communication, metabolic process, cell proliferation, and cellular component organization. Hub Genes linked with upregulated miRNA had an enrichment ratio of 11.828, whereas hub genes linked with downregulated miRNA had an enrichment ratio of 45.912. We identified common deregulated miRNAs between OSMF and OM patients, which were further analyzed to find out associations with the genes correlated to cancer pathways. The hub genes identified in this study were found to have a significant impact on tumor growth and carcinogenesis. Also, the enrichment of these genes has revealed that the genes are associated with cellular communication, metabolic processes and various biological regulation. These deregulated miRNAs can be used to make a panel of biomarkers to diagnose oral cancer from blood even before its onset.

Abstract 2774: Reference free transcriptomic characterization of chemoresistant triple negative breast cancers provides a promising reservoir of predictive biomarkers of early chemoresistance

Abstract Triple Negative Breast Cancers (TNBCs) are the most aggressive breast cancers and so far, benefited very little from personalized medicine. Unlike in hormone positive breast cancers, coding transcriptomic signatures like Lehmann’s failed to predict which TNBC patients will benefit from perioperative chemotherapy. Here, we analyzed the total transcriptome of a unique cohort of 44 TNBC tumors before neoadjuvant chemotherapy (NAC), including both patients that were chemosensitive (N=26) and chemoresistant (N=18) to following treatment. Differential gene expression analysis (DE-seq) was first performed on annotated genes and de novo Scallop RNA-profiled transcripts. Nearly half of detected differential transcripts were from non-coding genes that were mostly long non-coding (lnc)RNAs (90%). More than half differential lncRNAs were not annotated in gencode. Next, De-kupl, a new reference-free analysis of differential fragments of transcripts, was applied. This approach is based on 31-nucleotide long k-mers that are later merged into contigs without annotation bias. These differential contigs were able to separate correctly, in an unsupervised manner, all TNBC patients regarding their response to chemotherapy, outperforming our previous DE-seq analysis of gencode and scallop transcripts. Strikingly, 80% of the DE-kupl differential contigs were not derived from DE-seq differential genes. Furthermore, as BRCA 1/2 mutated TNBCs are now well known for their chemosensitivity, we excluded them from subsequent analysis and repeated the differential expression analysis. Our results showed that DE-kupl differential contigs once more perfectly classified all patients and outperformed the whole length transcripts from DE-seq. Finally, we applied the LASSO regression analysis machine learning method, on both all misregulated genes and all misregulated contigs in parallel, to generate minimal signatures characterizing pre-chemotherapy TNBC tumors that are going to be chemoresistant. We obtained an 8-contig-signature that overpassed the 11-gene-signature in the accuracy to discriminate chemosensitive and chemoresistant TNBC patients before chemotherapy. These results suggest that a contig based unreferenced differential analysis of TNBCs regarding their response status to NAC provides promising predictive biomarkers of early TNBC chemoresistance and thus need further confirmation in a larger and independent validation cohort. Citation Format: Nouritza Torossian, Dominika Foretek, Marc Gabriel, Linda Larbi Cherif, Charlotte Lecerf, Maud Kamal, Christophe Le Tourneau, Daniel Gautheret, Sergio Roman-Roman, Antonin Morillon. Reference free transcriptomic characterization of chemoresistant triple negative breast cancers provides a promising reservoir of predictive biomarkers of early chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2774.

The Effects of Alcohol Drinking on Oral Microbiota in the Chinese Population.

The dysbiosis of oral microbiota is linked to numerous diseases and is associated with personal lifestyles, such as alcohol drinking. However, there is inadequate data to study the effect of alcohol drinking on oral microbiota from the Chinese population. Here, we profiled the oral microbiota of 150 healthy subjects in the Chinese population by 16S rRNA gene sequencing. The results showed that drinkers had significantly higher alpha diversity than non-drinkers. A significant difference in overall microbiota composition was observed between non-drinkers and drinkers. Additionally, using DESeq analysis, we found genus Prevotella and Moryella, and species Prevotella melaninogenica and Prevotella tannerae were significantly enriched in drinkers; meanwhile, the genus Lautropia, Haemophilus and Porphyromonas, and species Haemophilus parainfluenzae were significantly depleted in drinkers. PICRUSt analysis showed that significantly different genera were mainly related to metabolism pathways. The oxygen-independent pathways, including galactose, fructose and mannose metabolism pathways, were enriched in drinkers and positively associated with genera enriched in drinkers; while the pyruvate metabolism pathway, an aerobic metabolism pathway, was decreased in drinkers and negatively associated with genera enriched in drinkers. Our results suggested that alcohol drinking may affect health by altering oral microbial composition and potentially affecting microbial functional pathways. These findings may have implications for better understanding the potential role those oral bacteria play in alcohol-related diseases.

