An efficient system for the generation of marked genetic mutants in members of the genus Burkholderia

To elucidate the function of a gene in bacteria it is vital that targeted gene inactivation (allelic replacement) can be achieved. Allelic replacement is often carried out by disruption of the gene of interest by insertion of an antibiotic-resistance marker followed by subsequent transfer of the mutant allele to the genome of the host organism in place of the wild-type gene. However, due to their intrinsic resistance to many antibiotics only selected antibiotic-resistance markers can be used in members of the genus Burkholderia, including the Burkholderia cepacia complex (Bcc). Here we describe the construction of improved antibiotic-resistance cassettes that specify resistance to kanamycin, chloramphenicol or trimethoprim effectively in the Bcc and related species. These were then used in combination with and/or to construct a series enhanced suicide vectors, pSHAFT2, pSHAFT3 and pSHAFT-GFP to facilitate effective allelic replacement in the Bcc. Validation of these improved suicide vectors was demonstrated by the genetic inactivation of selected genes in the Bcc species Burkholderia cenocepacia and B. lata, and in the non-Bcc species, B. thailandensis

An Association of Cancer Physicians' strategy for improving services and outcomes for cancer patients.

The Association of Cancer Physicians in the United Kingdom has developed a strategy to improve outcomes for cancer patients and identified the goals and commitments of the Association and its members.The ACP is very grateful to all of its members who have expressed views on the development of the strategy and to the sponsors of our workshops and publications, especially Cancer Research UK and Macmillan Cancer SupportThis is the final version of the article. It was first available from Cancer Intelligence via http://dx.doi.org/10.3332/ecancer.2016.60

Deciphering the Role of RND Efflux Transporters in Burkholderia cenocepacia

Burkholderia cenocepacia J2315 is representative of a highly problematic group of cystic fibrosis (CF) pathogens. Eradication of B. cenocepacia is very difficult with the antimicrobial therapy being ineffective due to its high resistance to clinically relevant antimicrobial agents and disinfectants. RND (Resistance-Nodulation-Cell Division) efflux pumps are known to be among the mediators of multidrug resistance in Gram-negative bacteria. Since the significance of the 16 RND efflux systems present in B. cenocepacia (named RND-1 to -16) has been only partially determined, the aim of this work was to analyze mutants of B. cenocepacia strain J2315 impaired in RND-4 and RND-9 efflux systems, and assess their role in the efflux of toxic compounds. The transcriptomes of mutants deleted individually in RND-4 and RND-9 (named D4 and D9), and a double-mutant in both efflux pumps (named D4-D9), were compared to that of the wild-type B. cenocepacia using microarray analysis. Microarray data were confirmed by qRT-PCR, phenotypic experiments, and by Phenotype MicroArray analysis. The data revealed that RND-4 made a significant contribution to the antibiotic resistance of B. cenocepacia, whereas RND-9 was only marginally involved in this process. Moreover, the double mutant D4-D9 showed a phenotype and an expression profile similar to D4. The microarray data showed that motility and chemotaxis-related genes appeared to be up-regulated in both D4 and D4–D9 strains. In contrast, these gene sets were down-regulated or expressed at levels similar to J2315 in the D9 mutant. Biofilm production was enhanced in all mutants. Overall, these results indicate that in B. cenocepacia RND pumps play a wider role than just in drug resistance, influencing additional phenotypic traits important for pathogenesis

Evolutionary History of the Clostridium difficile Pathogenicity Locus

The symptoms of Clostridium difficile infection are caused by toxins expressed from its 19kb pathogenicity locus (PaLoc). Stable integration of the PaLoc is suggested by its single chromosomal location and the clade-specificity of its different genetic variants. However, the PaLoc is variably present, even among closely related strains, and thus resembles a mobile genetic element. Our aim was to explain these apparently conflicting observations by reconstructing the evolutionary history of the PaLoc. Phylogenetic analyses and annotation of the regions spanning the PaLoc were performed using C. difficile population-representative genomes chosen from a collection of 1,693 toxigenic (PaLoc present) and non-toxigenic (PaLoc absent) isolates. Comparison of the core genome and PaLoc phylogenies demonstrated an eventful evolutionary history, with distinct PaLoc variants acquired clade-specifically after divergence. In particular, our data suggest a relatively recent PaLoc acquisition in clade 4. Exchanges and losses of the PaLoc DNA have also occurred, via long homologous recombination events involving flanking chromosomal sequences. The most recent loss event occurred ~30 years ago within a clade 1 genotype. The genetic organisation of the clade 3 PaLoc was unique in containing a stably integrated novel transposon (designated Tn6218), variants of which were found at multiple chromosomal locations. Tn6218 elements were Tn916-related, but non-conjugative, and occasionally contained genes conferring resistance to clinically relevant antibiotics. The evolutionary histories of two contrasting, but clinically important genetic elements were thus characterised: the PaLoc, mobilised rarely via homologous recombination, and Tn6218, mobilised frequently through transposition

