A complete lipopolysaccharide inner core oligosaccharide is required for resistance of Burkholderia cenocepacia to antimicrobial peptides and bacterial survival in vivo

Burkholderia cenocepacia is an important opportunistic pathogen of patients with cystic fibrosis. This bacterium is inherently resistant to a wide range of antimicrobial agents, including high concentrations of antimicrobial peptides. We hypothesized that the lipopolysaccharide (LPS) of B. cenocepacia is important for both virulence and resistance to antimicrobial peptides. We identified hldA and hldD genes in B. cenocepacia strain K56-2. These two genes encode enzymes involved in the modification of heptose sugars prior to their incorporation into the LPS core oligosaccharide. We constructed a mutant, SAL1, which was defective in expression of both hldA and hldD, and by performing complementation studies we confirmed that the functions encoded by both of these B. cenocepacia genes were needed for synthesis of a complete LPS core oligosaccharide. The LPS produced by SAL1 consisted of a short lipid A-core oligosaccharide and was devoid of O antigen. SAL1 was sensitive to the antimicrobial peptides polymyxin B, melittin, and human neutrophil peptide 1. In contrast, another B. cenocepacia mutant strain that produced complete lipid A-core oligosaccharide but lacked polymeric O antigen was not sensitive to polymyxin B or melittin. As determined by the rat agar bead model of lung infection, the SAL1 mutant had a survival defect in vivo since it could not be recovered from the lungs of infected rats 14 days postinfection. Together, these data show that the B. cenocepacia LPS inner core oligosaccharide is needed for in vitro resistance to three structurally unrelated antimicrobial peptides and for in vivo survival in a rat model of chronic lung infection.

Defective O-antigen polymerization in tolA and pal mutants of Escherichia coli in response to extracytoplasmic stress

We have previously shown that the TolA protein is required for the correct surface expression of the Escherichia coli O7 antigen lipopolysaccharide (LPS). In this work, delta tolA and delta pal mutants of E. coli K-12 W3110 were transformed with pMF19 (encoding a rhamnosyltransferase that reconstitutes the expression of O16-specific LPS), pWQ5 (encoding the Klebsiella pneumoniae O1 LPS gene cluster), or pWQ802 (encoding the genes necessary for the synthesis of Salmonella enterica O:54). Both DeltatolA and delta pal mutants exhibited reduced surface expression of O16 LPS as compared to parental W3110, but no significant differences were observed in the expression of K. pneumoniae O1 LPS and S. enterica O:54 LPS. Therefore, TolA and Pal are required for the correct surface expression of O antigens that are assembled in a wzy (polymerase)-dependent manner (like those of E. coli O7 and O16) but not for O antigens assembled by wzy-independent pathways (like K. pneumoniae O1 and S. enterica O:54). Furthermore, we show that the reduced surface expression of O16 LPS in delta tolA and delta pal mutants was associated with a partial defect in O-antigen polymerization and it was corrected by complementation with intact tolA and pal genes, respectively. Using derivatives of W3110 delta tolA and W3110 delta pal containing lacZ reporter fusions to fkpA and degP, we also demonstrate that the RpoE-mediated extracytoplasmic stress response is upregulated in these mutants. Moreover, an altered O16 polymerization was also detected under conditions that stimulate RpoE-mediated extracytoplasmic stress responses in tol+ and pal+ genetic backgrounds. A Wzy derivative with an epitope tag at the C-terminal end of the protein was stable in all the mutants, ruling out stress-mediated proteolysis of Wzy. We conclude that the absence of TolA and Pal elicits a sustained extracytoplasmic stress response that in turn reduces O-antigen polymerization but does not affect the stability of the Wzy O-antigen polymerase.

A population-based association study of SNPs of GSTP1, MnSOD, GPX2 and Barrett's esophagus and esophageal adenocarcinoma

