Genome-wide Gene Expression Profiling Research Articles

Morphological and transcriptomic effects of endocrine modulators on the gonadal differentiation of chicken embryos: The case of tributyltin (TBT)

Morphological malformations induced by tributyltin (TBT) exposure during embryonic development have already been characterized in various taxonomic groups, but, nonetheless, the molecular processes underlying these changes remain obscure. The present study provides the first genome-wide screening for differentially expressed genes that are linked to morphological alterations of gonadal tissue from chicken embryos after exposure to TBT. We applied a single injection of TBT (between 0.5 and 30 pg as Sn/g egg) into incubated fertile eggs to simulate maternal transfer of the endocrine disruptive compound. Methyltestosterone (MT) served as a positive control (30 pg/g egg). After 19 days of incubation, structural features of the gonads as well as genome-wide gene expression profiles were assessed simultaneously. TBT induced significant morphological and histological malformations of gonadal tissue from female embryos that show a virilization of the ovaries. This phenotypical virilization was mirrored by altered expression profiles of sex-dependent genes. Among these are several transcription and growth factors (e.g. FGF12, CTCF, NFIB), whose altered expression might serve as a set of markers for early identification of endocrine active chemicals that affect embryonic development by transcriptome profiling without the need of elaborate histological analyses.

Modulation of fatty acid metabolism and immune suppression are features of in vitro tumour sphere formation in ontogenetically distinct dog cancers

Non-adherent, 3-dimensional sphere formation is used as an in vitro surrogate to evaluate cellular potential for tumour initiation and self-renewal. To determine if a shared molecular program underlies the capacity for sphere formation by cells originating from diverse tumour types, we characterized molecular and functional properties of 10 independent cell lines derived from 3 ontogenetically distinct dog cancers: hemangiosarcoma, osteosarcoma and glial brain tumours. Genome-wide gene expression profiling identified tumour-of-origin-dependent patterns of adjustment to sphere formation in a uniform culture condition. However, expression of the stem/progenitor markers CD34 and CD117, resistance to cytotoxic drugs and dye efflux (side population assays) showed no association with these gene expression profiles. Instead, primary sphere-forming capacity was inversely correlated with the ability to reform secondary spheres, regardless of tumour ontogeny. Primary sphere formation seemed to be proportional to the number of pre-existing cells with sphere-forming capacity in the cell lines. Cell lines where secondary sphere formation was more proficient than primary sphere formation showed enrichment of genes involved in fatty acid synthesis and immunosuppressive cytokines. In contrast, cell lines where secondary sphere formation was approximately equivalent to or less proficient than primary sphere formation showed upregulation of CD40 and enrichment of genes involved in fatty acid oxidation. Our data suggest that in vitro sphere formation is associated with upregulation of gene clusters involved in metabolic and immunosuppressive functions, which might be necessary for self-renewal and for tumour initiation and/or tumour propagation in vivo.

Transcriptomic and epigenetic responses to short-term nutrient-exercise stress in humans

High fat feeding impairs skeletal muscle metabolic flexibility and induces insulin resistance, whereas exercise training exerts positive effects on substrate handling and improves insulin sensitivity. To identify the genomic mechanisms by which exercise ameliorates some of the deleterious effects of high fat feeding, we investigated the transcriptional and epigenetic response of human skeletal muscle to 9 days of a high-fat diet (HFD) alone (Sed-HFD) or in combination with resistance exercise (Ex-HFD), using genome-wide profiling of gene expression and DNA methylation. HFD markedly induced expression of immune and inflammatory genes, which was not attenuated by Ex. Conversely, Ex markedly remodelled expression of genes associated with muscle growth and structure. We detected marked DNA methylation changes following HFD alone and in combination with Ex. Among the genes that showed a significant association between DNA methylation and gene expression changes were PYGM, which was epigenetically regulated in both groups, and ANGPTL4, which was regulated only following Ex. In conclusion, while short-term Ex did not prevent a HFD-induced inflammatory response, it provoked a genomic response that may protect skeletal muscle from atrophy. These epigenetic adaptations provide mechanistic insight into the gene-specific regulation of inflammatory and metabolic processes in human skeletal muscle.

