Correlates of Menarcheal Age in a Psychiatric Sample of Adolescents.

ABSTRACT: Early pubertal timing is associated with more adverse childhood experiences (ACEs) and increased risk for psychopathology during adolescence. However, most work to date has used community or epidemiological samples, and it remains unclear whether these associations persist in acute clinical samples. The present study examined associations between age at menarche and ACEs, psychiatric symptoms, and emotion regulation difficulties in a sample of N = 140 adolescents on a psychiatric inpatient unit. Youth with early menarche reported higher levels of depressive symptoms, more severe suicidal ideation, and greater difficulty with emotion regulation than youth with normative age at menarche. There was a marginal effect of youth with early menarche reporting more ACEs and more anxiety symptoms. These results suggest menarcheal age, and ACEs may be useful risk factors to assess in inpatient settings to predict risk for more severe outcomes, and future research on pubertal timing in high acuity settings is warranted.

Investigating Substance Use as a Coping Strategy Among Adolescent Psychiatric Inpatients: A Comparative Analysis Before and During the COVID-19 Pandemic.

The COVID-19 pandemic resulted in significant changes in daily life, potentially impacting mental health and substance use behavior. Research on COVID-related changes in adolescent substance use have yielded mixed findings. The current cross-sectional chart review study compared rates of past-year substance use before and during COVID-19 among adolescent psychiatric inpatients, and investigated how motives for coping with COVID-19 changes were related to psychiatric acuity, and past-year substance use. Count models assessed if the number of past-year days of alcohol and cannabis use was higher among adolescents (n = 491, 11-18 years, 61% female) hospitalized during COVID-19 (3/14/20 to 4/5/21) versus adolescents hospitalized before COVID-19 (8/30/2019 to 3/13/20). For a subsample of COVID-19 inpatients (n = 124; 75% female), we evaluated psychiatric correlates of endorsing substances to cope with COVID-19 changes/rules. Results indicated adolescents admitted during COVID-19 reported significantly more past-year alcohol and cannabis use days than adolescents admitted before COVID-19. Adolescents endorsed using alcohol (19%), cannabis (33%), and e-cigarettes/vaping (25%) to cope with COVID-19. E-cigarette/vaping to cope with COVID-19 was significantly related to lifetime suicide attempt. Endorsing alcohol or cannabis to cope with COVID-19 was associated with a significantly greater number of past-year use days for each respective substance. Adolescent psychiatric inpatients admitted during COVID-19 reported more substance use days than adolescents admitted before COVID-19. Using substances to cope was linked to psychiatric correlates (e.g., suicidality). Assessing the presence and function of substance use in this population may be important to identify, treat, and prevent compounding negative outcomes during times of community stress.

Management Considerations for Air Medical Transport Programs Transfusing RhD-Positive Red Blood Cell-Containing Products to Females of Childbearing Potential.

Recent years have seen increased discussion surrounding the benefits of damage control resuscitation, prehospital transfusion (PHT) of blood products, and the use of whole blood over component therapy. Concurrent shortages of blood products with the desire to provide PHT during air medical transport have prompted reconsideration of the traditional approach of administering RhD-negative red cell-containing blood products first-line to females of childbearing potential (FCPs). Given that only 7% of the US population has blood type O negative and 38% has O positive, some programs may be limited to offering RhD-positive blood products to FCPs. Adopting the practice of giving RhD-positive blood products first-line to FCPs extends the benefits of PHT to such patients, but this practice does incur the risk of future hemolytic disease of the fetus and newborn (HDFN). Although the risk of future fetal mortality after an RhD-incompatible transfusion is estimated to be low in the setting of acute hemorrhage, the number of FCPs who are affected by this disease will increase as more air medical transport programs adopt this practice. The process of monitoring and managing HDFN can also be time intensive and costly regardless of the rates of fetal mortality. Air medical transport programs planning on performing PHT of RhD-positive red cell-containing products to FCPs should have a basic understanding of the pathophysiology, prevention, and management of hemolytic disease of the newborn before introducing this practice. Programs should additionally ensure there is a reliable process to notify receiving centers of potentially RhD-incompatible PHT because alloimmunization prophylaxis is time sensitive. Facilities receiving patients who have had PHT must be prepared to identify, counsel, and offer alloimmunization prophylaxis to these patients. This review aims to provide air medical transport professionals with an understanding of the pathophysiology and management of HDFN and provide a template for the early management of FCPs who have received an RhD-positive red cell-containing PHT. This review also covers the initial workup and long-term anticipatory guidance that receiving trauma centers must provide to FCPs who have received RhD-positive red cell-containing PHT.

