The role of ethylene and cold temperature in the regulation of the apple POLYGALACTURONASE1 gene and fruit softening

Fruit softening in apple (Malus 3 domestica) is associated with an increase in the ripening hormone ethylene. Here, we show that in cv Royal Gala apples that have the ethylene biosynthetic gene ACC OXIDASE1 suppressed, a cold treatment preconditions the apples to soften independently of added ethylene. When a cold treatment is followed by an ethylene treatment, a more rapid softening occurs than in apples that have not had a cold treatment. Apple fruit softening has been associated with the increase in the expression of cell wall hydrolase genes. One such gene, POLYGALACTURONASE1 (PG1), increases in expression both with ethylene and following a cold treatment. Transcriptional regulation of PG1 through the ethylene pathway is likely to be through an ETHYLENE-INSENSITIVE3-like transcription factor, which increases in expression during apple fruit development and transactivates the PG1 promoter in transient assays in the presence of ethylene. A coldrelated gene that resembles a COLD BINDING FACTOR (CBF) class of gene also transactivates the PG1 promoter. The transactivation by the CBF-like gene is greatly enhanced by the addition of exogenous ethylene. These observations give a possible molecular mechanism for the coldand ethylene-regulated control of fruit softening and suggest that either these two pathways act independently and synergistically with each other or cold enhances the ethylene response such that background levels of ethylene in the ethylene-suppressed apples is sufficient to induce fruit softening in apples.

High temperature reduces apple fruit colour via modulation of the anthocyanin regulatory complex

The biosynthesis of anthocyanin in many plants is affected by environmental conditions. In apple (Malus×domestica Borkh.), concentrations of fruit anthocyanins are lower under hot climatic conditions. We examined the anthocyanin accumulation in the peel of maturing 'Mondial Gala' and 'Royal Gala' apples, grown in both temperate and hot climates, and using artificial heating of on-tree fruit. Heat caused a dramatic reduction of both peel anthocyanin concentration and transcripts of the genes of the anthocyanin biosynthetic pathway. Heating fruit rapidly reduced expression of the R2R3 MYB transcription factor (MYB10) responsible for coordinative regulation for red skin colour, as well as expression of other genes in the transcriptional activation complex. A single night of low temperatures is sufficient to elicit a large increase in transcription of MYB10 and consequently the biosynthetic pathway. Candidate genes that can repress anthocyanin biosynthesis did not appear to be responsible for reductions in anthocyanin content. We propose that temperature-induced regulation of anthocyanin biosynthesis is primarily caused by altered transcript levels of the activating anthocyanin regulatory complex.

Identification of a cis-regulatory element by transient analysis of co-ordinately regulated genes

Background Transcription factors (TFs) co-ordinately regulate target genes that are dispersed throughout the genome. This co-ordinate regulation is achieved, in part, through the interaction of transcription factors with conserved cis-regulatory motifs that are in close proximity to the target genes. While much is known about the families of transcription factors that regulate gene expression in plants, there are few well characterised cis-regulatory motifs. In Arabidopsis, over-expression of the MYB transcription factor PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT 1) leads to transgenic plants with elevated anthocyanin levels due to the co-ordinated up-regulation of genes in the anthocyanin biosynthetic pathway. In addition to the anthocyanin biosynthetic genes, there are a number of un-associated genes that also change in expression level. This may be a direct or indirect consequence of the over-expression of PAP1. Results Oligo array analysis of PAP1 over-expression Arabidopsis plants identified genes co-ordinately up-regulated in response to the elevated expression of this transcription factor. Transient assays on the promoter regions of 33 of these up-regulated genes identified eight promoter fragments that were transactivated by PAP1. Bioinformatic analysis on these promoters revealed a common cis-regulatory motif that we showed is required for PAP1 dependent transactivation. Conclusion Co-ordinated gene regulation by individual transcription factors is a complex collection of both direct and indirect effects. Transient transactivation assays provide a rapid method to identify direct target genes from indirect target genes. Bioinformatic analysis of the promoters of these direct target genes is able to locate motifs that are common to this sub-set of promoters, which is impossible to identify with the larger set of direct and indirect target genes. While this type of analysis does not prove a direct interaction between protein and DNA, it does provide a tool to characterise cis-regulatory sequences that are necessary for transcription activation in a complex list of co-ordinately regulated genes.