Advanced surrogate target development for evaluating pre-collision systems

This paper presents the requirements, design, and resulting performance of a new surrogate target for evaluating pre-collision systems (PCS) that include crash imminent braking and dynamic brake support. The design addresses several criteria for rear-end crash scenarios, including three critical and conflicting criteria: enabling high relative impact speed, maintaining a radar signature that is representative of real vehicles, and minimizing expenses and complexity of fabrication and usage. Test scenarios for evaluating PCS are developed using analyses of US crash data sets, including General Estimates Systems and the Crashworthiness Data System (including its electronic data recording data). Test procedures were developed, leveraging previous work in this area. Surrogate target design requirements were developed, notably new requirements for “impactability,” or the ability to be struck repeatedly without damage or safety risks, and 77 GHz radar scattering characteristics. Robustness to higher impact speeds allow testing in severe crash scenarios. Radar characteristics were addressed using a new data set generated using instrumentation-grade radar to scan the rear ends of 25 actual vehicles at numerous angles. The radar scan data was used to create a recommended set of scattering elements to be applied to a radarneutral structure. A novel approach to the structure was used, emphasizing low cost, weight, resilience, and, safety in higher speed impacts. This approach is intended to present a radarrepresentative vehicle target to the PCS under test, while allowing the target to be fabricated easily and inexpensively. The target is intended for PCS development as well as formal testing. Work continues to refine the target’s signature for vision- and LIDAR-based systems, and an instrumentation system is being fitted to test PCS vehicles in extended use. This work is significant for its contribution to surrogate target work in the emerging area of PCS with crash-imminent braking and dynamic brake support systems. Higher impact speeds and improved radar signatures will improve the correlation between pre-crash system performance in the real world and the outcome of performance testing on the track. Simpler and lower-cost targets allow wider use and perhaps better PCS designs

The impact of Michigan's partial repeal of the universal motorcycle helmet law on helmet use, fatalities, and head injuries

Objectives. To evaluate the impact of the partial repeal of Michigan's universal motorcycle helmet law on helmet use, fatalities, and head injuries. Methods. We compared helmet use rates and motorcycle crash fatality risk for the 12 months before and after the April 13, 2012, repeal with a statewide police-reported crash data set. We linked police-reported crashes to injured riders in a statewide trauma registry.We compared head injury before and after the repeal. Regression examined the effect of helmet use on fatality and head injury risk. Results. Helmet use decreased in crash (93.2% vs 70.8%; P < .001) and trauma data (91.1% vs 66.2%; P < .001) after the repeal. Although fatalities did not change overall (3.3% vs 3.2%; P = .87), head injuries (43.4% vs 49.6%; P < .05) and neurosurgical intervention increased (3.7% vs 6.5%; P < .05). Male gender (adjusted odds ratio [AOR] = 1.65), helmet nonuse (AOR = 1.84), alcohol intoxication (AOR = 11.31), intersection crashes (AOR = 1.62), and crashes at higher speed limits (AOR = 1.04) increased fatality risk. Helmet nonuse (AOR = 2.31) and alcohol intoxication (AOR = 2.81) increased odds of head injury. Conclusions. Michigan's helmet law repeal resulted in a 24% to27% helmet use decline among riders in crashes and a 14% increase in head injury

SHOT is affiliated to the Royal College of Pathologists. This report is produced by SHOT working with MHRA.