Association of Gender With Outcome and Host Response in Critically Ill Sepsis Patients.

To determine the association of gender with the presentation, outcome, and host response in critically ill patients with sepsis. A prospective observational cohort study in the ICU of two tertiary hospitals between January 2011 and January 2014. All consecutive critically ill patients admitted with sepsis, involving 1,815 admissions (1,533 patients). The host response was evaluated on ICU admission by measuring 19 plasma biomarkers reflecting organ systems implicated in sepsis pathogenesis (1,205 admissions) and by applying genome-wide blood gene expression profiling (582 admissions). Sepsis patients admitted to the ICU were more frequently males (61.0%; p < 0.0001 vs females). Baseline characteristics were not different between genders. Urosepsis was more common in females; endocarditis and mediastinitis in men. Disease severity was similar throughout ICU stay. Mortality was similar up to 1 year after ICU admission, and gender was not associated with 90-day mortality in multivariate analyses in a variety of subgroups. Although plasma proteome analyses (including systemic inflammatory and cytokine responses, and activation of coagulation) were largely similar between genders, females showed enhanced endothelial cell activation; this difference was virtually absent in patients more than 55 years old. More than 80% of the leukocyte blood gene expression response was similar in male and female patients. The host response and outcome in male and female sepsis patients requiring ICU admission are largely similar.

A molecular morphometric approach to diabetic kidney disease can link structure to function and outcome

Diabetic kidney disease is the leading cause of kidney failure. However, studies of molecular mechanisms of early kidney damage are lacking. Here we examined for possible linkage between transcriptional regulation and quantitative structural damage in early diabetic kidney disease in Pima Indians with type 2 diabetes. Tissue obtained from protocol kidney biopsies underwent genome-wide compartment-specific gene expression profiling and quantitative morphometric analysis. The ultrastructural lesion most strongly associated with transcriptional regulation was cortical interstitial fractional volume (VvInt), an index of tubule-interstitial damage. Transcriptional co-expression network analysis identified 1843 transcripts that correlated significantly with VvInt. These transcripts were enriched for pathways associated with mitochondrial dysfunction, inflammation, migratory mechanisms, and tubular metabolic functions. Pathway network analysis identified IL-1β as a key upstream regulator of the inflammatory response and five transcription factors cooperating with p53 to regulate metabolic functions. VvInt-associated transcripts showed significant correlation with the urine albumin to creatinine ratio and measured glomerular filtration rate 10 years after biopsy, establishing a link between the early molecular events and long-term disease progression. Thus, molecular mechanisms active early in diabetic kidney disease were revealed by correlating intrarenal transcripts with quantitative morphometry and long-term outcomes. This provides a starting point for identification of urgently needed therapeutic targets and non-invasive biomarkers of early diabetic kidney disease.

DNA Methylation and Transcription Patterns in Intestinal Epithelial Cells From Pediatric Patients With Inflammatory Bowel Diseases Differentiate Disease Subtypes and Associate With Outcome

Background & AimsWe analyzed DNA methylation patterns and transcriptomes of primary intestinal epithelial cells (IEC) of children newly diagnosed with inflammatory bowel diseases (IBD) to learn more about pathogenesis.MethodsWe obtained mucosal biopsies (N = 236) collected from terminal ileum and ascending and sigmoid colons of children (median age 13 years) newly diagnosed with IBD (43 with Crohn’s disease [CD], 23 with ulcerative colitis [UC]), and 30 children without IBD (controls). Patients were recruited and managed at a hospital in the United Kingdom from 2013 through 2016. We also obtained biopsies collected at later stages from a subset of patients. IECs were purified and analyzed for genome-wide DNA methylation patterns and gene expression profiles. Adjacent microbiota were isolated from biopsies and analyzed by 16S gene sequencing. We generated intestinal organoid cultures from a subset of samples and genome-wide DNA methylation analysis was performed.ResultsWe found gut segment-specific differences in DNA methylation and transcription profiles of IECs from children with IBD vs controls; some were independent of mucosal inflammation. Changes in gut microbiota between IBD and control groups were not as large and were difficult to assess because of large amounts of intra-individual variation. Only IECs from patients with CD had changes in DNA methylation and transcription patterns in terminal ileum epithelium, compared with controls. Colon epithelium from patients with CD and from patients with ulcerative colitis had distinct changes in DNA methylation and transcription patterns, compared with controls. In IECs from patients with IBD, changes in DNA methylation, compared with controls, were stable over time and were partially retained in ex-vivo organoid cultures. Statistical analyses of epithelial cell profiles allowed us to distinguish children with CD or UC from controls; profiles correlated with disease outcome parameters, such as the requirement for treatment with biologic agents.ConclusionsWe identified specific changes in DNA methylation and transcriptome patterns in IECs from pediatric patients with IBD compared with controls. These data indicate that IECs undergo changes during IBD development and could be involved in pathogenesis. Further analyses of primary IECs from patients with IBD could improve our understanding of the large variations in disease progression and outcomes.