Oxidative stress appears to be important in the pathogenesis of Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). Single-nucleotide polymorphisms (SNPs) of antioxidant enzyme genes may play a part in determining individual susceptibility to these diseases. The Factors Influencing the Barrett's Adenocarcinoma Relationship (FINBAR) study is a population-based, case-control study of BE and EAC in Ireland. DNA from EAC (n = 207), BE (> or =3 cm BE at endoscopy with specialized intestinal metaplasia on biopsy, n = 189) and normal population controls (n = 223) were analyzed. Several SNPs spanning the genes for glutathione S-transferase P1 (GSTP1), manganese superoxide dismutase (MnSOD) and glutathione peroxidase 2 (GPX2) were genotyped using multiplex polymerase chain reaction and SNaPshottrade mark. The chi(2) test was used to compare genotype and allele frequencies between case and control subjects. Linkage disequilibrium between SNPs was quantified using Lewontin's D' value and haplotype frequency estimates obtained using Haploview. Eleven SNPs were genotyped (six for GSTP1, three for MnSOD and two for GPX2); all were in Hardy-Weinberg equilibrium. None was significantly associated with EAC or BE even before Bonferroni correction. Odds ratios for EAC for individual SNPs ranged from 0.68 [95% confidence interval (CI) 0.43-1.08] to 1.25 (95% CI 0.73-2.16), and for BE from 0.84 (95% CI 0.52-1.30) to 1.30 (95% CI 0.85-1.97). SNPs in all three genes were in strong linkage disequilibrium (D' > 0.887) but haplotype analysis did not show any significant association with EAC or BE. SNPs involving the GSTP1, MnSOD and GPX2 genes were not associated with BE or EAC. Further studies aimed at identifying susceptibility genes should focus on different antioxidant genes or different pathways.

Progressive lung cancer determined by expression profiling and transcriptional regulation

Clinically, our ability to predict disease outcome for patients with early stage lung cancer is currently poor. To address this issue, tumour specimens were collected at surgery from non-small cell lung cancer (NSCLC) patients as part of the European Early Lung Cancer (EUELC) consortium. The patients were followed-up for three years post-surgery and patients who suffered progressive disease (PD, tumour recurrence, metastasis or a second primary) or remained disease-free (DF) during follow-up were identified. RNA from both tumour and adjacent-normal lung tissue was extracted from patients and subjected to microarray expression profiling. These samples included 36 adenocarcinomas and 23 squamous cell carcinomas from both PD and DF patients. The microarray data was subject to a series of systematic bioinformatics analyses at gene, network and transcription factor levels. The focus of these analyses was 2-fold: firstly to determine whether there were specific biomarkers capable of differentiating between PD and DF patients, and secondly, to identify molecular networks which may contribute to the progressive tumour phenotype. The experimental design and analyses performed permitted the clear differentiation between PD and DF patients using a set of biomarkers implicated in neuroendocrine signalling and allowed the inference of a set of transcription factors whose activity may differ according to disease outcome. Potential links between the biomarkers, the transcription factors and the genes p21/CDKN1A and Myc, which have previously been implicated in NSCLC development, were revealed by a combination of pathway analysis and microarray meta-analysis. These findings suggest that neuroendocrine-related genes, potentially driven through p21/CDKN1A and Myc, are closely linked to whether or not a NSCLC patient will have poor clinical outcome.

Vasculogenic and osteogenesis-enhancing potential of human umbilical cord blood endothelial colony-forming cells

Umbilical cord blood-derived endothelial colony-forming cells (UCB-ECFC) show utility in neovascularization, but their contribution to osteogenesis has not been defined. Cocultures of UCB-ECFC with human fetal-mesenchymal stem cells (hfMSC) resulted in earlier induction of alkaline phosphatase (ALP) (Day 7 vs. 10) and increased mineralization (1.9×; p <.001) compared to hfMSC monocultures. This effect was mediated through soluble factors in ECFC-conditioned media, leading to 1.8-2.2× higher ALP levels and a 1.4-1.5× increase in calcium deposition (p <.01) in a dose-dependent manner. Transcriptomic and protein array studies demonstrated high basal levels of osteogenic (BMPs and TGF-ßs) and angiogenic (VEGF and angiopoietins) regulators. Comparison of defined UCB and adult peripheral blood ECFC showed higher osteogenic and angiogenic gene expression in UCB-ECFC. Subcutaneous implantation of UCB-ECFC with hfMSC in immunodeficient mice resulted in the formation of chimeric human vessels, with a 2.2-fold increase in host neovascularization compared to hfMSC-only implants (p = .001). We conclude that this study shows that UCB-ECFC have potential in therapeutic angiogenesis and osteogenic applications in conjunction with MSC. We speculate that UCB-ECFC play an important role in skeletal and vascular development during perinatal development but less so in later life when expression of key osteogenesis and angiogenesis genes in ECFC is lower.

