Major Regulatory Genes Research Articles

Critical cis-Acting Elements and Interacting Transcription Factors: Key Players Associated with Abiotic Stress Responses in Plants

Plants are constantly confronted with various abiotic stresses, such as drought, extreme temperature, salinity, nutrient element deficiency and heavy metal contamination. In the long process of evolution, plants have developed a suite of sophisticated strategies to adapt to and to survive under the harsh environmental cues, among which transcriptional alteration constitutes an overriding mechanism. Stress-responsive genes can be classified into two major groups, functional genes and regulatory genes. Transcription factors play an essential role in the abiotic stress response by regulating the mRNA abundance of a large spectrum of downstream target genes via interaction with the cis-acting elements on the promoters of the genes. So far, several families of TFs and their interacting cis-acting elements, collectively called as regulons, have been well characterized to be key players closely implicated in abiotic stress responses. In the current overview, the major TFs and the cis-acting elements that have been extensively investigated in the last decades are introduced. In addition, future perspectives pertinent to their research directions are proposed, with the intention of gaining insight into better understanding and desirable application of the transcription factors.

Iron deficiency modifies gene expression variation induced by augmented hypoxia sensing

In congenital Chuvash polycythemia (CP), VHL(R200W) homozygosity leads to elevated hypoxia inducible factor (HIF) levels at normoxia. CP is often treated by phlebotomy resulting in iron deficiency, permitting us to examine the separate and synergistic effects of iron deficiency and HIF signaling on gene expression. We compared peripheral blood mononuclear cell gene expression profiles of eight VHL(R200W) homozygotes with 17 wildtype individuals with normal iron status and found 812 up-regulated and 2120 down-regulated genes at false discovery rate of 0.05. Among differential genes we identified three major gene regulation modules involving induction of innate immune responses, alteration of carbohydrate and lipid metabolism, and down-regulation of cell proliferation, stress-induced apoptosis and T-cell activation. These observations suggest molecular mechanisms for previous observations in CP of lower blood sugar without increased insulin and low oncogenic potential. Studies including 16 additional VHL(R200W) homozygotes with low ferritin indicated that iron deficiency enhanced the induction effect of VHL(R200W) for 50 genes including hemoglobin synthesis loci but suppressed the effect for 107 genes enriched for HIF-2 targets. This pattern is consistent with potentiation of HIF-1α protein stability by iron deficiency but a trend for down-regulation of HIF-2α translation by iron deficiency overriding an increase in HIF-2α protein stability.

Eukaryotic Initiation Factor 4H Is under Transcriptional Control of p65/NF-κB

Protein synthesis is mainly regulated at the initiation step, allowing the fast, reversible and spatial control of gene expression. Initiation of protein synthesis requires at least 13 translation initiation factors to assemble the 80S ribosomal initiation complex. Loss of translation control may result in cell malignant transformation. Here, we asked whether translational initiation factors could be regulated by NF-κB transcription factor, a major regulator of genes involved in cell proliferation, survival, and inflammatory response. We show that the p65 subunit of NF-κB activates the transcription of eIF4H gene, which is the regulatory subunit of eIF4A, the most relevant RNA helicase in translation initiation. The p65-dependent transcriptional activation of eIF4H increased the eIF4H protein content augmenting the rate of global protein synthesis. In this context, our results provide novel insights into protein synthesis regulation in response to NF-κB activation signalling, suggesting a transcription-translation coupled mechanism of control.

