Evolution of a Plasmid Regulatory Circuit Ameliorates Plasmid Fitness Cost.

Plasmids play a major role in rapid adaptation of bacteria by facilitating horizontal transfer of diverse genes, most notably those conferring antibiotic resistance. While most plasmids that replicate in a broad range of bacteria also persist well in diverse hosts, there are exceptions that are poorly understood. We investigated why a broad-host range plasmid, pBP136, originally found in clinical Bordetella pertussis isolates, quickly became extinct in laboratory Escherichia coli populations. Through experimental evolution we found that inactivation of a previously uncharacterized plasmid gene, upf31, drastically improved plasmid maintenance in E. coli. This gene inactivation resulted in decreased transcription of the global plasmid regulators (korA, korB, and korC) and numerous genes in their regulons. It also caused transcriptional changes in many chromosomal genes primarily related to metabolism. In silico analyses suggested that the change in plasmid transcriptome may be initiated by Upf31 interacting with the plasmid regulator KorB. Expression of upf31 in trans negatively affected persistence of pBP136Δupf31 as well as the closely related archetypal IncP-1ß plasmid R751, which is stable in E. coli and natively encodes a truncated upf31 allele. Our results demonstrate that while the upf31 allele in pBP136 might advantageously modulate gene expression in its original host, B. pertussis, it has harmful effects in E. coli. Thus, evolution of a single plasmid gene can change the range of hosts in which that plasmid persists, due to effects on the regulation of plasmid gene transcription.

Babesia bovis Rad51 ortholog influences switching of ves genes but is not essential for segmental gene conversion in antigenic variation.

The tick-borne apicomplexan parasite, Babesia bovis, a highly persistent bovine pathogen, expresses VESA1 proteins on the infected erythrocyte surface to mediate cytoadhesion. The cytoadhesion ligand, VESA1, which protects the parasite from splenic passage, is itself protected from a host immune response by rapid antigenic variation. B. bovis relies upon segmental gene conversion (SGC) as a major mechanism to vary VESA1 structure. Gene conversion has been considered a form of homologous recombination (HR), a process for which Rad51 proteins are considered pivotal components. This could make BbRad51 a choice target for development of inhibitors that both interfere with parasite genome integrity and disrupt HR-dependent antigenic variation. Previously, we knocked out the Bbrad51 gene from the B. bovis haploid genome, resulting in a phenotype of sensitivity to methylmethane sulfonate (MMS) and apparent loss of HR-dependent integration of exogenous DNA. In a further characterization of BbRad51, we demonstrate here that ΔBbrad51 parasites are not more sensitive than wild-type to DNA damage induced by γ-irradiation, and repair their genome with similar kinetics. To assess the need for BbRad51 in SGC, RT-PCR was used to observe alterations to a highly variant region of ves1α transcripts over time. Mapping of these amplicons to the genome revealed a significant reduction of in situ transcriptional switching (isTS) among ves loci, but not cessation. By combining existing pipelines for analysis of the amplicons, we demonstrate that SGC continues unabated in ΔBbrad51 parasites, albeit at an overall reduced rate, and a reduction in SGC tract lengths was observed. By contrast, no differences were observed in the lengths of homologous sequences at which recombination occurred. These results indicate that, whereas BbRad51 is not essential to babesial antigenic variation, it influences epigenetic control of ves loci, and its absence significantly reduces successful variation. These results necessitate a reconsideration of the likely enzymatic mechanism(s) underlying SGC and suggest the existence of additional targets for development of small molecule inhibitors.

Knockout of Babesia bovis rad51 ortholog and its complementation by expression from the BbACc3 artificial chromosome platform.

Babesia bovis establishes persistent infections of long duration in cattle, despite the development of effective anti-disease immunity. One mechanism used by the parasite to achieve persistence is rapid antigenic variation of the VESA1 cytoadhesion ligand through segmental gene conversion (SGC), a phenomenon thought to be a form of homologous recombination (HR). To begin investigation of the enzymatic basis for SGC we initially identified and knocked out the Bbrad51 gene encoding the B. bovis Rad51 ortholog. BbRad51 was found to be non-essential for in vitro growth of asexual-stage parasites. However, its loss resulted in hypersensitivity to methylmethane sulfonate (MMS) and an apparent defect in HR. This defect rendered attempts to complement the knockout phenotype by reinsertion of the Bbrad51 gene into the genome unsuccessful. To circumvent this difficulty, we constructed an artificial chromosome, BbACc3, into which the complete Bbrad51 locus was inserted, for expression of BbRad51 under regulation by autologous elements. Maintenance of BbACc3 makes use of centromeric sequences from chromosome 3 and telomeric ends from chromosome 1 of the B. bovis C9.1 line. A selection cassette employing human dihydrofolate reductase enables recovery of transformants by selection with pyrimethamine. We demonstrate that the BbACc3 platform is stably maintained once established, assembles nucleosomes to form native chromatin, and expands in telomere length over time. Significantly, the MMS-sensitivity phenotype observed in the absence of Bbrad51 was successfully complemented at essentially normal levels. We provide cautionary evidence, however, that in HR-competent parasites BbACc3 can recombine with native chromosomes, potentially resulting in crossover. We propose that, under certain circumstances this platform can provide a useful alternative for the genetic manipulation of this group of parasites, particularly when regulated gene expression under the control of autologous elements may be important.

Patient-reported outcomes after open carpal tunnel release using a standard protocol with 1 hand therapy visit.

