Plasmid expression of Deinococcus radiodurans RecA confers UV-A protection to Escherichia coli with an inverse protein dose dependence, which does not exceed conspecific RecA protection.

Low level expression in Escherichia coli of the RecA protein from the radiation resistant bacterium Deinococcus radiodurans protects a RecA deficient strain of E. coli from UV-A irradiation by up to ∼160% over basal UV-A resistance. The protection effect is inverse protein dose dependent: increasing the expression level of the D. radiodurans RecA (DrRecA) protein decreases the protection factor. This inverse protein dose dependence effect helps resolve previously conflicting reports of whether DrRecA expression is protective or toxic for E. coli. In contrast to the D. radiodurans protein effect, conspecific plasmid expression of E. coli RecA protein in RecA deficient E. coli is consistently protective over several protein expression levels, as well as consistently more protective to higher levels of UV-A exposure than that provided by the D. radiodurans protein. The results indicate that plasmid expression of D. radiodurans RecA can modestly enhance the UV resistance of living E. coli, but that the heterospecific protein shifts from protective to toxic as expression is increased.

Intraobserver and Interobserver Reliability of the Snyder and Expanded SLAP Classification System: A Video Study.

Background: Superior labral anterior and posterior (SLAP) tears are a common finding in overhead athletes. The original classification system produced by Snyder in 1990 contained 4 types of SLAP tears and was later expanded to 10 types. The classification has been challenging because of inconsistencies between surgeons making diagnoses and treatments based on the diagnosis. Furthermore, patient factors-such as age and sports played-affect the treatment algorithms, even across similarly classified SLAP tears. Purpose: To (1) assess the interobserver and intraobserver reliability of the Snyder and expanded SLAP (ESLAP) classification systems and (2) determine the consistency of treatment for a given SLAP tear depending on different clinical scenarios. Study Design: Cohort study (diagnosis); Level of evidence, 3. Methods: A total of 20 arthroscopic surgical videos and magnetic resonance imaging scans of patients with SLAP tears were sent to 20 orthopaedic sports medicine surgeons at various stages of training. Surgeons were asked to identify the type of SLAP tear using the Snyder and ESLAP classifications. Surgeons were then asked to determine the treatment for a SLAP tear using 4 clinical scenarios: (1) in the throwing arm of an 18-year-old pitcher; (2) in the dominant arm of an 18-year-old overhead athlete; (3) a 35-year-old overhead athlete; (4) or a 50-year-old overhead athlete. Responses were recorded, and the cases were shuffled and sent back 6 weeks after the initial responses. Results were then analyzed using the Fleiss kappa coefficient (κ) to determine interobserver and intraobserver degrees of agreement. Results: There was moderate intraobserver reliability in both the Snyder and ESLAP classifications (κ = 0.52) and fair interobserver reliability for both classification systems (Snyder, κ = 0.31; ESLAP, κ = 0.30; P < .0001) among all surgeons. Additionally, there was only fair agreement (κ = 0.30; P < .0001) for the treatment modalities chosen by the reviewers for each case. Conclusion: This study demonstrated that SLAP tears remain a challenging problem for orthopaedic surgeons in diagnostics and treatment plans. Therefore, care should be taken in the preoperative discussion with the patient to consider all the possible treatment options because this may affect the postoperative recovery period and patient expectations.

Functional mobility, employment and safety benefits of seat elevating devices.

STATUS OF RESEARCH PROCESS: Study completed. INVOLVEMENT OF ASSISTIVE TECHNOLOGY USERS: Participants were power wheelchair users.

The Use of Suture Augmentation for Graft Protection in ACL Reconstruction: A Biomechanical Study in Porcine Knees.

PURPOSE: To biomechanically evaluate the use of the suture augmentation construct at time 0 of ACL reconstruction. METHODS: Eighty porcine knees underwent ACL reconstruction using 2 techniques for graft fixation: a single suspensory construct (SSC), performed with a femoral button and tibial interference screw; and a double suspensory construct (DSC), with a femoral and tibial button. Each fixation technique was performed on 40 porcine knees divided into 4 subgroups. The first group had a nonaugmented ACL reconstruction, the second group had an ACL reconstruction with suture augmentation, and the third and fourth groups were the same as the first and second groups, with the graft resected 80% to simulate graft weakening. Ultimate load, yield load, stiffness, cyclic displacement values, and mode of failure were recorded for each graft. RESULTS: In a weakened graft model with 80% graft resection, there was a significant increase in ultimate strength (P < .001), yield strength (P < .001), and cyclic displacement (P < .001) with suture augmentation. There was no significant increase in stiffness with suture augmentation with either construct (P = .278). In the setting of an intact graft, there were no differences in either SSC or DCS groups with or without suture augmentation. CONCLUSIONS: The addition of a suture to ACL reconstruction techniques resulted in minimal changes in baseline biomechanical characteristics while improving ultimate load, yield load, and cyclic displacement in a weakened graft model. CLINICAL RELEVANCE: Suture augmentation of ACL reconstruction may confer improved integrity of the graft and is worth consideration and future clinical study.