Establishment of an immortal chicken embryo liver-derived cell line

A continuously growing immortal cell substrate can be used for virus propagation, diagnostic purposes, and vaccine production. The aim of this study was to develop an immortal chicken cell line for efficient propagation of avian infectious viruses. From the various chicken embryo cells that were tested for life span extension, an immortalized chicken embryo liver (CEL) cell line, named CEL-im, was derived spontaneously without either oncogenic viruses or carcinogenic chemical treatment. Currently, CEL-im cells are growing 0.8 to 1.1 population doublings per day and have reached 120 passages. The CEL-im cell line is permissive for poultry infectious viruses, including avian metapneumovirus (AMPV), Marek's disease virus serotype 1 (MDV-1), and infectious laryngotracheitis virus. The CEL-im cells produced high AMPV titer (>10(5) pfu/mL), whereas very low titers (~10 pfu/mL) for MDV-1 and infectious laryngotracheitis virus were produced. To identify genetic alterations in the immortal CEL-im cell line, telomerase activity and mRNA expression for major cell cycle regulatory genes were determined during the immortalizing process. The CEL-im cell line has negative telomerase activity, and when compared with the primary passage 2 CEL cell counterpart, mRNA expression of tumor suppressor protein p53, mouse double minute 2 (Mdm2), cyclin dependent kinase (CDK) inhibitor p21 (p21(WAF)), and CDK inhibitor p16 (p16(INK4)) were downregulated in the CEL-im cell line, whereas retinoblastoma (Rb), transcription factor E2F, member 1 (E2F-1), and alternative reading frame of p16(INK4) (ARF) were upregulated. These results are similar to genetic alterations found previously in immortal chicken embryo fibroblast (CEF) cell lines that showed efficient propagation of MDV-1. Therefore, this newly established CEL-im cell line can serve as an alternative cell substrate for the propagation of poultry viruses, such as AMPV.

The maize lipoxygenase, ZmLOX10, mediates green leaf volatile, jasmonate and herbivore‐induced plant volatile production for defense against insect attack

Fatty acid derivatives are of central importance for plant immunity against insect herbivores; however, major regulatory genes and the signals that modulate these defense metabolites are vastly understudied, especially in important agro-economic monocot species. Here we show that products and signals derived from a single Zea mays (maize) lipoxygenase (LOX), ZmLOX10, are critical for both direct and indirect defenses to herbivory. We provide genetic evidence that two 13-LOXs, ZmLOX10 and ZmLOX8, specialize in providing substrate for the green leaf volatile (GLV) and jasmonate (JA) biosynthesis pathways, respectively. Supporting the specialization of these LOX isoforms, LOX8 and LOX10 are localized to two distinct cellular compartments, indicating that the JA and GLV biosynthesis pathways are physically separated in maize. Reduced expression of JA biosynthesis genes and diminished levels of JA in lox10 mutants indicate that LOX10-derived signaling is required for LOX8-mediated JA. The possible role of GLVs in JA signaling is supported by their ability to partially restore wound-induced JA levels in lox10 mutants. The impaired ability of lox10 mutants to produce GLVs and JA led to dramatic reductions in herbivore-induced plant volatiles (HIPVs) and attractiveness to parasitoid wasps. Because LOX10 is under circadian rhythm regulation, this study provides a mechanistic link to the diurnal regulation of GLVs and HIPVs. GLV-, JA- and HIPV-deficient lox10 mutants display compromised resistance to insect feeding, both under laboratory and field conditions, which is strong evidence that LOX10-dependent metabolites confer immunity against insect attack. Hence, this comprehensive gene to agro-ecosystem study reveals the broad implications of a single LOX isoform in herbivore defense.

Bioinformatics of varicella-zoster virus: Single nucleotide polymorphisms define clades and attenuated vaccine genotypes

Varicella zoster virus (VZV) is one of the human herpesviruses. To date, over 40 complete VZV genomes have been sequenced and analyzed. The VZV genome contains around 125,000 base pairs including 70 open reading frames (ORFs). Enumeration of single nucleotide polymorphisms (SNPs) has determined that the following ORFs are the most variable (in descending order): 62, 22, 29, 28, 37, 21, 54, 31, 1 and 55. ORF 62 is the major immediate early regulatory VZV gene. Further SNP analysis across the entire genome has led to the observation that VZV strains can be broadly grouped into clades within a phylogenetic tree. VZV strains collected in Singapore provided important sequence data for construction of the phylogenetic tree. Currently five VZV clades are recognized; they have been designated clades 1 through 5. Clades 1 and 3 include European/North American strains; clade 2 includes Asian strains, especially from Japan; and clade 5 includes strains from India. Clade 4 includes some strains from Europe, but its geographic origins need further documentation. Within clade 1, five variant viruses have been isolated with a missense mutation in the gE (ORF 68) glycoprotein; these strains have an altered increased cell spread phenotype. Bioinformatics analyses of the attenuated vaccine strains have also been performed, with a subsequent discovery of a stop-codon SNP in ORFO as a likely attenuation determinant. Taken together, these VZV bioinformatics analyses have provided enormous insights into VZV phylogenetics as well as VZV SNPs associated with attenuation.

