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The localization, mass, and dynamics of microtubules are important in many processes. Cells may actively monitor the state of their microtubules and respond to perturbation, but how this occurs outside mitosis is poorly understood. We used gene-expression analysis in quiescent cells to analyze responses to subtle and strong perturbation of microtubules. Genes encoding α-, β, and γ-tubulins (TUBAs, TUBBs, and TUBGs), but not δ- or ε-tubulins (TUBDs or TUBEs), exhibited the strongest differential expression response to microtubule-stabilizing versus destabilizing drugs. Quantitative PCR of exon versus intron sequences confirmed that these changes were caused by regulation of tubulin mRNA stability and not transcription. Using tubulin mRNA stability as a signature to query the Gene Expression Omnibus (GEO) database, we find that tubulin genes respond to toxins known to damage microtubules. Importantly, we find many other experimental perturbations, including multiple signaling and metabolic inputs that trigger tubulin differential expression, suggesting their novel, to our knowledge, role in the regulation of the microtubule cytoskeleton. Mechanistic follow-up confirms that one important physiological signal, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) activity, indeed regulates tubulin mRNA stability via changes in microtubule dynamics. We propose that that tubulin gene expression is regulated as part of many coordinated biological responses, with wide implications in physiology and toxicology. Furthermore, we present a new way to discover microtubule regulation using transcriptomics. [ABSTRACT FROM AUTHOR]

Necroptosis is a lytic programmed cell death mediated by the RIPK1-RIPK3-MLKL pathway. The loss of Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) expression and necroptotic potential have been previously reported in several cancer cell lines; however, the extent of this loss across cancer types, as well as its mutational drivers, were unknown. Here, we show that RIPK3 expression loss occurs progressively during tumor growth both in patient tumor biopsies and tumor xenograft models. Using a cell-based necroptosis sensitivity screen of 941 cancer cell lines, we find that escape from necroptosis is prevalent across cancer types, with an incidence rate of 83%. Genome-wide bioinformatics analysis of this differential necroptosis sensitivity data in the context of differential gene expression and mutation data across the cell lines identified various factors that correlate with resistance to necroptosis and loss of RIPK3 expression, including oncogenes BRAF and AXL. Inhibition of these oncogenes can rescue the RIPK3 expression loss and regain of necroptosis sensitivity. This genome-wide analysis also identifies that the loss of RIPK3 expression is the primary factor correlating with escape from necroptosis. Thus, we conclude that necroptosis resistance of cancer cells is common and is oncogene driven, suggesting that escape from necroptosis could be a potential hallmark of cancer, similar to escape from apoptosis. [ABSTRACT FROM AUTHOR]

We characterized the establishment of an Epidermal Growth Factor Receptor (EGFR) organizing center (EOC) during leg development in Drosophila melanogaster. Initial EGFR activation occurs in the center of leg discs by expression of the EGFR ligand Vn and the EGFR ligand-processing protease Rho, each through single enhancers, vnE and rhoE, that integrate inputs from Wg, Dpp, Dll and Sp1. Deletion of vnE and rhoE eliminates vn and rho expression in the center of the leg imaginal discs, respectively. Animals with deletions of both vnE and rhoE (but not individually) show distal but not medial leg truncations, suggesting that the distal source of EGFR ligands acts at short-range to only specify distal-most fates, and that multiple additional ‘ring’ enhancers are responsible for medial fates. Further, based on the cis-regulatory logic of vnE and rhoE we identified many additional leg enhancers, suggesting that this logic is broadly used by many genes during Drosophila limb development. [ABSTRACT FROM AUTHOR]

Disproportionate high intake of n-6 polyunsaturated fatty acids (PUFAs) in the diet is considered as a major human health concern. The present study examines changes in the hepatic gene expression pattern of adult male zebrafish progeny associated with high levels of the n-6 PUFA arachidonic acid (ARA) in the parental diet. The parental generation (F0) was fed a diet which was either low (control) or high in ARA (high ARA). Progenies of both groups (F1) were given the control diet. No differences in body weight were found between the diet groups within adult stages of either F0 or F1 generation. Few differentially expressed genes were observed between the two dietary groups in the F0 in contrast to the F1 generation. Several links were found between the previous metabolic analysis of the parental fish and the gene expression analysis in their adult progeny. Main gene expression differences in the progeny were observed related to lipid and retinoid metabolism by PPARα/RXRα playing a central role in mediating changes to lipid and long-chain fatty acid metabolism. The enrichment of genes involved in β-oxidation observed in the progeny, corresponded to the increase in peroxisomal β-oxidative degradation of long-chain fatty acids in the parental fish metabolomics data. Similar links between the F0 and F1 generation were identified for the methionine cycle and transsulfuration pathway in the high ARA group. In addition, estrogen signalling was found to be affected by parental high dietary ARA levels, where gene expression was opposite directed in F1 compared to F0. This study shows that the dietary n-3/n-6 PUFA ratio can alter gene expression patterns in the adult progeny. Whether the effect is mediated by permanent epigenetic mechanisms regulating gene expression in developing gametes needs to be further investigated. [ABSTRACT FROM AUTHOR]

Aluminum (Al) stress is becoming the major limiting factor in crop production in acidic soils. Rice has been reported as the most Al-tolerant crop and the capacity of Al toxicity tolerance is generally evaluated by comparing root growth under Al stress. Here, we performed an association mapping of Al toxicity tolerance using a core collection of 211 indica rice accessions with 700 K high quality SNP data. A total of 21 putative QTL affecting shoot height (SH), root length (RL), shoot fresh weight (SFW), shoot dry weight (SDW), root dry weight (RDW) and shoot water content (SWC) were identified at seedling stage, including three QTL detected only under control condition, eight detected only under Al stress condition, ten simultaneously detected in both control and Al stress conditions, and seven were identified by stress tolerance index of their corresponding traits. Total of 21 candidate genes for 7 important QTL regions associated with Al toxicity tolerance were identified based on combined haplotype analysis and functional annotation, and the most likely candidate gene(s) for each important QTL were also discussed. Also a candidate gene Nrat1 on chromosome 2 was further fine-mapped using BSA-seq and linkage analysis in the F2 population derived from the cross of Al tolerant accession CC105 and super susceptible accession CC180. A new non-synonymous SNP variation was observed at Nrat1 between CC105 and CC180, which resulted in an amino-acid substitution from Ala (A) in CC105 to Asp (D) in CC180. Haplotype analysis of Nrat1 using 327 3K RGP accessions indicated that minor allele variations in aus and indica subpopulations decreased Al toxicity tolerance in rice. The candidate genes identified in this study provide valuable information for improvement of Al toxicity tolerance in rice. Our research indicated that minor alleles are important for QTL mapping and its application in rice breeding when natural gene resources are used. [ABSTRACT FROM AUTHOR]

Background: BRAFV600E is the most common mutation in papillary thyroid carcinoma (PTC), and it is associated with high-risk prognostic factors. However, the significance of the BRAF mRNA level in PTC remains unknown. We evaluated the significance of BRAF mRNA expression level by analyzing PTC data from The Cancer Genome Atlas (TCGA) database. Methods: Data from 499 patients were downloaded from the TCGA database. After excluding other PTC variants, we selected 353 cases of classic PTC, including 193 cases with BRAFV600E and 160 cases with the wild-type BRAF. mRNA abundances were measured using RNA-Seq with the Expectation Maximization algorithm. Results: The mean BRAF mRNA level was significantly higher in BRAFV600E patients than in patients with wild-type BRAF (197.6 vs. 179.3, p = 0.031). In wild-type BRAF patients, the mean BRAF mRNA level was higher in cases with a tumor > 2 cm than those with a tumor ≤ 2.0 cm (189.4 vs. 163.8, p = 0.046), and was also higher in cases with lymph node metastasis than in those without lymph node metastasis (188.5 vs. 157.9, p = 0.040). Within BRAFV600E patients, higher BRAF mRNA expression was associated with extrathyroidal extension (186.4 vs. 216.4, p = 0.001) and higher T stage (188.1 vs. 210.2, p = 0.016). Conclusions: A higher BRAF mRNA expression level was associated with tumor aggressiveness in classic PTC regardless of BRAF mutational status. Evaluation of BRAF mRNA level may be helpful in prognostic risk stratification of PTC. [ABSTRACT FROM AUTHOR]

