Transcriptional responses of Burkholderia cenocepacia to polymyxin B in isogenic strains with diverse polymyxin B resistance phenotypes

<p>Abstract</p> <p>Background</p> <p><it>Burkholderia cenocepacia </it>is a Gram-negative opportunistic pathogen displaying high resistance to antimicrobial peptides and polymyxins. We identified mechanisms of resistance by analyzing transcriptional changes to polymyxin B treatment in three isogenic <it>B. cenocepacia </it>strains with diverse polymyxin B resistance phenotypes: the polymyxin B-resistant parental strain K56-2, a polymyxin B-sensitive K56-2 mutant strain with heptoseless lipopolysaccharide (LPS) (RSF34), and a derivative of RSF34 (RSF34 4000B) isolated through multiple rounds of selection in polymyxin B that despite having a heptoseless LPS is highly polymyxin B-resistant.</p> <p>Results</p> <p>A heptoseless LPS mutant of <it>B. cenocepacia </it>was passaged through multiple rounds of selection to regain high levels of polymyxin B-resistance. This process resulted in various phenotypic changes in the isolate that could contribute to polymyxin B resistance and are consistent with LPS-independent changes in the outer membrane. The transcriptional response of three <it>B. cenocepacia </it>strains to subinhibitory concentrations of polymyxin B was analyzed using microarray analysis and validated by quantitative Real Time-PCR. There were numerous baseline changes in expression between the three strains in the absence of polymyxin B. In both K56-2 and RSF34, similar transcriptional changes upon treatment with polymyxin B were found and included upregulation of various genes that may be involved in polymyxin B resistance and downregulation of genes required for the synthesis and operation of flagella. This last result was validated phenotypically as both swimming and swarming motility were impaired in the presence of polymyxin B. RSF34 4000B had altered the expression in a larger number of genes upon treatment with polymyxin B than either K56-2 or RSF34, but the relative fold-changes in expression were lower.</p> <p>Conclusions</p> <p>It is possible to generate polymyxin B-resistant isolates from polymyxin B-sensitive mutant strains of <it>B. cenocepacia</it>, likely due to the multifactorial nature of polymyxin B resistance of this bacterium. Microarray analysis showed that <it>B. cenocepacia </it>mounts multiple transcriptional responses following exposure to polymyxin B. Polymyxin B-regulated genes identified in this study may be required for polymyxin B resistance, which must be tested experimentally. Exposure to polymyxin B also decreases expression of flagellar genes resulting in reduced swimming and swarming motility.</p

Detection of copy number variations in rice using array-based comparative genomic hybridization

<p>Abstract</p> <p>Background</p> <p>Copy number variations (CNVs) can create new genes, change gene dosage, reshape gene structures, and modify elements regulating gene expression. As with all types of genetic variation, CNVs may influence phenotypic variation and gene expression. CNVs are thus considered major sources of genetic variation. Little is known, however, about their contribution to genetic variation in rice.</p> <p>Results</p> <p>To detect CNVs, we used a set of NimbleGen whole-genome comparative genomic hybridization arrays containing 718,256 oligonucleotide probes with a median probe spacing of 500 bp. We compiled a high-resolution map of CNVs in the rice genome, showing 641 CNVs between the genomes of the rice cultivars 'Nipponbare' (from <it>O. sativa </it>ssp. <it>japonica</it>) and 'Guang-lu-ai 4' (from <it>O. sativa </it>ssp. <it>indica</it>). The CNVs identified vary in size from 1.1 kb to 180.7 kb, and encompass approximately 7.6 Mb of the rice genome. The largest regions showing copy gain and loss are of 37.4 kb on chromosome 4, and 180.7 kb on chromosome 8. In addition, 85 DNA segments were identified, including some genic sequences. Contracted genes greatly outnumbered duplicated ones. Many of the contracted genes corresponded to either the same genes or genes involved in the same biological processes; this was also the case for genes involved in disease and defense.</p> <p>Conclusion</p> <p>We detected CNVs in rice by array-based comparative genomic hybridization. These CNVs contain known genes. Further discussion of CNVs is important, as they are linked to variation among rice varieties, and are likely to contribute to subspecific characteristics.</p

Amplified Genes May Be Overexpressed, Unchanged, or Downregulated in Cervical Cancer Cell Lines