Transfusion delays, particularly in major haemorrhage and major trauma situations, must be prevented. Delays in provision and administration of blood components including delays in anticoagulant reversal, particularly in patients with intracranial haemorrhage, can result in death, or serious sequelae. Every minute counts in these situations. Effective and reliable transfusion information technology systems should be implemented to reduce the risk of errors at all steps in the transfusion pathway, provided they are configured and used correctly. Effective investigation of all incidents and near miss events, application of effective corrective and preventive actions, and closing the loop by measuring the effectiveness of interventions should be carried out to optimise learning from incidents Taken from Key recommendations from summary: https://www.shotuk.org/wp-content/uploads/myimages/SHOT-Summary-ZCard-2020-v2.2.pdf Full text available at: https://www.shotuk.org/wp-content/uploads/myimages/SHOT-REPORT-2020.pd

Does unbelted safety requirement affect protection for belted occupants?

<p><b>Objective</b>: Federal regulations in the United States require vehicles to meet occupant performance requirements with unbelted test dummies. Removing the test requirements with unbelted occupants might encourage the deployment of seat belt interlocks and allow restraint optimization to focus on belted occupants. The objective of this study is to compare the performance of restraint systems optimized for belted-only occupants with those optimized for both belted and unbelted occupants using computer simulations and field crash data analyses.</p> <p><b>Methods</b>: In this study, 2 validated finite element (FE) vehicle/occupant models (a midsize sedan and a midsize SUV) were selected. Restraint design optimizations under standardized crash conditions (U.S.-NCAP and FMVSS 208) with and without unbelted requirements were conducted using Hybrid III (HIII) small female and midsize male anthropomorphic test devices (ATDs) in both vehicles on both driver and right front passenger positions. A total of 10 to 12 design parameters were varied in each optimization using a combination of response surface method (RSM) and genetic algorithm. To evaluate the field performance of restraints optimized with and without unbelted requirements, 55 frontal crash conditions covering a greater variety of crash types than those in the standardized crashes were selected. A total of 1,760 FE simulations were conducted for the field performance evaluation. Frontal crashes in the NASS-CDS database from 2002 to 2012 were used to develop injury risk curves and to provide the baseline performance of current restraint system and estimate the injury risk change by removing the unbelted requirement.</p> <p><b>Results</b>: Unbelted requirements do not affect the optimal seat belt and airbag design parameters in 3 out of 4 vehicle/occupant position conditions, except for the SUV passenger side. Overall, compared to the optimal designs with unbelted requirements, optimal designs without unbelted requirements generated the same or lower total injury risks for belted occupants depending on statistical methods used for the analysis, but they could also increase the total injury risks for unbelted occupants.</p> <p><b>Conclusions</b>: This study demonstrated potential for reducing injury risks to belted occupants if the unbelted requirements are eliminated. Further investigations are necessary to confirm these findings.</p

Phylogeography of the Koala, (Phascolarctos cinereus), and Harmonising Data to Inform Conservation.

The Australian continent exhibits complex biogeographic patterns but studies of the impacts of Pleistocene climatic oscillation on the mesic environments of the Southern Hemisphere are limited. The koala (Phascolarctos cinereus), one of Australia's most iconic species, was historically widely distributed throughout much of eastern Australia but currently represents a complex conservation challenge. To better understand the challenges to koala genetic health, we assessed the phylogeographic history of the koala. Variation in the maternally inherited mitochondrial DNA (mtDNA) Control Region (CR) was examined in 662 koalas sampled throughout their distribution. In addition, koala CR haplotypes accessioned to Genbank were evaluated and consolidated. A total of 53 unique CR haplotypes have been isolated from koalas to date (including 15 haplotypes novel to this study). The relationships among koala CR haplotypes were indicative of a single Evolutionary Significant Unit and do not support the recognition of subspecies, but were separated into four weakly differentiated lineages which correspond to three geographic clusters: a central lineage, a southern lineage and two northern lineages co-occurring north of Brisbane. The three geographic clusters were separated by known Pleistocene biogeographic barriers: the Brisbane River Valley and Clarence River Valley, although there was evidence of mixing amongst clusters. While there is evidence for historical connectivity, current koala populations exhibit greater structure, suggesting habitat fragmentation may have restricted female-mediated gene flow. Since mtDNA data informs conservation planning, we provide a summary of existing CR haplotypes, standardise nomenclature and make recommendations for future studies to harmonise existing datasets. This holistic approach is critical to ensuring management is effective and small scale local population studies can be integrated into a wider species context