Reconstructing regulatory and signaling response networks is one of the major goals of systems biology. While several successful methods have been suggested for this task, some integrating large and diverse datasets, these methods have so far been applied to reconstruct a single response network at a time, even when studying and modeling related conditions. To improve network reconstruction we developed MT-SDREM, a multi-task learning method which jointly models networks for several related conditions. In MT-SDREM, parameters are jointly constrained across the networks while still allowing for condition-specific pathways and regulation. We formulate the multi-task learning problem and discuss methods for optimizing the joint target function. We applied MT-SDREM to reconstruct dynamic human response networks for three flu strains: H1N1, H5N1 and H3N2. Our multi-task learning method was able to identify known and novel factors and genes, improving upon prior methods that model each condition independently. The MT-SDREM networks were also better at identifying proteins whose removal affects viral load indicating that joint learning can still lead to accurate, condition-specific, networks. Supporting website with MT-SDREM implementation: [ABSTRACT FROM AUTHOR]

Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888), which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis. [ABSTRACT FROM AUTHOR]

Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on fresh-cut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genes involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. gigantea's extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes. [ABSTRACT FROM AUTHOR]

Background: Ulcerative colitis (UC) is associated with differential colonic expression of genes involved in immune response (e.g. IL8) and barrier integrity (e.g. cadherins). MicroRNAs (miRNAs) are regulators of gene expression and are involved in various immune-related diseases. In this study, we investigated (1) if miRNA expression in UC mucosa is altered and (2) if any of these changes correlate with mucosal mRNA expression. Integration of mRNA and miRNA expression profiling may allow the identification of functional links between dysregulated miRNAs and their target mRNA. Methodology: Colonic mucosal biopsies were obtained from 17 UC (10 active and 7 inactive) patients and 10 normal controls. Total RNA was used to analyze miRNA and mRNA expression via Affymetrix miRNA 2.0 and Affymetrix Human Gene 1.0ST arrays, respectively. Both miRNA and gene expression profiles were integrated by correlation analysis to identify dysregulated miRNAs with their corresponding predicted target mRNA. Microarray data were validated with qRT-PCR. Regulation of IL8 and CDH11 expression by hsa-miR-200c-3p was determined by luciferase reporter assays. Results: When comparing active UC patients vs. controls, 51 miRNAs and 1543 gene probe sets gave significantly different signals. In contrast, in inactive UC vs. controls, no significant miRNA expression differences were found while 155 gene probe sets had significantly different signals. We then identified potential target genes of the significantly dysregulated miRNAs and genes in active UC vs. controls and found a highly significant inverse correlation between hsa-miR-200c-3p and IL8, an inflammatory marker, and between hsa-miR-200c-3p and CDH11, a gene related to intestinal epithelial barrier function. We could demonstrate that hsa-miR-200c-3p directly regulates IL8 and CDH11 expression. Conclusion: Differential expression of immune- and barrier-related genes in inflamed UC mucosa may be influenced by altered expression of miRNAs. Integrated analysis of miRNA and mRNA expression profiles revealed hsa-miR-200c-3p for use of miRNA mimics as therapeutics. [ABSTRACT FROM AUTHOR]

Sequencing the transcriptome can answer various questions such as determining the transcripts expressed in a given species for a specific tissue or condition, evaluating differential expression, discovering variants, and evaluating allele-specific expression. Differential expression evaluates the expression differences between different strains, tissues, and conditions. Allele-specific expression evaluates expression differences between parental alleles. Both differential expression and allele-specific expression have been studied for heterosis (hybrid vigor), where the hybrid has improved performance over the parents for one or more traits. The Allele Workbench software was developed for a heterosis study that evaluated allele-specific expression for a mouse F1 hybrid using libraries from multiple tissues with biological replicates. This software has been made into a distributable package, which includes a pipeline, a Java interface to build the database, and a Java interface for query and display of the results. The required input is a reference genome, annotation file, and one or more RNA-Seq libraries with optional replicates. It evaluates allelic imbalance at the SNP and transcript level and flags transcripts with significant opposite directional allele-specific expression. The Java interface allows the user to view data from libraries, replicates, genes, transcripts, exons, and variants, including queries on allele imbalance for selected libraries. To determine the impact of allele-specific SNPs on protein folding, variants are annotated with their effect (e.g., missense), and the parental protein sequences may be exported for protein folding analysis. The Allele Workbench processing results in transcript files and read counts that can be used as input to the previously published Transcriptome Computational Workbench, which has a new algorithm for determining a trimmed set of gene ontology terms. The software with demo files is available from . Additionally, all software is ready for immediate use from an Atmosphere Virtual Machine Image available from the iPlant Collaborative (). [ABSTRACT FROM AUTHOR]

The ability of commensal bacteria to influence gene expression in host cells under the influence of pathogenic bacteria has previously been demonstrated, however the extent of this interaction is important for understanding how bacteria can be used as probiotics. Real-time quantitative polymerase chain reaction is the most sensitive tool for evaluating relative changes to gene expression levels. However as a result of its sensitivity an appropriate method of normalisation should be used to account for any variation incurred in preparatory experimental procedures. These variations may result from differences in the amount of starting material, quality of extracted RNA, or in the efficiency of the reverse transcriptase or polymerase enzymes. Selection of an endogenous control gene is the preferred method of normalisation, and ideally a proper validation of the gene's appropriateness for the study in question should be performed. In this study we used quantitative polymerase chain reaction data and applied four different algorithms (geNorm, BestKeeper, NormFinder, and comparative ΔCq) to evaluate eleven different genes as to their suitability as endogenous controls for use in studies involving colonic (HT-29) and vaginal (VK2/E6E7) human mucosal epithelial cells treated with probiotic and pathogenic bacteria. We found phosphoglycerate kinase 1 to be most appropriate for HT-29 cells, and ribosomal protein large P0 to be the best choice for VK2/E6E7 cells. We also showed that use of less stable reference genes can lead to less accurate quantification of expression levels of gene of interest (GOI) and also can result in decreased statistical significance for GOI expression levels when compared to control. Additionally, we found the cell type being analysed had greater influence on reference gene selection than the treatment performed. This study provides recommendations for stable endogenous control genes for use in further studies involving colonic and vaginal cell lines after bacterial challenge. [ABSTRACT FROM AUTHOR]

Necrotic enteritis (NE) is an important intestinal infectious disease of commercial poultry flocks caused by Clostridium perfringens. Using an experimental model of NE involving co-infection with C. perfringens and Eimeria maxima, transcriptome profiling and functional genomics approaches were applied to identify the genetic mechanisms that might regulate the host response to this disease. Microarray hybridization identified 1,049 transcripts whose levels were altered (601 increased, 448 decreased) in intestinal lymphocytes from C. perfringens/E. maxima co-infected Ross chickens compared with uninfected controls. Five biological functions, all related to host immunity and inflammation, and 11 pathways were identified from this dataset. To further elucidate the role of host genetics in NE susceptibility, two inbred chicken lines, ADOL line 6 and line 7 which share an identical B2 major histocompatibility complex haplotype but differ in their susceptibility to virus infection, were compared for clinical symptoms and the expression levels of a panel of immune-related genes during experimental NE. Line 6 chickens were more susceptible to development of experimental NE compared with line 7, as revealed by decreased body weight gain and increased E. maxima oocyst shedding. Of 21 immune-related genes examined, 15 were increased in C. perfringens/E. maxima co-infected line 6 vs. line 7 chickens. These results suggest that immune pathways are activated in response to experimental NE infection and that genetic determinants outside of the chicken B complex influence resistance to this disease. [ABSTRACT FROM AUTHOR]

Sugarcane interacts with particular types of beneficial nitrogen-fixing bacteria that provide fixed-nitrogen and plant growth hormones to host plants, promoting an increase in plant biomass. Other benefits, as enhanced tolerance to abiotic stresses have been reported to some diazotrophs. Here we aim to study the effects of the association between the diazotroph Gluconacetobacter diazotrophicus PAL5 and sugarcane cv. SP70-1143 during water depletion by characterizing differential transcriptome profiles of sugarcane. RNA-seq libraries were generated from roots and shoots of sugarcane plants free of endophytes that were inoculated with G. diazotrophicus and subjected to water depletion for 3 days. A sugarcane reference transcriptome was constructed and used for the identification of differentially expressed transcripts. The differential profile of non-inoculated SP70-1143 suggests that it responds to water deficit stress by the activation of drought-responsive markers and hormone pathways, as ABA and Ethylene. qRT-PCR revealed that root samples had higher levels of G. diazotrophicus 3 days after water deficit, compared to roots of inoculated plants watered normally. With prolonged drought only inoculated plants survived, indicating that SP70-1143 plants colonized with G. diazotrophicus become more tolerant to drought stress than non-inoculated plants. Strengthening this hypothesis, several gene expression responses to drought were inactivated or regulated in an opposite manner, especially in roots, when plants were colonized by the bacteria. The data suggests that colonized roots would not be suffering from stress in the same way as non-inoculated plants. On the other hand, shoots specifically activate ABA-dependent signaling genes, which could act as key elements in the drought resistance conferred by G. diazotrophicus to SP70-1143. This work reports for the first time the involvement of G. diazotrophicus in the promotion of drought-tolerance to sugarcane cv. SP70-1143, and it describes the initial molecular events that may trigger the increased drought tolerance in the host plant. [ABSTRACT FROM AUTHOR]