Genome-wide identification of key modulators of gene-gene interaction networks in breast cancer

BackgroundWith the advances in high-throughput gene profiling technologies, a large volume of gene interaction maps has been constructed. A higher-level layer of gene-gene interaction, namely modulate gene interaction, is composed of gene pairs of which interaction strengths are modulated by (i.e., dependent on) the expression level of a key modulator gene. Systematic investigations into the modulation by estrogen receptor (ER), the best-known modulator gene, have revealed the functional and prognostic significance in breast cancer. However, a genome-wide identification of key modulator genes that may further unveil the landscape of modulated gene interaction is still lacking.ResultsWe proposed a systematic workflow to screen for key modulators based on genome-wide gene expression profiles. We designed four modularity parameters to measure the ability of a putative modulator to perturb gene interaction networks. Applying the method to a dataset of 286 breast tumors, we comprehensively characterized the modularity parameters and identified a total of 973 key modulator genes. The modularity of these modulators was verified in three independent breast cancer datasets. ESR1, the encoding gene of ER, appeared in the list, and abundant novel modulators were illuminated. For instance, a prognostic predictor of breast cancer, SFRP1, was found the second modulator. Functional annotation analysis of the 973 modulators revealed involvements in ER-related cellular processes as well as immune- and tumor-associated functions.ConclusionsHere we present, as far as we know, the first comprehensive analysis of key modulator genes on a genome-wide scale. The validity of filtering parameters as well as the conservativity of modulators among cohorts were corroborated. Our data bring new insights into the modulated layer of gene-gene interaction and provide candidates for further biological investigations.

Stromal regulation of prostate cancer cell growth by mevalonate pathway enzymes HMGCS1 and HMGCR.

It has been suggested that the tumor microenvironment plays an important role in tumor progression, acquisition of androgen independence, and distant metastasis in prostate cancer (PC). However, little is known about the transcriptional basis of cellular interactions in the human PC microenvironment. To clarify the mechanism of PC progression and metastasis, we investigated the interaction of PC, epithelial, and stromal cells using genome-wide gene expression profiling. We hypothesized that PC cells could induce stromal cells to differentiate into so-called cancer-associated fibroblasts (CAFs), which might contribute to cancer invasion and metastasis. Genes upregulated in normal human prostate stromal cells (PrSC) co-cultured with human PC cells (LNCaP) included the mevalonate pathway enzymes 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR). Knockdown of endogenous HMGCS1 or HMGCR in PC cells by shRNA resulted in a significant reduction of PC cell viability. Importantly, exogenous overexpression of HMGCS1 or HMGCR in either PC cells or prostate stromal cells stimulated PC cell growth, suggesting a possible autocrine/paracrine mechanism of action. Immunohistochemical analysis confirmed that HMGCS1 and HMGCR were overexpressed in PC stroma, especially in early stage PC. These results provide clues to the molecular mechanisms underlying PC invasion and metastasis, and suggest that HMGCS1 and HMGCR in PC, as well as in PC stroma, might serve as molecular targets for the treatment of PC.