The relationship between N2O, NO, and N2 fluxes from fertilized and irrigated dryland soils of the Aral Sea Basin, Uzbekistan

Microbial respiratory reduction of nitrous oxide (N2O) to dinitrogen (N2) via denitrification plays a key role within the global N-cycle since it is the most important process for converting reactive nitrogen back into inert molecular N2. However, due to methodological constraints, we still lack a comprehensive, quantitative understanding of denitrification rates and controlling factors across various ecosystems. We investigated N2, N2O and NO emissions from irrigated cotton fields within the Aral Sera Basin using the He/O2 atmosphere gas flow soil core technique and an incubation assay. NH4NO3 fertilizer, equivalent to 75 kg ha−1 and irrigation water, adjusting the water holding capacity to 70, 100 and 130% were applied to the incubation vessels to assess its influence on gaseous N emissions. Under soil conditions as they are naturally found after concomitant irrigation and fertilization, denitrification was the dominant process and N2 the main end product of denitrification. The mean ratios of N2/N2O emissions increased with increasing soil moisture content. N2 emissions exceeded N2O emissions by a factor of 5 ± 2 at 70% soil water holding capacity (WHC) and a factor of 55 ± 27 at 130% WHC. The mean ratios of N2O/NO emissions varied between 1.5 ± 0.4 (70% WHC) and 644 ± 108 (130% WHC). The magnitude of N2 emissions for irrigated cotton was estimated to be in the range of 24 ± 9 to 175 ± 65 kg-N ha−1season−1, while emissions of NO were only of minor importance (between 0.1 to 0.7 kg-N ha−1 season−1). The findings demonstrate that for irrigated dryland soils in the Aral Sera Basin, denitrification is a major pathway of N-loss and that substantial amounts of N-fertilizer are lost as N2 to the atmosphere for irrigated dryland soils.

Ibuprofen supplementation and its effects on NF-KB activation in skeletal muscle following resistance exercise

Resistance exercise triggers a subclinical inflammatory response that plays a pivotal role in skeletal muscle regeneration. Nuclear factor‐κB (NF‐κB) is a stress signalling transcription factor that regulates acute and chronic states of inflammation. The classical NF‐κB pathway regulates the early activation of post‐exercise inflammation; however there remains scope for this complex transcription factor to play a more detailed role in post‐exercise muscle recovery. Sixteen volunteers completed a bout of lower body resistance exercise with the ingestion of three 400 mg doses of ibuprofen or a placebo control. Muscle biopsy samples were obtained prior to exercise and at 0, 3 and 24 h post‐exercise and analysed for key markers of NF‐κB activity. Phosphorylated p65 protein expression and p65 inflammatory target genes were elevated immediately post‐exercise independent of the two treatments. These changes did not translate to an increase in p65 DNA binding activity. NF‐κB p50 protein expression and NF‐κB p50 binding activity were lower than pre‐exercise at 0 and 3 h post‐exercise, but were elevated at 24 h post‐exercise. These findings provide novel evidence that two distinct NF‐κB pathways are active in skeletal muscle after resistance exercise. The initial wave of activity involving p65 resembles the classical pathway and is associated with the onset of an acute inflammatory response. The second wave of NF‐κB activity comprises the p50 subunit, which has been previously shown to resolve an acute inflammatory program. The current study showed no effect of the ibuprofen treatment on markers of the NF‐κB pathway, however examination of the within group effects of the exercise protocol suggests that this pathway warrants further research.

Plasmid DNA purification via the use of a dual affinity protein

Methods are presented for the production, affinity purification and analysis of plasmid DNA (pDNA). Batch fermentation is used for the production of the pDNA, and expanded bed chromatography, via the use of a dual affinity glutathione S-transferase (GST) fusion protein, is used for the capture and purification of the pDNA. The protein is composed of GST, which displays affinity for glutathione immobilized to a solid-phase adsorbent, fused to a zinc finger transcription factor, which displays affinity for a target 9-base pair sequence contained within the target pDNA. A Picogreen™ fluorescence assay and/or anx ethidium bromide agarose gel electrophoresis assay can be used to analyze the eluted pDNA.