Demographic and Functional Characteristics of National Veterans Wheelchair Games Participants: A Cross-sectional Study.

INTRODUCTION: Although many studies report the impact of adaptive sports and recreation on quality of life for people with disabilities across several age groups, few have focused on the Veteran population. The purpose of this study was to establish a baseline of common characteristics of the Veteran population that participated in the National Veterans Wheelchair Games (NVWG) in 2017 and 2018, including their perception on how their participation is associated with function and social factors. MATERIALS AND METHODS: A cross-sectional study was implemented as part of a quality assurance collaboration between the University of Pittsburgh and the Veterans Administration National Veterans Sports Programs and Special Events. Demographic and quality-of-life data were collected through the Functional Mobility Assessment (FMA) and associated Uniform Dataset as well as the Sports Participation Outcome Research Tool and Comprehensive Uniform Survey (SPORTACUS). This report provides and discusses the descriptive analyses that were performed on the data and establishes a framework to assess the impact of sports and exercise for Veterans with disabilities. RESULTS: A sample of 426 Veterans, 87% who were male and an average population age of 56 years old, reported high FMA scores on each of 10 items (daily routine, comfort, health, operate, reach, transfer, personal care, indoor mobility, outdoor mobility, and transportation) along with SPORTACUS scores scoring above 5, based on a 1-6 scoring scale (1 being "completely disagree" and 6 being "completely agree"), on each domain indicating sports participation is associated with their ability to function and participate in the community. CONCLUSION: Based on these results, it can be concluded for this military Veteran population that participation in a large, organized adaptive sports programs such as the NVWG has a positive association with daily function, quality of life, community participation, and use of higher quality assistive technology.

Methodology for the identification of small molecule inhibitors of the Fanconi Anaemia ubiquitin E3 ligase complex.

DNA inter-strand crosslinks (ICLs) threaten genomic stability by creating a physical barrier to DNA replication and transcription. ICLs can be caused by endogenous reactive metabolites or from chemotherapeutics. ICL repair in humans depends heavily on the Fanconi Anaemia (FA) pathway. A key signalling step of the FA pathway is the mono-ubiquitination of Fanconi Anaemia Complementation Group D2 (FANCD2), which is achieved by the multi-subunit E3 ligase complex. FANCD2 mono-ubiquitination leads to the recruitment of DNA repair proteins to the site of the ICL. The loss of FANCD2 mono-ubiquitination is a common clinical feature of FA patient cells. Therefore, molecules that restore FANCD2 mono-ubiquitination could lead to a potential drug for the management of FA. On the other hand, in some cancers, FANCD2 mono-ubiquitination has been shown to be essential for cell survival. Therefore, inhibition of FANCD2 mono-ubiquitination represents a possible therapeutic strategy for cancer specific killing. We transferred an 11-protein FANCD2 mono-ubiquitination assay to a high-throughput format. We screened 9,067 compounds for both activation and inhibition of the E3 ligase complex. The use of orthogonal assays revealed that candidate compounds acted via non-specific mechanisms. However, our high-throughput biochemical assays demonstrate the feasibility of using sophisticated and robust biochemistry to screen for small molecules that modulate a key step in the FA pathway. The future identification of FA pathway modulators is anticipated to guide future medicinal chemistry projects with drug leads for human disease.

Orchestrating single-cell analysis with Bioconductor.

Recent technological advancements have enabled the profiling of a large number of genome-wide features in individual cells. However, single-cell data present unique challenges that require the development of specialized methods and software infrastructure to successfully derive biological insights. The Bioconductor project has rapidly grown to meet these demands, hosting community-developed open-source software distributed as R packages. Featuring state-of-the-art computational methods, standardized data infrastructure and interactive data visualization tools, we present an overview and online book (https://osca.bioconductor.org) of single-cell methods for prospective users.

Publisher Correction: Orchestrating single-cell analysis with Bioconductor.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Is there an oxidative cost of acute stress? Characterization, implication of glucocorticoids and modulation by prior stress experience.

