Differential Gene Regulation Research Articles

Transcriptomic stability or lability explains sensitivity to climate stressors in coralline algae

BackgroundCrustose coralline algae (CCA) are calcifying red macroalgae that play important ecological roles including stabilisation of reef frameworks and provision of settlement cues for a range of marine invertebrates. Previous research into the responses of CCA to ocean warming (OW) and ocean acidification (OA) have found magnitude of effect to be species-specific. Response to OW and OA could be linked to divergent underlying molecular processes across species.ResultsHere we show Sporolithon durum, a species that exhibits low sensitivity to climate stressors, had little change in metabolic performance and did not significantly alter the expression of any genes when exposed to temperature and pH perturbations. In contrast, Porolithon onkodes, a major coral reef builder, reduced photosynthetic rates and had a labile transcriptomic response with over 400 significantly differentially expressed genes, with differential regulation of genes relating to physiological processes such as carbon acquisition and metabolism. The differential gene expression detected in P. onkodes implicates possible key metabolic pathways, including the pentose phosphate pathway, in the stress response of this species.ConclusionsWe suggest S. durum is more resistant to OW and OA than P. onkodes, which demonstrated a high sensitivity to climate stressors and may have limited ability for acclimatisation. Understanding changes in gene expression in relation to physiological processes of CCA could help us understand and predict how different species will respond to, and persist in, future ocean conditions predicted for 2100.

Little skate genome provides insights into genetic programs essential for limb-based locomotion.

The little skate Leucoraja erinacea, a cartilaginous fish, displays pelvic fin driven walking-like behavior using genetic programs and neuronal subtypes similar to those of land vertebrates. However, mechanistic studies on little skate motor circuit development have been limited, due to a lack of high-quality reference genome. Here, we generated an assembly of the little skate genome, with precise gene annotation and structures, which allowed post-genome analysis of spinal motor neurons (MNs) essential for locomotion. Through interspecies comparison of mouse, skate and chicken MN transcriptomes, shared and divergent gene expression profiles were identified. Comparison of accessible chromatin regions between mouse and skate MNs predicted shared transcription factor (TF) motifs with divergent ones, which could be used for achieving differential regulation of MN-expressed genes. A greater number of TF motif predictions were observed in MN-expressed genes in mouse than in little skate. These findings suggest conserved and divergent molecular mechanisms controlling MN development of vertebrates during evolution, which might contribute to intricate gene regulatory networks in the emergence of a more sophisticated motor system in tetrapods.

Identification of putative transcriptomic biomarkers in irritable bowel syndrome (IBS): Differential gene expression and regulation of TPH1 and SERT by vitamin D.

Irritable bowel syndrome (IBS) is one of the most common gastrointestinal disorders and affects approximately 4% of the global population. The diagnosis of IBS can be made based on symptoms using the validated Rome criteria and ruling out commonly occurring organic diseases. Although biomarkers exist for “IBS mimickers” such as celiac disease and inflammatory bowel disease (IBD), no such test exists for IBS. DNA microarrays of colonic tissue have been used to identify disease-associated variants in other gastrointestinal (GI) disorders. In this study, our objective was to identify biomarkers and unique gene expression patterns that may define the pathological state of IBS. Mucosal tissue samples were collected from the sigmoid colon of 29 participants (11 IBS and 18 healthy controls). DNA microarray analysis was used to assess gene expression profiling. Extraction and purification of RNA were then performed and used to synthesize cDNA. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was employed to identify differentially expressed genes in patients diagnosed with IBS compared to healthy, non-IBS patient-derived cDNA. Additional testing probed vitamin D-mediated regulation of select genes associated with serotonergic metabolism. DNA microarray analyses led to the identification of 858 differentially expressed genes that may characterize the IBS pathological state. After screening a series of genes using a combination of gene ontological analysis and RT-qPCR, this spectrum of potential IBS biomarkers was narrowed to 23 genes, some of which are regulated by vitamin D. Seven putative IBS biomarkers, including genes involved in serotonin metabolism, were identified. This work further supports the hypothesis that IBS pathophysiology is evident within the human transcriptome and that vitamin D modulates differential expression of genes in IBS patients. This suggests that IBS pathophysiology may also involve vitamin D deficiency and/or an irregularity in serotonin metabolism.