Effects of Short-Chain Fatty Acid Modulation on Potentially Diarrhea-Causing Pathogens in Yaks Through Metagenomic Sequencing.

Short-chain fatty acids (SCFA) are principal nutrient substrates of intestinal epithelial cells that regulate the epithelial barrier in yaks. Until now, metagenomics sequencing has not been reported in diarrheal yaks. Scarce information is available regarding the levels of fecal SCFA and diarrhea in yaks. So, our study aims to identify the potential pathogens that cause the emerging diarrhea and explore the potential relationship of short-chain fatty acids in this issue. We estimated diarrhea rate in yaks after collecting an equal number of fecal samples from affected animals. Metagenomics sequencing and quantitative analysis of SCFA were performed, which revealed 15%–25% and 5%–10% prevalence of diarrhea in yak’s calves and adults, respectively. Violin box plot also showed a higher degree of dispersion in gene abundance distribution of diarrheal yaks, as compared to normal yaks. We found 366,163 significant differential abundance genes in diarrheal yaks, with 141,305 upregulated and 224,858 downregulated genes compared with normal yaks via DESeq analysis. Metagenomics binning analysis indicated the higher significance of bin 33 (Bacteroidales) (p < 0.05) in diarrheal animals, while bin 10 (p < 0.0001), bin 30 (Clostridiales) (p < 0.05), bin 51 (Lactobacillales) (p < 0.05), bin 8 (Lachnospiraceae) (p < 0.05), and bin 47 (Bacteria) (p < 0.05) were significantly higher in normal yaks. At different levels, a significant difference in phylum (n = 4), class (n = 8), oder (n = 8), family (n = 16), genus (n = 17), and species (n = 30) was noticed, respectively. Compared with healthy yaks, acetic acid (p < 0.01), propionic acid (p < 0.01), butyric acid (p < 0.01), isobutyric acid (p < 0.01), isovaleric acid (p < 0.05), and caproic acid (p < 0.01) were all observed significantly at a lower rate in diarrheal yaks. In conclusion, besides the increased Staphylococcus aureus, Babesia ovata, Anaplasma phagocytophilum, Bacteroides fluxus, viruses, Klebsiella pneumonia, and inflammation-related bacteria, the decrease of SCFA caused by the imbalance of intestinal microbiota was potentially observed in diarrheal yaks.

Scaffold compound L971 exhibits anti-inflammatory activities through inhibition of JAK/STAT and NFκB signalling pathways.

JAK/STAT and NFκB signalling pathways play essential roles in regulating inflammatory responses, which are important pathogenic factors of various serious immune‐related diseases, and function individually or synergistically. To find prodrugs that can treat inflammation, we performed a preliminary high‐throughput screening of 18 840 small molecular compounds and identified scaffold compound L971 which significantly inhibited JAK/STAT and NFκB driven luciferase activities. L971 could inhibit the constitutive and stimuli‐dependent activation of STAT1, STAT3 and IκBα and could significantly down‐regulate the proinflammatory gene expression in mouse peritoneal macrophages stimulated by LPS. Gene expression profiles upon L971 treatment were determined using high‐throughput RNA sequencing, and significant differentially up‐regulated and down‐regulated genes were identified by DESeq analysis. The bioinformatic studies confirmed the anti‐inflammatory effects of L971. Finally, L971 anti‐inflammatory character was further verified in LPS‐induced sepsis shock mouse model in vivo. Taken together, these data indicated that L971 could down‐regulate both JAK/STAT and NFκB signalling activities and has the potential to treat inflammatory diseases such as sepsis shock.

Transcriptional Changes in Potato Sprouts upon Interaction with Rhizoctonia solani Indicate Pathogen-Induced Interference in the Defence Pathways of Potato.