As corals decline and macroalgae proliferate on coral reefs, coral-macroalgal competition becomes more frequent and ecologically important. Whether corals are damaged by these interactions depends on susceptibility of the coral and traits of macroalgal competitors. Investigating changes in gene expression of corals and their intracellular symbiotic algae, Symbiodinium, in response to contact with different macroalgae provides insight into the biological processes and cellular pathways affected by competition with macroalgae. We evaluated the gene expression profiles of coral and Symbiodinium genes from two confamilial corals, Acropora millepora and Montipora digitata, after 6 h and 48 h of contact with four common macroalgae that differ in their allelopathic potency to corals. Contacts with macroalgae affected different biological pathways in the more susceptible (A. millepora) versus the more resistant (M. digitata) coral. Genes of coral hosts and of their associated Symbiodinium also responded in species-specific and time-specific ways to each macroalga. Changes in number and expression intensity of affected genes were greater after 6 h compared to 48 h of contact and were greater following contact with Chlorodesmis fastigiata and Amphiroa crassa than following contact with Galaxaura filamentosa or Turbinaria conoides. We documented a divergence in transcriptional responses between two confamilial corals and their associated Symbiodinium, as well as a diversity of dynamic responses within each coral species with respect to the species of macroalgal competitor and the duration of exposure to that competitor. These responses included early initiation of immune processes by Montipora, which is more resistant to damage after long-term macroalgal contact. Activation of the immune response by corals that better resist algal competition is consistent with the hypothesis that some macroalgal effects on corals may be mediated by microbial pathogens. [ABSTRACT FROM AUTHOR]

The pluripotency of embryonic stem cells (ESCs) is maintained by a small group of master transcription factors including Oct4, Sox2 and Nanog. These core factors form a regulatory circuit controlling the transcription of a number of pluripotency factors including themselves. Although previous studies have identified transcriptional regulators of this core network, the cis-regulatory DNA sequences required for the transcription of these key pluripotency factors remain to be defined. We analyzed epigenomic data within the 1.5 Mb gene-desert regions around the Sox2 gene and identified a 13kb-long super-enhancer (SE) located 100kb downstream of Sox2 in mouse ESCs. This SE is occupied by Oct4, Sox2, Nanog, and the mediator complex, and physically interacts with the Sox2 locus via DNA looping. Using a simple and highly efficient double-CRISPR genome editing strategy we deleted the entire 13-kb SE and characterized transcriptional defects in the resulting monoallelic and biallelic deletion clones with RNA-seq. We showed that the SE is responsible for over 90% of Sox2 expression, and Sox2 is the only target gene along the chromosome. Our results support the functional significance of a SE in maintaining the pluripotency transcription program in mouse ESCs. [ABSTRACT FROM AUTHOR]

Approximately 30% of rheumatoid arthritis patients achieve inadequate response to anti-TNF biologics. Attempts to identify molecular biomarkers predicting response have met with mixed success. This may be attributable, in part, to the variable and subjective disease assessment endpoints with large placebo effects typically used to classify patient response. Sixty-one patients with active RA despite methotrexate treatment, and with MRI-documented synovitis, were randomized to receive infliximab or placebo. Blood was collected at baseline and genome-wide transcription in whole blood was measured using microarrays. The primary endpoint in this study was determined by measuring the transfer rate constant (Ktrans) of a gadolinium-based contrast agent from plasma to synovium using MRI. Secondary endpoints included repeated clinical assessments with DAS28(CRP), and assessments of osteitis and synovitis by the RAMRIS method. Infliximab showed greater decrease from baseline in DCE-MRI Ktrans of wrist and MCP at all visits compared with placebo (P<0.001). Statistical analysis was performed to identify genes associated with treatment-specific 14-week change in Ktrans. The 256 genes identified were used to derive a gene signature score by averaging their log expression within each patient. The resulting score correlated with improvement of Ktrans in infliximab-treated patients and with deterioration of Ktrans in placebo-treated subjects. Poor responders showed high expression of activated B-cell genes whereas good responders exhibited a gene expression pattern consistent with mobilization of neutrophils and monocytes and high levels of reticulated platelets. This gene signature was significantly associated with clinical response in two previously published whole blood gene expression studies using anti-TNF therapies. These data provide support for the hypothesis that anti-TNF inadequate responders comprise a distinct molecular subtype of RA characterized by differences in pre-treatment blood mRNA expression. They also highlight the importance of placebo controls and robust, objective endpoints in biomarker discovery. Trial Registration: ClinicalTrials.gov [ABSTRACT FROM AUTHOR]

: Minnie Sarwal and colleagues developed a gene expression assay using peripheral blood samples to detect patients with renal transplant at high risk for acute rejection. Please see later in the article for the Editors' Summary [ABSTRACT FROM AUTHOR]

Oxidative stress is an important mechanism of chemical toxicity, contributing to teratogenesis and to cardiovascular and neurodegenerative diseases. Developing animals may be especially sensitive to chemicals causing oxidative stress. The developmental expression and inducibility of anti-oxidant defenses through activation of NF-E2-related factor 2 (NRF2) affect susceptibility to oxidants, but the embryonic response to oxidants is not well understood. To assess the response to chemically mediated oxidative stress and how it may vary during development, zebrafish embryos, eleutheroembryos, or larvae at 1, 2, 3, 4, 5, and 6 days post fertilization (dpf) were exposed to DMSO (0.1%), tert-butylhydroquinone (tBHQ; 10 µM) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 2 nM) for 6 hr. Transcript abundance was assessed by real-time qRT-PCR and microarray. qRT-PCR showed strong (4- to 5-fold) induction of gstp1 by tBHQ as early as 1 dpf. tBHQ also induced gclc (2 dpf), but not sod1, nqo1, or cyp1a. TCDD induced cyp1a but none of the other genes. Microarray analysis showed that 1477 probes were significantly different among the DMSO-, tBHQ-, and TCDD-treated eleutheroembryos at 4 dpf. There was substantial overlap between genes induced in developing zebrafish and a set of marker genes induced by oxidative stress in mammals. Genes induced by tBHQ in 4-dpf zebrafish included those involved in glutathione synthesis and utilization, signal transduction, and DNA damage/stress response. The strong induction of hsp70 determined by microarray was confirmed by qRT-PCR and by use of transgenic zebrafish expressing enhanced green fluorescent protein (EGFP) under control of the hsp70 promoter. Genes strongly down-regulated by tBHQ included mitfa, providing a molecular explanation for the loss of pigmentation in tBHQ-exposed embryos. These data show that zebrafish embryos are responsive to oxidative stress as early as 1 dpf, that responsiveness varies with development in a gene-specific manner, and that the oxidative stress response is substantially conserved in vertebrate animals. [ABSTRACT FROM AUTHOR]

A main challenge of data-driven sciences is how to make maximal use of the progressively expanding databases of experimental datasets in order to keep research cumulative. We introduce the idea of a modeling-based dataset retrieval engine designed for relating a researcher's experimental dataset to earlier work in the field. The search is (i) data-driven to enable new findings, going beyond the state of the art of keyword searches in annotations, (ii) modeling-driven, to include both biological knowledge and insights learned from data, and (iii) scalable, as it is accomplished without building one unified grand model of all data. Assuming each dataset has been modeled beforehand, by the researchers or automatically by database managers, we apply a rapidly computable and optimizable combination model to decompose a new dataset into contributions from earlier relevant models. By using the data-driven decomposition, we identify a network of interrelated datasets from a large annotated human gene expression atlas. While tissue type and disease were major driving forces for determining relevant datasets, the found relationships were richer, and the model-based search was more accurate than the keyword search; moreover, it recovered biologically meaningful relationships that are not straightforwardly visible from annotations—for instance, between cells in different developmental stages such as thymocytes and T-cells. Data-driven links and citations matched to a large extent; the data-driven links even uncovered corrections to the publication data, as two of the most linked datasets were not highly cited and turned out to have wrong publication entries in the database. [ABSTRACT FROM AUTHOR]