Global and gene-specific translational regulation in Escherichia coli across different conditions

How well mRNA transcript levels represent protein abundances has been a controversial issue. Particularly across different environments, correlations between mRNA and protein exhibit remarkable variability from gene to gene. Translational regulation is likely to be one of the key factors contributing to mismatches between mRNA level and protein abundance in bacteria. Here, we quantified genome-wide transcriptome and relative translation efficiency (RTE) under 12 different conditions in Escherichia coli. By quantifying the mRNA-RTE correlation both across genes and across conditions, we uncovered a diversity of gene-specific translational regulations, cooperating with transcriptional regulations, in response to carbon (C), nitrogen (N), and phosphate (P) limitations. Intriguingly, we found that many genes regulating translation are themselves subject to translational regulation, suggesting possible feedbacks. Furthermore, a random forest model suggests that codon usage partially predicts a gene's cross-condition variability in translation efficiency; such cross-condition variability tends to be an inherent quality of a gene, independent of the specific nutrient limitations. These findings broaden the understanding of translational regulation under different environments and provide novel strategies for the control of translation in synthetic biology. In addition, our data offers a resource for future multi-omics studies.

An update on the role of Hfq RNA Chaperone in resistance and virulence of Acinetobacter baumannii

Introduction: The difficulty in treating Acinetobacter baumannii infections due to its high rate of resistance to antibiotics has led to the study of mechanisms inherent to the pathogen itself that can be used as effective targets in the treatment. Host Factor I Protein (Hfq) is an RNA chaperone generally necessary to assist in the connection between sRNAs and their mRNA target acting in the regulation of different genes, studies carried out in a range of bacterial species have shown that Hfq acts in a pleiotropic manner, contributing to virulence and response stress. Aim: To summarize current knowledge about the role of the Hfq RNA chaperone in the virulence and antibiotic resistance of Acinetobacter baumannii. Outlining: This is an integrative review developed from articles published in any language on Science Direct and PubMed platforms. Data collection and analysis were carried out between the period of April 2020 and February 2021. Results: Hfq shown to play important roles in cell growth, OMVs, metabolism of carbon sources, tolerance to physical and chemical stress, virulence through biofilm formation, fimbriae modulation, among others. Implications: Our work shows data that strengthen the role of Hfq in different aspects of virulence and environmental adaptation, including antimicrobial resistance of this pathogen, warning about the importance of Hfq as a possible future effective target in the treatment of these infections.

Revisiting the significance of keratin expression in complex epithelia.

A large group of keratin genes (n=54 in the human genome) code for intermediate filament (IF)-forming proteins and show differential regulation in epithelial cells and tissues. Keratin expression can be highly informative about the type of epithelial tissue, differentiation status of constituent cells and biological context (e.g. normal versus diseased settings). The foundational principles underlying the use of keratin expression to gain insight about epithelial cells and tissues primarily originated in pioneering studies conducted in the 1980s. The recent emergence of single cell transcriptomics provides an opportunity to revisit these principles and gain new insight into epithelial biology. Re-analysis of single-cell RNAseq data collected from human and mouse skin has confirmed long-held views regarding the quantitative importance and pairwise regulation of specific keratin genes in keratinocytes of surface epithelia. Furthermore, such analyses confirm and extend the notion that changes in keratin gene expression occur gradually as progenitor keratinocytes commit to and undergo differentiation, and challenge the prevailing assumption that specific keratin combinations reflect a mitotic versus a post-mitotic differentiating state. Our findings provide a blueprint for similar analyses in other tissues, and warrant a more nuanced approach in the use of keratin genes as biomarkers in epithelia.

Differential Regulation of Genes by the Glucogenic Hormone Asprosin in Ovarian Cancer.