Helicopter Emergency Medical Services Outcomes Research 1983 to 2022: Evidence Overview and Longitudinal Trends.

OBJECTIVE: Helicopter emergency medical services (HEMS) literature has been assessed in reviews focusing on various diagnoses, but there are few, if any, summaries of the entire body of HEMS outcomes evidence. Our goal was to summarize the existing research addressing patient-centered outcomes potentially accrued with HEMS. METHODS: As part of the Critical Care Transport Collaborative Outcomes Research Effort, we generated the HEMS Outcomes Assessment Research Database and executed descriptive analyses of longitudinal trends from 1983 to 2022. Both indexed and gray literature sources were incorporated in the HEMS Outcomes Assessment Research Database. Studies were reviewed by at least 2 authors to select those that addressed a patient-centered outcome. Studies addressing solely HEMS logistics were excluded. Categoric analyses were executed with the Fisher exact test, and continuous variables were evaluated for normality with normal quantile plotting and a comparison of medians and 95% confidence intervals. RESULTS: We found that HEMS outcomes study sample sizes increased steadily from 1983 to 2012, with the most recent decade demonstrating a marked increase in the rate of publication of HEMS outcomes studies. Most research (70.6%) addressed trauma patient outcomes, but recent decades have seen a significant increase in non-trauma studies. Recent decades have also been characterized by an increase in the production of HEMS outcomes research outside of North America and Europe. CONCLUSION: This study summarizes the current state of the HEMS outcome literature. We highlight increasing contributions from worldwide researchers and increasing focus on HEMS benefits in non-trauma cases, particularly time-critical cases such as cardiac or stroke diagnoses. This provides a basis for further investigations into patient-oriented benefits potentially accrued with HEMS.

Relationship between cognitive flexibility and subsequent course of mood symptoms and suicidal ideation in young adults with childhood-onset bipolar disorder.

Neurocognitive deficits, such as cognitive flexibility impairments, are common in bipolar disorder (BD) and predict poor academic, occupational, and functional outcomes. However, the association between neurocognition and illness trajectory is not well understood, especially across developmental transitions. This study examined cognitive flexibility and subsequent mood symptom and suicidal ideation (SI) course in young adults with childhood-onset BD-I (with distinct mood episodes) vs. BD-not otherwise specified (BD-NOS) vs. typically-developing controls (TDCs). Sample included 93 young adults (ages 18-30) with prospectively verified childhood-onset DSM-IV BD-I (n = 34) or BD-NOS (n = 15) and TDCs (n = 44). Participants completed cross-sectional neuropsychological tasks and clinical measures. Then participants with BD completed longitudinal assessments of mood symptoms and SI at 6-month intervals (M = 39.18 ± 16.57 months of follow-up data). Analyses included ANOVAs, independent-samples t tests, chi-square analyses, and multiple linear regressions. Participants with BD-I had significant deficits in cognitive flexibility and executive functioning vs. BD-NOS and TDCs, and impaired spatial working memory vs. TDCs only. Two significant BD subtype-by-cognitive flexibility interactions revealed that cognitive flexibility deficits were associated with subsequent percentage of time depressed and with SI in BD-I but not BD-NOS, regardless of other neurocognitive factors (full-scale IQ, executive functioning, spatial working memory) and clinical factors (current and prior mood and SI symptoms, age of BD onset, global functioning, psychiatric medications, comorbidity). Thus, cognitive flexibility may be an important etiological brain/behavior mechanism, prognostic indicator, and intervention target for childhood-onset BD-I, as this deficit appears to endure into young adulthood and is associated with worse prognosis for subsequent depression and SI.

Adolescent Sibling Associations among Alcohol, Cannabis, and Sexual Risk Behavior: A Test of Interdependence.