Quantitative real-time reverse transcription PCR (qRT-PCR) has become a widely used method for gene expression analysis; however, its data interpretation largely depends on the stability of reference genes. The transcriptomics of Panax ginseng, one of the most popular and traditional ingredients used in Chinese medicines, is increasingly being studied. Furthermore, it is vital to establish a series of reliable reference genes when qRT-PCR is used to assess the gene expression profile of ginseng. In this study, we screened out candidate reference genes for ginseng using gene expression data generated by a high-throughput sequencing platform. Based on the statistical tests, 20 reference genes (10 traditional housekeeping genes and 10 novel genes) were selected. These genes were tested for the normalization of expression levels in five growth stages and three distinct plant organs of ginseng by qPCR. These genes were subsequently ranked and compared according to the stability of their expressions using geNorm, NormFinder, and BestKeeper computational programs. Although the best reference genes were found to vary across different samples, CYP and EF-1α were the most stable genes amongst all samples. GAPDH/30S RPS20, CYP/60S RPL13 and CYP/QCR were the optimum pair of reference genes in the roots, stems, and leaves. CYP/60S RPL13, CYP/eIF-5A, aTUB/V-ATP, eIF-5A/SAR1, and aTUB/pol IIa were the most stably expressed combinations in each of the five developmental stages. Our study serves as a foundation for developing an accurate method of qRT-PCR and will benefit future studies on gene expression profiles of Panax Ginseng. [ABSTRACT FROM AUTHOR]

Currently, there is no efficient therapy for patients with peripheral T cell lymphoma (PTCL). The Proviral Integration site of Moloney murine leukemia virus (PIM) kinases are important mediators of cell survival. We aimed to determine the therapeutic value of PIM kinases because they are overexpressed in PTCL patients, T cell lines and primary tumoral T cells. PIM kinases were inhibited genetically (using small interfering and short hairpin RNAs) and pharmacologically (mainly with the pan-PIM inhibitor (PIMi) ETP-39010) in a panel of 8 PTCL cell lines. Effects on cell viability, apoptosis, cell cycle, key proteins and gene expression were evaluated. Individual inhibition of each of the PIM genes did not affect PTCL cell survival, partially because of a compensatory mechanism among the three PIM genes. In contrast, pharmacological inhibition of all PIM kinases strongly induced apoptosis in all PTCL cell lines, without cell cycle arrest, in part through the induction of DNA damage. Therefore, pan-PIMi synergized with Cisplatin. Importantly, pharmacological inhibition of PIM reduced primary tumoral T cell viability without affecting normal T cells ex vivo. Since anaplastic large cell lymphoma (ALK+ ALCL) cell lines were the most sensitive to the pan-PIMi, we tested the simultaneous inhibition of ALK and PIM kinases and found a strong synergistic effect in ALK+ ALCL cell lines. Our findings suggest that PIM kinase inhibition could be of therapeutic value in a subset of PTCL, especially when combined with ALK inhibitors, and might be clinically beneficial in ALK+ ALCL. [ABSTRACT FROM AUTHOR]

Advances in sequencing technology have allowed for detailed analyses of the transcriptome at single-nucleotide resolution, facilitating the study of RNA editing or sequence differences between RNA and DNA genome-wide. In humans, two types of post-transcriptional RNA editing processes are known to occur: A-to-I deamination by ADAR and C-to-U deamination by APOBEC1. In addition to these sequence differences, researchers have reported the existence of all 12 types of RNA-DNA sequence differences (RDDs); however, the validity of these claims is debated, as many studies claim that technical artifacts account for the majority of these non-canonical sequence differences. In this study, we used a detection theory approach to evaluate the performance of RNA-Sequencing (RNA-Seq) and associated aligners in accurately identifying RNA-DNA sequence differences. By generating simulated RNA-Seq datasets containing RDDs, we assessed the effect of alignment artifacts and sequencing error on the sensitivity and false discovery rate of RDD detection. Overall, we found that even in the presence of sequencing errors, false negative and false discovery rates of RDD detection can be contained below 10% with relatively lenient thresholds. We also assessed the ability of various filters to target false positive RDDs and found them to be effective in discriminating between true and false positives. Lastly, we used the optimal thresholds we identified from our simulated analyses to identify RDDs in a human lymphoblastoid cell line. We found approximately 6,000 RDDs, the majority of which are A-to-G edits and likely to be mediated by ADAR. Moreover, we found the majority of non A-to-G RDDs to be associated with poorer alignments and conclude from these results that the evidence for widespread non-canonical RDDs in humans is weak. Overall, we found RNA-Seq to be a powerful technique for surveying RDDs genome-wide when coupled with the appropriate thresholds and filters. [ABSTRACT FROM AUTHOR]

Recent high-throughput techniques have generated a flood of biological data in all aspects. The transformation and visualization of multi-dimensional and numerical gene or protein expression data in a single heatmap can provide a concise but comprehensive presentation of molecular dynamics under different conditions. In this work, we developed an easy-to-use tool named HemI (Heat map Illustrator), which can visualize either gene or protein expression data in heatmaps. Additionally, the heatmaps can be recolored, rescaled or rotated in a customized manner. In addition, HemI provides multiple clustering strategies for analyzing the data. Publication-quality figures can be exported directly. We propose that HemI can be a useful toolkit for conveniently visualizing and manipulating heatmaps. The stand-alone packages of HemI were implemented in Java and can be accessed at . [ABSTRACT FROM AUTHOR]

Detecting periodicity signals from time-series microarray data is commonly used to facilitate the understanding of the critical roles and underlying mechanisms of regulatory transcriptomes. However, time-series microarray data are noisy. How the temporal data structure affects the performance of periodicity detection has remained elusive. We present a novel method based on empirical mode decomposition (EMD) to examine this effect. We applied EMD to a yeast microarray dataset and extracted a series of intrinsic mode function (IMF) oscillations from the time-series data. Our analysis indicated that many periodically expressed genes might have been under-detected in the original analysis because of interference between decomposed IMF oscillations. By validating a protein complex coexpression analysis, we revealed that 56 genes were newly determined as periodic. We demonstrated that EMD can be used incorporating with existing periodicity detection methods to improve their performance. This approach can be applied to other time-series microarray studies. [ABSTRACT FROM AUTHOR]

This study investigated whether there are marked differences in surface markers between rabbit and human mesenchymal stem cells (MSCs). Murine and rabbit MSCs have been reported to be CD90-negative. Rat MSCs have been reported to be CD71-negative. Our previous study also shows that rabbit MSCs are CD29-negative. However, human MSCs are generally considered to be CD29-, CD71-, and CD90-positive. Therefore, the surface markers of human MSCs might differ from those of other species. Rabbit bone marrow MSCs were obtained that had a multi-differentiation potential. The phenotype of these cells was studied using flow cytometry antibodies for 25 rabbit surface markers, namely, CD13, CD14, CD29, CD31, CD34, CD44, CD45, CD49d, CD49f, CD51, CD54, CD59, CD71, CD73, CD90, CD105, CD106, CD133, CD166, MHC I, MHC II, α-smooth muscle actin (α-SMA), cytokeratin, desmin, and vimentin. The phenotype of commercially available human MSCs was similarly studied using antibodies for human surface markers. CD14, CD31, CD34, CD45, CD49d, CD49f, CD51, CD54, CD71, CD106, CD133, MHC II, and cytokeratin were absent from both rabbit and human MSCs, while CD44, α-SMA, and vimentin were present on both cell lines. CD13, CD29, CD59, CD73, CD90, CD105, CD166, and MHC I were present on human MSCs, but not on rabbit MSCs. However, desmin was present on rabbit MSCs, but not on human MSCs. In total, the surface expression of nine markers differed between human and rabbit MSCs, whereas the surface expression of 16 markers was the same in the two cell lines. [ABSTRACT FROM AUTHOR]