Background: Ovarian cancer (OvCa) is one of the most lethal forms of gynaecological malignancy. Altered energy metabolism and increased aerobic glycolysis in OvCa are hallmarks that demand attention. The glucogenic hormone asprosin is often dysregulated in metabolic disorders such as insulin resistance, diabetes (type 2 and gestational), and preeclampsia. Despite association with metabolic disorders, its role in energy metabolism within the tumour microenvironment is yet to be explored. Here, we study the role of asprosin in OvCa using transcriptomics and expand on functional studies with clinical samples. Methods: RNA sequencing, functional gene enrichment analysis, Western blotting and ImageStream. Results: Following treatment with 100 nM of asprosin, the serous OvCa cell line, SKOV-3, displayed 160 and 173 gene regulatory changes, at 4 and 12 h respectively, when compared with control samples (p < 0.05 and Log2FC > 1). In addition to energy metabolism and glucose-related pathways, asprosin was shown to alter pathways associated with cell communication, TGF-β signalling, and cell proliferation. Moreover, asprosin was shown to induce phosphorylation of ERK1/2 in the same in vitro model. Using liquid biopsies, we also report for novel expression of asprosin’s predicted receptors OR4M1 and TLR4 in cancer-associated circulating cells; with significant reduction seen between pre-chemotherapy and end of first line chemotherapy, in addition to patients under maintenance with bevacizumab +/− olaparib for OR4M1. Conclusions: In relation to OvCa, asprosin appears to regulate numerous signalling pathways in-vitro. The prognostic potential of OR4M1 in liquid biopsies should also be explored further.

A Screening of Epigenetic Therapeutic Targets for Non-Small Cell Lung Cancer Reveals PADI4 and KDM6B as Promising Candidates.

Despite advances in diagnostic and therapeutic approaches for lung cancer, new therapies targeting metastasis by the specific regulation of cancer genes are needed. In this study, we screened a small library of epigenetic inhibitors in non-small-cell lung cancer (NSCLC) cell lines and evaluated 38 epigenetic targets for their potential role in metastatic NSCLC. The potential candidates were ranked by a streamlined approach using in silico and in vitro experiments based on publicly available databases and evaluated by real-time qPCR target gene expression, cell viability and invasion assays, and transcriptomic analysis. The survival rate of patients with lung adenocarcinoma is inversely correlated with the gene expression of eight epigenetic targets, and a systematic review of the literature confirmed that four of them have already been identified as targets for the treatment of NSCLC. Using nontoxic doses of the remaining inhibitors, KDM6B and PADI4 were identified as potential targets affecting the invasion and migration of metastatic lung cancer cell lines. Transcriptomic analysis of KDM6B and PADI4 treated cells showed altered expression of important genes related to the metastatic process. In conclusion, we showed that KDM6B and PADI4 are promising targets for inhibiting the metastasis of lung adenocarcinoma cancer cells.

Follicle-Stimulating Hormone Glycosylation Variants Distinctly Modulate Pre-antral Follicle Growth and Survival.

Follicle-stimulating hormone (FSH) is a key endocrine regulator of ovarian function. FSH is secreted as 2 macroglycosylation variants: partially glycosylated FSH (FSH21/18) and fully glycosylated FSH (FSH24). FSH21/18 is more potent than FSH24 at binding to and activating the FSH receptor (R). The ratio of FSH21/18:FSH24 has been shown to change with age, with FSH21/18 predominant at reproductive prime, and FSH24 predominant during perimenopause/menopause. How these FSH glycosylation variants modulate ovarian follicle functions remains largely unknown. The aim of this study was to investigate the effect of FSH glycosylation variants of pre-antral follicle function. Pre-antral follicles were isolated from 3- to 5-week-old C57BL/6 mice and treated ±10 ng/mL FSH21/18, FSH24, a ratio of 80:20 FSH21/18:FSH24 (to mimic reproductive prime), 50:50 FSH21/18:FSH24 (perimenopause), or 20:80 FSH21/18:FSH24 (menopause) for up to 96 hours. FSH21/18 and 80:20 FSH21/18:FSH24 increased follicle growth, in comparison with control, contrasting with FSH24 and 20:80 FSH21/18:FSH24. Survival rates were decreased in follicles treated with FSH24 or 20:80 FSH21/18:FSH24, with follicles undergoing basement membrane rupture and oocyte extrusion, increased Caspase3 gene and protein expression, and decreased markers of cell proliferation in FSH24 or 20:80 FSH21/18:FSH24-treated follicles. Moreover, this correlated with differential regulation of key genes modulating follicular functions. Pharmacological inhibitors of key FSH signal pathways suggests FSH21/18 and FSH24 initiate different FSHR signal pathway activation, which may determine their differential effects on follicle growth and survival. These data suggest that the nature of FSH glycosylation modulates the follicular cellular environment to regulate follicle growth and survival and may underpin the increasing ovarian resistance to FSH observed during aging.

A germline SNP in BRMS1 predisposes patients with lung adenocarcinoma to metastasis and can be ameliorated by targeting c-fos.