Genome expression profile analysis of two types of new roots in apple rootstock via Solexa sequencing

Currently, the molecular regulation mechanisms involved in the early development of new roots are poorly known in apple. To gain insight into the transcriptome dynamics that are associated with its development, genome-wide gene expression profiling was conducted by Solexa sequencing. More than five million tags were generated from two type new roots, long root and short root, including 5,913,044 and 6,236,711 clean tags in the two libraries, respectively. Of these, 44,513 (26.61%) and 43,117 (20.02%) tags were matched to the reference genes. The most differentially expressed tags with FDR≤0.001 and |log2Ratio|≥1 were analyzed further, representing 815 up-regulated and 831 down-regulated genes, except for unknown transcripts. These genes participated in biosynthesis of secondary metabolites, plant-pathogen interaction, plant hormone signal transduction, phenylpropanoid biosynthesis, ribosomes and flavonoid biosynthesis. Most genes were identified in the cell part function (82.7%), catalytic activity (71.1%), ion binding (59.2%) and membrane transcription (24.5%), etc. Next, the expression patterns of 20 genes were assessed by quantitative real-time PCR, and the results obtained showed general agreement with the Solexa analysis. Moreover, a visual database was constructed in order to browse and query the results, and the website is http://www.applegene.org/deg/. In conclusion, we first reveal the complex changes in the transcriptome during the early development of apple new root and provide a comprehensive set of data that are essential for understanding its molecular regulation.

The dynamic landscape of gene regulation during Bombyx mori oogenesis

BackgroundOogenesis in the domestic silkworm (Bombyx mori) is a complex process involving previtellogenesis, vitellogenesis and choriogenesis. During this process, follicles show drastic morphological and physiological changes. However, the genome-wide regulatory profiles of gene expression during oogenesis remain to be determined.ResultsIn this study, we obtained time-series transcriptome data and used these data to reveal the dynamic landscape of gene regulation during oogenesis. A total of 1932 genes were identified to be differentially expressed among different stages, most of which occurred during the transition from late vitellogenesis to early choriogenesis. Using weighted gene co-expression network analysis, we identified six stage-specific gene modules that correspond to multiple regulatory pathways. Strikingly, the biosynthesis pathway of the molting hormone 20-hydroxyecdysone (20E) was enriched in one of the modules. Further analysis showed that the ecdysteroid 20-hydroxylase gene (CYP314A1) of steroidgenesis genes was mainly expressed in previtellogenesis and early vitellogenesis. However, the 20E–inactivated genes, particularly the ecdysteroid 26-hydroxylase encoding gene (Cyp18a1), were highly expressed in late vitellogenesis. These distinct expression patterns between 20E synthesis and catabolism-related genes might ensure the rapid decline of the hormone titer at the transition point from vitellogenesis to choriogenesis. In addition, we compared landscapes of gene regulation between silkworm (Lepidoptera) and fruit fly (Diptera) oogeneses. Our results show that there is some consensus in the modules of gene co-expression during oogenesis in these insects.ConclusionsThe data presented in this study provide new insights into the regulatory mechanisms underlying oogenesis in insects with polytrophic meroistic ovaries. The results also provide clues for further investigating the roles of epigenetic reconfiguration and circadian rhythm in insect oogenesis.

GENOMICS SYMPOSIUM: Using genomic approaches to uncover sources of variation in age at puberty and reproductive longevity in sows1,2

Genetic variants associated with traits such as age at puberty and litter size could provide insight into the underlying genetic sources of variation impacting sow reproductive longevity and productivity. Genomewide characterization and gene expression profiling were used using gilts from the University of Nebraska-Lincoln swine resource population ( = 1,644) to identify genetic variants associated with age at puberty and litter size traits. From all reproductive traits studied, the largest fraction of phenotypic variation explained by the Porcine SNP60 BeadArray was for age at puberty (27.3%). In an evaluation data set, the predictive ability of all SNP from high-ranked 1-Mb windows (1 to 50%), based on genetic variance explained in training, was greater (12.3 to 36.8%) compared with the most informative SNP from these windows (6.5 to 23.7%). In the integrated data set ( = 1,644), the top 1% of the 1-Mb windows explained 6.7% of the genetic variation of age at puberty. One of the high-ranked windows detected (SSC2, 12-12.9 Mb) showed pleiotropic features, affecting both age at puberty and litter size traits. The RNA sequencing of the hypothalami arcuate nucleus uncovered 17 differentially expressed genes (adjusted 180 d of age). Twelve of the differentially expressed genes are upregulated in the late pubertal gilts. One of these genes is involved in energy homeostasis (), a function in which the arcuate nucleus plays an important contribution, linking nutrition with reproductive development. Energy restriction during the gilt development period delayed age at puberty by 7 d but increased the probability of a sow to produce up to 3 parities ( < 0.05). Identification of pleotropic functional polymorphisms may improve accuracy of genomic prediction while facilitating a reduction in sow replacement rates and addressing welfare concerns.