Isolation and characterization of a novel simazine-degrading bacterium from agricultural soil of central Chile, Pseudomonas sp MHP41

s-Triazine herbicides are used extensively in South America in agriculture and forestry. In this study, a bacterium designated as strain MHP41, capable of degrading simazine and atrazine, was isolated from agricultural soil in the Quillota valley, central Chile. Strain MHP41 is able to grow in minimal medium, using simazine as the sole nitrogen source. In this medium, the bacterium exhibited a growth rate of mu = 0.10 h(-1), yielding a high biomass of 4.2 x 10(8) CFU mL(-1). Resting cells of strain MHP41 degrade more than 80% of simazine within 60 min. The atzA, atzB, atzC, atzD, atzE and atzF genes encoding the enzymes of the simazine upper and lower pathways were detected in strain MHP41. The motile Gram-negative bacterium was identified as a Pseudomonas sp., based on the Biolog microplate system and comparative sequence analyses of the 16S rRNA gene. Amplified ribosomal DNA restriction analysis allowed the differentiation of strain MHP41 from Pseudomonas sp. ADP. The comparative 16S rRNA gene sequence analyses suggested that strain MHP41 is closely related to Pseudomonas nitroreducens and Pseudomonas multiresinovorans. This is the first s-triazine-degrading bacterium isolated in South America. Strain MHP41 is a potential biocatalyst for the remediation of s-triazine-contaminated environments.

Dam Methylation Participates in the Regulation of PmrA/PmrB and RcsC/RcsD/RcsB Two Component Regulatory Systems in Salmonella enterica Serovar Enteritidis

The absence of Dam in Salmonella enterica serovar Enteritidis causes a defect in lipopolysaccharide (LPS) pattern associated to a reduced expression of wzz gene. Wzz is the chain length regulator of the LPS O-antigen. Here we investigated whether Dam regulates wzz gene expression through its two known regulators, PmrA and RcsB. Thus, the expression of rcsB and pmrA was monitored by quantitative real-time RT-PCR and Western blotting using fusions with 3×FLAG tag in wild type (wt) and dam strains of S. Enteritidis. Dam regulated the expression of both rcsB and pmrA genes; nevertheless, the defect in LPS pattern was only related to a diminished expression of RcsB. Interestingly, regulation of wzz in serovar Enteritidis differed from that reported earlier for serovar Typhimurium; RcsB induces wzz expression in both serovars, whereas PmrA induces wzz in S. Typhimurium but represses it in serovar Enteritidis. Moreover, we found that in S. Enteritidis there is an interaction between both wzz regulators: RcsB stimulates the expression of pmrA and PmrA represses the expression of rcsB. Our results would be an example of differential regulation of orthologous genes expression, providing differences in phenotypic traits between closely related bacterial serovars.

Molecular similarity between myelodysplastic form of chronic myelomonocytic leukemia and refractory anemia with ring sideroblasts

Chronic myelomonocytic leukemia is similar to but a separate entity from both myeloproliferative neoplasms and myelodysplastic syndromes, and shows either myeloproliferative or myelodysplastic features. We ask whether this distinction may have a molecular basis. We established the gene expression profiles of 39 samples of chronic myelomonocytic leukemia (including 12 CD34-positive) and 32 CD34-positive samples of myelodysplastic syndromes by using Affymetrix microarrays, and studied the status of 18 genes by Sanger sequencing and array-comparative genomic hybridization in 53 samples. Analysis of 12 mRNAS from chronic myelomonocytic leukemia established a gene expression signature of 122 probe sets differentially expressed between proliferative and dysplastic cases of chronic myelomonocytic leukemia. As compared to proliferative cases, dysplastic cases over-expressed genes involved in red blood cell biology. When applied to 32 myelodysplastic syndromes, this gene expression signature was able to discriminate refractory anemias with ring sideroblasts from refractory anemias with excess of blasts. By comparing mRNAS from these two forms of myelodysplastic syndromes we derived a second gene expression signature. This signature separated the myelodysplastic and myeloproliferative forms of chronic myelomonocytic leukemias. These results were validated using two independent gene expression data sets. We found that myelodysplastic chronic myelomonocytic leukemias are characterized by mutations in transcription/epigenetic regulators (ASXL1, RUNX1, TET2) and splicing genes (SRSF2) and the absence of mutations in signaling genes. Myelodysplastic chronic myelomonocytic leukemias and refractory anemias with ring sideroblasts share a common expression program suggesting they are part of a continuum, which is not totally explained by their similar but not, however, identical mutation spectrum. © 2013 Ferrata Storti Foundation.