About 50% of patients with early-stage, surgically resected lung cancer will develop distant metastasis. There remains an unmet need to identify patients likely to develop recurrence and to design innovative therapies to decrease this risk. Two primary isoforms of BRMS1, v1 and v2, are present in humans. Using next-generation sequencing of BRMS1 on matched human noncancerous lung tissue and non-small cell lung cancer (NSCLC) specimens, we identified single-nucleotide polymorphism (SNP) rs1052566 that results in an A273V mutation of BRMS1v2. This SNP is homozygous (BRMS1v2A273V/A273V) in 8% of the population and correlates with aggressive biology in lung adenocarcinoma (LUAD). Mechanistically, we show that BRMS1v2 A273V abolishes the metastasis suppressor function of BRMS1v2 and promotes robust cell invasion and metastases by activation of c-fos-mediated gene-specific transcriptional regulation. BRMS1v2 A273V increases cell invasion in vitro and increases metastases in both tail-vein injection xenografts and LUAD patient-derived organoid (PDO) intracardiac injection metastasis in vivo models. Moreover, we show that BRMS1v2 A273V fails to interact with nuclear Src, thereby activating intratumoral c-fos in vitro. Higher c-fos results in up-regulation of CEACAM6, which drives metastases in vitro and in vivo. Using both xenograft and PDO metastasis models, we repurposed T5224 for treatment, a c-fos pharmacologic inhibitor investigated in clinical trials for arthritis, and observed suppression of metastases in BRMS1v2A273V/A273V LUAD in mice. Collectively, we elucidate the mechanism of BRMS1v2A273V/A273V-induced metastases and offer a putative therapeutic strategy for patients with LUAD who have this germline alteration.

Molecular Process of Stem Cells in Modern Biology

Stem cells have a wide range of traits, including the ability to self-renewal and differential interactions between various cell types that vary with their location within tissue and their immediate surroundings. Inhibitors of both are influenced by the cell cycle, genes involved in chromosomal rearrangements, critical developmental proteins, and signaling pathways. Wnt, Notch, Hedgehog Pathways, BMI-1, OCT3/4, ARF, NANOG, p16Ink4a, and this includes HOXB4, SOX2, and their homologous paralog, govern self-renewal. Stem cells and their molecular pathways that regulate self-renewal and differentiation hold great promise for theoretical and practical studies. Keyword: gene regulation of stem cell self-renewal and differentiation genes for BMI-1, NANOG, OCT3, OCT4, p16Ink4a, SOX2, microenvironment, ARF protein proteins 1 (hedgehog), proteins 2 (Wnt.), and receptors 3 (notch).

Association between cancer genes and germ layer specificity.

Cancer signaling pathways defining cell fates are related to differentiation. During the developmental process, three germ layers (endoderm, mesoderm, and ectoderm) are formed during embryonic development that differentiate into organs via the epigenetic regulation of specific genes. To examine the relationship, the specificities of cancer gene mutations that depend on the germ layers are studied. The major organs affected by cancer were determined based on statistics from the National Cancer Information Center of Korea, and were grouped according to their germ layer origins. Then, the gene mutation frequencies were evaluated to identify any bias based on the differentiation group using the Catalogue of Somatic Mutations in Cancer (COSMIC) database. The chi-square test showed that the p-value of 152 of 166 genes was less than 0.05, and 151 genes showed p-values of less than 0.05 even after adjusting for the false discovery rate (FDR). The germ layer-specific genes were evaluated using visualization based on basic statistics, and the results matched the top ranking genes depending on organs in the COSMIC database.The current study confirmed the germ layer specificity of major cancer genes. The germ layer specificity of mutated driver genes is possibly important in cancer treatments because each mutated gene may react differently depending on the germ layer of origin. By understanding the mechanism of gene mutation in the development and progression of cancer in the context of cell-fate pathways, a more effective therapeutic strategy for cancer can be established.

