COS1: an Arabidopsis coronatine insensitive1 suppressor essential for regulation of jasmonate-mediated plant defense and senescence.

The Arabidopsis thaliana CORONATINE INSENSITIVE1 (COI1) gene encodes an F-box protein to assemble SCF(COI1) complexes essential for response to jasmonates (JAs), which are a family of plant signaling molecules required for many essential functions, including plant defense and reproduction. To better understand the molecular basis of JA action, we screened for suppressors of coi1 and isolated a coi1 suppressor1 (cos1) mutant. The cos1 mutation restores the coi1-related phenotypes, including defects in JA sensitivity, senescence, and plant defense responses. The COS1 gene was cloned through a map-based approach and found to encode lumazine synthase, a key component in the riboflavin pathway that is essential for diverse yet critical cellular processes. We demonstrated a novel function for the riboflavin pathway that acts downstream of COI1 in the JA signaling pathway and is required for suppression of the COI1-mediated root growth, senescence, and plant defense.

The ASK1 and ASK2 genes are essential for Arabidopsis early development.

The requirement of CUL1 for Arabidopsis embryogenesis suggests that Skp1-CUL1-F-box protein (SCF) complexes play important roles during embryo development. Among the 21 Arabidopsis Skp1-like genes (ASKs), it is unknown which ASK gene(s) is essential for embryo development. In this study, we demonstrate a vital role for ASK1 and ASK2 in Arabidopsis embryogenesis and postembryonic development through analysis of the ask1 ask2 double mutant. Our detailed analysis indicates that the double mutations in both ASK1 and ASK2 affect cell division and cell expansion/elongation and cause a developmental delay during embryogenesis and lethality in seedling growth. The expression patterns of ASK1 and ASK2 were examined further and found to be consistent with their roles in embryogenesis and seedling development. We propose that mutations in ASK1 and ASK2 abolish all of the ASK1- and ASK2-based SCF and non-SCF complexes, resulting in alteration of gene expression and leading to defects in growth and development.

Fosfomycin and tobramycin in combination downregulate nitrate reductase genes narG and narH, resulting in increased activity against Pseudomonas aeruginosa under anaerobic conditions

The activity of aminoglycosides, used to treat Pseudomonas aeruginosa respiratory infection in cystic fibrosis (CF) patients, is reduced under the anaerobic conditions that reflect the CF lung in vivo. In contrast, a 4:1 (w/w) combination of fosfomycin and tobramycin (F:T), under investigation for use in the treatment of CF lung infection, has increased activity against P. aeruginosa under anaerobic conditions. The aim of this study was to elucidate the mechanisms underlying the increased activity of F:T under anaerobic conditions. Microarray analysis was used to identify the transcriptional basis of increased F:T activity under anaerobic conditions, and key findings were confirmed by microbiological tests including nitrate utilization assays, growth curves and susceptibility testing. Notably, growth in sub-inhibitory concentrations of F:T, but not tobramycin or fosfomycin alone, significantly downregulated (p <0.05) nitrate reductase genes narG and narH, essential for normal anaerobic growth of P. aeruginosa. Under anaerobic conditions, F:T significantly decreased (p

A novel single base deletion in the LDLR gene (211delG):Effect on serum lipid profiles and the influence of other genetic polymorphisms in the ACE, APOE and APOB genes

A single base deletion (211delG) in the low density lipoprotein receptor (LDLR) gene was shown to cause familial hypercholesterolaemia (FH) in a large family from Northern Ireland. Twenty-four of 52 family members tested had this mutation, 13 of which were newly diagnosed. Mutation-positive individuals had significantly higher mean total-cholesterol (TC) and LDL-cholesterol (LDL-C) than those without 211delG. LDL-C was a more accurate indicator of disease status than TC, When TC levels alone were considered, in individuals over 16 years, a false negative rate (TC <7.5 mmol/l) of 40% was found; however, this fell to 13% based on inclusion of LDL-C levels. Individuals with coronary artery disease (CAD) had significantly higher TC levels than those without CAD and tended to have tendinous xanthomas (TX) and corneal arcus (CA). Genetic polymorphisms in the angiotensin converting enzyme (ACE) and apolipoprotein (ape) B genes did not appear to be associated with lipid levels or with the clinical severity of the disease; however, the apo E e4 allele did show a lipid-raising effect in individuals with the mutation.