Expression profiling of genes regulated by sphingosine kinase1 signaling in a murine model of hyperoxia induced neonatal bronchopulmonary dysplasia

BackgroundSphingosine- 1-Phosphate (S1P) is a bioactive lipid and an intracellular as well as an extracellular signaling molecule. S1P ligand specifically binds to five related cell surface G-protein-coupled receptors (S1P1-5). S1P levels are tightly regulated by its synthesis catalyzed by sphingosine kinases (SphKs) 1 & 2 and catabolism by S1P phosphatases, lipid phosphate phosphatases and S1P lyase. We previously reported that knock down of SphK1 (Sphk1−/−) in a neonatal mouse BPD model conferred significant protection against hyperoxia induced lung injury. To better understand the underlying molecular mechanisms, genome-wide gene expression profiling was performed on mouse lung tissue using Affymetrix MoGene 2.0 array.ResultsTwo-way ANOVA analysis was performed and differentially expressed genes under hyperoxia were identified using Sphk1−/− mice and their wild type (WT) equivalents. Pathway (PW) enrichment analyses identified several signaling pathways that are likely to play a key role in hyperoxia induced lung injury in the neonates. These included signaling pathways that were anticipated such as those involved in lipid signaling, cell cycle regulation, DNA damage/apoptosis, inflammation/immune response, and cell adhesion/extracellular matrix (ECM) remodeling. We noted hyperoxia induced downregulation of the expression of genes related to mitotic spindle formation in the WT which was not observed in Sphk1−/− neonates. Our data clearly suggests a role for SphK1 in neonatal hyperoxic lung injury through elevated inflammation and apoptosis in lung tissue. Further, validation by RT-PCR on 24 differentially expressed genes showed 83% concordance both in terms of fold change and vectorial changes. Our findings are in agreement with previously reported human BPD microarray data and completely support our published in vivo findings. In addition, the data also revealed a significant role for additional unanticipitated signaling pathways involving Wnt and GADD45.ConclusionUsing SphK1 knockout mice and differential gene expression analysis, we have shown here that S1P/SphK1 signaling plays a key role in promoting hyperoxia induced DNA damage, inflammation, apoptosis and ECM remodeling in neonatal lungs. It also appears to suppress pro-survival cellular responses involved in normal lung development. We therefore propose SphK1 as a therapeutic target for the development drugs to combat BPD.

Classification of patients with sepsis according to blood genomic endotype: a prospective cohort study