Polystyrene nanoparticles enhance the adverse effects of di-(2-ethylhexyl) phthalate on male reproductive system in mice

Coexposure of nanoplastics (NPs) with other pollutants adsorbed from the surroundings has received extensive attention. Currently, the combined effects of NPs and plasticizers remain unclear. Di-(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer that has raised much concern owing to its ubiquitous pollution and endocrine-disrupting potential. This study aimed to investigate the toxic effects on the male reproductive system upon coexposure to NPs and DEHP. The C57BL/6J mice were orally administrated with polystyrene nanoparticles (PSNPs), DEHP or both for 35 days to evaluate their effects on sperm quality, histology of testes and epididymides, testicular transcriptomic characteristics as well as expression of some important genes in the epididymides. The low-dose PSNPs used here did not induce significant changes in sperm quality, while DEHP alone or cotreatment with DEHP and PSNPs caused notable impairment, mainly manifesting as decreased sperm quality and aberrant structure of the testis and epididymis. Moreover, enhanced toxic effects were found in the cotreatment group when compared with the individual DEHP treatment group, as manifested by more obvious alterations in the sperm parameters as well as histological changes in the testis and epididymis. Testicular transcriptomic analysis revealed differential regulation of genes involved in immune response, cytoplasmic pattern recognition receptor signaling pathways, protein ubiquitination, oxidative stress, necrotic cell death, ATP synthesis and the cellular respiratory chain. RT-qPCR verified that the expression patterns of Cenpb, Crisp1 and Mars were changed in testes, and genes relevant to epididymal function including Aqp9 and Octn2 were downregulated in epididymides, particularly in the cotreatment group. Collectively, our results emphasize that DEHP at an environmentally relevant dose can induce male reproductive toxicity, and PSNPs may aggravate the toxic effects.

Transcriptional regulation of cell proliferation competence-associated Arabidopsis genes, CDKA;1, RID1 and SRD2, by phytohormones in tissue culture.

During organ regeneration, differentiated cells acquire cell proliferation competence before the re-start of cell division. In Arabidopsis thaliana (Arabidopsis), CDKA;1, a cyclin-dependent kinase, RID1, a DEAH-box RNA helicase, and SRD2, a small nuclear RNA transcription factor, are implicated in the regulation of cell proliferation competence. Here, we report phytohormonal transcriptional regulation of these cell proliferation competence-associated genes during callus initiation. We can induce the callus initiation from Arabidopsis hypocotyl explants by the culture on the auxin-containing medium. By RT-quantitative PCR analysis, we observed higher mRNA accumulation of CDKA;1, RID1, and SRD2 in culture on the auxin-containing medium than in culture on the auxin-free medium. Promoter-reporter analysis showed that the CDKA;1, RID1, and SRD2 expression was induced in the stele regions containing pericycle cells, where cell division would be resumed to make callus, by the culture in the medium containing auxin and/or cytokinin. However, the expression levels of these genes in cortical and epidermal cells, which would not originate callus cells, were variable by genes and phytohormonal conditions. We also found that the rid1-1 mutation greatly decreased the expression levels of CDKA;1 and SRD2 during callus initiation specifically at 28°C (restrictive temperature), while the srd2-1 mutation did not obviously decrease the expression levels of CDKA;1 and RID1 regardless of temperature conditions but rather even increased them at 22°C (permissive temperature). Together, our results implicated the phytohormonal and differential regulation of cell proliferation competence-associated genes in the multistep regulation of cell proliferation competence.

RNAa-mediated epigenetic attenuation of the cell senescence via locus specific induction of endogenous SIRT1

SIRT1, a known regulator of cellular senescence, is a therapeutic target for age related disorders and its upregulation is a strategy to improve the cell therapeutic potentials of human mesenchymal stem cell (MSCs). Knockdown of natural antisense transcripts via small activating RNAs (RNAa) is an emerging approach for safe and locus specific gene regulation. We have recently identified a natural antisense transcript at human SIRT1 locus (SIRT1-NAT), the expression of which shows a negative correlation with that of SIRT1. To test the hypothetic upregulation of SIRT1 via knockdown of SIRT1-NAT, in this study we designed a single stranded oligonucleotide (SIRT1-antagoNAT) against the antisense transcript, transfection of which efficiently knocked down the SIRT1-NAT and induced SIRT1 transcription in human MSCs. In addition, activation of SIRT1 transfection via knockdown of SIRT1-NAT in human MSCs enhanced their proliferation and differentiation potentials, reduced senescence associated β-galactosidase activity and reversed the senescence associated molecular alterations. Our findings introduce an RNAa mediated approach for epigenetic induction of endogenous SIRT1 and the consequent attenuation of senescence. Further studies should evaluate the therapeutic potentials of this approach against various age related disorders.

