We present a capture-based approach for bisulfite-converted DNA that allows interrogation of pre-defined genomic locations, allowing quantitative and qualitative assessments of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) at CG dinucleotides and in non-CG contexts (CHG, CHH) in mammalian and plant genomes. We show the technique works robustly and reproducibly using as little as 500 ng of starting DNA, with results correlating well with whole genome bisulfite sequencing data, and demonstrate that human DNA can be tested in samples contaminated with microbial DNA. This targeting approach will allow cell type-specific designs to maximize the value of 5mC and 5hmC sequencing.

The Salmonella enterica serovars Enteritidis, Dublin, and Gallinarum are closely related but differ in virulence and host range. To identify the genetic elements responsible for these differences and to better understand how these serovars are evolving, we sequenced the genomes of Enteritidis strain LK5 and Dublin strain SARB12 and compared these genomes to the publicly available Enteritidis P125109, Dublin CT 02021853 and Dublin SD3246 genome sequences. We also compared the publicly available Gallinarum genome sequences from biotype Gallinarum 287/91 and Pullorum RKS5078. Using bioinformatic approaches, we identified single nucleotide polymorphisms, insertions, deletions, and differences in prophage and pseudogene content between strains belonging to the same serovar. Through our analysis we also identified several prophage cargo genes and pseudogenes that affect virulence and may contribute to a host-specific, systemic lifestyle. These results strongly argue that the Enteritidis, Dublin and Gallinarum serovars of Salmonella enterica evolve by acquiring new genes through horizontal gene transfer, followed by the formation of pseudogenes. The loss of genes necessary for a gastrointestinal lifestyle ultimately leads to a systemic lifestyle and niche exclusion in the host-specific serovars.

Rationale: Upper respiratory tract infection is a guideline accepted risk domain for the loss of asthma control. The ionotrophic nucleotide receptor P2X 7 regulates compartmentalized acute inflammation and the immune response to airway pathogens. Objectives: We hypothesized that variability in P2X 7 function contributes to neutrophilic airway inflammation during a cold and thereby is linked to acute asthma. Methods: Research volunteers with asthma were enrolled at the onset of a naturally occurring cold and monitored through convalescence, assessing symptoms, lung function, and airway inflammation. P2X 7 pore activity in whole blood samples was measured using a genomically validated flow cytometric assay. Measurements and Main Results: Thirty-five participants with mild to moderate allergic asthma were enrolled and 31 completed all visits. P2X 7 pore function correlated with the change in nasal lavage neutrophil counts during the cold (R s 5 0.514, P 5 0.004) and was inversely related to the change in asthma symptoms (R s 5 -0.486, P 5 0.009). The change in peak expiratory flow recordings, precold use of inhaled corticosteroids, and P2X 7 pore function were multivariate predictors of asthma symptoms (P 5 0.001, , 0.001 and 5 0.003 respectively). Attenuated P2X 7 activity was associated with the risk of losing asthma control (crude odds ratio, 11.0; 95% confidence interval, 1.1-106.4) even after adjustment for inhaled corticosteroids and rhinovirus (odds ratio, 15.0). Conclusions: A whole blood P2X 7 pore assay robustly identifies participants with loss-of-function genotypes. Using this assay as an epidemiologic tool, attenuated P2X 7 pore activity may be a novel biomarker of virus-induced loss of asthma control.

The importance of accessory signaling pathways amplifying endotoxin responses has recently been highlighted by genetic studies describing LPS-hyporesponsive individuals despite carrying the common allele for TLR4. The nucleotide receptor P2X7 modulates the production of numerous LPS-stimulated inflammatory mediators. We have recently described the largest phenotypic screen known for genetic polymorphisms associated with the nucleotide receptor P2X7, a global regulator of leukocyte function. This required the development of a novel monocyte pore assay with numerous advantages over previous methods and with the potential to facilitate rapid (< 3 h), multiplex analysis of clinical samples. This paper addresses aspects pertinent to the development of the monocyte pore assay, briefly summarizes our results suggesting that P2X7 alleles modulate LPS-stimulated cytokine production, and discusses a model wherein P2X7 may serve as an amplification loop of innate immunity.

Robot-assisted radical cystectomy (RARC) for bladder cancer is increasingly becoming popular in specialist centres around the world. RARC has the advantage of being minimally invasive and also the dexterity of the instruments allow reconstruction such as ileal conduit urinary diversion or neobladder formation. Starting from the initial series demonstrating the feasibility of RARC and extended pelvic lymph node dissection, we now have mature series demonstrating equal oncological and functional outcomes in the medium term follow-up. In addition, literature suggests decreased hospital stay, less blood loss equating to less blood transfusion and a trend towards decreased complications as well. In the near future we would anticipate further refinement and reduced operating times with increased benefits for the patient undergoing RARC.