BACKGROUND: Although siblings are conceptualized as a salient social influence during adolescence, few studies have examined how adolescent siblings influence each other's substance use and risky sexual behavior. Objectives: In this study, we investigated the influence of alcohol use days, cannabis use days, and cannabis and alcohol co-use days on the sexual risk behavior of siblings while accounting for dyadic influence. METHODS: At the baseline visit for a randomized controlled trial for adolescents referred due to parents' concerns about their substance use ("referred adolescents"; n = 99; Mage=15.95; 38.38% female), we assessed alcohol and cannabis use days as well as sexual risk behavior of the referred adolescents and their sibling (Mage=15.03; 51.52% female). We computed the number of days in the 30 days prior to the baseline that alcohol and cannabis use occurred on the same day. Using a cross-sectional actor partner interdependence model, we tested two models of how adolescents' substance use is associated with their own ("actor effect") and their siblings' ("partner effect") sexual risk behavior-one model for alcohol and cannabis use, and one model for daily co-use. RESULTS: For referred adolescents and their siblings, within an individual, greater alcohol, cannabis, and daily co-use was significantly associated with sexual risk behavior (actor effects). Furthermore, more sibling co-use days was positively associated with referred adolescent sexual risk behavior (partner effect), representing interdependence. CONCLUSION: These findings confirm the influence siblings have on one another's risky behavior in adolescence and have implications for prevention and intervention efforts for adolescent substance use.

Adolescent alcohol use predicts cannabis use over a three year follow-up period.

Background: Alcohol and cannabis use frequently co-occur, which can result in problems from social and academic impairment to dependence (i.e., alcohol use disorder [AUD] and/or cannabis use disorder [CUD]). The Emergency Department (ED) is an excellent site to identify adolescents with alcohol misuse, conduct a brief intervention, and refer to treatment; however, given time constraints, alcohol use may be the only substance assessed due to its common role in unintentional injury. The current study, a secondary data analysis, assessed the relationship between adolescent alcohol and cannabis use by examining the National Institute of Alcohol Abuse and Alcoholism (NIAAA) two question screen's (2QS) ability to predict future CUD at one, two, and three years post-ED visit. Methods: At baseline, data was collected via tablet self-report surveys from medically and behaviorally stable adolescents 12-17 years old (n = 1,689) treated in 16 pediatric EDs for non-life-threatening injury, illness, or mental health condition. Follow-up surveys were completed via telephone or web-based survey. Logistic regression compared CUD diagnosis odds at one, two, or three-year follow-up between levels constituting a single-level change in baseline risk categorization on the NIAAA 2QS (nondrinker versus low-risk, low- versus moderate-risk, moderate- versus high-risk). Receiver operating characteristic curve methods examined the predictive ability of the baseline NIAAA 2QS cut points for CUD at one, two, or three-year follow-up. Results: Adolescents with low alcohol risk had significantly higher rates of CUD versus nondrinkers (OR range: 1.94-2.76, p < .0001). For low and moderate alcohol risk, there was no difference in CUD rates (OR range: 1.00-1.08). CUD rates were higher in adolescents with high alcohol risk versus moderate risk (OR range: 2.39-4.81, p < .05). Conclusions: Even low levels of baseline alcohol use are associated with risk for a later CUD. The NIAAA 2QS is an appropriate assessment measure to gauge risk for future cannabis use.

COVID-19-Specific Suicidal Thoughts and Behaviors in Psychiatrically Hospitalized Adolescents.

This study examined the presence and correlates of COVID-specific suicidal thoughts and behaviors (i.e., thoughts of or engaging in intentional COVID-19 exposure with associated suicidal intent) among psychiatrically hospitalized adolescents. Adolescents (N = 143) completed study measures as part of the standard intake process between March 13th and August 14th, 2020. Participants answered questionnaires assessing COVID-specific passive and active suicidal ideation (SI) and suicidal behavior, as well as COVID-related stressors and emotions, and public health guidance compliance. Findings highlights that COVID-specific SI is common in high-risk youth. COVID-specific SI was associated with COVID-19-related negative emotions, elevated stress, and decreased public health guidance compliance. Results suggest that COVID-specific suicidal thoughts and behaviors, and risk correlates, should be assessed within high-risk populations to facilitate prevention of risky behavior associated with intentional COVID-19 exposure.

