Key aspects of papillomavirus infection influence the host cervicovaginal microbiome in a preclinical murine papillomavirus (MmuPV1) infection model.

Human papillomaviruses (HPVs) are the most common sexually transmitted infection in the United States and are a major etiological agent of cancers in the anogenital tract and oral cavity. Growing evidence suggests changes in the host microbiome are associated with the natural history and ultimate outcome of HPV infection. We sought to define changes in the host cervicovaginal microbiome during papillomavirus infection, persistence, and pathogenesis using the murine papillomavirus (MmuPV1) cervicovaginal infection model. Cervicovaginal lavages were performed over a time course of MmuPV1 infection in immunocompetent female FVB/N mice and extracted DNA was analyzed by qPCR to track MmuPV1 viral copy number. 16S ribosomal RNA (rRNA) gene sequencing was used to determine the composition and diversity of microbial communities throughout this time course. We also sought to determine whether specific microbial communities exist across the spectrum of MmuPV1-induced neoplastic disease. We, therefore, performed laser-capture microdissection to isolate regions of disease representing all stages of neoplastic disease progression (normal, low- and high-grade dysplasia, and cancer) from female reproductive tract tissue sections from MmuPV1-infected mice and performed 16S rRNA sequencing. Consistent with other studies, we found that the natural murine cervicovaginal microbiome is highly variable across different experiments. Despite these differences in initial microbiome composition between experiments, we observed that MmuPV1 persistence, viral load, and severity of disease influenced the composition of the cervicovaginal microbiome. These studies demonstrate that papillomavirus infection can alter the cervicovaginal microbiome.IMPORTANCEHuman papillomaviruses (HPVs) are the most common sexually transmitted infection in the United States. A subset of HPVs that infect the anogenital tract (cervix, vagina, anus) and oral cavity cause at least 5% of cancers worldwide. Recent evidence indicates that the community of microbial organisms present in the human cervix and vagina, known as the cervicovaginal microbiome, plays a role in HPV-induced cervical cancer. However, the mechanisms underlying this interplay are not well-defined. In this study, we infected the female reproductive tract of mice with a murine papillomavirus (MmuPV1) and found that key aspects of papillomavirus infection and disease influence the host cervicovaginal microbiome. This is the first study to define changes in the host microbiome associated with MmuPV1 infection in a preclinical animal model of HPV-induced cervical cancer. These results pave the way for using MmuPV1 infection models to further investigate the interactions between papillomaviruses and the host microbiome.

HIV-1 virological synapse formation enhances infection spread by dysregulating Aurora Kinase B.

HIV-1 spreads efficiently through direct cell-to-cell transmission at virological synapses (VSs) formed by interactions between HIV-1 envelope proteins (Env) on the surface of infected cells and CD4 receptors on uninfected target cells. Env-CD4 interactions bring the infected and uninfected cellular membranes into close proximity and induce transport of viral and cellular factors to the VS for efficient virion assembly and HIV-1 transmission. Using novel, cell-specific stable isotope labeling and quantitative mass spectrometric proteomics, we identified extensive changes in the levels and phosphorylation states of proteins in HIV-1 infected producer cells upon mixing with CD4+ target cells under conditions inducing VS formation. These coculture-induced alterations involved multiple cellular pathways including transcription, TCR signaling and, unexpectedly, cell cycle regulation, and were dominated by Env-dependent responses. We confirmed the proteomic results using inhibitors targeting regulatory kinases and phosphatases in selected pathways identified by our proteomic analysis. Strikingly, inhibiting the key mitotic regulator Aurora kinase B (AURKB) in HIV-1 infected cells significantly increased HIV activity in cell-to-cell fusion and transmission but had little effect on cell-free infection. Consistent with this, we found that AURKB regulates the fusogenic activity of HIV-1 Env. In the Jurkat T cell line and primary T cells, HIV-1 Env:CD4 interaction also dramatically induced cell cycle-independent AURKB relocalization to the centromere, and this signaling required the long (150 aa) cytoplasmic C-terminal domain (CTD) of Env. These results imply that cytoplasmic/plasma membrane AURKB restricts HIV-1 envelope fusion, and that this restriction is overcome by Env CTD-induced AURKB relocalization. Taken together, our data reveal a new signaling pathway regulating HIV-1 cell-to-cell transmission and potential new avenues for therapeutic intervention through targeting the Env CTD and AURKB activity.