As next generation sequencing technologies are becoming more economical, large-scale ChIP-seq studies are enabling the investigation of the roles of transcription factor binding and epigenome on phenotypic variation. Studying such variation requires individual level ChIP-seq experiments. Standard designs for ChIP-seq experiments employ a paired control per ChIP-seq sample. Genomic coverage for control experiments is often sacrificed to increase the resources for ChIP samples. However, the quality of ChIP-enriched regions identifiable from a ChIP-seq experiment depends on the quality and the coverage of the control experiments. Insufficient coverage leads to loss of power in detecting enrichment. We investigate the effect of in silico pooling of control samples within multiple biological replicates, multiple treatment conditions, and multiple cell lines and tissues across multiple datasets with varying levels of genomic coverage. Our computational studies suggest guidelines for performing in silico pooling of control experiments. Using vast amounts of ENCODE data, we show that pairwise correlations between control samples originating from multiple biological replicates, treatments, and cell lines/tissues can be grouped into two classes representing whether or not in silico pooling leads to power gain in detecting enrichment between the ChIP and the control samples. Our findings have important implications for multiplexing samples. [ABSTRACT FROM AUTHOR]

The model plant Arabidopsis has been well-studied using high-throughput genomics technologies, which usually generate lists of differentially expressed genes under various conditions. Our group recently collected 1065 gene lists from 397 gene expression studies as a knowledgebase for pathway analysis. Here we systematically analyzed these gene lists by computing overlaps in all-vs.-all comparisons. We identified 16,261 statistically significant overlaps, represented by an undirected network in which nodes correspond to gene lists and edges indicate significant overlaps. The network highlights the correlation across the gene expression signatures of the diverse biological processes. We also partitioned the main network into 20 sub-networks, representing groups of highly similar expression signatures. These are common sets of genes that were co-regulated under different treatments or conditions and are often related to specific biological themes. Overall, our result suggests that diverse gene expression signatures are highly interconnected in a modular fashion. [ABSTRACT FROM AUTHOR]

Background: Paragonimiasis is a food-borne trematode infection acquired by eating raw or undercooked crustaceans. It is a major public health problem in the far East, but it also occurs in South Asia, Africa, and in the Americas. Paragonimus worms cause chronic lung disease with cough, fever and hemoptysis that can be confused with tuberculosis or other non-parasitic diseases. Treatment is straightforward, but diagnosis is often delayed due to a lack of reliable parasitological or serodiagnostic tests. Hence, the purpose of this study was to use a systems biology approach to identify key parasite proteins that may be useful for development of improved diagnostic tests. Methodology/Principal Findings: The transcriptome of adult Paragonimus kellicotti was sequenced with Illumina technology. Raw reads were pre-processed and assembled into 78,674 unique transcripts derived from 54,622 genetic loci, and 77,123 unique protein translations were predicted. A total of 2,555 predicted proteins (from 1,863 genetic loci) were verified by mass spectrometric analysis of total worm homogenate, including 63 proteins lacking homology to previously characterized sequences. Parasite proteins encoded by 321 transcripts (227 genetic loci) were reactive with antibodies from infected patients, as demonstrated by immunoaffinity purification and high-resolution liquid chromatography-mass spectrometry. Serodiagnostic candidates were prioritized based on several criteria, especially low conservation with proteins in other trematodes. Cysteine proteases, MFP6 proteins and myoglobins were abundant among the immunoreactive proteins, and these warrant further study as diagnostic candidates. Conclusions: The transcriptome, proteome and immunolome of adult P. kellicotti represent a major advance in the study of Paragonimus species. These data provide a powerful foundation for translational research to develop improved diagnostic tests. Similar integrated approaches may be useful for identifying novel targets for drugs and vaccines in the future. [ABSTRACT FROM AUTHOR]

Gene expression is a combinatorial function of genetic/epigenetic factors such as copy number variation (CNV), DNA methylation (DM), transcription factors (TF) occupancy, and microRNA (miRNA) post-transcriptional regulation. At the maturity of microarray/sequencing technologies, large amounts of data measuring the genome-wide signals of those factors became available from Encyclopedia of DNA Elements (ENCODE) and The Cancer Genome Atlas (TCGA). However, there is a lack of an integrative model to take full advantage of these rich yet heterogeneous data. To this end, we developed RACER (egression nalysis of ombined xpression egulation), which fits the mRNA expression as response using as explanatory variables, the TF data from ENCODE, and CNV, DM, miRNA expression signals from TCGA. Briefly, RACER first infers the sample-specific regulatory activities by TFs and miRNAs, which are then used as inputs to infer specific TF/miRNA-gene interactions. Such a two-stage regression framework circumvents a common difficulty in integrating ENCODE data measured in generic cell-line with the sample-specific TCGA measurements. As a case study, we integrated Acute Myeloid Leukemia (AML) data from TCGA and the related TF binding data measured in K562 from ENCODE. As a proof-of-concept, we first verified our model formalism by 10-fold cross-validation on predicting gene expression. We next evaluated RACER on recovering known regulatory interactions, and demonstrated its superior statistical power over existing methods in detecting known miRNA/TF targets. Additionally, we developed a feature selection procedure, which identified 18 regulators, whose activities clustered consistently with cytogenetic risk groups. One of the selected regulators is miR-548p, whose inferred targets were significantly enriched for leukemia-related pathway, implicating its novel role in AML pathogenesis. Moreover, survival analysis using the inferred activities identified C-Fos as a potential AML prognostic marker. Together, we provided a novel framework that successfully integrated the TCGA and ENCODE data in revealing AML-specific regulatory program at global level. [ABSTRACT FROM AUTHOR]

Noncoding RNAs are integral to a wide range of biological processes, including translation, gene regulation, host-pathogen interactions and environmental sensing. While genomics is now a mature field, our capacity to identify noncoding RNA elements in bacterial and archaeal genomes is hampered by the difficulty of de novo identification. The emergence of new technologies for characterizing transcriptome outputs, notably RNA-seq, are improving noncoding RNA identification and expression quantification. However, a major challenge is to robustly distinguish functional outputs from transcriptional noise. To establish whether annotation of existing transcriptome data has effectively captured all functional outputs, we analysed over 400 publicly available RNA-seq datasets spanning 37 different Archaea and Bacteria. Using comparative tools, we identify close to a thousand highly-expressed candidate noncoding RNAs. However, our analyses reveal that capacity to identify noncoding RNA outputs is strongly dependent on phylogenetic sampling. Surprisingly, and in stark contrast to protein-coding genes, the phylogenetic window for effective use of comparative methods is perversely narrow: aggregating public datasets only produced one phylogenetic cluster where these tools could be used to robustly separate unannotated noncoding RNAs from a null hypothesis of transcriptional noise. Our results show that for the full potential of transcriptomics data to be realized, a change in experimental design is paramount: effective transcriptomics requires phylogeny-aware sampling. [ABSTRACT FROM AUTHOR]

Systemic dimorphic fungi cause more than one million new infections each year, ranking them among the significant public health challenges currently encountered. Penicillium marneffei is a systemic dimorphic fungus endemic to Southeast Asia. The temperature-dependent dimorphic phase transition between mycelium and yeast is considered crucial for the pathogenicity and transmission of P. marneffei, but the underlying mechanisms are still poorly understood. Here, we re-sequenced P. marneffei strain PM1 using multiple sequencing platforms and assembled the genome using hybrid genome assembly. We determined gene expression levels using RNA sequencing at the mycelial and yeast phases of P. marneffei, as well as during phase transition. We classified 2,718 genes with variable expression across conditions into 14 distinct groups, each marked by a signature expression pattern implicated at a certain stage in the dimorphic life cycle. Genes with the same expression patterns tend to be clustered together on the genome, suggesting orchestrated regulations of the transcriptional activities of neighboring genes. Using qRT-PCR, we validated expression levels of all genes in one of clusters highly expressed during the yeast-to-mycelium transition. These included madsA, a gene encoding MADS-box transcription factor whose gene family is exclusively expanded in P. marneffei. Over-expression of madsA drove P. marneffei to undergo mycelial growth at 37°C, a condition that restricts the wild-type in the yeast phase. Furthermore, analyses of signature expression patterns suggested diverse roles of secreted proteins at different developmental stages and the potential importance of non-coding RNAs in mycelium-to-yeast transition. We also showed that RNA structural transition in response to temperature changes may be related to the control of thermal dimorphism. Together, our findings have revealed multiple molecular mechanisms that may underlie the dimorphic transition in P. marneffei, providing a powerful foundation for identifying molecular targets for mechanism-based interventions. [ABSTRACT FROM AUTHOR]