PDGFRα and CD51 Mark Human Nestin+ Sphere-Forming Mesenchymal Stem Cells Capable of Robust Hematopoietic Stem Cell Expansion

AbstractAbstract 505Few markers have thus far been identified on native mesenchymal stem cells (MSCs), both in the mouse and human systems. Most markers cited in the literature are indeed based on expression analyses on heterogeneous cultured cell populations, which may not have self-renewal properties if rigorously tested by transplantation assays. Previous studies using Nestin (Nes)-Gfp transgenic mice showed that Nes-GFP+ cells are self-renewing MSCs, a major constituent of the hematopoietic stem cell (HSC) niche in the bone marrow (BM) (Nature 2010; 466:829). However, the cytoplasmic location of Nestin precludes prospective live cell isolation outside of the transgenic mice. Hence, finding a combination of surface markers labeling Nestin+ cells in situ would be valuable to isolate bona fide MSCs and characterize niche cells. Screening analyses toward this end revealed that PDGFRα and CD51 expression among CD45− Ter119− CD31− BM stromal cells comprised a large fraction (∼60%) of Nes-GFP+ cells. Upon gating first on PDGFRα+ and CD51+, double-positive cells were also highly enriched in Nes-GFP+ cells (∼75%), and represented a rare fraction (∼2%) of the stromal population. Endogenous Nestin expression was also enriched in PDGFRα+ CD51+ cells, compared to single-positive or double-negative stromal cells (control subsets). Cell sorting of BM PDGFRα+ CD51+ and control subsets revealed that PDGFRα+ CD51+ significantly enriched (> 10-fold, p<0.05) for colony forming unit-fibroblastic (CFU-F) and multipotent clonal mesenspheres (> 7-fold, p<0.01) that differentiate robustly along the osteoblastic, chondrocytic and adipocytic lineages. To test in vivo self-renewal capacity, clonal spheres or polyclonal freshly sorted PDGFRα+ CD51+ cells and control subsets were transplanted into recipient mice by different approaches (renal capsule implants, collagen and/or HA/TCP carrier grafts). After 2 months, secondary sphere formation assays and histological analyses revealed the in vivo self-renewal and heterotopic BM niche regeneration capacity of PDGFRα+ CD51+ cells, but not the control subsets. In addition, the PDGFRα+ CD51+ fraction of Nestin+ cells was markedly enriched in major HSC regulatory genes (Cxcl12, Vcam1, Angpt1, Opn and Scf), supporting the notion that niche activity co-segregates with MSC activity in the BM. Next, we investigated whether PDGFRα+ CD51+ cells also labeled putative Nestin+ MSCs in the human BM. To this end, we analyzed the fetal human BM (13–19 gw), a period during which hematopoietic activity is nascent. At this stage, we found that PDGFRα+ CD51+ cells comprised ∼3% of stromal cells, contained most of the CFU-F activity (6.3 ± 0.8 CFU-Fs/102 cells) in the BM, and also expressed Nestin and HSC regulatory factors. PDGFRα+ CD51+ cells could also form mesenspheres that can self-renew in vivo after heterotopic transplantation. Furthermore, we found that human BM PDGFRα+ CD51+ cells represented a subset of CD146+ cells previously suggested to mark human MSCs (Cell 2007; 131:324), as ∼30% of the CD146high cells also expressed PDGFRα and CD51, and ∼65% of PDGFRα+ CD51+ cells were CD146high. To evaluate functionally the HSC niche properties of human PDGFRα+ CD51+ cells, we set up a co-culture system of human BM CD34+ cells with PDGFRα+ CD51+ mesenspheres. We found that mesenspheres were capable of expanding the number of human CD45+ Lin− CD38− CD34+ CD90+ CD49f+ cells (hHSCs) by 11-fold (p<0.05) compared to input (day 0). In addition, hHSC expansion was 2-fold greater (p<0.05) using mesenspheres compared to serum-free media alone with hematopoietic growth factors (SCF, TPO, Flt3L). Recent studies have suggested that SCF production in the BM niche is derived from perivascular and endothelial cells distinct from Nestin+ cells (Nature 2012; 481:457), although Nestin+ MSCs express high levels of SCF (Nature 2010; 466:829). Immunofluorescence analyses of human PDGFRα+ CD51+ mesenspheres showed that all cells forming the sphere uniformly expressed both Nestin and SCF. Moreover, in the absence of SCF from the media, PDGFRα+ CD51+ mesenspheres rescued hHSCs expansion, yielding a 46- and 5-fold (p<0.001) expansion, as compared to control media alone and input, respectively. These results thus indicate that the HSC niche is conserved between the murine and human species and suggest that highly purified non-adherent cultures of niche cells may represent a useful novel technology to expand hHSCs in vitro. Disclosures:No relevant conflicts of interest to declare.