Nodavirus RNA replication crown architecture reveals proto-crown precursor and viral protein A conformational switching.

Positive-strand RNA viruses replicate their genomes in virus-induced membrane vesicles, and the resulting RNA replication complexes are a major target for virus control. Nodavirus studies first revealed viral RNA replication proteins forming a 12-fold symmetric "crown" at the vesicle opening to the cytosol, an arrangement recently confirmed to extend to distantly related alphaviruses. Using cryoelectron microscopy (cryo-EM), we show that mature nodavirus crowns comprise two stacked 12-mer rings of multidomain viral RNA replication protein A. Each ring contains an ~19 nm circle of C-proximal polymerase domains, differentiated by strikingly diverged positions of N-proximal RNA capping/membrane binding domains. The lower ring is a "proto-crown" precursor that assembles prior to RNA template recruitment, RNA synthesis, and replication vesicle formation. In this proto-crown, the N-proximal segments interact to form a toroidal central floor, whose 3.1 Å resolution structure reveals many mechanistic details of the RNA capping/membrane binding domains. In the upper ring, cryo-EM fitting indicates that the N-proximal domains extend radially outside the polymerases, forming separated, membrane-binding "legs." The polymerase and N-proximal domains are connected by a long linker accommodating the conformational switch between the two rings and possibly also polymerase movements associated with RNA synthesis and nonsymmetric electron density in the lower center of mature crowns. The results reveal remarkable viral protein multifunctionality, conformational flexibility, and evolutionary plasticity and insights into (+)RNA virus replication and control.

Hazard Perception Skill and Driver Behavior in Patients With Functional Neurologic Disorders.

BACKGROUND AND OBJECTIVES: Driving in patients with functional neurologic disorders (FND) is a major concern, but current guidelines (where they exist) are based on expert consensus only due to a lack of relevant empirical evidence. This study aimed to provide such evidence by comparing drivers with FND with healthy controls on aspects of driving performance and behavior important to crash risk, including hazard perception skill. METHODS: Participants completed validated self-report questionnaires of driving behaviors (assessing lapses, errors, violations, and attentional issues) and 2 computer-based measures of hazard perception skill (both known to be associated with crash risk). RESULTS: We compared 43 patients who experience dissociative attacks or functional motor symptoms and 43 healthy controls. Patients with FND self-reported significantly more driving lapses and driving errors compared with healthy controls. However, there were no significant between-group differences in self-reports of ordinary violations, aggressive violations, or attention-related errors. Participants in the FND group and healthy controls exhibited a similar performance on a response time hazard perception test (6.27 vs 5.51 seconds, p = 0.245). However, participants with FND remarkably outperformed the controls in the number of plausible predictions they made in a verbal response hazard prediction test (1.55 vs 1.18 predictions per clip, p = 0.006). DISCUSSION: Our findings suggest that the ability of drivers with FND to predict traffic hazards in between attacks or flares is not worse than that of healthy individuals, with the possibility that it might even be better under some circumstances. Further studies with various populations are needed to replicate our findings.

Multifunctional Protein A Is the Only Viral Protein Required for Nodavirus RNA Replication Crown Formation.