Host responses during sepsis are highly heterogeneous, which hampers the identification of patients at high risk of mortality and their selection for targeted therapies. In this study, we aimed to identify biologically relevant molecular endotypes in patients with sepsis. This was a prospective observational cohort study that included consecutive patients admitted for sepsis to two intensive care units (ICUs) in the Netherlands between Jan 1, 2011, and July 20, 2012 (discovery and first validation cohorts) and patients admitted with sepsis due to community-acquired pneumonia to 29 ICUs in the UK (second validation cohort). We generated genome-wide blood gene expression profiles from admission samples and analysed them by unsupervised consensus clustering and machine learning. The primary objective of this study was to establish endotypes for patients with sepsis, and assess the association of these endotypes with clinical traits and survival outcomes. We also established candidate biomarkers for the endotypes to allow identification of patient endotypes in clinical practice. The discovery cohort had 306 patients, the first validation cohort had 216, and the second validation cohort had 265 patients. Four molecular endotypes for sepsis, designated Mars1-4, were identified in the discovery cohort, and were associated with 28-day mortality (log-rank p=0·022). In the discovery cohort, the worst outcome was found for patients classified as having a Mars1 endotype, and at 28 days, 35 (39%) of 90 people with a Mars1 endotype had died (hazard ratio [HR] vs all other endotypes 1·86 [95% CI 1·21-2·86]; p=0·0045), compared with 23 (22%) of 105 people with a Mars2 endotype (HR 0·64 [0·40-1·04]; p=0·061), 16 (23%) of 71 people with a Mars3 endotype (HR 0·71 [0·41-1·22]; p=0·19), and 13 (33%) of 40 patients with a Mars4 endotype (HR 1·13 [0·63-2·04]; p=0·69). Analysis of the net reclassification improvement using a combined clinical and endotype model significantly improved risk prediction to 0·33 (0·09-0·58; p=0·008). A 140-gene expression signature reliably stratified patients with sepsis to the four endotypes in both the first and second validation cohorts. Only Mars1 was consistently significantly associated with 28-day mortality across the cohorts. To facilitate possible clinical use, a biomarker was derived for each endotype; BPGM and TAP2 reliably identified patients with a Mars1 endotype. This study provides a method for the molecular classification of patients with sepsis to four different endotypes upon ICU admission. Detection of sepsis endotypes might assist in providing personalised patient management and in selection for trials. Center for Translational Molecular Medicine, Netherlands.

Hypoxia in Combination With Muscle Contraction Improves Insulin Action and Glucose Metabolism in Human Skeletal Muscle via the HIF-1α Pathway.

Skeletal muscle insulin resistance is the hallmark of type 2 diabetes and develops long before the onset of the disease. It is well accepted that physical activity improves glycemic control, but the knowledge on underlying mechanisms mediating the beneficial effects remains incomplete. Exercise is accompanied by a decrease in intramuscular oxygen levels, resulting in induction of HIF-1α. HIF-1α is a master regulator of gene expression and might play an important role in skeletal muscle function and metabolism. Here we show that HIF-1α is important for glucose metabolism and insulin action in skeletal muscle. By using a genome-wide gene expression profiling approach, we identified RAB20 and TXNIP as two novel exercise/HIF-1α-regulated genes in skeletal muscle. Loss of Rab20 impairs insulin-stimulated glucose uptake in human and mouse skeletal muscle by blocking the translocation of GLUT4 to the cell surface. In addition, exercise/HIF-1α downregulates the expression of TXNIP, a well-known negative regulator of insulin action. In conclusion, we are the first to demonstrate that HIF-1α is a key regulator of glucose metabolism in skeletal muscle by directly controlling the transcription of RAB20 and TXNIP These results hint toward a novel function of HIF-1α as a potential pharmacological target to improve skeletal muscle insulin sensitivity.

Shared and Distinct Aspects of the Sepsis Transcriptomic Response to Fecal Peritonitis and Pneumonia.

Heterogeneity in the septic response has hindered efforts to understand pathophysiology and develop targeted therapies. Source of infection, with different causative organisms and temporal changes, might influence this heterogeneity. To investigate individual and temporal variations in the transcriptomic response to sepsis due to fecal peritonitis, and to compare these with the same parameters in community-acquired pneumonia. We performed genome-wide gene expression profiling in peripheral blood leukocytes of adult patients admitted to intensive care with sepsis due to fecal peritonitis (n = 117) or community-acquired pneumonia (n = 126), and of control subjects without sepsis (n = 10). A substantial portion of the transcribed genome (18%) was differentially expressed compared with that of control subjects, independent of source of infection, with eukaryotic initiation factor 2 signaling being the most enriched canonical pathway. We identified two sepsis response signature (SRS) subgroups in fecal peritonitis associated with early mortality (P = 0.01; hazard ratio, 4.78). We defined gene sets predictive of SRS group, and serial sampling demonstrated that subgroup membership is dynamic during intensive care unit admission. We found that SRS is the major predictor of transcriptomic variation; a small number of genes (n = 263) were differentially regulated according to the source of infection, enriched for IFN signaling and antigen presentation. We define temporal changes in gene expression from disease onset involving phagosome formation as well as natural killer cell and IL-3 signaling. The majority of the sepsis transcriptomic response is independent of the source of infection and includes signatures reflecting immune response state and prognosis. A modest number of genes show evidence of specificity. Our findings highlight opportunities for patient stratification and precision medicine in sepsis.