Advancing Palliative Care in Patients With CKD: From Ideas to Practice.

A palliative approach to care focuses on what matters most to patients with life-limiting illness, including chronic kidney disease (CKD). Despite recent publication of related clinical practice guidelines in nephrology, there is limited information about how to practically implement these recommendations. In this Perspective, we describe our experience integrating a palliative approach within routine care of patients with CKD glomerular filtration rate categories 4 and 5 (G4-G5) across a provincial kidney care network during the past 15 years. The effort was led by a multidisciplinary group, tasked with building capacity and developing tools and resources for practical integration within a provincial network structure. We used an evidence-based framework that includes recommendations for 4 pillars of palliative care to guide our work: (1) patient identification, (2) advance care planning, (3) symptom assessment and management, and (4) caring of the dying patient and bereavement. Activities within each pillar have been iteratively implemented across all kidney care programs using existing committees and organizational structures. Key quality indicators were used to guide strategic planning and improvement. We supported culture change through the use of multiple strategies simultaneously. Altogether, we established and integrated palliative care activities into routine CKD G4-G5 care across the continuum from nondialysis to dialysis populations.

Increasing consultant-level staffing as a proportion of overall physician coverage improves emergency department length of stay targets.

OBJECTIVES: One goal of Emergency Department (ED) operations is achieving an overall length of stay (LOS) that is less than four hours. The goal of the current study was to assess for association between increasing number of on-duty EM Consultants and LOS, while adjusting for overall (all-grade) on-duty emergency doctors' numbers and other operational factors. METHODS: This was a retrospective analysis of three years (2016-2019) of data, employing a unit of analysis of 3276 eight-hour ED shifts. The study was conducted using a prospectively populated ED database in a busy (annual census 420,000) Middle Eastern ED with staffing by Consultants and multiple non-Consultant grades (Specialists, fellows, and residents). Using logistic regression, the main predictor variable of "on-duty Consultant n" was assessed for association with the study's primary (dichotomous) endpoint: whether a shift's median LOS met the target of < 240 min. Linear regression was used to assess for association between on-duty Consultant n and the study's secondary (continuous) endpoint: median LOS for the ED shift. RESULTS: Multivariate logistic regression adjusting for a number of operations factors (including total EP on-duty complement) identified an association between increasing n of on-duty Consultants and the likelihood of a shift's meeting the 4-h ED LOS target (OR 1.27, 95% CI 1.20 to 1.34, p < .0001). Multiple linear regression, which also adjusted for total on-duty EP n and other operational factors, also indicated LOS benefit from more on-duty Consultants: each additional on-duty Consultant was associated with a shift's median LOS improving by 5.4 min (95% CI 4.3 to 6.5, p < .0001). CONCLUSIONS: At the study site, in models that adjusted for overall on-duty EP numbers as well as myriad other operational factors, increasing numbers of on-duty Consultants was associated with a statistically and operationally significant reduction in ED LOS.

Qatar Prediction Rule Using ED Indicators of COVID-19 at Triage.

INTRODUCTION: The presence of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and its associated disease, COVID-19 has had an enormous impact on the operations of the emergency department (ED), particularly the triage area. The aim of the study was to derive and validate a prediction rule that would be applicable to Qatar's adult ED population to predict COVID-19-positive patients. METHODS: This is a retrospective study including adult patients. The data were obtained from the electronic medical records (EMR) of the Hamad Medical Corporation (HMC) for three EDs. Data from the Hamad General Hospital ED were used to derive and internally validate a prediction rule (Q-PREDICT). The Al Wakra Hospital ED and Al Khor Hospital ED data formed an external validation set consisting of the same time frame. The variables in the model included the weekly ED COVID-19-positivity rate and the following patient characteristics: region (nationality), age, acuity, cough, fever, tachypnea, hypoxemia, and hypotension. All statistical analyses were executed with Stata 16.1 (Stata Corp). The study team obtained appropriate institutional approval. RESULTS: The study included 45,663 adult patients who were tested for COVID-19. Out of these, 47% (n = 21461) were COVID-19 positive. The derivation-set model had very good discrimination (c = 0.855, 95% Confidence intervals (CI) 0.847-0.861). Cross-validation of the model demonstrated that the validation-set model (c = 0.857, 95% CI 0.849-0.863) retained high discrimination. A high Q-PREDICT score ( ≥ 13) is associated with a nearly 6-fold increase in the likelihood of being COVID-19 positive (likelihood ratio 5.9, 95% CI 5.6-6.2), with a sensitivity of 84.7% (95% CI, 84.0%-85.4%). A low Q-PREDICT ( ≤ 6) is associated with a nearly 20-fold increase in the likelihood of being COVID-19 negative (likelihood ratio 19.3, 95% CI 16.7-22.1), with a specificity of 98.7% (95% CI 98.5%-98.9%). CONCLUSION: The Q-PREDICT is a simple scoring system based on information readily collected from patients at the front desk of the ED and helps to predict COVID-19 status at triage. The scoring system performed well in the internal and external validation on datasets obtained from the state of Qatar.