Positive-strand RNA virus RNA genome replication occurs in membrane-associated RNA replication complexes (RCs). Nodavirus RCs are outer mitochondrial membrane invaginations whose necked openings to the cytosol are "crowned" by a 12-fold symmetrical proteinaceous ring that functions as the main engine of RNA replication. Similar protein crowns recently visualized at the openings of alphavirus and coronavirus RCs highlight their broad conservation and functional importance. Using cryo-EM tomography, we earlier showed that the major nodavirus crown constituent is viral protein A, whose polymerase, RNA capping, membrane interaction and multimerization domains drive RC formation and function. Other viral proteins are strong candidates for unassigned EM density in the crown. RNA-binding RNAi inhibitor protein B2 co-immunoprecipitates with protein A and could form crown subdomains that protect nascent viral RNA and dsRNA templates. Capsid protein may interact with the crown since nodavirus virion assembly has spatial and other links to RNA replication. Using cryoelectron tomography and complementary approaches, we show that, even when formed in mammalian cells, nodavirus RC crowns generated without B2 and capsid proteins are functional and structurally indistinguishable from mature crowns in infected Drosophila cells expressing all viral proteins. Thus, the only nodaviral factors essential to form functional RCs and crowns are RNA replication protein A and an RNA template. We also resolve apparent conflicts in prior results on B2 localization in infected cells, revealing at least two distinguishable pools of B2. The results have significant implications for crown structure, assembly, function and control as an antiviral target.

How distracting is chronic pain? The impact of chronic pain on driving behaviour and hazard perception.

In road safety research, few studies have examined driving behaviour in chronic pain cohorts. The aim of this study was to investigate driving behaviour among drivers experiencing chronic pain. We compared individuals with chronic pain with age-gender matched healthy controls. Participants completed: (i) an anonymous online survey that included participant demographics, transport characteristics, self-reported driving behaviour, and pain characteristics (ii) a response-time hazard perception test and a verbal-response hazard prediction test for drivers, and (iii) a driving diary in which participants recorded their driving over two weeks. The results showed that participants with chronic pain were not significantly worse than controls for hazard perception and prediction test scores, self-reported attention-related errors, driving errors, driving violations, and involuntary distraction. Drivers with chronic pain did report significantly more driving lapses but this effect became non-significant when variables confounded with chronic pain, such as fatigue, were adjusted for. We also found that participants who reported particularly high levels of chronic pain performed worse in the hazard prediction test compared to the control group (and this effect could not be accounted for by other variables associated with chronic pain). In addition, participants with chronic pain reported significantly higher driving workload (mental demand, physical demand, effort, and frustration) compared with controls. The findings of this study provide new insights into driving behaviour in individuals with chronic pain and recommendations for future research in terms of driving assessment and self-regulation strategies are provided.

Exploring driving behaviour from the perspectives of individuals with chronic pain and health professionals.

Chronic pain affects one in five Australians, and this could impact daily activities such as driving. Driving is a complex task, which requires the cognitive and physical ability to predict, identify, and respond to hazards to avoid crashing. However, research exploring the factors that influence safe driving behaviour for chronic pain individuals is limited. A qualitative study was conducted which involved semi-structured interviews with 23 people who had experienced persistent pain for at least three months and 17 health professionals who had experience working with individuals with chronic pain. The aim of this study was to obtain a deeper understanding of the experiences and challenges that people with chronic pain may have in their day-to-day driving. Participants were also asked about currently available driving assessments and strategies for individuals with chronic pain in the Australian healthcare system. The themes emerging from the interviews highlighted the need for clearer guidelines and educational materials regarding the impact of chronic pain on an individual's ability to drive. These themes included the physical and cognitive challenges resulting from chronic pain, as well as the potential side effects of pain medications. In addition, participants identified a number of self-regulation strategies and driving assessments currently available for monitoring safe driving behaviour in Australia. This study improves our understanding of how chronic pain affects driving behaviour, as reported by individuals experiencing the pain and relevant health professionals. Recommendations for improving the safety of drivers with chronic pain are discussed, including possible technological interventions and better public education.

Aptitude and attitude: predictors of performance during and after basic laparoscopic skills training.