Rhizoctonia solani is the causer of black scurf disease on potatoes and is responsible for high economical losses in global agriculture. In order to increase the limited knowledge of the plants’ molecular response to this pathogen, we inoculated potatoes with R. solani AG3-PT isolate Ben3 and carried out RNA sequencing with total RNA extracted from potato sprouts at three and eight days post inoculation (dpi). In this dual RNA-sequencing experiment, the necrotrophic lifestyle of R. solani AG3-PT during early phases of interaction with its host has already been characterised. Here the potato plants’ comprehensive transcriptional response to inoculation with R. solani AG3 was evaluated for the first time based on significantly different expressed plant genes extracted with DESeq analysis. Overall, 1640 genes were differentially expressed, comparing control (−Rs) and with R. solani AG3-PT isolate Ben3 inoculated plants (+Rs). Genes involved in the production of anti-fungal proteins and secondary metabolites with antifungal properties were significantly up regulated upon inoculation with R. solani. Gene ontology (GO) terms involved in the regulation of hormone levels (i.e., ethylene (ET) and jasmonic acid (JA) at 3 dpi and salicylic acid (SA) and JA response pathways at 8 dpi) were significantly enriched. Contrastingly, the GO term “response to abiotic stimulus” was down regulated at both time points analysed. These results may support future breeding efforts toward the development of cultivars with higher resistance level to black scurf disease or the development of new control strategies.

Oral microbiome and risk of malignant esophageal lesions in a high-risk area of China: A nested case-control study.

ObjectiveWe aimed to prospectively evaluate the association of oral microbiome with malignant esophageal lesions and its predictive potential as a biomarker of risk.MethodsWe conducted a case-control study nested within a population-based cohort with up to 8 visits of oral swab collection for each subject over an 11-year period in a high-risk area for esophageal cancer in China. The oral microbiome was evaluated with 16S ribosomal RNA (rRNA) gene sequencing in 428 pre-diagnostic oral specimens from 84 cases with esophageal lesions of severe squamous dysplasia and above (SDA) and 168 matched healthy controls. DESeq analysis was performed to identify taxa of differential abundance. Differential oral species together with subject characteristics were evaluated for their potential in predicting SDA risk by constructing conditional logistic regression models.ResultsA total of 125 taxa including 37 named species showed significantly different abundance between SDA cases and controls (all P<0.05 & false discovery rate-adjusted Q<0.10). A multivariate logistic model including 11 SDA lesion-related species and family history of esophageal cancer provided an area under the receiver operating characteristic curve (AUC) of 0.89 (95% CI, 0.84−0.93). Cross-validation and sensitivity analysis, excluding cases diagnosed within 1 year of collection of the baseline specimen and their matched controls, or restriction to screen-endoscopic-detected or clinically diagnosed case-control triads, or using only bacterial data measured at the baseline, yielded AUCs>0.84.ConclusionsThe oral microbiome may play an etiological and predictive role in esophageal cancer, and it holds promise as a non-invasive early warning biomarker for risk stratification for esophageal cancer screening programs.

Human Eosinophils Express a Distinct Gene Expression Program in Response to IL-3 Compared with Common β-Chain Cytokines IL-5 and GM-CSF.

Despite recent advances in asthma management with anti-IL-5 therapies, many patients have eosinophilic asthma that remains poorly controlled. IL-3 shares a common β subunit receptor with both IL-5 and GM-CSF but, through α-subunit-specific properties, uniquely influences eosinophil biology and may serve as a potential therapeutic target. We aimed to globally characterize the transcriptomic profiles of GM-CSF, IL-3, and IL-5 stimulation on human circulating eosinophils and identify differences in gene expression using advanced statistical modeling. Human eosinophils were isolated from the peripheral blood of healthy volunteers and stimulated with either GM-CSF, IL-3, or IL-5 for 48 h. RNA was then extracted and bulk sequencing performed. DESeq analysis identified differentially expressed genes and weighted gene coexpression network analysis independently defined modules of genes that are highly coexpressed. GM-CSF, IL-3, and IL-5 commonly upregulated 252 genes and downregulated 553 genes, producing a proinflammatory and survival phenotype that was predominantly mediated through TWEAK signaling. IL-3 stimulation yielded the most numbers of differentially expressed genes that were also highly coexpressed (n = 119). These genes were enriched in pathways involving JAK/STAT signaling. GM-CSF and IL-5 stimulation demonstrated redundancy in eosinophil gene expression. In conclusion, IL-3 produces a distinct eosinophil gene expression program among the β-chain receptor cytokines. IL-3-upregulated genes may provide a foundation for research into therapeutics for patients with eosinophilic asthma who do not respond to anti-IL-5 therapies.