The regulatory logic underlying global transcriptional programs controlling development of visceral organs like the pancreas remains undiscovered. Here, we profiled gene expression in 12 purified populations of fetal and adult pancreatic epithelial cells representing crucial progenitor cell subsets, and their endocrine or exocrine progeny. Using probabilistic models to decode the general programs organizing gene expression, we identified co-expressed gene sets in cell subsets that revealed patterns and processes governing progenitor cell development, lineage specification, and endocrine cell maturation. Purification of Neurog3 mutant cells and module network analysis linked established regulators such as Neurog3 to unrecognized gene targets and roles in pancreas development. Iterative module network analysis nominated and prioritized transcriptional regulators, including diabetes risk genes. Functional validation of a subset of candidate regulators with corresponding mutant mice revealed that the transcription factors Etv1, Prdm16, Runx1t1 and Bcl11a are essential for pancreas development. Our integrated approach provides a unique framework for identifying regulatory genes and functional gene sets underlying pancreas development and associated diseases such as diabetes mellitus. [ABSTRACT FROM AUTHOR]

In addition to their protein coding function, exons can also serve as transcriptional enhancers. Mutations in these exonic-enhancers (eExons) could alter both protein function and transcription. However, the functional consequence of eExon mutations is not well known. Here, using massively parallel reporter assays, we dissect the enhancer activity of three liver eExons (SORL1 exon 17, TRAF3IP2 exon 2, PPARG exon 6) at single nucleotide resolution in the mouse liver. We find that both synonymous and non-synonymous mutations have similar effects on enhancer activity and many of the deleterious mutation clusters overlap known liver-associated transcription factor binding sites. Carrying a similar massively parallel reporter assay in HeLa cells with these three eExons found differences in their mutation profiles compared to the liver, suggesting that enhancers could have distinct operating profiles in different tissues. Our results demonstrate that eExon mutations could lead to multiple phenotypes by disrupting both the protein sequence and enhancer activity and that enhancers can have distinct mutation profiles in different cell types. [ABSTRACT FROM AUTHOR]

Neurofibromatosis type 1 (NF1) is an autosomal dominant, monogenic disorder of dysregulated neurocutaneous tissue growth. Pleiotropy, variable expressivity and few NF1 genotype-phenotype correlates limit clinical prognostication in NF1. Phenotype complexity in NF1 is hypothesized to derive in part from genetic modifiers unlinked to the NF1 locus. In this study, we hypothesized that normal variation in germline gene expression confers risk for certain phenotypes in NF1. In a set of 79 individuals with NF1, we examined the association between gene expression in lymphoblastoid cell lines with NF1-associated phenotypes and sequenced select genes with significant phenotype/expression correlations. In a discovery cohort of 89 self-reported European-Americans with NF1 we examined the association between germline sequence variants of these genes with café-au-lait macule (CALM) count, a tractable, tumor-like phenotype in NF1. Two correlated, common SNPs (rs4660761 and rs7161) between DPH2 and ATP6V0B were significantly associated with the CALM count. Analysis with tiled regression also identified SNP rs4660761 as significantly associated with CALM count. SNP rs1800934 and 12 rare variants in the mismatch repair gene MSH6 were also associated with CALM count. Both SNPs rs7161 and rs4660761 (DPH2 and ATP6V0B) were highly significant in a mega-analysis in a combined cohort of 180 self-reported European-Americans; SNP rs1800934 (MSH6) was near-significant in a meta-analysis assuming dominant effect of the minor allele. SNP rs4660761 is predicted to regulate ATP6V0B, a gene associated with melanosome biology. Individuals with homozygous mutations in MSH6 can develop an NF1-like phenotype, including multiple CALMs. Through a multi-platform approach, we identified variants that influence NF1 CALM count. [ABSTRACT FROM AUTHOR]

Embryogenic tissue culture systems are utilized in propagation and genetic engineering of crop plants, but applications are limited by genotype-dependent culture response. To date, few genes necessary for embryogenic callus formation have been identified or characterized. The goal of this research was to enhance our understanding of gene expression during maize embryogenic tissue culture initiation. In this study, we highlight the expression of candidate genes that have been previously regarded in the literature as having important roles in somatic embryogenesis. We utilized RNA based sequencing (RNA-seq) to characterize the transcriptome of immature embryo explants of the highly embryogenic and regenerable maize genotype A188 at 0, 24, 36, 48, and 72 hours after placement of explants on tissue culture initiation medium. Genes annotated as functioning in stress response, such as glutathione-S-transferases and germin-like proteins, and genes involved with hormone transport, such as PINFORMED, increased in expression over 8-fold in the study. Maize genes with high sequence similarity to genes previously described in the initiation of embryogenic cultures, such as transcription factors BABY BOOM, LEAFY COTYLEDON, and AGAMOUS, and important receptor-like kinases such as SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASES and CLAVATA, were also expressed in this time course study. By combining results from whole genome transcriptome analysis with an in depth review of key genes that play a role in the onset of embryogenesis, we propose a model of coordinated expression of somatic embryogenesis-related genes, providing an improved understanding of genomic factors involved in the early steps of embryogenic culture initiation in maize and other plant species. [ABSTRACT FROM AUTHOR]

Background: Rheum nobile is an alpine plant with translucent bracts concealing the inflorescence which produce a “glasshouse” effect promoting the development of fertile pollen grains in such conditions. The current understanding of the adaptation of such bracts to alpine environments mainly focuses on the phenotypic and physiological changes while the genetic basis is very limited. By sequencing the upper bract and the lower rosulate leaf from the same R. nobile stem, we identified candidate genes that may be involved in alpine adaption of the translucent bract in “glasshouse” plants and illustrated the changes in gene expression underlying the adaptive and complex evolution of the bracts phenotype. Results: A total of 174.2 million paired-end reads from each transcriptome were assembled into 25,249 unigenes. By comparing the gene expression profiles, we identified 1,063 and 786 genes up-regulated respectively in the upper bract and the lower leaf. Functional enrichment analyses of these genes recovered a number of differential important pathways, including flavonoid biosynthesis, mismatch repair and photosynthesis related pathways. These pathways are mainly involved in three types of functions: 9 genes in the UV protective process, 9 mismatch repair related genes and 88 genes associated with photosynthesis. Conclusions: This study provides the first comprehensive dataset characterizing Rheum nobile gene expression at the transcriptomic scale, and provides novel insights into the gene expression profiles associated with the adaptation of the “glasshouse” plant bracts. The dataset will be served as a public genetic resources for further functional and evolutionary studies of “glasshouse” plants. [ABSTRACT FROM AUTHOR]

Objective: The immune inflammatory disorders rheumatoid arthritis (RA), psoriatic arthritis (PsA) and psoriasis (Ps) share common pathologic features and show responsiveness to anti-tumor necrosis factor (TNF) agents yet they are phenotypically distinct. The aim of this study was to examine if anti-TNF therapy is associated with divergent gene expression profiles in circulating cells and target tissues of patients with these diseases. Methods: Peripheral blood CD14+ and CD14− cells were isolated from 9 RA, 12 PsA and 10 Ps patients before and after infliximab (IFX) treatment. Paired synovial (n = 3, RA, PsA) and skin biopsies (n = 5, Ps) were also collected. Gene expression was analyzed by microarrays. Results: 26 out of 31 subjects responded to IFX. The transcriptional response of CD14+ cells to IFX was unique for the three diseases, with little overlap (<25%) in significantly changed gene lists (with PsA having the largest number of changed genes). In Ps, altered gene expression was more pronounced in lesional skin (relative to paired, healthy skin) compared to blood (relative to healthy controls). Marked suppression of up-regulated genes in affected skin was noted 2 weeks after therapy but the expression patterns differed from uninvolved skin. Divergent patterns of expression were noted between the blood cells and skin or synovial tissues in individual patients. Functions that promote cell differentiation, proliferation and apoptosis in all three diseases were enriched. RA was enriched in functions in CD14− cells, PsA in CD14+ cells and Ps in both CD14+ and CD14− cells, however, the specific functions showed little overlap in the 3 disorders. Conclusion: Divergent patterns of altered gene expression are observed in RA, PsA and Ps patients in blood cells and target organs in IFX responders. Differential gene expression profiles in the blood do not correlate with those in target organs. [ABSTRACT FROM AUTHOR]