BACKGROUND: Manual dexterity and visual-spatial ability are considered key to the development of superior laparoscopic skills. Nevertheless, these abilities do not reliably explain all the variance found in the technical performance of surgical trainees. Consequently, we must look beyond these abilities to improve our understanding of laparoscopic skills and to better identify/develop surgical potential earlier on. PURPOSE: To assess the individual and collective impact of physical, cognitive, visual, and psychological variables on performance during and after basic simulation-based laparoscopic skills training. METHOD: Thirty-four medical students (laparoscopic novices) completed a proficiency-based laparoscopic skills training program (using either a 2D or 3D viewing mode). This was followed by one testing session, a follow-up testing session with new (yet similar) tasks, and a series of physical, cognitive, visual, and psychological measures. RESULTS: The statistical models that best predicted variance in training performance metrics included four variables: viewing mode (2D vs 3D), psychological flexibility, perceived task demands, and manual dexterity (bimanual). In subsequent testing, a model that included viewing mode and manual dexterity (assembly) best predicted performance on the pre-practiced tasks. However, for a highly novel, spatially complex laparoscopic task, performance was best predicted by a model that comprised viewing mode, visual-spatial ability, and perceived task demands. At follow-up, manual dexterity (assembly) alone was the best predictor of performance on new (yet similar) tasks. CONCLUSION: By focussing exclusively on physical/cognitive abilities, we may overlook other important predictors of surgical performance (e.g. psychological variables). The present findings suggest that laparoscopic performance may be more accurately explained through the combined effects of physical, cognitive, visual, and psychological variables. Further, the results suggest that the predictors may change with both task demands and the development of the trainee. This study highlights the key role of psychological skills in overcoming initial training challenges, with far-reaching implications for practice.

Impact of chronic pain on driving behaviour: a systematic review.

ABSTRACT: Driving is a complex task that requires both the ability to rapidly identify potential hazards and respond appropriately to driving situations to avoid crashing. A great deal of research has sought to increase road safety by focusing on risky behaviours, very few of which have explored the effects of chronic pain (CP) on driving behaviour. This systematic review aimed to assess driving behaviour and motor vehicle crash risk in drivers with CP. Four databases (Embase, PubMed, Scopus, and PsycINFO) were searched using relevant search terms. From 8543 studies, 22 studies met the eligibility criteria for inclusion in this review. A driving behaviour framework, based on the Michon model of driving behaviour, is proposed to map the effect of CP on driving behaviour. Findings suggest that drivers with CP engage in risk-compensatory strategies that are positive from a precautionary perspective. However, there is considerable variability in the use of such strategies across different samples, suggesting that there are significant barriers and facilitators involved in these decisions. Moreover, our findings provide some evidence that CP could increase crash risk and change driving behaviour. Evidence-based recommendations for practitioners and policymakers are proposed regarding the risks of driving in individuals experiencing CP. Future research into CP in driving could benefit from having a unified evidence-based approach to determine behaviour at all levels of the driving task.

Learner drivers (and their parent-supervisors) benefit from an online hazard perception course incorporating evidence-based training strategies and extensive crash footage.

We previously found that a six-session online hazard perception training course, which incorporates evidence-based learning strategies and footage of over a hundred real crashes, improved hazard perception skill and reduced risk-taking intentions in novice drivers who had passed their on-road driving test within the previous three years. However, one issue with targeting crash-prevention training at individuals who are already driving unsupervised is that drivers are at their highest crash risk immediately after they pass their on-road driving test. That is, the training may arrive too late to protect drivers while they are at their most vulnerable. It is also possible that it may prove difficult to persuade drivers to complete an unsupervised training course if they are already licensed to drive independently. Given that learner drivers cannot drive unsupervised, and that they are typically supervised by a parent, one potential strategy is to target the training at learners and to ask their parents to provide one-on-one mentoring throughout the course. We therefore recruited learner driver/parent-supervisor dyads to participate in a randomized control study, with the objective of examining the effects of the hazard perception training course on aspects of driving behaviour associated with crash risk (as measured using validated computer-based tests). Outcome measures included two hazard perception skill assessments (a response time hazard perception test and a verbal response hazard prediction test), and three tests assessing aspects of risk-taking propensity in driving (speed choice, following distance, and gap acceptance). Learners who completed the course (N = 26) significantly improved their scores on both hazard perception skill measures, and also chose safer following distances, compared with a waitlist control group (N = 23). However, the training did not significantly reduce learners' speed choice or gap acceptance propensity. The hazard perception skill of parent-supervisors, who observed the course but did not complete it, also improved on both hazard perception measures, relative to controls. Additionally, both learners and their parent-supervisors reported a range of positive effects on the learners' real-world driving performance. These results suggest that this type of hazard perception training could be beneficial if deployed during the learner phase of driver licensing.