STUDY DESIGN: Retrospective case series. INTRODUCTION: Open carpal tunnel release (OCTR) is a common treatment for carpal tunnel syndrome, but there is no consensus on the number of hand therapy visits needed to achieve optimal patient outcomes. PURPOSE OF THE STUDY: The purpose is to examine changes in patient-reported symptoms and function over a 12-week period after OCTR with 1 postoperative hand therapy visit. METHODS: Eligible subjects were consecutive patients treated with a standard OCTR protocol by a fellowship trained hand surgeon that included 1 hand therapy visit at 10-14 days postoperatively. Patients were excluded from participation if they had additional surgery at the time of OCTR, had another upper extremity diagnosis that required therapeutic intervention, or received more or less than 1 visit of hand therapy. Responses on the Boston Carpal Tunnel Questionnaire (BCTQ) were collected at preoperative and 3 postoperative time points: at the hand therapy visit, 6 weeks, and 12 weeks. Change over time in the BCTQ Symptom Severity Scale and Functional Status Scale was assessed. RESULTS: A total of 134 patients who were treated with the standard protocol had a complete BCTQ data set. Both BCTQ scales showed significant improvement over time. The Symptom Severity Scale showed significant improvement by the hand therapy visit at 10-14 days postoperatively, whereas significant improvement on the Functional Status Scale did not occur until 6 weeks postoperatively. The magnitude of change from preoperative to 12 weeks postoperative was 1.51 points on the Symptom Severity Scale and 0.91 points on the Functional Status Scale. Complication rates were low with an incidence of 13% for pillar pain and palm pain combined. CONCLUSIONS: Patient-reported symptoms and function improved significantly up to 12 weeks after OCTR. Moreover, there was a low incidence of pillar and palm pain. In a retrospective review of patients with a favorable prognosis based on having no need for extra surgical procedures or additional therapy visits, one therapy visit associated with improvements in symptoms and function, a low incidence of pillar/palm pain and favorable 12-weeks outcomes. There appears to be a subset of less complicated patients for whom one visit can allow for favorable outcomes. LEVEL OF EVIDENCE: 2B.

Just-in-Time to Save Lives: A Pilot Study of Layperson Tourniquet Application.

OBJECTIVES: The objective was to determine whether just-in-time (JiT) instructions increase successful tourniquet application by laypersons. METHODS: This was a randomized pilot study conducted in August 2014. The study occurred at the Uniformed Services University campus in Bethesda, Maryland. A total of 194 volunteers without prior military service or medical training completed the study. The participant stood in front of a waist-down mannequin that had an exposed leg. An observer read a scenario card aloud that described a mass casualty event. The observer then asked the participant to apply a Combat Application Tourniquet (C-A-T) to the mannequin. Test participants received a 4 × 6-inch card, with JiT instructions, in addition to their C-A-T; controls received no instructions. Participants were randomized in a 3:1 ratio of instructions to no instructions. The study's primary outcome was the proportion of successfully applied tourniquets by participants receiving JiT instructions compared to participants not receiving instructions. Secondary outcomes included the time for successful tourniquet placement, reasons for failed tourniquet application, and participants' self-reported willingness and comfort using tourniquets in real-life settings. RESULTS: Just-in-time instructions more than doubled successful tourniquet placement. Participants supplied with JiT instructions placed a tourniquet successfully 44.14% of the time, compared to 20.41% of the time for controls without instructions (risk ratio = 2.16; 95% confidence interval = 1.21 to 3.87; p = 0.003). CONCLUSIONS: Just-in-time instructions increase laypeople's successful application of C-A-T. This pilot study provides evidence that JiT instructions may assist the lay public in providing effective point-of-injury hemorrhage control.

'Poisonous, filthy, loathsome, damnable stuff': the rhetorical ecology of vaccination concern.

In this article, we analyze newspaper articles and advertisements mentioning vaccination from 1915 to 1922 and refer to historical studies of vaccination practices and attitudes in the early 20th century in order to assess historical continuities and discontinuities in vaccination concern. In the Progressive Era period, there were a number of themes or features that resonated with contemporary issues and circumstances: 1) fears of vaccine contamination; 2) distrust of medical professionals; 3) resistance to compulsory vaccination; and 4) the local nature of vaccination concern. Such observations help scholars and practitioners understand vaccine skepticism as longstanding, locally situated, and linked to the sociocultural contexts in which vaccination occurs and is mandated for particular segments of the population. A rhetorical approach offers a way to understand how discourses are engaged and mobilized for particular purposes in historical contexts. Historically situating vaccine hesitancy and addressing its articulation with a particular rhetorical ecology offers scholars and practitioners a robust understanding of vaccination concerns that can, and should, influence current approaches to vaccination skepticism.

Failed exstrophy closure: management and outcome.

OBJECTIVE: In a series of failed exstrophy closures, to identify determinants of successful repeat closure and the impact of failed closure on the fate of the lower urinary tract and continence status. PATIENTS AND METHODS: We performed a retrospective review of operative notes and medical records of patients with a history of one or more failed exstrophy closures in 1978-2007. The primary surgical endpoints were failure rate of repeat closure attempts, mode of continence surgery and continence outcome. Continence was defined as achieving a dry interval of >3h and voiding through the urethra. RESULTS: We identified 122 patients (85 male/37 female) who had undergone repeat closure following failure. The success rate of repeat closure attempts at our institution was 98%. Of the 94 patients who had undergone successful repeat closure, definitive continence management and had their dryness evaluated, 38 were candidates for bladder neck reconstruction and 17(18%) were continent. Of the remaining patients, 90% were able to attain dryness, but at the expense of clean intermittent catheterization and continent urinary diversion. CONCLUSION: A failed exstrophy closure has significant implications for long-term surgical outcome. Reclosure can be accomplished in the majority of cases. In comparison to patients with successful primary closure, the rates of urethral continence following successful repeat closure were lower.