Genome-wide scans of three independent sets of 90 Irish multiplex schizophrenia families and follow-up of selected regions in all families provides evidence for multiple susceptibility genes

From our linkage study of Irish families with a high density of schizophrenia, we have previously reported evidence for susceptibility genes in regions 5q21-31, 6p24-21, 8p22-21, and 10p15-p11. In this report, we describe the cumulative results from independent genome scans of three a priori random subsets of 90 families each, and from multipoint analysis of all 270 families in ten regions. Of these ten regions, three (13q32, 18p11-q11, and 18q22-23) did not generate scores above the empirical baseline pairwise scan results, and one (6q13-26) generated a weak signal. Six other regions produced more positive pairwise and multipoint results. They showed the following maximum multipoint H-LOD (heterogeneity LOD) and NPL scores: 2p14-13: 0.89 (P = 0.06) and 2.08 (P = 0.02), 4q24-32: 1.84 (P = 0.007) and 1.67 (P = 0.03), 5q21-31: 2.88 (P= 0.0007), and 2.65 (P = 0.002), 6p25-24: 2.13 (P = 0.005) and 3.59 (P = 0.0005), 6p23: 2.42 (P = 0.001) and 3.07 (P = 0.001), 8p22-21: 1.57 (P = 0.01) and 2.56 (P = 0.005), 10p15-11: 2.04 (P = 0.005) and 1.78 (P = 0.03). The degree of 'internal replication' across subsets differed, with 5q, 6p, and 8p being most consistent and 2p and 10p being least consistent. On 6p, the data suggested the presence of two susceptibility genes, in 6p25-24 and 6p23-22. Very few families were positive on more than one region, and little correlation between regions was evident, suggesting substantial locus heterogeneity. The levels of statistical significance were modest, as expected from loci contributing to complex traits. However, our internal replications, when considered along with the positive results obtained in multiple other samples, suggests that most of these six regions are likely to contain genes that influence liability to schizophrenia.

Haplotypes spanning SPEC2, PDZ-GEF2 and ACSL6 genes are associated with schizophrenia

Chromosome 5q22-33 is a region where studies have repeatedly found evidence for linkage to schizophrenia. In this report, we took a stepwise approach to systematically map this region in the Irish Study of High Density Schizophrenia Families (ISHDSF, 267 families, 1337 subjects) sample. We typed 289 SNPs in the critical interval of 8 million basepairs and found a 758 kb interval coding for the SPEC2/PDZ-GEF2/ACSL6 genes to be associated with the disease. Using sex and genotype-conditioned transmission disequilibrium test analyses, we found that 19 of the 24 typed markers were associated with the disease and the associations were sex-specific. We replicated these findings with an Irish case-control sample (657 cases and 414 controls), an Irish parent-proband trio sample (187 families, 564 subjects), a German nuclear family sample (211 families, 751 subjects) and a Pittsburgh nuclear family sample (247 families, 729 subjects). In all four samples, we replicated the sex-specific associations at the levels of both individual markers and haplotypes using sex- and genotype-conditioned analyses. Three risk haplotypes were identified in the five samples, and each haplotype was found in at least two samples. Consistent with the discovery of multiple estrogen-response elements in this region, our data showed that the impact of these haplotypes on risk for schizophrenia differed in males and females. From these data, we concluded that haplotypes underlying the SPEC2/PDZ-GEF2/ACSL6 region are associated with schizophrenia. However, due to the extended high LD in this region, we were unable to distinguish whether the association signals came from one or more of these genes.

S100A2 is a BRCA1/p63 coregulated tumour suppressor gene with roles in the regulation of mutant p53 stability

Here, we show for the first time that the familial breast/ovarian cancer susceptibility gene, BRCA1, along with interacting ΔNp63 proteins, transcriptionally upregulate the putative tumour suppressor protein, S100A2. Both BRCA1 and ΔNp63 proteins are required for S100A2 expression. BRCA1 requires ΔNp63 proteins for recruitment to the S100A2 proximal promoter region, while exogenous expression of individual ΔNp63 proteins cannot activate S100A2 transcription in the absence of a functional BRCA1. Consequently, mutation of the ΔNp63/p53 response element within the S100A2 promoter completely abrogates the ability of BRCA1 to upregulate S100A2. S100A2 shows growth control features in a range of cell models. Transient or stable exogenous S100A2 expression inhibits the growth of BRCA1 mutant and basal-like breast cancer cell lines, while short interfering RNA (siRNA) knockdown of S100A2 in non-tumorigenic cells results in enhanced proliferation. S100A2 modulates binding of mutant p53 to HSP90, which is required for efficient folding of mutant p53 proteins, by competing for binding to HSP70/HSP90 organising protein (HOP). HOP is a cochaperone that is required for the efficient transfer of proteins from HSP70 to HSP90. Loss of S100A2 leads to an HSP90-dependent stabilisation of mutant p53 with a concomitant loss of p63. Accordingly, S100A2-deficient cells are more sensitive to the HSP-90 inhibitor, 17-N-allylamino-17-demethoxygeldanamycin, potentially representing a novel therapeutic strategy for S100A2- and BRCA1-deficient cancers. Taken together, these data demonstrate the importance of S100A2 downstream of the BRCA1/ΔNp63 signalling axis in modulating transcriptional responses and enforcing growth control mechanisms through destabilisation of mutant p53.

The prognostic significance of the aberrant extremes of p53 immunophenotypes in breast cancer

Aims: The utility of p53 as a prognostic assay has been elusive. The aims of this study were to describe a novel, reproducible scoring system and assess the relationship between differential p53 immunohistochemistry (IHC) expression patterns, TP53 mutation status and patient outcomes in breast cancer.