A human factors approach to subcutaneous insulin chart design improves user-performance: An experimental study.

Insulin is a high-risk medicine that has been implicated in serious adverse events for hospital inpatients, including medication-error related deaths. Most insulin errors occur during administration, and "wrong dose" is the most common type. A paper-based subcutaneous insulin chart (the "NSIC") was developed for the Australian Commission on Safety and Quality in Health Care, using a range of human factors methods, with the aim of reducing the opportunity for errors. The present lab-based study empirically assessed whether the NSIC's human factors design translates into improved user-performance in the determination of insulin doses, compared with a pre-existing chart. Forty-one experienced nurses and 48 novice chart-users completed 60 experimental trials (30 per chart), in which they determined doses to administer to patients. Both groups determined insulin doses faster, and made fewer dose errors, when using the NSIC. These results support the utility of the usability heuristics employed in developing the chart.

Hazard Perception in Older Drivers With Eye Disease.

Purpose: Timely detection of hazards is a key driving skill; however, the hazard perception of drivers with eye disease and related visual changes and the visual predictors of hazard perception are poorly understood. Methods: Participants included drivers aged 65 years and older with a range of eye diseases, including cataract, age-related maculopathy (AMD), and glaucoma (n = 99; mean age, 75.4 ± 6.4 years) and controls (n = 118; mean age, 72.2 ± 5.5 years). Visual performance was assessed using clinical measures (visual acuity, contrast sensitivity, visual fields) and non-clinical measures (useful field of view, motion sensitivity). Participants completed a computer-based hazard perception test (HPT) that has been related to driving performance and crash risk. Results: Participants with eye disease exhibited a 0.73-second delay in HPT response times compared to controls (6.61 ± 1.62 seconds vs. 5.88 ± 1.38 seconds; age-adjusted P = 0.012). Participants with glaucoma exhibited significantly delayed responses compared to those with AMD (P = 0.038) and controls (P = 0.004). Poorer motion sensitivity (standardized ß = 0.27; P < 0.001), visual acuity (ß = 0.21; P = 0.002), and better-eye mean defect (ß = -0.17; P = 0.009) were most strongly associated with delayed HPT responses. Motion sensitivity remained significantly associated with HPT responses, adjusted for visual acuity and visual fields. Conclusions: HPT responses of older drivers with eye disease were delayed compared to controls and translate to an estimated 16-meter longer stopping distance when traveling at 80 km/hr. Decreased motion sensitivity was most strongly associated with delayed HPT responses. Translational Relevance: HPT tests can provide insight into difficulties regarding road hazard detection of older drivers with eye disease and provide a potential avenue for interventions to improve road safety.

A thousand years of crash experience in three hours: An online hazard perception training course for drivers.

A key goal of driver training is to teach drivers to avoid crashes. However, in traditional driver training, drivers are unlikely to see even a single example of the class of event that we want them to learn to avoid. We developed a six-session automated online hazard perception training course for drivers, which incorporates a range of evidence-based strategies and employs extensive video footage of real crashes. We evaluated this course in a randomized control trial by examining its effects on previously-validated computer-based measures of hazard perception, hazard prediction, speed choice, following distance, and gap acceptance propensity, as well as self-rated measures of driver skill, safety, and real world transfer. We found that the course resulted in significant improvements in hazard perception response time and hazard prediction scores, and significantly longer vehicle following distances. Additionally, all participants in the trained group reported that their real world driving behaviour had improved. No significant training effects were found for the other measures. The results suggest that the course can improve key behaviours associated with crash risk.

Laparoscopic skills training: the effects of viewing mode (2D vs. 3D) on skill acquisition and transfer.