Elevated NRD1 metalloprotease expression plays a role in breast cancer growth and proliferation

Understanding the molecular etiology of cancer and increasing the number of drugs and their targets are critical to cancer management. In our attempt to unravel novel breast-cancer associated proteins, we previously conducted protein expression profiling of the MCF10AT model, which comprises a series of isogenic cell lines that mimic different stages of breast cancer progression. NRD1 expression was found to increase during breast cancer progression. Here, we attempted to confirm the relevance of NRD1 in clinical breast cancer and understand the functional role and mechanism of NRD1 in breast cancer cells. Immunohistochemistry data show that NRD1 expression was elevated in ductal carcinoma in situ and invasive ductal carcinomas compared with normal tissues in 30% of the 26 matched cases studied. Examination of NRD1 expression in tissue microarray comprising >100 carcinomas and subsequent correlation with clinical data revealed that NRD1 expression was significantly associated with tumor size, grade, and nodal status (P <0.05). Silencing of NRD1 reduced MCF10CA1h and MDA-MD-231 breast-cancer-cell proliferation and growth. Probing the oncogenic EGF signaling pathways revealed that NRD1 knock down did not affect overall downstream tyrosine phosphorylation cascades including AKT and MAPK activation. Instead, silencing of NRD1 resulted in a reduction of overall cyclin D1 expression, a reduction of EGF-induced increase in cyclin D1 expression and an increase in apoptotic cell population compared with control cells.

Prognostic value of TP53, KRAS and EGFR mutations in nonsmall cell lung cancer:The EUELC cohort

Nonsmall cell lung cancer samples from the European Early Lung Cancer biobank were analysed to assess the prognostic significance of mutations in the TP53, KRAS and EGFR genes. The series included 11 never-smokers, 86 former smokers, 152 current smokers and one patient without informed smoking status. There were 110 squamous cell carcinomas (SCCs), 133 adenocarcinomas (ADCs) and seven large cell carcinomas or mixed histologies. Expression of p53 was analysed by immunohistochemistry. DNA was extracted from frozen tumour tissues. TP53 mutations were detected in 48.8% of cases and were more frequent among SCCs than ADCs (p

DNA hypermethylation and DNA hypomethylation is present at different loci in chronic kidney disease

Genetic risk factors for chronic kidney disease (CKD) are being identified through international collaborations. By comparison, epigenetic risk factors for CKD have only recently been considered using population-based approaches. DNA methylation is a major epigenetic modification that is associated with complex diseases, so we investigated methylome-wide loci for association with CKD. A total of 485,577 unique features were evaluated in 255 individuals with CKD (cases) and 152 individuals without evidence of renal disease (controls). Following stringent quality control, raw data were quantile normalized and β values calculated to reflect the methylation status at each site. The difference in methylation status was evaluated between cases and controls with resultant P values adjusted for multiple testing. Genes with significantly increased and decreased levels of DNA methylation were considered for biological relevance by functional enrichment analysis using KEGG pathways in Partek Genomics Suite. Twenty-three genes, where more than one CpG per loci was identified with Padjusted < 10−8, demonstrated significant methylation changes associated with CKD and additional support for these associated loci was sought from published literature. Strong biological candidates for CKD that showed statistically significant differential methylation include CUX1, ELMO1, FKBP5, INHBA-AS1, PTPRN2, and PRKAG2 genes; several genes are differentially methylated in kidney tissue and RNA-seq supports a functional role for differential methylation in ELMO1 and PRKAG2 genes. This study reports the largest, most comprehensive, genome-wide quantitative evaluation of DNA methylation for association with CKD. Evidence confirming methylation sites influence development of CKD would stimulate research to identify epigenetic therapies that might be clinically useful for CKD.

Variants in the 1q21 risk region are associated with a visual endophenotype of autism and schizophrenia

Deficits in sensitivity to visual stimuli of low spatial frequency and high temporal frequency (so-called frequency-doubled gratings) have been demonstrated both in schizophrenia and in autism spectrum disorder (ASD). Such basic perceptual functions are ideal candidates for molecular genetic study, because the underlying neural mechanisms are well characterized; but they have sometimes been overlooked in favor of cognitive and neurophysiological endophenotypes, for which neural substrates are often unknown. Here, we report a genome-wide association study of a basic visual endophenotype associated with psychological disorder. Sensitivity to frequency-doubled gratings was measured in 1060 healthy young adults, and analyzed for association with genotype using linear regression at 642758 single nucleotide polymorphism (SNP) markers. A significant association (P=7.9×10) was found with the SNP marker rs1797052, situated in the 5′-untranslated region of PDZK1; each additional copy of the minor allele was associated with an increase in sensitivity equivalent to more than half a standard deviation. A permutation procedure, which accounts for multiple testing, showed that the association was significant at the α=0.005 level. The region on chromosome 1q21.1 surrounding PDZK1 is an established susceptibility locus both for schizophrenia and for ASD, mirroring the common association of the visual endophenotype with the two disorders. PDZK1 interacts with N-methyl-d-aspartate receptors and neuroligins, which have been implicated in the etiologies of schizophrenia and ASD. These findings suggest that perceptual abnormalities observed in two different disorders may be linked by common genetic elements. © 2013 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