The RESPONSIVE TO DEHYDRATION 22 (RD22) gene is a molecular link between abscisic acid (ABA) signalling and abiotic stress responses. Its expression has been used as a reliable ABA early response marker. In Arabidopsis, the single copy RD22 gene possesses a BURP domain also located at the C-terminus of USP embryonic proteins and the beta subunit of polygalacturonases. In grapevine, a RD22 gene has been identified but putative paralogs are also found in the grape genome, possibly forming a large RD22 family in this species. In this work, we searched for annotations containing BURP domains in the Vitis vinifera genome. Nineteen proteins were defined by a comparative analysis between the two genome predictions and RNA-Seq data. These sequences were compared to other plant BURPs identified in previous genome surveys allowing us to reconceive group classifications based on phylogenetic relationships and protein motif occurrence. We observed a lineage-specific evolution of the RD22 family, with the biggest expansion in grapevine and poplar. In contrast, rice, sorghum and maize presented highly expanded monocot-specific groups. The Vitis RD22 group may have expanded from segmental duplications as most of its members are confined to a region in chromosome 4. The inspection of transcriptomic data revealed variable expression of BURP genes in vegetative and reproductive organs. Many genes were induced in specific tissues or by abiotic and biotic stresses. Three RD22 genes were further studied showing that they responded oppositely to ABA and to stress conditions. Our results show that the inclusion of RNA-Seq data is essential while describing gene families and improving gene annotations. Robust phylogenetic analyses including all BURP members from other sequenced species helped us redefine previous relationships that were erroneously established. This work provides additional evidence for RD22 genes serving as marker genes for different organs or stresses in grapevine. [ABSTRACT FROM AUTHOR]

Integrated analysis of genomic and transcriptomic level changes holds promise for a better understanding of colorectal cancer (CRC) biology. There is a pertinent need to explain the functional effect of genome level changes by integrating the information at the transcript level. Using high resolution cytogenetics array, we had earlier identified driver genes by ‘Genomic Identification of Significant Targets In Cancer (GISTIC)’ analysis of paired tumour-normal samples from colorectal cancer patients. In this study, we analyze these driver genes at three levels using exon array data – gene, exon and network. Gene level analysis revealed a small subset to experience differential expression. These results were reinforced by carrying out separate differential expression analyses (SAM and LIMMA). ATP8B1 was found to be the novel gene associated with CRC that shows changes at cytogenetic, gene and exon levels. Splice index of 29 exons corresponding to 13 genes was found to be significantly altered in tumour samples. Driver genes were used to construct regulatory networks for tumour and normal groups. There were rearrangements in transcription factor genes suggesting the presence of regulatory switching. The regulatory pattern of AHR gene was found to have the most significant alteration. Our results integrate data with focus on driver genes resulting in highly enriched novel molecules that need further studies to establish their role in CRC. [ABSTRACT FROM AUTHOR]

Quantitative Real-time PCR (qRT-PCR) is a powerful technique to investigate comparative gene expression. In general, normalization of results using a highly stable housekeeping gene (HKG) as an internal control is recommended and necessary. However, there are several reports suggesting that regulation of some HKGs is affected by different conditions. The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a serious pest of corn in the United States and Europe. The expression profile of target genes related to insecticide exposure, resistance, and RNA interference has become an important experimental technique for study of western corn rootworms; however, lack of information on reliable HKGs under different conditions makes the interpretation of qRT-PCR results difficult. In this study, four distinct algorithms (Genorm, NormFinder, BestKeeper and delta-CT) and five candidate HKGs to genes of reference (β-actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; β-tubulin; RPS9, ribosomal protein S9; EF1a, elongation factor-1α) were evaluated to determine the most reliable HKG under different experimental conditions including exposure to dsRNA and Bt toxins and among different tissues and developmental stages. Although all the HKGs tested exhibited relatively stable expression among the different treatments, some differences were noted. Among the five candidate reference genes evaluated, β-actin exhibited highly stable expression among different life stages. RPS9 exhibited the most similar pattern of expression among dsRNA treatments, and both experiments indicated that EF1a was the second most stable gene. EF1a was also the most stable for Bt exposure and among different tissues. These results will enable researchers to use more accurate and reliable normalization of qRT-PCR data in WCR experiments. [ABSTRACT FROM AUTHOR]

Background: Trastuzumab improves survival outcomes in patients with HER2+ metastatic breast cancer. The Long-Her study was designed to identify clinical and molecular markers that could differentiate long-term survivors from patients having early progression after trastuzumab treatment. Methods: Data were collected from women with HER2-positive metastatic breast cancer treated with trastuzumab that experienced a response or stable disease during at least 3 years. Patients having a progression in the first year of therapy with trastuzumab were used as a control. Genes related with trastuzumab resistance were identified and investigated for network and gene functional interrelation. Models predicting poor response to trastuzumab were constructed and evaluated. Finally, a mutational status analysis of selected genes was performed in HER2 positive breast cancer samples. Results: 103 patients were registered in the Long-HER study, of whom 71 had obtained a durable complete response. Median age was 58 years. Metastatic disease was diagnosed after a median of 24.7 months since primary diagnosis. Metastases were present in the liver (25%), lungs (25%), bones (23%) and soft tissues (23%), with 20% of patients having multiple locations of metastases. Median duration of response was 55 months. The molecular analysis included 35 patients from the group with complete response and 18 patients in a control poor-response group. Absence of trastuzumab as part of adjuvant therapy was the only clinical factor associated with long-term survival. Gene ontology analysis demonstrated that PI3K pathway was associated with poor response to trastuzumab-based therapy: tumours in the control group usually had four or five alterations in this pathway, whereas tumours in the Long-HER group had two alterations at most. Conclusions: Trastuzumab may provide a substantial long-term survival benefit in a selected group of patients. Whole genome expression analysis comparing long-term survivors vs. a control group predicted early progression after trastuzumab-based therapy. Multiple alterations in genes related to the PI3K-mTOR pathway seem to be required to confer resistance to this therapy. [ABSTRACT FROM AUTHOR]

Background: Octopus vulgaris is a highly valuable species of great commercial interest and excellent candidate for aquaculture diversification; however, the octopus’ well-being is impaired by pathogens, of which the gastrointestinal coccidian parasite Aggregata octopiana is one of the most important. The knowledge of the molecular mechanisms of the immune response in cephalopods, especially in octopus is scarce. The transcriptome of the hemocytes of O. vulgaris was de novo sequenced using the high-throughput paired-end Illumina technology to identify genes involved in immune defense and to understand the molecular basis of octopus tolerance/resistance to coccidiosis. Results: A bi-directional mRNA library was constructed from hemocytes of two groups of octopus according to the infection by A. octopiana, sick octopus, suffering coccidiosis, and healthy octopus, and reads were de novo assembled together. The differential expression of transcripts was analysed using the general assembly as a reference for mapping the reads from each condition. After sequencing, a total of 75,571,280 high quality reads were obtained from the sick octopus group and 74,731,646 from the healthy group. The general transcriptome of the O. vulgaris hemocytes was assembled in 254,506 contigs. A total of 48,225 contigs were successfully identified, and 538 transcripts exhibited differential expression between groups of infection. The general transcriptome revealed genes involved in pathways like NF-kB, TLR and Complement. Differential expression of TLR-2, PGRP, C1q and PRDX genes due to infection was validated using RT-qPCR. In sick octopuses, only TLR-2 was up-regulated in hemocytes, but all of them were up-regulated in caecum and gills. Conclusion: The transcriptome reported here de novo establishes the first molecular clues to understand how the octopus immune system works and interacts with a highly pathogenic coccidian. The data provided here will contribute to identification of biomarkers for octopus resistance against pathogens, which could improve octopus farming in the near future. [ABSTRACT FROM AUTHOR]

We previously carried out the design and testing of a custom-built Haemophilus influenzae supragenome hybridization (SGH) array that contains probe sequences to 2,890 gene clusters identified by whole genome sequencing of 24 strains of H. influenzae. The array was originally designed as a tool to interrogate the gene content of large numbers of clinical isolates without the need for sequencing, however, the data obtained is quantitative and is thus suitable for transcriptomic analyses. In the current study RNA was extracted from H. influenzae strain CZ4126/02 (which was not included in the design of the array) converted to cDNA, and labelled and hybridized to the SGH arrays to assess the quality and reproducibility of data obtained from these custom-designed chips to serve as a tool for transcriptomics. Three types of experimental replicates were analyzed with all showing very high degrees of correlation, thus validating both the array and the methods used for RNA profiling. A custom filtering pipeline for two-condition unpaired data using five metrics was developed to minimize variability within replicates and to maximize the identification of the most significant true transcriptional differences between two samples. These methods can be extended to transcriptional analysis of other bacterial species utilizing supragenome-based arrays. [ABSTRACT FROM AUTHOR]