BACKGROUND: Three-dimensional (3D) visual displays have been suggested to aid laparoscopic skills training by providing the depth cues not present in traditional two-dimensional (2D) displays. However, few studies have robustly investigated the impact of viewing mode (2D vs. 3D) on learning outcomes. PURPOSE: To examine how viewing mode (2D vs. 3D) impacts the acquisition and transferability of basic laparoscopic skills by comparing performance between transfer and control groups on a complete proficiency-based training program. METHOD: A counterbalanced between-subjects design was employed. Each participant was randomly allocated to one of four groups, comprising two transfer groups (trained in one viewing mode and tested in the alternate mode: the 2D → 3D and 3D → 2D groups) and two control groups (trained and tested in one viewing mode: the 2D → 2D and 3D → 3D groups). Participants completed proficiency-based training in six laparoscopic training tasks. Testing included two further repetitions of all tasks under test conditions. Objective performance measures included the total number of repetitions to reach proficiency, and total performance scores (i.e. time + error penalties across all repetitions) in training and testing. RESULTS: The groups trained in 3D demonstrated superior training performance (i.e. less time + errors) and took fewer repetitions to reach proficiency than the groups trained in 2D. The groups tested in 3D also demonstrated superior test performance compared to those tested in 2D. However, training mode did not yield significant test differences between the groups tested in 2D (i.e. 2D → 2D vs. 3D → 2D), or between the groups tested in 3D (i.e. 3D → 3D vs. 2D → 3D). CONCLUSION: Novices demonstrate superior performance in laparoscopic skills training using a 3D viewing mode compared to 2D. However, this does not necessarily translate to superior performance in subsequent testing or enhanced learning overall. Rather, test performance appears to be dictated by the viewing mode used during testing, not that of prior training.

Quantitative systematic review: Sources of inaccuracy in manually measured adult respiratory rate data.

AIMS: To identify the potential sources of inaccuracy in manually measured adult respiratory rate (RR) data and quantify their effects. DESIGN: Quantitative systematic review with meta-analyses where appropriate. DATA SOURCES: Medline, CINAHL, and Cochrane Library (from database inception to 31 July 2019). REVIEW METHODS: Studies presenting data on individual sources of inaccuracy in the manual measurement of adult RR were analysed, assessed for quality, and grouped according to the source of inaccuracy investigated. Quantitative data were extracted and synthesized and meta-analyses performed where appropriate. RESULTS: Included studies (N = 49) identified five sources of inaccuracy. The awareness effect creates an artefactual reduction in actual RR, and observation methods involving shorter counts cause systematic underscoring. Individual RR measurements can differ substantially in either direction between observations due to inter- or intra-observer variability. Value bias, where particular RRs are over-represented (suggesting estimation), is a widespread problem. Recording omission is also widespread, with higher average rates in inpatient versus triage/admission contexts. CONCLUSION: This review demonstrates that manually measured RR data are subject to several potential sources of inaccuracy. IMPACT: RR is an important indicator of clinical deterioration and commonly included in track-and-trigger systems. However, the usefulness of RR data depends on the accuracy of the observations and documentation, which are subject to five potential sources of inaccuracy identified in this review. A single measurement may be affected by several factors. Hence, clinicians should interpret recorded RR data cautiously unless systems are in place to ensure its accuracy. For nurses, this includes counting rather than estimating RRs, employing 60-s counts whenever possible, ensuring patients are unaware that their RR is being measured, and documenting the resulting value. For any given site, interventions to improve measurement should take into account the local organizational and cultural context, available resources, and the specific measurement issues that need to be addressed.

The development and validation of video-based measures of drivers' following distance and gap acceptance behaviours.

The distance at which drivers follow other vehicles has been found to be linked to crash risk. Tailgating (i.e. driving at an unsafe following distance) is both endemic and a leading cause of rear-end crashes. Similarly, drivers' decisions about when to merge with a stream of traffic are likely to influence crash risk. Consistent with this, it has been shown that crashes are more common at intersections where drivers more frequently have to slow for vehicles pulling out into insufficient gaps. Therefore, the development of reliable and valid measures of both of these driving behaviours would facilitate further crash prevention research. Given the problems associated with assessing these behaviours during real driving, we developed new video-based measures. In our new following distance measure, participants view videos shot from the perspective of a driver who is following another vehicle at a range of distances across a variety of traffic environments. On each trial, participants report their own minimum comfortable following distance relative to the following distance depicted in the video. In our new test of gap acceptance behaviour, participants view a series of video clips and indicate when they would pull out into the approaching stream of traffic shown in each clip. The two new measures each yielded reliable data, and we found that young drivers made riskier choices than older drivers for both following distance and gap acceptance. These age-related differences are consistent with those found in observational studies of real driving, supporting the proposal that the new tests could potentially be used as proxies for these crash-related driving behaviours in both lab-based research and large-scale online studies.