Effects of mechanical ventilation on gene expression profiles in renal allografts from brain dead rats

Pathophysiological changes of brain death (BD) are impairing distal organ function and harming potential renal allografts. Whether ventilation strategies influence the quality of renal allografts from BD donors has not been thoroughly studied.28 adult male Wistar rats were randomly assigned to four groups: 1) no brain death (NBD) with low tidal volume/low positive endexpiratory pressure (PEEP) titrated to minimal static elastance of the respiratory system (LVT/OLPEEP); 2) NBD with high tidal volume/low PEEP (HVT/LPEEP); 3) brain death (BD) with LVT/OLPEEP; and 4) BD with HVT/LPEEP. We hypothesized that HVT/LPEEP in BD leads to increased interleukin 6 (IL-6) gene expression and impairs potential renal allografts after six hours of mechanical ventilation.We assessed inflammatory cytokines in serum, genome wide gene expression profiles and quantitative PCR (qPCR) in kidney tissue.The influence of BD on renal gene-expression profiles was greater than the influence of the ventilation strategy. In BD, LVT ventilation did not influence the inflammatory parameters or kidney function in our experimental model.

Molecular characteristics of the KCNJ5 mutated aldosterone-producing adenomas.

The pathophysiology of aldosterone-producing adenomas (APAs) has been investigated via genetic approaches and the pathogenic significance of a series of somatic mutations, including KCNJ5, has been uncovered. However, how the mutational status of an APA is associated with its molecular characteristics, including its transcriptome and methylome, has not been fully understood. This study was undertaken to explore the molecular characteristics of APAs, specifically focusing on APAs with KCNJ5 mutations as opposed to those without KCNJ5 mutations, by comparing their transcriptome and methylome status. Cortisol-producing adenomas (CPAs) were used as reference. We conducted transcriptome and methylome analyses of 29 APAs with KCNJ5 mutations, 8 APAs without KCNJ5 mutations and 5 CPAs. Genome-wide gene expression and CpG methylation profiles were obtained from RNA and DNA samples extracted from these 42 adrenal tumors. Cluster analysis of the transcriptome and methylome revealed molecular heterogeneity in APAs depending on their mutational status. DNA hypomethylation and gene expression changes in Wnt signaling and inflammatory response pathways were characteristic of APAs with KCNJ5 mutations. Comparisons between transcriptome data from our APAs and that from normal adrenal cortex obtained from the Gene Expression Omnibus suggested similarities between APAs with KCNJ5 mutations and zona glomerulosa. The present study, which is based on transcriptome and methylome analyses, indicates the molecular heterogeneity of APAs depends on their mutational status. Here, we report the unique characteristics of APAs with KCNJ5 mutations.

Comparative transcript profiling explores differentially expressed genes associated with sexual phenotype in kiwifruit.

BackgroundKiwifruit is a perennial, deciduous and functionally dioecious plant. However, very little is known about the whole-genome molecular mechanisms contributing to distinct sexual phenotypes. To gain a global view of genes differentially expressed between male and female flowers, we analyzed genome-wide gene expression profiles in the flowers of male and female plants using high-throughput RNA sequencing.ResultsA total of 53.5 million reads were generated. Based on the alignments of unigenes to kiwifruit genome predicted genes, a total of 39,040 unique genes with a mean length of 970 bp were identified. There were 2,503 UniGenes differentially expressed between female and male flowers, with 1,793 up-regulated and 710 down-regulated in the female flowers. Moreover, the gene expression pattern of 17 out of 19 unigenes differentially expressed between male and female flowers revealed by RNA-Seq was confirmed by real-time quantitative PCR (qRT-PCR).ConclusionsHere, we obtained a large number of EST sequences from female and male flowers of kiwifruit. This comparative transcriptome analysis provides an invaluable resource for gene expression, genomics, and functional genomic studies in A. chinensis and its related species. This study also represents a first step toward the investigation of genes involved in kiwifruit sex determination.