Q-DEPICT: Qatar Determining Emergency Physician Incidence of COVID-Positive Testing.

Despite protective measures such as personal protective equipment (PPE) and a COVID airway management program (CAMP), some emergency physicians will inevitably test positive for COVID. We aim to develop a model predicting weekly numbers of emergency physician COVID converters to aid operations planning. The data were obtained from the electronic medical record (EMR) used throughout the national healthcare system. Hamad Medical Corporation's internal emergency medicine workforce data were used as a source of information on emergency physician COVID conversion and numbers of emergency physicians completing CAMP training. The study period included the spring and summer months of 2020 and started on March 7 and ran for 21 whole weeks through July 31. Data were extracted from the system's EMR database into a spreadsheet (Excel, Microsoft, Redmond, USA). The statistical software used for all analyses and plots was Stata (version 16.1 MP, StataCorp, College Station, USA). All data definitions were made a priori. A total of 35 of 250 emergency physicians (14.0%, 95% CI 9.9%-19.9%) converted to a positive real-time reverse transcriptase-polymerase chain reaction (PCR) during the study's 21-week period. Of these. only two were hospitalized for having respiratory-only disease, and none required respiratory support. Both were discharged within a week of admission. The weekly number of newly COVID-positive emergency physicians was zero and was seen in eight of 21 (38.1%) weeks. The peak weekly counts of six emergency physicians with new COVID-positive were seen in week 14. The mean weekly number of newly COVID-positive emergency physicians was 1.7 ± 1.9, and the median was 1 (IQR, 0 to 3). This study demonstrates that in the State of Qatar's Emergency Department (ED) system, knowing only four parameters allows the reliable prediction of the number of emergency physicians likely to convert COVID PCR tests within the next week. The results also suggest that attention to the details of minimizing endotracheal intubation (ETI) risk can eliminate the expected finding of the association between ETI numbers and emergency physician COVID numbers.

Parent-adolescent relationship characteristics and adolescent cannabis use: A growth curve analysis.

Background: Adolescent cannabis misuse may be associated with serious academic, conduct, and health problems. Identifying factors associated with adolescent cannabis misuse over time may provide insight to address these factors in interventions. Parent-adolescent relationship characteristics (i.e., attachment, discipline) have been linked to adolescent cannabis misuse and may be important factors to study. Objectives: We investigated time-varying associations between parent-adolescent relationship domains and weekly adolescent-reported cannabis misuse. We hypothesized that during times when parents reported less positive aspects of their relationship with their adolescents, adolescents would report higher levels of cannabis misuse. Methods: Data were drawn from a community clinic treatment study for adolescents with substance use and co-occurring psychiatric disorders (n=110; average age=15.71; 57.3% male). Latent growth modeling with time-varying predictors (parent-adolescent relationship characteristics) was used to examine if the associations between adolescent cannabis misuse and relational frustration, discipline, and attachment varied across the study period (baseline, 3-, 6-, and 12-months). Results: Weekly cannabis misuse significantly increased over time, even after accounting for parental relationship characteristics. When parents rated higher levels of relational frustration relative to their average level of frustration, adolescents reported higher cannabis misuse at all study periods except 12-month follow-up. Conclusion: Results support the importance of considering how specific aspects of the parentadolescent relationship, in this case elevated parental frustration, are associated with adolescent cannabis misuse during treatment and after its completion. Findings suggest parental relationship frustration is a key factor to assess and address within individually tailored interventions for co-occurring cannabis misuse and psychiatric disorders.