Abstract 1975: Multivariate exome analysis identifies CXCR5-CXCL13 signaling as a key driver in early breast cancer development and prognosis

Abstract Breast cancer (BrCa) is the most frequently diagnosed cancer and the second leading cause of cancer-related death among women worldwide despite new developments in prevention and treatment. More recently, rapid increases in BrCa incidence have been reported in countries of the Asia-Pacific region. Age of diagnosis in Asian BrCa patients is much younger than age of diagnosis in western countries, such as the United States, and Asian early BrCa patients tend to be pre-menopausal, whereas only 15-30% of western BrCas are pre-menopausal. The mechanisms responsible for early BrCa remain unknown. Furthermore, it remains controversial whether early breast cancer has unique tumor biology. The goal of this study was to characterize the molecular phenotype of early BrCa in the context of CXCL13, CXCR5 and associated gene expression. We hypothesized that CXCR5-CXCL13 signaling contributes to the aggressive phenotype of early BrCa. A bioinformatic approach was used to aid in characterizing this condition. Our patient cohort data (50 females of Korean descent, age ≤ 35 years) was obtained from The International Cancer Genome Consortium (ICGC). DESeq analysis was used to identify genes differentially expressed among normal tissue and primary tumor groups. Weighted Gene Network Co-expression (WGCNA) analysis was carried-out to identify gene networks associated with factors influencing BrCa prognosis. Finally, canonical pathway, upstream regulator, and gene interaction analysis was conducted using Ingenuity Pathway Analysis. Our data show CXCL13, CXCR5, and associated genes (LTA, LTB, IL10, IL27RA, TNFRSF13B, TNFRSF17, and TNFRSF9), which have been previously shown to drive tertiary lymphoid structure (TLS) formation, were significantly associated with the molecular phenotype of early BrCa in our patient cohort. Taken together, our findings suggest TLSs contribute to the tumor immune microenvironment and contribute to early BrCa progression. Citation Format: Courtney D. Dill, Tiara Griffen, Corey Young, Kaylin Carey, James W. Lillard. Multivariate exome analysis identifies CXCR5-CXCL13 signaling as a key driver in early breast cancer development and prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1975.

Abstract LB-132: The role of CXCL13, its sole receptor CXCR5, and associated genes in multiple myeloma progression

Abstract Multiple myeloma (MM) is characterized by the neoplastic proliferation of plasma cells producing monoclonal immunoglobulin in blood, bone marrow, and urine. In 2018, it was estimated that approximately 30,770 new cases would be diagnosed (16,400 in men and 14,370 in women) and about 12,770 deaths were expected to occur (6,830 in men and 5,940 in women). MM is a heterogenous disease; thus, characterizing their molecular phenotypes, plays a significant role in prognosis and effective treatment of MM and relapse cases. Certain chemokine and it sole receptor (CXCL13 and CXCR5) has been shown to contribute to the progression and metastasis of several types of cancers. CXCL13 and CXCR5 are expressed by cells in the microenvironment of bone marrow and function in malignancy cell homing, adhesion, signal transduction, and calcium flux, all of which promote MM progression. To identify the potential function of relevant somatic mutations and gene expression in MM causing clonal evolution and lethality, we analyzed RNA-sequence data from matched normal, primary and relapsed MM cases. Patient control mRNA samples, matched primary tumor and relapse samples were acquired from dbGap (database of Genotypes and Phenotypes) to evaluate the mRNA expression of CXCL13-CXCR5 and associated genes in MM progression. A bioinformatics strategy was used to integrate published genomic data from these MM patients (n=240) and teased out genes associated with CXCL13-CXCR5 signaling. DESeq analysis was used to determine differentially expressed genes between normal tissues, primary tumor and relapse groups. Our preliminary analysis done with Weighted Gene Co-expression Network Analysis (WGCNA) identified clusters of genes; transcription factors (NF-κB and JUN) known to be induced by the CXCL13-CXCR5 axis, and plasma cell signaling pathways. Moreover, Ingenuity pathway analysis (IPA) was performed to analyze upstream regulators, gene interaction networks and canonical pathways, which identified biological processes associated with the differentially expressed genes. We suggested that, the doubling effect of the activation of transcription factors (NF-κB and JUN) by plasma cell signaling and CXCR5-CXCL13 signaling are most likely responsible for the pathogenesis and plasma clonality of MM. This study provided a better understanding of the heterogeneous nature of MM and the role of the CXCL13-CXCR5 axis plays in MM. Therefore, CXCL13-CXCR5 and associated genes serves as a potential clinical application and a new therapeutic target for MM. Note: This abstract was not presented at the meeting. Citation Format: Olayinka O. Adebayo, Tiara Griffin, Corey Young, Courtney D. Dill, Sha'Kayla Nunez, Kaylin Carey, James W. Lillard. The role of CXCL13, its sole receptor CXCR5, and associated genes in multiple myeloma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-132.