Acute rises in glucocorticoid hormones allow individuals to adaptively respond to environmental challenges but may also have negative consequences, including oxidative stress. While the effects of chronic glucocorticoid exposure on oxidative stress have been well characterized, those of acute stress or glucocorticoid exposure have mostly been overlooked. We examined the relationship between acute stress exposure, glucocorticoids and oxidative stress in Japanese quail (Coturnix japonica). We (i) characterized the pattern of oxidative stress during an acute stressor in two phenotypically distinct breeds; (ii) determined whether corticosterone ingestion, in the absence of acute stress, increased oxidative stress, which we call glucocorticoid-induced oxidative stress (GiOS); and (iii) explored how prior experience to stressful events affected GiOS. Both breeds exhibited an increase in oxidative stress in response to an acute stressor. Importantly, in the absence of acute stress, ingesting corticosterone caused an acute rise in plasma corticosterone and oxidative stress. Lastly, birds exposed to no previous acute stress or numerous stressful events had high levels of GiOS in response to acute stress, while birds with moderate prior exposure did not. Together, these findings suggest that an acute stress response results in GiOS, but prior experience to stressors may modulate that oxidative cost.

Drug-resistant tuberculosis diagnosis since Xpert® MTB/RIF introduction in Papua New Guinea, 2012-2017.

SETTING: Xpert® MTB/RIF was introduced in Papua New Guinea in 2012 for the diagnosis of tuberculosis (TB) and of rifampicin-resistant TB (RR-TB), a marker of multi-drug-resistant TB (MDR-TB). OBJECTIVE: To assess the concordance of Xpert with phenotypic drug susceptibility testing (DST) performed at the supranational reference laboratory and to describe the patterns of drug-resistant TB observed. DESIGN: This was a retrospective descriptive study of laboratory data collected from April 2012 to December 2017. RESULTS: In 69 months, 1408 specimens with Xpert results were sent for mycobacterial culture and DST; Mycobacterium tuberculosis was cultured from 63% (884/1408) and DST was completed in 99.4%. The concordance between Xpert and culture for M. tuberculosis detection was 98.6%. Of 760 RR-TB cases, 98.7% were detected using Xpert; 98.5% of 620 MDR-TB cases were identified using phenotypic DST. Phenotypic resistance to second-line drugs was detected in 59.4% (522/879) of specimens tested, including 29 with fluoroquinolone resistance; the majority were from the National Capital District and Daru Island. CONCLUSION: The high concordance between phenotypic DST and Xpert in identifying RR-TB cases supports the scale-up of initial Xpert testing in settings with high rates of drug resistance. However, rapid DST in addition to the detection of RR-TB is required.

Evaluation of Service Delivery Effectiveness Through Patient-Reported Outcome Measures.

OBJECTIVE: The aims of the study were to report the demographic characteristics and functional mobility for individuals accessing an academic medical center mobility device clinic and to compare functional mobility data across demographic characteristics and mobility device type. DESIGN: This study used a retrospective, cross-sectional design. Demographic, mobility type, and patient-reported outcome measure data for 833 clients were analyzed. The Functional Mobility Assessment was used as the patient-reported outcome measure to determine satisfaction. RESULTS: The mean (SD) baseline Functional Mobility Assessment score was 0.59 (0.25) on a 0-1 scale. Significant differences with the Functional Mobility Assessment scores were found across the mobility device types, with scooter and power wheelchair groups reporting higher satisfaction scores than those in the cane/crutch/walker or manual wheelchair groups. Device type, sex, and age were each found to be significant predictors of satisfaction scores (P < 0.01). CONCLUSIONS: Mobility device type is associated with satisfaction level. Mobility devices that offer higher levels of assistance are associated with increased satisfaction. The Functional Mobility Assessment, mobility device type, and demographic data provide baseline information for evaluating the effectiveness of an academic medical center mobility device clinic.

Development and Implementation of a Wheelchair Outcomes Registry.