PL01.3.A Radiomic features and DNA methylation attributes in primary CNS lymphoma

Abstract Background Clinical and laboratory markers have been exploited to model risk in patients with primary CNS lymphoma (PCNSL), but the derived risk models do not fully explain the observed variation in outcome. Here we present an extended framework of phenotype-epigenotype correlations that reveal novel prognostic constellations and enable prioritizing epigenetic therapy. Material and Methods In this retrospective discovery and validation study, we leverage radiomic feature-driven analysis of medical images and supervised bioinformatic integration of DNA methylation profiles. We integrate both data modalities synergistically using machine learning-based prediction and cross-domain alignment. Ultimately, we validate the most relevant biological associations in tumor tissues and cell lines. Results We leverage a cohort of 191 patients across 9 sites in Austria and an external validation site in South Korea, and use T1-weighted contrast-enhanced magnetic resonance imaging to derive a radiomic risk score that consists of 20 mostly textural features. We determine the risk score as strong and independent predictive factor (multivariate HR=6.56), and confirm its prognostic value in an external validation cohort. Radiomic features align with DNA methylation sites in distinct, biologically meaningful ways, and radiomic risk is predictable from selected DNA methylation sites (AUC=0.78). Ultimately, gene-regulatory differences between radiomically-defined risk groups converge on bcl6 binding activity, which is posed as testable treatment strategy in a subset of patients. Conclusion The radiomic risk score is a robust and complementary predictor of survival and is reflected at the level of DNA methylation in PCNSL. Assessing risk and selecting epigenetic treatment based on imaging phenotypes represents a huge step forward, and the ability to define radiomic risk groups provides a concept on which to advance prognostic modeling and precision therapy for this aggressive brain cancer.

Methylation of genes and regulation of inflammatory processes on emotional response in young adults with alcoholic parents

Many Americans are adult children of an alcoholic parent (ACoA), which can confer an increased risk of trauma and hazardous alcohol use, as well as heritable and environmental genetic influence. Psychological health and related neural activity can be influenced by inflammation responses, but it is not clear how these factors interact regarding risk or resilience to hazardous alcohol use. The goals of this study were to better understand the relationships between current alcohol use and inflammation, how these are modified by single nucleotide polymorphisms (SNPs) and/or epigenetic modifications of inflammation-associated genes; and how these alter neural reactivity to emotionally-salient stimuli. To do so, ACoA participants were dichotomized as resilient (not engaged in hazardous alcohol use) or vulnerable (currently engaged in hazardous alcohol use). Measures of blood-oxygen-level-dependent (BOLD) activity within regions of interest (ROIs), SNPs and DNA methylation of specific inflammation regulatory genes, and biological markers of inflammation were compared between these groups. Vulnerable ACoAs exhibited higher plasma C-reactive protein (CRP) and greater BOLD activity in the right hippocampus and ventral anterior cingulate cortex in response to emotional cues as well as reduced methylation of CRP and glucocorticoid-related genes. Path analysis revealed significant relationships between alcohol use, SNPs, DNA methylation of inflammatory-related genes, CRP levels, and BOLD activity to emotional stimuli. Taken together, these findings suggest a complex association related to hazardous alcohol use in ACoAs that may predict current inflammation and neural reactivity to emotional stimuli. A better understanding of these associations could direct the future of individual treatment options.

Evidence for Light and Tissue Specific Regulation of Genes Involved in Fructan Metabolism in Agave tequilana.

Plant Glycoside Hydrolase Family 32 (PGHF32) contains the fructosyltransferases and fructan exohydrolase enzymes responsible for fructan metabolism, in addition to closely related vacuolar and cell wall acid invertases. Agave species produce complex and dynamic fructan molecules (agavins) requiring 4 different fructosyltransferase activities (1-SST, 1-FFT, 6G-FFT and 6-SFT) for their synthesis. Combined analysis of RNAseq and genome data for A. tequilana led to the characterization of the genes encoding 3 fructosyltransferases for this species and support the hypothesis that no separate 6-SFT type enzyme exists in A. tequilana, suggesting that at least one of the fructosyltransferases identified may have multiple enzymatic activities. Structures for PGHF32 genes varied for A. tequilana and between other plant species but were conserved for different enzyme types within a species. The observed patterns are consistent with the formation of distinct gene structures by intron loss. Promoter analysis of the PGHF32 genes identified abundant putative regulatory motifs for light regulation and tissue-specific expression, and these regulatory mechanisms were confirmed experimentally for leaf tissue. Motifs for phytohormone response, carbohydrate metabolism and dehydration responses were also uncovered. Based on the regulatory motifs, full-length cDNAs for MYB, GATA, DOF and GBF transcription factors were identified and their phylogenetic distribution determined by comparison with other plant species. In silico expression analysis for the selected transcription factors revealed both tissue-specific and developmental patterns of expression, allowing candidates to be identified for detailed analysis of the regulation of fructan metabolism in A. tequilana at the molecular level.