Clinical significance of CXCR5-CXCL13 signaling in multiple myeloma.

e19517 Background: Multiple myeloma (MM) is characterized by the neoplastic proliferation of plasma cells resulting in monoclonal immunoglobulins in the blood, bone marrow, and urine (1). According to a 2019 American Cancer Society (ACS) report, MM is now classified as subtypes of non-Hodgkin lymphoma (NHL). ACS expects there will be ~32,110 new MM cases (18,130 in men and 13,980 in women) and ~12,960 (6,990 in men and 5,970 in women) anticipated deaths in 2019. MM is a heterogenous disease; characterizing its molecular phenotypes is important for effective treatment of MM and predicting relapse. CXCL13-CXCR5 interactions are involved in malignancy cell homing, adhesion, signal transduction, and calcium flux, all of which promote MM progression. Methods: We analyzed RNA-sequence data from matched normal, primary and relapsed MM cases. Patient control mRNA samples matched primary tumor and relapse samples were acquired from dbGap (database of Genotypes and Phenotypes) to evaluate the mRNA expression patterns of CXCL13, CXCR5 and associated genes in MM. A bioinformatics strategy was used to integrate published genomic data from MM patients (n = 480) and identify genes associated with CXCL13-CXCR5 signaling. DESeq analysis was used to determine differentially expressed genes between normal tissues, primary tumor and relapse groups. Weighted gene co-expression network analysis (WGCNA) identified clusters of genes significantly associated with the molecular phenotypes of MM. Results: Notably, gene expression driven by NFAT and JUN, known to be activated by the CXCL13-CXCR5 axis, and plasma cell signaling pathways significantly correlated with select MM molecular phenotypes and patient survival. Ingenuity pathway analysis (IPA) was performed to analyze upstream regulators, gene interaction networks and canonical pathways. Taken together, our data show CXCR5-CXCL13 signaling networks are significantly expressed and associated with MM pathogenesis and plasma clonality. Conclusions: This study provides a better understanding of the heterogeneous nature of MM and the role of the CXCL13-CXCR5 axis in MM.

Weighted Gene Co-expression Network Analysis of CXCL13, CXCR5, and associated genes in multiple myeloma

Abstract Multiple myeloma (MM) is characterized by the neoplastic proliferation of plasma cells. In 2018, there were approximately 30,770 new cases diagnosed and about 12,770 dead. Characterizing its molecular phenotypes is important for effective treatment of MM and predicting relapse. CXCL13-CXCR5 interactions are involved in malignancy cell homing, adhesion, signal transduction, and calcium flux, all of which promote MM progression. Here, we analyzed RNA-sequence data from matched normal, primary and relapsed MM cases. Patient control mRNA samples matched primary tumor and relapse samples were acquired from the database of Genotypes and Phenotypes to evaluate the mRNA expression patterns of CXCL13, CXCR5 and associated genes in MM. Bioinformatics tools were to integrate published genomic data from MM patients (n=480) and identify genes associated with CXCL13-CXCR5 signaling. DESeq analysis was used to determine differentially expressed genes between normal tissues, primary tumor, and relapse groups. Weighted gene co-expression network analysis identified clusters of genes significantly associated with the molecular phenotypes of MM. Notably, gene expression is driven by NFAT and JUN, known to be activated by the CXCL13-CXCR5 axis, and plasma cell signaling pathways significantly correlated with select MM molecular phenotypes and patient survival. Ingenuity pathway analysis was performed to analyze upstream regulators, gene interaction and canonical pathways. Taken together, our data show CXCR5-CXCL13 signaling networks are significantly expressed and associated with MM pathogenesis and plasma clonality. This study provides a better understanding of the heterogeneous nature of MM and the role of CXCL13-CXCR5 axis in MM.