OBJECTIVE: To describe the development of a patient registry related to wheeled mobility and seating (WMS) device interventions to accumulate large datasets for clinical quality assurance and research purposes. DESIGN: Accepted guidelines for registry development were applied and anchored around the Functional Mobility Assessment (FMA) questionnaire and a uniform dataset (UDS). SETTING: The FMA and UDS were developed under a corporate research agreement between clinical researchers and commercial providers. The questionnaires are administered in rehabilitation clinics to patients at the time of assessment for new device interventions (baseline) and readministered by telephone or other remote strategies periodically thereafter (follow-up). PARTICIPANTS: The FMA and UDS can be administered to any patient with a mobility impairment in need of a WMS device. INTERVENTIONS: WMS interventions include manual wheelchairs, power wheelchairs, scooters, seating, and other accessories. MAIN OUTCOME MEASURES: The FMA is a validated 10-item patient-centered outcome measure that investigates satisfaction in performing common mobility-related activities of daily living. The UDS includes variables related to age, diagnosis, and type of device used, as well as health, participation, and environmental factors. RESULTS: Currently there are over 1500 complete FMA and UDS cases at baseline and more than 600 follow-up datasets from 45 providers nationwide. Feedback indicates use of the FMA and UDS does not add burden to the clinical routine. CONCLUSIONS: A registry in the field of WMS has been developed and shown to be feasible in a clinical setting. This has created an opportunity to collect large datasets to increase sample sizes for future analyses to more scientifically evaluate what types of WMS devices work best for what types of patients under varying circumstances to promote health and participation.

Risk factors associated with acute in-hospital delirium for patients diagnosed with a hip fracture in the emergency department.

OBJECTIVE: The primary objective was to identify risk factors independently associated with acute in-hospital delirium within 72 hours of emergency department (ED) arrival for patients diagnosed with a hip fracture. METHODS: This was a retrospective chart review of patients ages 65 years and older presenting to one of two academic EDs with a discharge diagnosis of a hip fracture from January 1, 2014, to December 31, 2015. A multivariable logistic regression analysis was used to determine variables independently associated with the development of acute in-hospital delirium within 72 hours of ED arrival. RESULTS: Of the 668 included patients, 181 (27.1%) developed delirium within 72 hours of ED arrival. History of neurodegenerative disease or dementia (odds ratio [OR]: 5.7, 95% confidence interval [CI]: 3.9, 8.4), age > 75 (OR: 2.8, 95% CI: 1.4, 5.6), and absence of analgesia (no opioid or nerve block) in the ED (OR: 2.1, 95% CI: 1.3, 3.2) were independently associated with the development of acute in-hospital delirium; 525 (78.6%) patients received opioid analgesia in the ED. The most common analgesics used in the ED were intravenous (IV) morphine (35.8%), IV hydromorphone (35.2%), or dual therapy with both IV hydromorphone and IV morphine (2.2%). Femoral nerve blocks were initiated for 36 (5.4%) patients and successfully completed in 35 (5.2%) patients in the ED. CONCLUSIONS: Advanced age and signs of dementia or neurodegenerative disease are predictors of 72-hour delirium that can be screened for during triage. Improved pain control in the ED may reduce the risk of acute in-hospital delirium.

Pol I DNA polymerases stimulate DNA end-joining by Escherichia coli DNA ligase.

Klenow and Klentaq are the large fragment domains of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus, respectively. Herein, we show that both polymerases can significantly stimulate complementary intermolecular end-joining ligations by E.coli DNA ligase when the polymerases are present at concentrations lower than that of the DNA substrates. In contrast, high polymerase concentrations relative to the DNA substrates inhibit the intermolecular ligation activity of DNA ligase. Neither polymerase was able to stimulate the DNA ligase from T4 bacteriophage. Additionally, nick-closure by E. coli DNA ligase (but not T4 ligase) is slightly stimulated by both polymerases, but only at about 10% of the magnitude seen for end-joining enhancement. The data represent one of the first observations of direct polymerase-ligase interactions in prokaryotes, and suggest that the polymerases stabilize the associated DNA ends during intermolecular ligation, and that such a complex can be taken advantage of by some, but not all, DNA ligases.

Cell death is not essential for caspase-1-mediated interleukin-1ß activation and secretion.

Caspase-1 cleaves and activates the pro-inflammatory cytokine interleukin-1 beta (IL-1ß), yet the mechanism of IL-1ß release and its dependence on cell death remains controversial. To address this issue, we generated a novel inflammasome independent system in which we directly activate caspase-1 by dimerization. In this system, caspase-1 dimerization induced the cleavage and secretion of IL-1ß, which did not require processing of caspase-1 into its p20 and p10 subunits. Moreover, direct caspase-1 dimerization allowed caspase-1 activation of IL-1ß to be separated from cell death. Specifically, we demonstrate at the single cell level that IL-1ß can be released from live, metabolically active, cells following caspase-1 activation. In addition, we show that dimerized or endogenous caspase-8 can also directly cleave IL-1ß into its biologically active form, in the absence of canonical inflammasome components. Therefore, cell death is not obligatory for the robust secretion of bioactive IL-1ß.