Radial glia phagocytose axonal debris from degenerating overextending axons in the developing olfactory bulb

Axon targeting during the development of the olfactory system is not always accurate, and numerous axons overextend past the target layer into the deeper layers of the olfactory bulb. To date, the fate of the mis-targeted axons has not been determined. We hypothesized that following overextension, the axons degenerate, and cells within the deeper layers of the olfactory bulb phagocytose the axonal debris. We utilized a line of transgenic mice that expresses ZsGreen fluorescent protein in primary olfactory axons. We found that overextending axons closely followed the filaments of radial glia present in the olfactory bulb during embryonic development. Following overextension into deeper layers of the olfactory bulb, axons degenerated and radial glia responded by phagocytosing the resulting debris. We used in vitro analysis to confirm that the radial glia had phagocytosed debris from olfactory axons. We also investigated whether the fate of overextending axons was altered when the development of the olfactory bulb was perturbed. In mice that lacked Sox10, a transcription factor essential for normal olfactory bulb development, we observed a disruption to the morphology and positioning of radial glia and an accumulation of olfactory axon debris within the bulb. Our results demonstrate that during early development of the olfactory system, radial glia play an important role in removing overextended axons from the deeper layers of the olfactory bulb.

Novel loci affecting iron homeostasis and their effects in individuals at risk for hemochromatosis

Variation in body iron is associated with or causes diseases, including anaemia and iron overload. Here, we analyse genetic association data on biochemical markers of iron status from 11 European-population studies, with replication in eight additional cohorts (total up to 48,972 subjects). We find 11 genome-wide-significant (P<5 × 10−8) loci, some including known iron-related genes (​HFE, ​SLC40A1, ​TF, ​TFR2, ​TFRC, ​TMPRSS6) and others novel (​ABO, ​ARNTL, ​FADS2, ​NAT2, ​TEX14). SNPs at ​ARNTL, ​TF, and ​TFR2 affect iron markers in ​HFE C282Y homozygotes at risk for hemochromatosis. There is substantial overlap between our iron loci and loci affecting erythrocyte and lipid phenotypes. These results will facilitate investigation of the roles of iron in disease.

Genome-wide association study using extreme truncate selection identifies novel genes affecting bone mineral density and fracture risk

Osteoporotic fracture is a major cause of morbidity and mortality worldwide. Low bone mineral density (BMD) is a major predisposing factor to fracture and is known to be highly heritable. Site-, gender-, and age-specific genetic effects on BMD are thought to be significant, but have largely not been considered in the design of genome-wide association studies (GWAS) of BMD to date. We report here a GWAS using a novel study design focusing on women of a specific age (postmenopausal women, age 55-85 years), with either extreme high or low hip BMD (age- and gender-adjusted BMD z-scores of +1.5 to +4.0, n = 1055, or -4.0 to -1.5, n = 900), with replication in cohorts of women drawn from the general population (n = 20,898). The study replicates 21 of 26 known BMD-associated genes. Additionally, we report suggestive association of a further six new genetic associations in or around the genes CLCN7, GALNT3, IBSP, LTBP3, RSPO3, and SOX4, with replication in two independent datasets. A novel mouse model with a loss-of-function mutation in GALNT3 is also reported, which has high bone mass, supporting the involvement of this gene in BMD determination. In addition to identifying further genes associated with BMD, this study confirms the efficiency of extreme-truncate selection designs for quantitative trait association studies. © 2011 Duncan et al.

