The Generation of Successive Unmarked Mutations and Chromosomal Insertion of Heterologous Genes in Actinobacillus pleuropneumoniae Using Natural Transformation

We have developed a simple method of generating scarless, unmarked mutations in Actinobacillus pleuropneumoniae by exploiting the ability of this bacterium to undergo natural transformation, and with no need to introduce plasmids encoding recombinases or resolvases. This method involves two successive rounds of natural transformation using linear DNA: the first introduces a cassette carrying cat (which allows selection by chloramphenicol) and sacB (which allows counter-selection using sucrose) flanked by sequences to either side of the target gene; the second transformation utilises the flanking sequences ligated directly to each other in order to remove the cat-sacB cassette. In order to ensure efficient uptake of the target DNA during transformation, A. pleuropneumoniae uptake sequences are added into the constructs used in both rounds of transformation. This method can be used to generate multiple successive deletions and can also be used to introduce targeted point mutations or insertions of heterologous genes into the A. pleuropneumoniae chromosome for development of live attenuated vaccine strains. So far, we have applied this method to highly transformable isolates of serovars 8 (MIDG2331), which is the most prevalent in the UK, and 15 (HS143). By screening clinical isolates of other serovars, it should be possible to identify other amenable strains

p53 Transactivation and the Impact of Mutations, Cofactors and Small Molecules Using a Simplified Yeast-Based Screening System

The p53 tumor suppressor, which is altered in most cancers, is a sequence-specific transcription factor that is able to modulate the expression of many target genes and influence a variety of cellular pathways. Inactivation of the p53 pathway in cancer frequently occurs through the expression of mutant p53 protein. In tumors that retain wild type p53, the pathway can be altered by upstream modulators, particularly the p53 negative regulators MDM2 and MDM4. promoter, ii) single copy, chromosomally located p53-responsive and control luminescence reporters, iii) enhanced chemical uptake using modified ABC-transporters, iv) small-volume formats for treatment and dual-luciferase assays, and v) opportunities to co-express p53 with other cofactor proteins. This robust system can distinguish different levels of expression of WT and mutant p53 as well as interactions with MDM2 or 53BP1.We found that the small molecules Nutlin and RITA could both relieve the MDM2-dependent inhibition of WT p53 transactivation function, while only RITA could impact p53/53BP1 functional interactions. PRIMA-1 was ineffective in modifying the transactivation capacity of WT p53 and missense p53 mutations. This dual-luciferase assay can, therefore, provide a high-throughput assessment tool for investigating a matrix of factors that can influence the p53 network, including the effectiveness of newly developed small molecules, on WT and tumor-associated p53 mutants as well as interacting proteins

Identification of novel fusion genes in lung cancer using breakpoint assembly of transcriptome sequencing data

Genomic translocation events frequently underlie cancer development through generation of gene fusions with oncogenic properties. Identification of such fusion transcripts by transcriptome sequencing might help to discover new potential therapeutic targets. We developed TRUP (Tumor-specimen suited RNA-seq Unified Pipeline) (https://github.com/ruping/TRUP), a computational approach that combines split-read and read-pair analysis with de novo assembly for the identification of chimeric transcripts in cancer specimens. We apply TRUP to RNA-seq data of different tumor types, and find it to be more sensitive than alternative tools in detecting chimeric transcripts, such as secondary rearrangements in EML4-ALK-positive lung tumors, or recurrent inactivating rearrangements affecting RASSF8

Circulating Monocyte Chemoattractant Protein-1 in Patients with Cardiogenic Shock Complicating Acute Myocardial Infarction Treated with Mild Hypothermia: A Biomarker Substudy of SHOCK-COOL Trial

Background: There is evidence that monocyte chemoattractant protein-1 (MCP-1) levels reflect the intensity of the inflammatory response in patients with cardiogenic shock (CS) complicating acute myocardial infarction (AMI) and have a predictive value for clinical outcomes. However, little is known about the effect of mild therapeutic hypothermia (MTH) on the inflammatory response in patients with CS complicating AMI. Therefore, we conducted a biomarker study to investigate the effect of MTH on MCP-1 levels in patients with CS complicating AMI. Methods: In the randomized mild hypothermia in cardiogenic shock (SHOCK-COOL) trial, 40 patients with CS complicating AMI were enrolled and assigned to MTH (33 °C) for 24 h or normothermia at a 1:1 ratio. Blood samples were collected at predefined time points at the day of admission/day 1, day 2 and day 3. Differences in MCP-1 levels between and within the MTH and normothermia groups were assessed. Additionally, the association of MCP-1 levels with the risk of all-cause mortality at 30 days was analyzed. Missing data were accounted for by multiple imputation as sensitivity analyses. Results: There were differences in MCP-1 levels over time between patients in MTH and normothermia groups (P for interaction = 0.013). MCP-1 levels on day 3 were higher than on day 1 in the MTH group (day 1 vs day 3: 21.2 [interquartile range, 0.25–79.9] vs. 125.7 [interquartile range, 87.3–165.4] pg/mL; p = 0.006) and higher than in the normothermia group at day 3 (MTH 125.7 [interquartile range, 87.3–165.4] vs. normothermia 12.3 [interquartile range, 0–63.9] pg/mL; p = 0.011). Irrespective of therapy, patients with higher levels of MCP-1 at hospitalization tended to have a decreased risk of all-cause mortality at 30 days (HR, 2.61; 95% CI 0.997–6.83; p = 0.051). Conclusions: The cooling phase of MTH had no significant effect on MCP-1 levels in patients with CS complicating AMI compared to normothermic control, whereas MCP-1 levels significantly increased after rewarming. Trial registration: NCT01890317

Analysis of the Actinobacillus pleuropneumoniae ArcA Regulon Identifies Fumarate Reductase as a Determinant of Virulence▿ †

The ability of the bacterial pathogen Actinobacillus pleuropneumoniae to grow anaerobically allows the bacterium to persist in the lung. The ArcAB two-component system is crucial for metabolic adaptation in response to anaerobic conditions, and we recently showed that an A. pleuropneumoniae arcA mutant had reduced virulence compared to the wild type (F. F. Buettner, A. Maas, and G.-F. Gerlach, Vet. Microbiol. 127:106-115, 2008). In order to understand the attenuated phenotype, we investigated the ArcA regulon of A. pleuropneumoniae by using a combination of transcriptome (microarray) and proteome (two-dimensional difference gel electrophoresis and subsequent mass spectrometry) analyses. We show that ArcA negatively regulates the expression of many genes, including those encoding enzymes which consume intermediates during fumarate synthesis. Simultaneously, the expression of glycerol-3-phosphate dehydrogenase, a component of the respiratory chain serving as a direct reduction equivalent for fumarate reductase, was upregulated. This result, together with the in silico analysis finding that A. pleuropneumoniae has no oxidative branch of the citric acid cycle, led to the hypothesis that fumarate reductase might be crucial for virulence by providing (i) energy via fumarate respiration and (ii) succinate and other essential metabolic intermediates via the reductive branch of the citric acid cycle. To test this hypothesis, an isogenic A. pleuropneumoniae fumarate reductase deletion mutant was constructed and studied by using a pig aerosol infection model. The mutant was shown to be significantly attenuated, thereby strongly supporting a crucial role for fumarate reductase in the pathogenesis of A. pleuropneumoniae infection