Project description:Global response to ciprofloxacin in low level quinolone resistant Escherichia coli: a shorter path to survival. Background: Bactericidal activity of quinolones in bacteria has been related to a combination of DNA fragmentation, ROS production and programmed dead cell systems. Subjacent molecular systems responsible for reduction of bactericidal effect in low-level quinolone resistance (LLQR) phenotypes remain to be clarified. To answer this question and to define new possible antimicrobial targets, the transcriptomic profile in isogenic Escherichia coli harbouring quinolone resistance mechanisms in the presence of ciprofloxacin was evaluated. Materials and methods: E. coli 25922 was used as background strain. Four LLQR isogenic strains were tested for transcriptomic assays: ATCC 25922 (wild-type), EC14 (coding for QnrS1), EC19 (marR deletion and coding for QnrS1) and EC24 (Ser83Leu substitution in GyrA and coding for QnrS1). Cells in exponential phase (DO600=0.4) were exposed to 1 mg/L of ciprofloxacin (breakpoint for reduced susceptibility according to CLSI) during 1 hour and, further, RNA was purified. Gene expression analysis was performed using AGILENT technology. Data obtained for each strain were always normalized to the wild-type E. coli ATCC 25922. Specific ROS modulation targets were validated by genetic and biochemical approach. Results: A radical differential response to ciprofloxacin in LLQR strains, either up or downregulation, was observed (proportional to the MIC values). Multiple genes implicated in ROS production (related to TCA cycle, aerobic respiration or detoxification systems) were upregulated (sdhC up to 63.5-folds) in LLQR mutants. SOS system components were downregulated (recA up to 30.7-folds). yihE, coding for a protective kinase of programmed cell death, was also upregulated (up to 5.2-folds). SdhC inhibition sensitized LLQR phenotypes (up to Log=2.3 after 24 hours). Conclusions: At clinical relevant concentration of ciprofloxacin, the pattern of genes expression of critical systems for bacterial survival and mutant development were significantly modified in LLQR phenotypes. This approach allowed validating ROS modulation as an interesting target in terms of bacterial sensitization.
Project description:The ideal genome sequence for medical interpretation is complete and diploid, capturing the full spectrum of genetic variation. Toward this end, there has been progress in discovery of single nucleotide polymorphism (SNP) and small (<10bp) insertion/deletions (indels), but annotation of larger structural variation (SV) including copy number variation (CNV) has been less comprehensive, even with available diploid sequence assemblies. We applied a multi-step sequence and microarray-based analysis to identify numerous previously unknown SVs within the first genome sequence reported from an individual. CGH experiments were performed with genomic DNA extracted from the HuRef and six HapMap lymphoblastoid cell lines, hybridized against the reference NA10851. A dye-swap experiment was performed for each experiment. The custom CGH microarray contains probes that target novel sequences that are not on the NCBI reference build 35. Instead, the probes target scaffold sequences that are unique to the Celera R27C assembly.
Project description:Genome assemblies can form the basis of comparative analyses fostering insight into the evolutionary genetics. Genome evolution influences a parasite’s pathogenicity, host–pathogen interactions, environmental constraints and invasion biology. Comparative genomics and epigenomics analyses will provide deep understanding of parasitism biology for future diagnosis and prevention of trchinellosis. We provide a near-complete new T.p reference genome using SMRT technology, first time confirmed and characterized T.p DNA methylome, enabling full annotation. Based on this new version, we show repetitive sequences play important role in genome expansion, in synergy with DNA methylation during evolution. We further portrait the genomic and epigenomic regulation on E-S products in relation with their parasitism differences, especially for two super-families, including DNase II and EGF-like domain proteins.
Project description:The ideal genome sequence for medical interpretation is complete and diploid, capturing the full spectrum of genetic variation. Toward this end, there has been progress in discovery of single nucleotide polymorphism (SNP) and small (<10bp) insertion/deletions (indels), but annotation of larger structural variation (SV) including copy number variation (CNV) has been less comprehensive, even with available diploid sequence assemblies. We applied a multi-step sequence and microarray-based analysis to identify numerous previously unknown SVs within the first genome sequence reported from an individual. The HuRef genomic DNA (from lymphoblastoid cell line) was co-hybridized with the female sample NA15510 (from lymphoblastoid cell line) from the Polymorphism Discovery Resources. The NimbleGen platform consists of 20 NimbleGen HD2 chips, each containing 2.1M probes, and each chip is further subdivided into 3 equal-sized subarrays containing about 726K probes. The probes target the NCBI Build 36 genome, with the exception that no homology filter was applied, allowing coverage of segmentally duplicated regions.
Project description:Synthetic dataset containing genome-wide genotypes of 500.000 individuals was generated using a hybrid approach combining coalescent approach and resampling based methods