Association of STAT3 and TNFRSF1A with ankylosing spondylitis in Han Chinese

Objectives: Recent association studies by the Australo-Anglo-American Spondyloarthritis Consortium (TASC) in Caucasian European populations from Australia, North America and the UK have identified a number of genes as being associated with ankylosing spondylitis (AS). A candidate gene study in a Han Chinese population was performed based on these findings to identify associated genes in this population. Methods: A case-control study was performed in a Han Chinese population of patients with AS (n=775) and controls (n=1587) from Shanghai and Nanjing. All patients met the modified New York criteria for AS. The cases and controls were genotyped for 115 single nucleotide polymorphisms (SNPs) tagging IL23R, ERAP1, STAT3, JAK2, TNFRSF1A and TRADD, as well as other confirmation SNPs from the TASC study, using the Sequenom iPlex and the ABI OpenArray platforms. Statistical analysis of genotyped SNPs was performed using the Cochran - Armitage test for trend and meta-analysis was performed using METAL. SNPs in AS-associated genes in this study were then imputed using MaCH, and association with AS tested by logistic regression. Results: SNPs in TNFRSF1A (rs4149577, p=8.2×10-4), STAT3 (rs2293152, p=0.0015; rs1053005, p=0.017) and ERAP1 (rs27038, p=0.0091; rs27037, p=0.0092) were significantly associated with AS in Han Chinese. Association was also observed between AS and the intergenic region 2p15 (rs10865331, p=0.023). The lack of association between AS and IL23R in Han Chinese was confirmed (all SNPs p>0.1). Conclusions: The study results demonstrate for the first time that genetic polymorphisms in STAT3, TNFRSF1A and 2p15 are associated with AS in Han Chinese, suggesting common pathogenic mechanisms for the disease in Chinese and Caucasian European populations. Furthermore, previous findings demonstrating that ERAP1, but not IL23R, is associated with AS in Chinese patients were confirmed.

Finnish HLA studies confirm the increased risk conferred by HLA-B27 homozygosity in ankylosing spondylitis

Objective: To determine the influence of HLA-B27 homozygosity and HLA-DRB1 alleles in the susceptibility to, and severity of, ankylosing spondylitis in a Finnish population. Methods: 673 individuals from 261 families with ankylosing spondylitis were genotyped for HLA-DRB1 alleles and HLA-B27 heterozygosity/ homozygosity. The frequencies of HLA-B27 homozygotes in probands from these families were compared with the expected number of HLA-B27 homozygotes in controls under Hardy-Weinberg equilibrium (HWE). The effect of HLA-DRB1 alleles was assessed using a logistic regression procedure conditioned on HLA-B27 and case-control analysis. Results: HLA-B27 was detected in 93% of cases of ankylosing spondylitis. An overrepresentation of HLA-B27 homozygotes was noted in ankylosing spondylitis (11%) compared with the expected number of HLA-B27 homozygotes under HWE (4%) (odds ratio (OR) = 3.3 (95% confidence interval, 1.6 to 6.8), p = 0.002). HLA-B27 homozygosity was marginally associated with reduced BASDAI (HLA-B27 homozygotes, 4.5 (1.6); HLA-B27 heterozygotes, 5.4 (1.8) (mean (SD)), p = 0.05). Acute anterior uveitis (AAU) was present in significantly more HLA-B27 positive cases (50%) than HLA-B27 negative cases (16%) (OR = 5.4 (1.7 to 17), p<0.004). HLA-B27 positive cases had a lower average age of symptom onset (26.7 (8.0) years) compared with HLA-B27 negative cases (35.7 (11.2) years) (p<0.0001). Conclusions: HLA-627 homozygosity is associated with a moderately increased risk of ankylosing spondylitis compared with HLA-β27 heterozygosity. HLA-B27 positive cases had an earlier age of onset of ankylosing spondylitis than HLA-B27 negative cases and were more likely to develop AAU. HLA-DRB1 alleles may influence the age of symptom onset of ankylosing spondylitis.

Induction of a Th1 immune response and suppression of IgE via immunotherapy with a recombinant hybrid molecule encapsulated in liposome-protamine-DNA nanoparticles in a model of experimental allergy

Liposome-protamine-DNA nanoparticles (LPD) are safe, effective, and non-toxic adjuvants that induce Th1-like immune responses. We hypothesized that encapsulation of allergens into liposomes could be an appropriate option for immunotherapy. The present study evaluated the immunotherapeutic potential of a recombinant hybrid molecule (rHM) encapsulated in LPD nanoparticles in a murine model of Chenopodium album allergy. BALB/c mice were sensitized with the allergen in alum, and the immunotherapy procedure was performed by subcutaneous injections of LPD-rHM, rHM, or empty LPD at weekly intervals. Sensitized mice developed a Th2-biased immune response characterized by strong specific IgG1 and IgE production, IL-4, and the transcription factor GATA3 in spleen cell cultures. Treatment with the LPD-rHM resulted in a reduction in IgE and a marked increase in IgG2a. The LPD-rHM induced allergen-specific responses with relatively high interferon-gamma production, as well as expression of the transcription factor T-bet in stimulated splenocytes. In addition, lymphoproliferative responses were higher in the LPD-rHM-treated mice than in the other groups. Removal of the nanoparticles from the rHM resulted in a decrease in the allergen's immunogenicity. These results indicate that the rHM complexed with LPD nanoparticles has a marked suppressive effect on the allergic response and caused a shift toward a Th1 pathway.