Several copy number-altered regions (CNAs) have been identified in the genome of cervical cancer, notably, amplifications of 3q and 5p. However, the contribution of copy-number alterations to cervical carcinogenesis is unresolved because genome-wide there exists a lack of correlation between copy-number alterations and gene expression. In this study, we investigated whether CNAs in the cell lines CaLo, CaSki, HeLa, and SiHa were associated with changes in gene expression. On average, 19.2% of the cell-line genomes had CNAs. However, only 2.4% comprised minimal recurrent regions (MRRs) common to all the cell lines. Whereas 3q had limited common gains (13%), 5p was entirely duplicated recurrently. Genome-wide, only 15.6% of genes located in CNAs changed gene expression; in contrast, the rate in MRRs was up to 3 times this. Chr 5p was confirmed entirely amplified by FISH; however, maximum 33.5% of the explored genes in 5p were deregulated. In 3q, this rate was 13.4%. Even in 3q26, which had 5 MRRs and 38.7% recurrently gained SNPs, the rate was only 15.1%. Interestingly, up to 19% of deregulated genes in 5p and 73% in 3q26 were downregulated, suggesting additional factors were involved in gene repression. The deregulated genes in 3q and 5p occurred in clusters, suggesting local chromatin factors may also influence gene expression. In regions amplified discontinuously, downregulated genes increased steadily as the number of amplified SNPs increased (p<0.01, Spearman's correlation). Therefore, partial gene amplification may function in silencing gene expression. Additional genes in 1q, 3q and 5p could be involved in cervical carcinogenesis, specifically in apoptosis. These include PARP1 in 1q, TNFSF10 and ECT2 in 3q and CLPTM1L, AHRR, PDCD6, and DAP in 5p. Overall, gene expression and copy-number profiles reveal factors other than gene dosage, like epigenetic or chromatin domains, may influence gene expression within the entirely amplified genome segments

Can asthma control be improved by understanding the patient's perspective?

Clinical trials show that asthma can be controlled in the majority of patients, but poorly controlled asthma still imposes a considerable burden. The level of asthma control achieved reflects the behaviour of both healthcare professionals and patients. A key challenge for healthcare professionals is to help patients to engage in self-management behaviours with optimal adherence to appropriate treatment. These issues are particularly relevant in primary care, where most asthma is managed. An international panel of experts invited by the International Primary Care Respiratory Group considered the evidence and discussed the implications for primary care practice

Tumor Transcriptome Sequencing Reveals Allelic Expression Imbalances Associated with Copy Number Alterations

Due to growing throughput and shrinking cost, massively parallel sequencing is rapidly becoming an attractive alternative to microarrays for the genome-wide study of gene expression and copy number alterations in primary tumors. The sequencing of transcripts (RNA-Seq) should offer several advantages over microarray-based methods, including the ability to detect somatic mutations and accurately measure allele-specific expression. To investigate these advantages we have applied a novel, strand-specific RNA-Seq method to tumors and matched normal tissue from three patients with oral squamous cell carcinomas. Additionally, to better understand the genomic determinants of the gene expression changes observed, we have sequenced the tumor and normal genomes of one of these patients. We demonstrate here that our RNA-Seq method accurately measures allelic imbalance and that measurement on the genome-wide scale yields novel insights into cancer etiology. As expected, the set of genes differentially expressed in the tumors is enriched for cell adhesion and differentiation functions, but, unexpectedly, the set of allelically imbalanced genes is also enriched for these same cancer-related functions. By comparing the transcriptomic perturbations observed in one patient to his underlying normal and tumor genomes, we find that allelic imbalance in the tumor is associated with copy number mutations and that copy number mutations are, in turn, strongly associated with changes in transcript abundance. These results support a model in which allele-specific deletions and duplications drive allele-specific changes in gene expression in the developing tumor

Live Recombinant Salmonella Typhi Vaccines Constructed to Investigate the Role of rpoS in Eliciting Immunity to a Heterologous Antigen

We hypothesized that the immunogenicity of live Salmonella enterica serovar Typhi vaccines expressing heterologous antigens depends, at least in part, on its rpoS status. As part of our project to develop a recombinant attenuated S. Typhi vaccine (RASTyV) to prevent pneumococcal diseases in infants and children, we constructed three RASTyV strains synthesizing the Streptococcus pneumoniae surface protein PspA to test this hypothesis. Each vector strain carried ten engineered mutations designed to optimize safety and immunogenicity. Two S. Typhi vector strains (χ9639 and χ9640) were derived from the rpoS mutant strain Ty2 and one (χ9633) from the RpoS+ strain ISP1820. In χ9640, the nonfunctional rpoS gene was replaced with the functional rpoS gene from ISP1820. Plasmid pYA4088, encoding a secreted form of PspA, was moved into the three vector strains. The resulting RASTyV strains were evaluated for safety in vitro and for immunogenicity in mice. All three RASTyV strains were similar to the live attenuated typhoid vaccine Ty21a in their ability to survive in human blood and human monocytes. They were more sensitive to complement and were less able to survive and persist in sewage and surface water than their wild-type counterparts. Adult mice intranasally immunized with any of the RASTyV strains developed immune responses against PspA and Salmonella antigens. The RpoS+ vaccines induced a balanced Th1/Th2 immune response while the RpoS− strain χ9639(pYA4088) induced a strong Th2 immune response. Immunization with any RASTyV provided protection against S. pneumoniae challenge; the RpoS+ strain χ9640(pYA4088) provided significantly greater protection than the ISP1820 derivative, χ9633(pYA4088). In the pre-clinical setting, these strains exhibited a desirable balance between safety and immunogenicity and are currently being evaluated in a Phase 1 clinical trial to determine which of the three RASTyVs has the optimal safety and immunogenicity profile in human hosts