Multivariate analysis of genes expressed in the colorectal tumor immune microenvironment driven by CXCL13

Abstract Colorectal cancer (CRC) is a heterogenous disease having numerous molecular phenotypes. Key tumor progression markers such as microsatellite instability, BRAF, and KRAS mutation status have been widely studied. Less is known regarding the influence of chemokines and chemokine receptors in CRC. CXCL13 and CXCR5 have been shown to be associated with poor prognosis of advanced CRC. The central hypothesis tested in this study was CXCR5-CXCL13 signaling mediates gene expressions associated with CRC progression. RNAseq patient control and matched primary tumor samples were acquired from The Cancer Genome Atlas (TCGA). DESeq analysis identified differentially expressed genes between normal tissue and primary tumor groups. Weighted Gene Co-expression Network Analysis (WGCNA) elucidated gene networks that correlated with factors influencing CRC prognosis. Canonical pathway, upstream regulator, and protein-protein interaction analyses were performed using Ingenuity Pathway Analysis (IPA). CXCL13 was identified in the WGCNA-designated green module, which also contained XCL1, TNFRSF17, FCRN1, CCL19, IL-17A and IFNG. These genes are associated with immunoregulatory signals found in lymphoid and non-lymphoid cells, BCR-signaling and poor prognosis of CRC. Genes associated with energy metabolism, apoptosis, hypoxia and angiogenesis, including GNG4, PRKCG, WNT11, and KNG1, were found in the WCGNA-designated yellow module. These factors are involved in Wnt/CTNNNB1 and PI3K/AKT signaling, which have previously shown to be driven by CXCR5-CXCL13 interactions. Taken together, our data suggest a role of the CXCL13-CXCR5 axis in shaping the tumor immune microenvironment of CRC.

RNA-Seq transcriptomic profiling of primary murine microglia treated with LPS or LPS\u2009+\u2009IFN\u03b3

Microglia, the main resident immune cells in the CNS, are thought to participate in the pathogenesis of various neurological disorders. LPS and LPS + IFNγ are stimuli that are widely used to activate microglia. However, the transcriptomic profiles of microglia treated with LPS and LPS + IFNγ have not been properly compared. Here, we treated murine primary microglial cultures with LPS or LPS + IFNγ for 6 hours and then performed RNA-Sequencing. Gene expression patterns induced by the treatments were obtained by WGCNA and 11 different expression profiles were found, showing differential responses to LPS and LPS + IFNγ in many genes. Interestingly, a subset of genes involved in Parkinson’s, Alzheimer’s and Huntington’s disease were downregulated by both treatments. By DESeq analysis we found differentially upregulated and downregulated genes that confirmed LPS and LPS + IFNγ as inducers of microglial pro-inflammatory responses, but also highlighted their involvement in specific cell functions. In response to LPS, microglia tended to be more proliferative, pro-inflammatory and phagocytic; whereas LPS + IFNγ inhibited genes were involved in pain, cell division and, unexpectedly, production of some inflammatory mediators. In summary, this study provides a detailed description of the transcriptome of LPS- and LPS + IFNγ treated primary microglial cultures. It may be useful to determine whether these in vitro phenotypes resemble microglia in in vivo pathological conditions.