The BXD genetic reference population is a recombinant inbred panel descended from crosses between the C57BL/6 (B6) and DBA/2 (D2) strains of mice, which segregate for about 5 million sequence variants. Recently, some of these variants have been established with effects on general metabolic phenotypes such as glucose response and bone strength. Here we phenotype 43 BXD strains and observe they have large variation (∼5-fold) in their spontaneous activity during waking hours. QTL analyses indicate that ∼40% of this variance is attributable to a narrow locus containing the aryl hydrocarbon receptor (Ahr), a basic helix-loop-helix transcription factor with well-established roles in development and xenobiotic metabolism. Strains with the D2 allele of Ahr have reduced gene expression compared to those with the B6 allele, and have significantly higher spontaneous activity. This effect was also observed in B6 mice with a congenic D2 Ahr interval, and in B6 mice with a humanized AHR allele which, like the D2 allele, is expressed much less and has less enzymatic activity than the B6 allele. Ahr is highly conserved in invertebrates, and strikingly inhibition of its orthologs in D. melanogaster and C. elegans (spineless and ahr-1) leads to marked increases in basal activity. In mammals, Ahr has numerous ligands, but most are either non-selective (e.g. resveratrol) or highly toxic (e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)). Thus, we chose to examine a major environmental influence—long term feeding with high fat diet (HFD)—to see if the effects of Ahr are dependent on major metabolic differences. Interestingly, while HFD robustly halved movement across all strains, the QTL position and effects of Ahr remained unchanged, indicating that the effects are independent. The highly consistent effects of Ahr on movement indicate that changes in its constitutive activity have a role on spontaneous movement and may influence human behavior. [ABSTRACT FROM AUTHOR]

The olfactory (OR) and vomeronasal receptor (VR) repertoires are collectively encoded by 1700 genes and pseudogenes in the mouse genome. Most OR and VR genes were identified by comparative genomic techniques and therefore, in many of those cases, only their protein coding sequences are defined. Some also lack experimental support, due in part to the similarity between them and their monogenic, cell-specific expression in olfactory tissues. Here we use deep RNA sequencing, expression microarray and quantitative RT-PCR in both the vomeronasal organ and whole olfactory mucosa to quantify their full transcriptomes in multiple male and female mice. We find evidence of expression for all VR, and almost all OR genes that are annotated as functional in the reference genome, and use the data to generate over 1100 new, multi-exonic, significantly extended receptor gene annotations. We find that OR and VR genes are neither equally nor randomly expressed, but have reproducible distributions of abundance in both tissues. The olfactory transcriptomes are only minimally different between males and females, suggesting altered gene expression at the periphery is unlikely to underpin the striking sexual dimorphism in olfactory-mediated behavior. Finally, we present evidence that hundreds of novel, putatively protein-coding genes are expressed in these highly specialized olfactory tissues, and carry out a proof-of-principle validation. Taken together, these data provide a comprehensive, quantitative catalog of the genes that mediate olfactory perception and pheromone-evoked behavior at the periphery. [ABSTRACT FROM AUTHOR]

DNA methylation is an important epigenetic regulator of gene expression. Recent studies have revealed widespread associations between genetic variation and methylation levels. However, the mechanistic links between genetic variation and methylation remain unclear. To begin addressing this gap, we collected methylation data at ∼300,000 loci in lymphoblastoid cell lines (LCLs) from 64 HapMap Yoruba individuals, and genome-wide bisulfite sequence data in ten of these individuals. We identified (at an FDR of 10%) 13,915 cis methylation QTLs (meQTLs)—i.e., CpG sites in which changes in DNA methylation are associated with genetic variation at proximal loci. We found that meQTLs are frequently associated with changes in methylation at multiple CpGs across regions of up to 3 kb. Interestingly, meQTLs are also frequently associated with variation in other properties of gene regulation, including histone modifications, DNase I accessibility, chromatin accessibility, and expression levels of nearby genes. These observations suggest that genetic variants may lead to coordinated molecular changes in all of these regulatory phenotypes. One plausible driver of coordinated changes in different regulatory mechanisms is variation in transcription factor (TF) binding. Indeed, we found that SNPs that change predicted TF binding affinities are significantly enriched for associations with DNA methylation at nearby CpGs. [ABSTRACT FROM AUTHOR]

To design novel therapeutics against bone loss, understanding the molecular mechanisms regulating osteoclastogenesis is critical. Osteoclast formation and function are tightly regulated by transcriptional, post-transcriptional and post-translational mechanisms. This stringent regulation is crucial to prevent excessive or insufficient bone resorption and to maintain bone homeostasis. microRNAs (miRNAs) are key post-transcriptional regulators that repress expression of target mRNAs controlling osteoclast proliferation, differentiation, and apoptosis. Disruption of miRNA-mediated regulation alters osteoclast formation and bone resorption. Prior studies profiled miRNA expression in murine osteoclast precursors treated with RANKL for 24 hours. However, a more complete miRNA signature, encompassing early, mid and late stages of osteoclastogenesis, is wanting. An Agilent microarray platform was used to analyze expression of mature miRNAs in an enriched population of murine bone marrow osteoclast precursors (depleted of B220+ and CD3+ cells) undergoing 1, 3, or 5 days of RANKL-driven differentiation. Expression of 93 miRNAs, changed by >2 fold during early, mid, and late stages of osteoclastogenesis, were identified and sorted into 7 clusters. We validated the function and expression of miR-365, miR-451, and miR-99b, which were found in distinct clusters. Inhibition of miR-365 increased osteoclast number but decreased osteoclast size, while miR-99b inhibition decreased both osteoclast number and size. In contrast, overexpression of miR-451 had no effect. Computational analyses predicted mTOR, PI3 kinase/AKT, cell-matrix interactions, actin cytoskeleton organization, focal adhesion, and axon guidance pathways to be top targets of several miRNA clusters. This suggests that many miRNA clusters differentially expressed during osteoclastogenesis converge on some key functional pathways. Overall, our study is unique in that we identified miRNAs differentially expressed during early, mid, and late osteoclastogenesis in a population of primary mouse bone marrow cells enriched for osteoclast progenitors. This novel data set contributes to our understanding of the molecular mechanisms regulating the complex process of osteoclast differentiation. [ABSTRACT FROM AUTHOR]

Cephalotaxus hainanensis, an endangered plant, is known to contain several metabolites with anti-cancer activity. Despite its clinical impact, the alkaloid metabolism of this species has remained largely uncharacterized. The potential of Cephalotaxus for metabolic engineering of medically interesting compounds has, so far, not been exploited, due to the almost complete lack of molecular information. We have therefore performed a high throughput RNA-seq analysis and assembled the transcriptome de novo. Raw reads comprising 4.3 Gbp were assembled de novo into 39,416 unique sequences (unigenes) with a mean length of 1,089.8 bp and a total assembly size of 45.8 Mbp, which equals to more than 50 times the number of Cephalotaxaceae sequences currently deposited in the GenBank (as of August 2013). As proof of principle for medically interesting pathways, gene fragments related to paclitaxel biosynthesis were searched and detected. To verify their functionality, the metabolic product paclitaxel, and its precursor baccatin III, were identified in the leaves of C. hainanensis by HPLC, and shown to be induced by MeJA. This finding demonstrates exemplarily the potential of the annotated transcriptome as information resource for the biotechnological exploitation of plant secondary metabolism. [ABSTRACT FROM AUTHOR]

Cancer and healthy cells have distinct distributions of molecular properties and thus respond differently to drugs. Cancer drugs ideally kill cancer cells while limiting harm to healthy cells. However, the inherent variance among cells in both cancer and healthy cell populations increases the difficulty of selective drug action. Here we formalize a classification framework based on the idea that an ideal cancer drug should maximally discriminate between cancer and healthy cells. More specifically, this discrimination should be performed on the basis of measurable cell markers. We divide the problem into three parts which we explore with examples. First, molecular markers should discriminate cancer cells from healthy cells at the single-cell level. Second, the effects of drugs should be statistically predicted by these molecular markers. Third, drugs should be optimized for classification performance. We find that expression levels of a handful of genes suffice to discriminate well between individual cells in cancer and healthy tissue. We also find that gene expression predicts the efficacy of some cancer drugs, suggesting that these cancer drugs act as suboptimal classifiers using gene profiles. Finally, we formulate a framework that defines an optimal drug, and predicts drug cocktails that may target cancer more accurately than the individual drugs alone. Conceptualizing cancer drugs as solving a discrimination problem in the high-dimensional space of molecular markers promises to inform the design of new cancer drugs and drug cocktails. [ABSTRACT FROM AUTHOR]