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.

Genome Wide Association Mapping of Grain Arsenic, Copper, Molybdenum and Zinc in Rice (Oryza sativa L.) Grown at Four International Field Sites

The mineral concentrations in cereals are important for human health, especially for individuals who consume a cereal subsistence diet. A number of elements, such as zinc, are required within the diet, while some elements are toxic to humans, for example arsenic. In this study we carry out genome-wide association (GWA) mapping of grain concentrations of arsenic, copper, molybdenum and zinc in brown rice using an established rice diversity panel of,300 accessions and 36.9 k single nucleotide polymorphisms (SNPs). The study was performed across five environments: one field site in Bangladesh, one in China and two in the US, with one of the US sites repeated over two years. GWA mapping on the whole dataset and on separate subpopulations of rice revealed a large number of loci significantly associated with variation in grain arsenic, copper, molybdenum and zinc. Seventeen of these loci were detected in data obtained from grain cultivated in more than one field location, and six co-localise with previously identified quantitative trait loci. Additionally, a number of candidate genes for the uptake or transport of these elements were located near significantly associated SNPs (within 200 kb, the estimated global linkage disequilibrium previously employed in this rice panel). This analysis highlights a number of genomic regions and candidate genes for further analysis as well as the challenges faced when mapping environmentally-variable traits in a highly genetically structured diversity panel.

Recombinant Subgroup B Human Respiratory Syncytial Virus Expressing Enhanced Green Fluorescent Protein Efficiently Replicates in Primary Human Cells and Is Virulent in Cotton Rats

Human respiratory syncytial virus (HRSV) is the most important viral cause of severe respiratory tract disease in infants. Two subgroups (A and B) have been identified, which cocirculate during, or alternate between, yearly epidemics and cause indistinguishable disease. Existing in vitro and in vivo models of HRSV focus almost exclusively on subgroup A viruses. Here, a recombinant (r) subgroup B virus (rHRSV(B05)) was generated based on a consensus genome sequence obtained directly from an unpassaged clinical specimen from a hospitalized infant. An additional transcription unit containing the gene encoding enhanced green fluorescent protein (EGFP) was introduced between the phosphoprotein and matrix genes (position 5) of the genome to generate rHRSV(B05)EGFP(5). The recombinant viruses replicated efficiently in both HEp-2 cells and in well-differentiated normal human bronchial cells grown at air-liquid interface. Intranasal infection of cotton rats (Sigmodon hispidus) resulted in high numbers of EGFP(+) cells in epithelia of the nasal septum and conchae. When administered in a relatively large inoculum volume, the virus also replicated efficiently in bronchiolar epithelial cells and spread extensively in both the upper and lower respiratory tracts. Virus replication was not observed in ciliated epithelial cells of the trachea. This is the first virulent rHRSV strain with the genetic composition of a currently circulating wild-type virus. In vivo tracking of infected cells by means of EGFP fluorescence in the absence of cytopathic changes increases the sensitivity of virus detection in HRSV pathogenesis studies.

IMPORTANCE

Virology as a discipline has depended on monitoring cytopathic effects following virus culture in vitro. However, wild-type viruses isolated from patients often do not cause significant changes to infected cells, necessitating blind passage. This can lead to genetic and phenotypic changes and the generation of high-titer, laboratory-adapted viruses with diminished virulence in animal models of disease. To address this, we determined the genome sequence of an unpassaged human respiratory syncytial virus from a sample obtained directly from an infected infant, assembled a molecular clone, and recovered a wild-type recombinant virus. Addition of a gene encoding enhanced green fluorescent protein allowed this wild-type virus to be tracked in primary human cells and living animals in the absence of significant cytopathic effects. Imaging of fluorescent cells proved to be a highly valuable tool for monitoring the spread of virus and may help improve assays for evaluating novel intervention strategies.