Comparative transcriptome profiling and characterization of gene expression for ovarian differentiation under RU486 treatment

17α, 20β-dihydroxypregn-4-en-3-one (17α, 20β-DP, DHP), a teleost specific biologically active progestin, has been proved to play a critical role in oocytes maturation, ovulation and spermiation. RU486 (Mifepristone, an antagonist of progestin receptor) has been applied in contraceptives, abortion and hormone therapy in clinical medicine. To get further insights into the molecular mechanisms of nuclear progestin receptor (Pgr) activated ovarian differentiation and maintenance, we conducted comparative gonadal transcriptome analysis, and investigated histological and transcriptional differences using 4 months after hatching (mah) RU486-treated XX and control XX/XY Nile tilapia (Oreochromis niloticus). DESeq analysis identified 7148 DEGs (differentially expressed genes) between RU486-treated and control XX gonads, while merely 442 DEGs were screened between the gonads of RU486-treated XX and control XY fish highlighting that RU486 treatment set forwards masculinity in XX fish. Comprehensive analysis of gene hierarchical clustering revealed that RU486 treatment in XX fish resulted in robust changes of gene expression profiles. In comparison with XX group, female-dominant genes were significantly repressed in RU486 treated XX fish gonads. Moreover, most parts of down-regulated genes in wild type female were evidently up-regulated genes in RU486-treated XX fish gonads. Comparing with control XY group, the majority of male-dominant genes represent a high level of expression. However, RU486-treatment led to an up-regulation of a cluster genes specifically which showed relative lower expression in both control XX and XY group. RU486-treatment mediated global changes of gene expression profiles in steroidogenesis, germ cell differentiation and follicular cell trans-differentiation were verified by quantitative PCR. Both morphological and immunohistochemistry results further proved that RU486 treatment initiates testicular-like gonads development in XX fish via simultaneously enhancing the male responsive genes and suppressing the female-dominant genes. Moreover, RU486 treatment caused significant decline of fshr, lhr and increase of ars. Taken together, our data confirms blocking of DHP physiology by RU486 treatment induces masculinization in XX gonad preferably via repressing of gonadotropin physiology, germ cell differentiation and promoting follicular trans-differentiation in teleosts.

P.2.010 - In search for predictive biomarkers: dissecting the molecular pathways in brain and blood underlying poor and good antidepressant treatment response

Major depression poses a serious social and economic threat to modern societies, as it accounts for more lost productivity compared with any other disorder. There are currently two major problems calling for innovative research approaches: 1. The absence of biomarkers predicting antidepressant response and 2. The lack of conceptually novel antidepressant compounds. Identification of biomarkers could allow patient stratification and enable the selection of pathophysiologically distinct patient subgroups to allow optimized treatment choices based on biology. In search for conceptually novel antidepressants, the hippocampal dentate gyrus is a region of particular interest, as there is a large body of evidence suggesting that stimulation of neurogenesis by antidepressant drugs could be one of their major common targets [1,2]. Aiming to identify the molecular pathways shaping response to antidepressant treatment and to identify predictors for antidepressant response, we used our animal experimental approach to model heterogeneity in response to antidepressant treatment (Carillo-Roa et al., PLoS BIOL, in revision). Male, adult DBA/2J mice show a strong innate anxiety and depression-like phenotype. Mice were treated for 14 days with customized pills containing either paroxetine or vehicle, providing a stress-free and oral treatment paradigm. Efficacy of antidepressant treatment was measured using the Forced Swim Test and results analyzed using a Student’s t-test. For the identification of peripheral biosignatures predicting response, we collected blood samples at baseline and following 14 days of treatment (longitudinal design), whereas for the identification of novel antidepressant targets, we specifically dissected the hippocampal dentate gyrus using a microdissecion protocol for native hippocampal tissue. We extracted stranded total RNA from dentate gyrus samples (paroxetine vs vehicle and good vs poor responders) and whole blood samples (baseline and 14 days in good vs poor responders) and sequenced. Analysis of differentially expressed genes (DESeq analysis) was conducted applying Benjamini-Hochberg correction with a padj 2.68, and fold changes >1.5. Pathway analysis was conducted using STRING software. qPCR of selected genes was conducted and analyzed using delta-delta-ct method. Paroxetine treatment led to antidepressant-like (p With this study, we provide additional strong evidence that neurogenesis drives antidepressant treatment effects and determines response status in rodents. We also expect to identify transcriptome signatures predictive of later antidepressant response in whole blood. Finally, we aim at validating our murine predictive biomarkers in human studies on antidepressant response.