Identification and Promoter Activity Analysis of Porcine miR-181 and miR-1

Since its discovery a decade ago, microRNA has been identified as one of the major regulatory gene families in eukaryotic cells. Many functions of microRNAs have been revealed both in flora and fauna in recent years, but the transcriptional regulation of microRNA genes is not well-understood. In the present study, a series of primers were designed in the 2000 nt upstream regions of porcine miR-181 and miR-1 and then the sequences were cloned into pGL3-basic vector to test their transcriptional activity. Dual-luciferase reporter assays showed that, the activity of 5′-flanking sequence of miR-181 started on construct −51, decreasing with the length of the fragment up to −444. The upstream 590 bp confer maximal transcriptional activity and the basal promoter activity is located within the −82 to +16 bp region. For miR-1, the activity starts on construct −50, decreasing with the length of the fragment up to −1254 in despite of a bit of fluctuation, and the basal promoter activity is located within the −50 to +47 bp region. Furthermore, some putative regulation elements of both miR-181 and miR-1 were located. In addition, tissue distribution revealed that miR-181 is expressed at a relatively low level.

Long-term memory CD4+ T cells - a survival network (47.12)

Abstract Memory CD4+ T cells specific for a particular antigen can survive for a lifetime of an individual through undefined mechanisms. Previously we have demonstrated that memory CD4+ T cells undergo a state of dormancy during the resolution of infection, and dormant memory T cells survive for the life of mice. To gain an understanding of the genetic programs involved, we examined gene expression profile of memory DO11.10 CD4+ T cells in mice infected with Vaccinia-OVA virus 10.5 months post infection and compared it with gene expression in age-matched old mice. Extensive literature search allowed reverse engineering of genetic network of differentially expressed genes in long-term memory T cells. Pattern of gene expression pointed to the induction of anti-apoptotic and genomic stability pathways. We found that in long term memory CD4+ T cells a shift from glucose to lipid metabolism occurs, along with upregulation of ceramide synthesis and utilization that may favor memory T cell survival. Insulin resistance appears to play a major role in downregulation of glucose metabolism. In conclusion, our data show that in long-term memory CD4+ T cells several major regulatory genes that regulate anti-apoptotic, genomic stability, glucose and lipid metabolism pathways are upregulated and possibly coordinate an overall pro-survival effect.