Abstract 4682: Genomic profiles in gene expression and methylation help define the molecular characteristics of hairy cell leukemia subtypes

Abstract Hairy cell leukemia (HCL) until recently was characterized as a single blood and bone marrow malignancy, even though clinically at least two pathologies seemed to be at work. This study sought to confirm the clinical observations using genomic evidence. HCL is a chronic mature B-cell malignancy with distinctive immunophenotype, typically expressing CD20, CD22, CD25, CD11c, CD103, CD123, annexin A1 (ANXA1), and tartrate-resistant acid phosphatase (TRAP). Purine analog therapy is highly effective, with most patients achieving durable remissions. HCL- variant (HCLv) was first identified by Cawley et al. (1980) and recently recognized by the World Health Organization as a separate cancer. HCLv lacks CD25, ANXA1, TRAP, and BRAF V600E expression, and patients respond poorly to purine analogs. In order to find detailed biological information involving these two diseases, we studied the genome-wide gene expression and methylation profiles of 75 HCL patient samples; 67 were tested by both methods. Our study results indicate that HCL and HCLv show very distinct gene expression and methylation patterns. Many genes that are differentially expressed are involved in immunological and inflammatory response pathways. Several cancer associated genes such as ANXA1 and FLT3 are down-regulated in HCLv compared to HCL. Correlation patterns between methylation and gene expression for many genes such as ANXA1 suggest that methylation plays an import role in gene expression regulation for these malignancies. This study has begun the process of identifying better biomarkers for disease discrimination between HCL and HCLv and providing potential new targets for strongly needed therapies for HCLv. Citation Format: Daniel C. Edelman, Holly S. Stevenson, Yonghong Wang, Evgeny Arons, Joshua J. Waterfall, David Petersen, Hong Zhou, Paul S. Meltzer, Robert J. Kreitman. Genomic profiles in gene expression and methylation help define the molecular characteristics of hairy cell leukemia subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4682. doi:10.1158/1538-7445.AM2017-4682

Abstract 3562: A novel TGF-β signaling pathway promotes a slow growing phenotype of cancer cells in response to glutamine insufficiency

Abstract Glutamine is a critical nutrient for proliferating cells and most tumor cells. Glutamine addiction as one of the dramatic tumor cell adaptive metabolisms is characterized by increased glutaminolysis. To better understand how tumor cells respond to glutamine deficiency, we cultured Hep3B cells in glutamine free media supplemented with ammonia, and isolated survival clones (MM01). These clones are capable of perpetual survival in glutamine free media with ammonia, but assume a slow growing phenotype, which represents the model adapted to long-term glutamine insufficiency. Comparing MM01 and Hep3B by microarray-based genome-wide gene expression profiling, we identified functional activation of Smad2/3 in MM01 cells, suggesting that long-term glutamine insufficiency activates a TGF-β signaling pathway. We have validated that glutamine insufficiency triggers Samd2 phosphorylation, which in turn, stimulates the expression of p15INK4B, providing a link to the slow growing phenotype. To elucidate how glutamine insufficiency triggers TGF-β signaling, we examined the effects of glutamine insufficiency on the expression levels of TGF-β family ligands, and found that Inhibin-βE, a newly discovered inhibin subunit isoform in the TGF-β superfamily, is up-regulated at both mRNA and protein levels in MM01. Luciferase assays demonstrate that glutamine insufficiency enhances the activity of Inhibin-βE promoter. Furthermore, we show that overexpressing Inhibin-βE is sufficient to induce Smad2/3 activation and growth inhibition in Hep3B cells. Taken together, our data suggest that Inhibin-βE likely plays a crucial role in cell adaptation to metabolic stress, facilitating cancer cell survival by slowing down biosynthesis and proliferation. A better understanding of the novel Inhibin-βE-triggered TGF-β signaling pathway may provide potential new therapeutic targets for cancer treatment. Citation Format: Dan He, Chengqian Yin, Shuo Qie, Shuyang Chen, Nianli Sang. A novel TGF-β signaling pathway promotes a slow growing phenotype of cancer cells in response to glutamine insufficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3562. doi:10.1158/1538-7445.AM2017-3562