Abstract P3117: Validating Genetic Risk Markers Causing Doxorubicin-induced Cardiotoxicity In African American And European American Pediatric Patients Using A Hipsc Model

The W.H.O. estimates that globally 400,000 children under 19 are diagnosed with cancer annually. 60% of pediatric cancers are treated with chemotherapy using anthracyclines, mainly doxorubicin. Survivors of childhood cancer are approximately 15 times more likely to be diagnosed with congestive heart failure. African American (AA) survivors had a 2.5-fold increased risk of severe or life-threatening cardiomyopathy vs European Americans (EA). In recent WGS studies, 2 novel loci in chromosome 1 &amp; 6 were identified to be correlated with an increased risk of doxorubicin-induced cardiotoxicity (DIC) in AA and EA population groups respectively and were reduced or absent in the other group. Our study aims to validate these loci using patient specific hiPSC-derived cardiomyocytes (hiPSC-CM), and for that we recruited and generated 20 hiPSC lines from the blood of AA and EA pediatric patients. Using a novel bioreactor system, a large-scale production of hiPSC-CM for this patient specific samples is underway, and the cells will be utilized for phenotypic and genotypic characterization. Our aim is to assess the effect of doxorubicin on cardiomyocyte viability, apoptosis, mitochondrial function, and calcium dynamics. Differential cardiac gene regulation after exposure to doxorubicin will be studied using RNA sequencing. In conclusion, our findings will validate the genetic variants predisposing AA and EA populations to DIC. This study will clinically impact therapeutic approaches on both AA and EA populations and at individual levels while using doxorubicin. The validated hiPSC-CMs could help in identifying personalized cardioprotectants to reduce DIC without modifying chemotherapy. This study could also potentially provide data for genomically-informed drug discovery for novel cardioprotectants.

CD44 Contributes to the Regulation of MDR1 Protein and Doxorubicin Chemoresistance in Osteosarcoma.

Osteosarcoma is the most common type of pediatric bone tumor. Despite great advances in chemotherapy during the past decades, the survival rates of osteosarcoma patients remain unsatisfactory. Drug resistance is one of the main reasons, leading to treatment failure and poor prognosis. Previous reports correlated expression of cluster of differentiation 44 (CD44) with drug resistance and poor survival of osteosarcoma patients, however the underlying mechanisms are poorly defined. Here, we investigated the role of CD44 in the regulation of drug chemoresistance, using osteosarcoma cells isolated from mice carrying a mutation of the tumor suppressor neurofibromatosis type 2 (Nf2) gene. CD44 expression was knocked-down in the cells using CRISPR/Cas9 approach. Subsequently, CD44 isoforms and mutants were re-introduced to investigate CD44-dependent processes. Sensitivity to doxorubicin was analyzed in the osteosarcoma cells with modified CD44 expression by immunoblot, colony formation- and WST-1 assay. To dissect the molecular alterations induced by deletion of Cd44, RNA sequencing was performed on Cd44-positive and Cd44-negative primary osteosarcoma tissues isolated from Nf2-mutant mice. Subsequently, expression of candidate genes was evaluated by quantitative reverse transcription PCR (qRT-PCR). Our results indicate that CD44 increases the resistance of osteosarcoma cells to doxorubicin by up-regulating the levels of multidrug resistance (MDR) 1 protein expression, and suggest the role of proteolytically released CD44 intracellular domain, and hyaluronan interactions in this process. Moreover, high throughput sequencing analysis identified differential regulation of several apoptosis-related genes in Cd44-positive and -negative primary osteosarcomas, including p53 apoptosis effector related to PMP-22 (Perp). Deletion of Cd44 in osteosarcoma cells led to doxorubicin-dependent p53 activation and a profound increase in Perp mRNA expression. Overall, our results suggest that CD44 might be an important regulator of drug resistance and suggest that targeting CD44 can sensitize osteosarcoma to standard chemotherapy.