Methods and Results: Tissue microarrays were used to study p53 IHC expression patterns: expression was defined as extreme positive (EP), extreme negative (EN), and non-extreme (NE; intermediate patterns). Overall survival (OS) was used to define patient outcome. A representative subgroup (n = 30) showing the various p53 immunophenotypes was analysed for TP53 hotspot mutation status (exons 4-9). Extreme expression of any type occurred in 176 of 288 (61%) cases. As compared with NE expression, EP expression was significantly associated (P = 0.039) with poorer OS. In addition, as compared with NE expression, EN expression was associated (P = 0.059) with poorer OS. Combining cases showing either EP or EN expression better predicted OS than either pattern alone (P = 0.028). This combination immunophenotype was significant in univariate but not multivariate analysis. In subgroup analysis, six substitution exon mutations were detected, all corresponding to extreme IHC phenotypes. Five missense mutations corresponded to EP staining, and the nonsense mutation corresponded to EN staining. No mutations were detected in the NE group.

Conclusions: Patients with extreme p53 IHC expression have a worse OS than those with NE expression. Accounting for EN as well as EP expression improves the prognostic impact. Extreme expression positively correlates with nodal stage and histological grade, and negatively with hormone receptor status. Extreme expression may relate to specific mutational status.

Genome-wide characterization reveals complex interplay between TP53 and TP63 in response to genotoxic stress

In response to genotoxic stress the TP53 tumour suppressor activates target gene expression to induce cell cycle arrest or apoptosis depending on the extent of DNA damage. These canonical activities can be repressed by TP63 in normal stratifying epithelia to maintain proliferative capacity or drive proliferation of squamous cell carcinomas, where TP63 is frequently overexpressed/amplified. Here we use ChIP-sequencing, integrated with microarray analysis, to define the genome-wide interplay between TP53 and TP63 in response to genotoxic stress in normal cells. We reveal that TP53 and TP63 bind to overlapping, but distinct cistromes of sites through utilization of distinctive consensus motifs and that TP53 is constitutively bound to a number of sites. We demonstrate that cisplatin and adriamycin elicit distinct effects on TP53 and TP63 binding events, through which TP53 can induce or repress transcription of an extensive network of genes by direct binding and/or modulation of TP63 activity. Collectively, this results in a global TP53-dependent repression of cell cycle progression, mitosis and DNA damage repair concomitant with activation of anti-proliferative and pro-apoptotic canonical target genes. Further analyses reveal that in the absence of genotoxic stress TP63 plays an important role in maintaining expression of DNA repair genes, loss of which results in defective repair.

Molecular surveillance for carbapenemase genes in carbapenem-resistant Pseudomonas aeruginosa in Australian patients with cystic fibrosis.

Summary: The aim of this study was to assess the prevalence of acquired carbapenemase genes amongst carbapenem non-susceptible Pseudomonas aeruginosa isolates in Australian patients with cystic fibrosis (CF). Cross-sectional molecular surveillance for acquired carbapenemase genes was performed on CF P. aeruginosa isolates from two isolate banks comprising: (i) 662 carbapenem resistant P. aeruginosa isolates from 227 patients attending 10 geographically diverse Australian CF centres (2007-2009), and (ii) 519 P. aeruginosa isolates from a cohort of 173 adult patients attending one Queensland CF clinic in 2011. All 1189 P. aeruginosa isolates were tested by polymerase chain reaction (PCR) protocols targeting ten common carbapenemase genes, as well the Class 1 integron intI1 gene and the aadB aminoglycoside resistance gene. No carbapenemase genes were identified among all isolates tested. The intI1 and aadB genes were frequently detected and were significantly associated with the AUST-02 strain (OR 24.6, 95% CI 9.3-65.6; p < 0.0001) predominantly from Queensland patients. Despite the high prevalence of carbapenem resistance in P. aeruginosa in Australian patients with CF, no acquired carbapenemase genes were detected in the study, suggesting chromosomal mutations remain the key resistance mechanism in CF isolates. Systematic surveillance for carbapenemase-producing P. aeruginosa in CF by molecular surveillance is ongoing.

Identification of Pseudomonas aeruginosa by a duplex real-time polymerase chain reaction assay targeting the ecfX and the gyrB genes

Phenotypic identification of Gram-negative bacteria from respiratory specimens of patients with cystic fibrosis carries a high risk of misidentification. Molecular identification techniques that use single-gene targets are also susceptible to error, including cross-reaction issues with other Gram-negative organisms. In this study, we have designed a Pseudomonas aeruginosa duplex real-time polymerase chain reaction (PCR) (PAduplex) assay targeting the ecfX and the gyrB genes. The PAduplex was evaluated against a panel of 91 clinical and environmental isolates that were presumptively identified as P. aeruginosa. The results were compared with those obtained using a commercial biochemical identification kit and several other P. aeruginosa PCR assays. The results showed that the PAduplex assay is highly suitable for routine identification of P. aeruginosa isolates from clinical or environmental samples. The 2-target format provides simultaneous confirmation of P. aeruginosa identity where both the ecfX and gyrB PCR reactions are positive and may also reduce the potential for false negatives caused by sequence variation in primer or probe targets.