A novel biclustering algorithm for the discovery of meaningful biological correlations between miRNAs and mRNAs

Motivations. microRNAs (miRNAs) are post-transcriptional regulators which represent one of the major regulatory gene families in animals, plants and viruses and that plays a key role in almost all main cellular processes. The computational prediction of miRNA target genes is important for the functional annotation of genomes and, on the other side, functional annotation of target genes can be of great help in suggesting specific biological functions of miRNAs [1]. This work aims to contribute to the elucidation of miRNAs role in the regulation of gene expression, by proposing a method for the hierarchical and overlapping biclustering of miRNAs and target messenger RNAs (mRNAs). The method allows to discover possible miRNA:mRNA functional relationships, at different granularity levels, in large datasets produced by miRNA target site prediction algorithms, thus reducing the impact of noise on the significance of the resulting biclusters. Methods. In order to properly work on miRNA:mRNA interactions, three important issues have to be considered. In particular, extracted biclusters should be: i) possibly overlapping, because miRNAs can be involved in different regulatory networks; ii) exhaustive, i.e. each miRNA or mRNA should belong to at least one bicluster, thus preventing the loss of possible co-regulations; iii) hierarchically organized, thus facilitating biological interpretability of results even when a high number of biclusters is extracted from large miRNA:mRNA datasets. We propose an algorithm for the efficient discovery of overlapping, exhaustive and hierarchically organized biclusters. Our algorithm effectively deals with a kind of relational imbalance (i.e. miRNAs and mRNAs participate with significantly different cardinalities in the interactions). Moreover, it combines the notions of bicluster separability and bicluster distance in order to extract significant biclusters according to both density and distance-based criteria. The performance of our method is evaluated on miRNAs target predictions in the human genome dataset extracted by miRNAMap 2.0 [2]. The strength of the miRNA:mRNA interactions is estimated on the basis of predictions provided by miRanda, RNAHybrid and TargetScan algorithms. It is computed as a linear combination of three criteria: 1) the number of algorithms which predict the miRNA target site; 2) the number of miRNA target sites found in the same UTR region; 3) the accessibility of the target site. Weights of the linear combination are selected such that criterion 1 dominates over the other two, while criterion 3, ceteris paribus on criterion 1, dominates over criterion 2. Results. The performance of our method is evaluated in terms of execution time, bicluster compactness (the intra-bicluster cohesion) and bicluster co-regulation (the ability to group together miRNAs that target the same mRNAs). A comparative analysis shows that our method is able to extract a smaller number of (hierarchically organized) biclusters, with higher compactness and co-regulation values than ROCC [3]. Execution times of our method and ROCC are comparable. The significance of the extracted hierarchies is evaluated in terms of the F-Measure, on a set of synthetic datasets generated at different levels of noise. The analysis reveals that the hierarchy structure is almost correctly discovered, even for high levels of noise. The effectiveness of the algorithm in extracting biologically related biclusters is tested on the basis of: i) classification of biclustered miRNAs in the same miRNA family or gene cluster; ii) validated functional associations of biclustered miRNAs reported in the literature and in major web specialised resources, iii) GO classification and functional clustering of biclustered mRNAs [4]. Results show that the proposed algorithm allows to extract a relatively small number of biclusters that preserve both compactness and co-regulation. Biclusters extracted by our method represent meaningful biological correlations between miRNAs and mRNAs. Availability http://dl.dropbox.com/u/66737165/biclustering/ index.html

RNA-seq Analysis Reveals That an ECF σ Factor, AcsS, Regulates Achromobactin Biosynthesis in Pseudomonas syringae pv. syringae B728a

Iron is an essential micronutrient for Pseudomonas syringae pv. syringae strain B728a and many other microorganisms; therefore, B728a has evolved methods of iron acquirement including the use of iron-chelating siderophores. In this study an extracytoplasmic function (ECF) sigma factor, AcsS, encoded within the achromobactin gene cluster is shown to be a major regulator of genes involved in the biosynthesis and secretion of this siderophore. However, production of achromobactin was not completely abrogated in the deletion mutant, implying that other regulators may be involved such as PvdS, the sigma factor that regulates pyoverdine biosynthesis. RNA-seq analysis identified 287 genes that are differentially expressed between the AcsS deletion mutant and the wild type strain. These genes are involved in iron response, secretion, extracellular polysaccharide production, and cell motility. Thus, the transcriptome analysis supports a role for AcsS in the regulation of achromobactin production and the potential activity of both AcsS and achromobactin in the plant-associated lifestyle of strain B728a.