Evolutionary divergence of the necroptosis effector MLKL.

The pseudokinase, MLKL (mixed-lineage kinase domain-like), is the most terminal obligatory component of the necroptosis cell death pathway known. Phosphorylation of the MLKL pseudokinase domain by the protein kinase, receptor interacting protein kinase-3 (RIPK3), is known to be the key step in MLKL activation. This phosphorylation event is believed to trigger a molecular switch, leading to exposure of the N-terminal four-helix bundle (4HB) domain of MLKL, its oligomerization, membrane translocation and ultimately cell death. To examine how well this process is evolutionarily conserved, we analysed the function of MLKL orthologues. Surprisingly, and unlike their mouse, horse and frog counterparts, human, chicken and stickleback 4HB domains were unable to induce cell death when expressed in murine fibroblasts. Forced dimerization of the human MLKL 4HB domain overcame this defect and triggered cell death in human and mouse cell lines. Furthermore, recombinant proteins from mouse, frog, human and chicken MLKL, all of which contained a 4HB domain, permeabilized liposomes, and were most effective on those designed to mimic plasma membrane composition. These studies demonstrate that the membrane-permeabilization function of the 4HB domain is evolutionarily conserved, but reveal that execution of necroptotic death by it relies on additional factors that are poorly conserved even among closely related species.

The potential of blue lupins as a protein source, in the diets of laying hens.

Layers diets typically contain 15-20% soya due to its high crude protein content (ca. 36%). Reliance on soya for protein can result in large increases in cost of feed due to the law of supply and demand as a global commodity. Lupin grains have high protein content (35-40%) but previous experience with white lupins has shown toxic effects in poultry due to high levels alkaloids and poor performance due to anti-nutritional Non-starch polysaccharides (NSP). Here blue lupins either processed or whole were trialled for their potential as a protein source. Point of lay chickens (64) at 16 weeks of age were weighed and allocated to 16 coops of four hens. Coops, as the experimental unit, were randomly allocated to four treatments: layers mash with soya (Control); or layers mash with 150 g of lupin/kg diet with the lupin either: whole (Whole); dehulled (Dehulled) or dehulled + a solid state fermentation enzyme extract (SSF; 150 g/tonne DM). All diets were ground and formulated to be balanced for energy, crude protein and essential amino acids using NIRS. No difference in growth rate, final hen weight, DM and water intake, eggs per day, mean egg weight, yellowness of yolk or chroma was found between treatments. There was a trend (P<0.1) for the SSF treatment to produce less heavy shells and a significant effect for the lupin treatments to have redder yolks (P<0.001). Fecal DM and bacterial counts were not different and there was no sign of enteritis or intestinal tissue hyperplasia from hen autopsies. Inclusion of blue lupins in the diet of laying hens at a rate of 150 g/kg DM resulted in no adverse effects in production or hen health and could be used as part of a balanced ration with inclusion of NSP degrading enzymes to reduce reliance on soya protein.

Enhanced DNA binding affinity of RecA protein from Deinococcus radiodurans.

Deinococcus radiodurans (Dr) has a significantly more robust DNA repair response than Escherichia coli (Ec), which helps it survive extremely high doses of ionizing radiation and prolonged periods of desiccation. DrRecA protein plays an essential part in this DNA repair capability. In this study we directly compare the binding of DrRecA and EcRecA to the same set of short, defined single (ss) and double stranded (ds) DNA oligomers. In the absence of cofactors (ATPγS or ADP), DrRecA binds to dsDNA oligomers more than 20 fold tighter than EcRecA, and binds ssDNA up to 9 fold tighter. Binding to dsDNA oligomers in the absence of cofactor presumably predominantly monitors DNA end binding, and thus suggests a significantly higher affinity of DrRecA for ds breaks. Upon addition of ATPγS, this species-specific affinity difference is nearly abolished, as ATPγS significantly decreases the affinity of DrRecA for DNA. Other findings include that: (1) both proteins exhibit a dependence of binding affinity on the length of the ssDNA oligomer, but not the dsDNA oligomer; (2) the salt dependence of binding is modest for both species of RecA, and (3) in the absence of DNA, DrRecA produces significantly shorter and/or fewer free-filaments in solution than does EcRecA. The results suggest intrinsic biothermodynamic properties of DrRecA contribute directly to the more robust DNA repair capabilities of D. radiodurans.