Identification of 15 new psoriasis susceptibility loci highlights the role of innate immunity

To gain further insight into the genetic architecture of psoriasis, we conducted a meta-analysis of 3 genome-wide association studies (GWAS) and 2 independent data sets genotyped on the Immunochip, including 10,588 cases and 22,806 controls. We identified 15 new susceptibility loci, increasing to 36 the number associated with psoriasis in European individuals. We also identified, using conditional analyses, five independent signals within previously known loci. The newly identified loci shared with other autoimmune diseases include candidate genes with roles in regulating T-cell function (such as RUNX3, TAGAP and STAT3). Notably, they included candidate genes whose products are involved in innate host defense, including interferon-mediated antiviral responses (DDX58), macrophage activation (ZC3H12C) and nuclear factor (NF)-κB signaling (CARD14 and CARM1). These results portend a better understanding of shared and distinctive genetic determinants of immune-mediated inflammatory disorders and emphasize the importance of the skin in innate and acquired host defense. © 2012 Nature America, Inc. All rights reserved.

Integrated genome-wide chromatin occupancy and expression analyses identify key myeloid pro-differentiation transcription factors repressed by Myb

To gain insight into the mechanisms by which the Myb transcription factor controls normal hematopoiesis and particularly, how it contributes to leukemogenesis, we mapped the genome-wide occupancy of Myb by chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) in ERMYB myeloid progenitor cells. By integrating the genome occupancy data with whole genome expression profiling data, we identified a Myb-regulated transcriptional program. Gene signatures for leukemia stem cells, normal hematopoietic stem/progenitor cells and myeloid development were overrepresented in 2368 Myb regulated genes. Of these, Myb bound directly near or within 793 genes. Myb directly activates some genes known critical in maintaining hematopoietic stem cells, such as Gfi1 and Cited2. Importantly, we also show that, despite being usually considered as a transactivator, Myb also functions to repress approximately half of its direct targets, including several key regulators of myeloid differentiation, such as Sfpi1 (also known as Pu.1), Runx1, Junb and Cebpb. Furthermore, our results demonstrate that interaction with p300, an established coactivator for Myb, is unexpectedly required for Myb-mediated transcriptional repression. We propose that the repression of the above mentioned key pro-differentiation factors may contribute essentially to Myb's ability to suppress differentiation and promote self-renewal, thus maintaining progenitor cells in an undifferentiated state and promoting leukemic transformation. © 2011 The Author(s).

Common variants in the region around Osterix are associated with bone mineral density and growth in childhood

Peak bone mass achieved in adolescence is a determinant of bone mass in later life. In order to identify genetic variants affecting bone mineral density (BMD), we performed a genome-wide association study of BMD and related traits in 1518 children from the Avon Longitudinal Study of Parents and Children (ALSPAC). We compared results with a scan of 134 adults with high or low hip BMD. We identified associations with BMD in an area of chromosome 12 containing the Osterix (SP7) locus, a transcription factor responsible for regulating osteoblast differentiation (ALSPAC: P = 5.8 × 10-4; Australia: P = 3.7 × 10-4). This region has previously shown evidence of association with adult hip and lumbar spine BMD in an Icelandic population, as well as nominal association in a UK population. A meta-analysis of these existing studies revealed strong association between SNPs in the Osterix region and adult lumbar spine BMD (P = 9.9 × 10-11). In light of these findings, we genotyped a further 3692 individuals from ALSPAC who had whole body BMD and confirmed the association in children as well (P = 5.4 × 10-5). Moreover, all SNPs were related to height in ALSPAC children, but not weight or body mass index, and when height was included as a covariate in the regression equation, the association with total body BMD was attenuated. We conclude that genetic variants in the region of Osterix are associated with BMD in children and adults probably through primary effects on growth.