Protective Role of Estrogen-induced miRNA-29 Expression in Carbon Tetrachloride-induced Mouse Liver Injury

Previous studies have indicated that female animals are more resistant to carbon tetrachloride (CCl(4))-induced liver fibrosis than male animals, and that estradiol (E(2)) treatment can inhibit CCl(4)-induced animal hepatic fibrosis. The underlying mechanism governing these phenomena, however, has not been fully elucidated. Here we reported the role of estrogen-induced miRNA-29 (miR-29) expression in CCl(4)-induced mouse liver injury. Hepatic miR-29 levels were differentially regulated in female and male mice during CCl(4) treatment. Specifically, the levels of miR-29a and miR-29b expression were significantly decreased in the livers of male, but not female, mice following 4 weeks of CCl(4) treatment. The down-regulation of miR-29a and miR-29b in male mouse livers correlated with the early development of liver fibrosis, as indicated by increased expressions of fibrotic markers in male mice relative to female mice. In addition, E(2) was maintained at a higher level in female mice than in male mice. In contrast to TGF-β1 that decreased miR-29a/b expression in murine hepatoma IAR20 cells and normal hepatocytes, E(2) enhanced the expression of miR-29a/b through suppression of the nuclear factor-κB (NF-κB) signal pathway, which negatively regulates miR-29 expression. Furthermore, both E(2) treatment and intravenous injection of the recombinant adenovirus expressing miR-29a/b markedly increased the miR-29a/b level and attenuated the expression of fibrotic markers in mouse livers during CCl(4) treatment, supporting the protective role of E(2)-induced miR-29 in CCl(4)-induced hepatic injury. In conclusion, our results collectively demonstrate that estrogen can inhibit CCl(4)-induced hepatic injury through the induction of hepatic miR-29.

The Role of microRNAs in Cardiovascular Disease

Cardiovascular disease, which is multifactorial and can be influenced by a multitude of environmental and heritable risk factors, remains a major health problem, even though its pathophysiology is far from been elucidated. Discovered just over a decade ago, microRNAs comprise short, non-coding RNAs, which have evoked a great deal of interest, due to their importance for many aspects of homeostasis and disease. Hundreds of different microRNAs are constantly being reported in various organisms. According to a growing body of literature, they have been implicated in the regulation of human physiological processes. More specifically, miRNAs are expressed in the cardiovascular system and could have crucial roles in normal development and physiology, as well as in disease development. Furthermore, they have been shown to participate in cardiovascular disease pathogenesis including atherosclerosis, coronary artery disease, myocardial infarction, heart failure and cardiac arrhythmias. In contrast to our original thought, miRNAs exist in circulating blood and are relatively stable, thus, they could be proved useful as biomarkers in that state. Understanding the underlying mechanisms, in which these major regulatory gene families are implicated, will provide novel opportunities for diagnosis and therapy of cardiovascular diseases.

Transcription Factor STOX1A Promotes Mitotic Entry by Binding to the CCNB1 Promotor

BackgroundIn this study we investigated the involvement of the transcription factor STOX1A in the regulation of the cell cycle.Methodology/Principal FindingsWe found that several major cell cycle regulatory genes were differentially expressed upon STOX1A stimulation and knockdown in the neuroblastoma cell line SH-SY5Y. This includes STOX1A dependent differential regulation of cyclin B1 expression, a cyclin which is known to regulate mitotic entry during the cell cycle. The differential regulation of cyclin B1 expression by STOX1A is direct as shown with chromatin immunoprecipitation. Results furthermore suggest that mitotic entry is enhanced through the direct upregulation of cyclin B1 expression effectuated by STOX1A.ConclusionsIn conclusion we hereby show that STOX1A is directly involved in the regulation of the cell cycle.