Subdomain cryo-EM structure of nodaviral replication protein A crown complex provides mechanistic insights into RNA genome replication.

For positive-strand RNA [(+)RNA] viruses, the major target for antiviral therapies is genomic RNA replication, which occurs at poorly understood membrane-bound viral RNA replication complexes. Recent cryoelectron microscopy (cryo-EM) of nodavirus RNA replication complexes revealed that the viral double-stranded RNA replication template is coiled inside a 30- to 90-nm invagination of the outer mitochondrial membrane, whose necked aperture to the cytoplasm is gated by a 12-fold symmetric, 35-nm diameter "crown" complex that contains multifunctional viral RNA replication protein A. Here we report optimizing cryo-EM tomography and image processing to improve crown resolution from 33 to 8.5 Å. This resolves the crown into 12 distinct vertical segments, each with 3 major subdomains: A membrane-connected basal lobe and an apical lobe that together comprise the ∼19-nm-diameter central turret, and a leg emerging from the basal lobe that connects to the membrane at ∼35-nm diameter. Despite widely varying replication vesicle diameters, the resulting two rings of membrane interaction sites constrain the vesicle neck to a highly uniform shape. Labeling protein A with a His-tag that binds 5-nm Ni-nanogold allowed cryo-EM tomography mapping of the C terminus of protein A to the apical lobe, which correlates well with the predicted structure of the C-proximal polymerase domain of protein A. These and other results indicate that the crown contains 12 copies of protein A arranged basally to apically in an N-to-C orientation. Moreover, the apical polymerase localization has significant mechanistic implications for template RNA recruitment and (-) and (+)RNA synthesis.

Validation of Brief Screening Tools to Identify Impaired Driving Among Older Adults in Australia.

Importance: There is an urgent need to develop evidence-based assessments to identify older individuals who may be unsafe drivers. Objective: To validate 8 off-road brief screening tests to predict on-road driving ability and to identify which combination of these provides the best prediction of older adults who will not pass an on-road driving test. Design, Setting, and Participants: This prognostic study was conducted between October 31, 2013, and May 10, 2017, using the criterion standard for screening tests, an on-road driving test, with analysis conducted from August 1, 2019, to April 2, 2020. A volunteer sample of older drivers was recruited from community advertisements, rehabilitation and driver assessment clinics, and an optometry clinic in Canberra and Brisbane, Australia. Exposures: Off-road driver screening measures, including the Useful Field of View, DriveSafe/DriveAware, Multi-D battery, Trails B, Maze test, Hazard Perception Test, DriveSafe Intersection test, and 14-item Road Law test. Main Outcomes and Measures: Classification as unsafe on a standardized 50-minute on-road driving assessment administered by a driving instructor and an occupational therapist masked to the participant's clinical diagnosis and off-road test performance. Results: A total of 560 drivers aged 63 to 94 years (mean [SD] age, 74.7 [6.2] years]; 350 [62.5%] men) were assessed. Logistic regression and receiver operating characteristic analyses indicated the area under the curve was largest for a multivariate model comprising the Multi-D, Useful Field of View, and Hazard Perception Test, with an area under the curve of 0.89 (95% CI, 0.85-0.94), sensitivity of 80.4%, and specificity of 84.1% for predicting unsafe drivers. The Multi-D battery was the most accurate individual assessment and had an area under the curve of 0.85 (95% CI, 0.79-0.90), sensitivity of 77.1%, and specificity of 82.1%. The multivariate model had sensitivity of 83.3% and specificity of 91.8% in the cognitively impaired group and sensitivity of 87.5% and specificity of 70.8% in the visually impaired group. Conclusions and Relevance: These findings suggest that off-road screening tests can reliably identify older drivers with a strong probability of failing an on-road driving test. Implementation of these measures could enable better targeting of resources for managing older driver licensing and support injury prevention strategies in this group.