KdgR, an IClR Family Transcriptional Regulator, Inhibits Virulence Mainly by Repression of hrp Genes in Xanthomonas oryzae pv. oryzae

KdgR has been reported to negatively regulate the genes involved in degradation and metabolization of pectic acid and other extracellular enzymes in soft-rotting Erwinia spp. through direct binding to their promoters. The possible involvement of a KdgR orthologue in virulence by affecting the expression of extracellular enzymes in Xanthomonas oryzae pv. oryzae, the causal agent of rice blight disease, was examined by comparing virulence and regulation of extracellular enzymes between the wild type (WT) and a strain carrying a mutation in putative kdgR (ΔXoo0310 mutant). This putative kdgR mutant of X. oryzae pv. oryzae showed increased pathogenicity on rice without affecting the regulation of extracellular enzymes, such as amylase, cellulase, xylanase, and protease. However, the mutant carrying a mutation in an ortholog of xpsL, which encodes the functional secretion machinery for the extracellular enzymes, showed a dramatic decrease in pathogenicity on rice. Both mutants of kdgR and of xpsL orthologs showed higher expression of two major hrp regulatory genes, hrpG and hrpX, and the genes in the hrp operons when grown in hrp-inducing medium. Thus, both genes were shown to be involved in repression of hrp genes. The kdgR ortholog was thought to suppress virulence mainly by repressing the expression of hrp genes without affecting the expression of extracellular enzymes, unlike findings for the kdgR gene in soft-rotting Erwinia spp. On the other hand, xpsL was confirmed to be involved in virulence by promoting the secretion of extracellular enzymes in spite of repressing the expression of the hrp genes.

Gene-environment interactions between CRHR1 variants and physical assault in suicide attempts

Risk factors for suicidal behaviors are partly heritable, including genetic variants that drive diathesis-stress in addition to, or by interaction with, exposure to certain stressful life events (SLEs). Hypothalamic-pituitary-adrenal (HPA) axis regulatory genes are candidates for association with suicide as well as its endophenotypes. Using a family-based design of offspring who attempted suicide (SA) and both parents, we investigated gene-environment interactions (G×Es) of SLE exposures with single nucleotide polymorphisms (SNPs) in corticotropin-releasing hormone receptor-1 (CRHR1), a major HPA axis regulatory gene. We observed a novel G×E among predominantly female SA between 5'-SNP rs7209436 and childhood/adolescence physical assault or attack (PA), as well as a second novel and male-specific G×E between 3'-SNP rs16940665 and adulthood PA exposure. A third male-specific G×E previously reported by us among depressed SA, between SNP rs4792887 and cumulative SLEs, was also further confirmed. The two novel G×Es presented here shared the SA characteristic of aggression, while showing differences on other aspects of SA heterogeneity. We conclude that different SA subjects were observed to differentially associate with two novel G×Es involving exposures to PA with different life timing and SNPs located in opposite ends of CRHR1. Concerning sex differences, we observed three subsets of distinct male SA that associated with each of the three observed G×Es, whereas female SAs were affected by only one of the G×Es. These results are consistent with a diathesis-stress model of suicidal behavior and may help to explain SA heterogeneity.

IKKβ phosphorylation regulates RPS3 nuclear translocation and NF-κB function during infection with Escherichia coli strain O157:H7

NF-κB is a major gene regulator in immune responses and ribosomal protein S3 (RPS3) is an NF-κB subunit that directs specific gene transcription. However, it is unknown how RPS3 nuclear translocation is regulated. Here we report that IKKβ phosphorylation of serine 209 (S209) was crucial for RPS3 nuclear localization in response to activating stimuli. Moreover, the foodborne pathogen Escherichia coli O157:H7 virulence protein NleH1 specifically inhibited RPS3 S209 phosphorylation and blocked RPS3 function, thereby promoting bacterial colonization and diarrhea but decreasing mortality in a gnotobiotic piglet infection model. Thus, the IKKβ-dependent modification of a specific amino acid in RPS3 promotes specific NF-κB functions that underlie the molecular pathogenetic mechanisms of E. coli O157:H7.

Analysis of osteoblastic gene expression in the early human mesenchymal cell response to a chemically modified implant surface: an in vitro study

The effect of a chemical modification of the SLA surface (SLActive surface) on human bone marrow-derived mesenchymal cells (hMSCs) on; (1) adhesion, (2) proliferation and (3) early transcriptional control of osteogenic differentiation was investigated. We are based on the hypothesis that expression patterns of genes responsible for osteogenesis might be dependent on the characteristics of the implant surface. hMSCs were allowed to grow on smooth (SMO-control), SLA and SLActive implant surfaces (chemically modified). Cell attachment and proliferation were assessed at 3 and 24 h using a MTT dye reduction assay. At 24 h of culture, DNA microarray analysis examined alterations in early gene expression using a human osteogenesis gene array, including 109 cDNAs in quadruplicates of major regulatory genes for osteogenesis. Initial attachment and proliferation were found to be significantly reduced. Nineteen genes were significantly upregulated when hMSCs were cultured on the SLA surfaces and 27 genes were significantly upregulated when hMSCs were cultured on the SLActive surfaces. Upregulated genes control cell differentiation, signal transduction, cell cycle regulation, angiogenesis, cell adhesion and extracellular matrix and bone formation. Chemical modification decreases further cell attachment and proliferation and upregulates early osteoblastic differentiation genes. Hence, a microenvironment is created around chemically modified implants that may enhance osseointegration.

Analysis of Immune Regulatory Genes' Copy Number Variants in Graves' Disease

Copy number variants (CNVs) have recently been reported to be associated with several autoimmune conditions. Moreover, loci involved in immunity are enriched in CNVs. Therefore, we hypothesized that CNVs in immune genes associated with Graves' disease (GD) may contribute to the etiology of disease. One hundred ninety-one North American Caucasian GD patients and 192 Caucasian controls were analyzed for CNVs in three major immune regulatory genes: CD40, PTPN22, and CTLA-4. Copy number was determined using quantitative-PCR (Q-PCR) assays specifically designed for determining copy numbers in genomic DNA. Additionally, a well-characterized CNV in the amylase gene was typed in a separate dataset of DNA samples that were derived from cell lines or blood. No CNVs could be confirmed in the CD40 and CTLA-4 genes, even though a CD40 CNV is cataloged in the Database of Genomic Variants. Only the PTPN22 CNV was confirmed in our cohort, but it was rare and appeared in only two individuals. A key finding was that the source of DNA has a significant effect on CNV typing. There was a statistically significant increase in amylase locus deletions in cell line-derived DNA compared to blood-derived DNA samples. We conclude that CNV analysis should be performed only using blood-derived DNA Samples. Additionally, the CTLA-4, CD40, and PTPN22 loci do not harbor CNVs that play a role in the etiology of GD.

Analysis of the Petunia hybrida double flower transcriptome using suppression subtractive hybridization

The aim of this study was to characterize the transcriptional response that is associated with the Petunia double flower phenotype with a view to understanding the underlying molecular mechanisms. We created forward and reverse subtracted cDNA libraries to represent the up-regulated genes found in the double and single flowers obtained from segregated progenies through backcross, respectively. This was achieved using suppression subtractive hybridization (SSH) followed by differential screening through dot blot hybridization. In total, 285 expressed sequence tags (ESTs) were identified. Analysis using Blastx searches showed that the libraries contained several important developmental genes. The expression patterns of selected genes, believed to be involved in the development of Petunia double flowers, were further analyzed by semi-quantitative or real-time reverse transcription-polymerase chain reaction (RT-PCR). These analyses were conducted using RNA extracts taken from specific floral organs and from flowers at various stages of development. Each of the selected genes showed differential expression levels between single and double flowers, and this was particularly clear in young flower buds, thereby suggesting that the genes are important for the early development of the double flower form. In particular, we discuss the expression patterns of genes with putative roles relating to cell expansion, organ fusion and organ number. Other genes, with less well-documented roles, may be involved in modulating the development of the Petunia double flower. Thus, such genes appear to function along with the major regulatory MADS-box genes as part of a complicated network of genetic factors that direct the formation of the Petunia double flower.