Cannabis Use and Emotional Awareness Difficulties in Adolescents with Co-Occurring Substance Use and Psychiatric Disorders.

Background: Assessing predictors of cannabis use following adolescent substance use treatment may inform essential treatment elements to be emphasized before discharge. Adolescents with low emotional awareness may have limited resources for identifying and overcoming negative emotions, and therefore, use cannabis to regulate emotions. Purpose/objectives: The purpose of this study was to test the hypothesis that emotional awareness difficulties are associated with increased cannabis use across the transition out of substance use treatment. This hypothesis was investigated by applying an autoregressive random-intercept cross-lagged panel-modeling framework to test the fit of alternative models and inform hypotheses about directional associations between cannabis use and emotional awareness difficulties over time. Methods: Participants were 110 adolescents with co-occurring disorders and their families participating in an intensive home-based treatment trial. Adolescents reported on past 7-day cannabis use and difficulties in emotional awareness at baseline and three follow-up assessments across 12 months. Results: At baseline, 54% of the sample reported past-week cannabis use. A directional effect was supported such that difficulties with emotional awareness at 3 months' post-baseline, which corresponded to the approximate end of the treatment program, were associated with increased cannabis use at 6 months' post-baseline, controlling for the stability of cannabis use, and emotional awareness over time. Cannabis use, however, was not associated with subsequent difficulties in emotional awareness (i.e., effects in the opposite direction were not supported). Conclusions/Importance: Emotional awareness difficulties toward the end of a course of intensive outpatient treatment may be associated with increased cannabis use after the completion of treatment.

ABC/2 estimation in intracerebral hemorrhage: A comparison study between emergency radiologists and emergency physicians.

OBJECTIVES: We aimed to define levels of agreement (LOA) between emergency radiologists (RAD) and emergency medicine (EM) physicians for estimating bleed volume in intracranial hemorrhages (ICH) using ABC/2 formula. METHODS: A prospective study of a curated sample of head CT's were performed in an emergency department. Raters independently reviewed the scans. Perpendicular maximal dimensions (A and B) were measured on an axial CT image. The 'C' dimension was a product of slice thickness and number of slices with visible bleed. RESULTS: A hundred CT head examinations were included with a median age of 50 years (IQR 43 to 57). The median bleed volume was 11.2 mL (IQR 6.6-18.6) per the index radiologist estimations. The overall mean of differences between the RAD mean and the EM mean estimated bleed volume was 0.3 (95% CI -1.5 to +1.7) in milliliters. The percentage difference between EM and RAD expressed as median was 1.9% (IQR -13.4% to +14.1%). Compared to the index RAD the mean of differences for bleed volume [rater, mean (95% CI) in milliliters] were: second RAD, 1.19 (1.14 to 1.24); EM attending, 1.05 (0.98 to 1.13); senior fellow, 1.05 (1.00 to 1.10); junior fellow, 1.19 (1.06 to 1.33); senior resident, 1.29 (1.19 to 1.39); junior resident, 1.11 (1.03 to 1.20). The difference between EM versus radiologist, junior versus senior EM physician estimation of bleed size was clinically insignificant. CONCLUSIONS: Excellent level of agreement was found between emergency physicians and emergency radiologists for estimating ICH bleed volumes using ABC/2 formula.

Helicopter Emergency Medical Services Literature 1972-2017: Characteristics and Trends.

Since its inception in the latter part of the 20th century, the rapid expansion of helicopter emergency medical services (HEMS) has been accompanied by remarkable growth in the relevant evidence base. There are many review articles describing lessons contained within the various arenas of HEMS literature, but there is little or no characterization of the numbers and types of publications comprising the HEMS-related evidence base. This study analyzed all indexed publications mentioning HEMS (with abstract included) in the United States National Library of Medicine's PubMed collection. The aims of the analysis were to provide quantitative, qualitative, and longitudinal trend information regarding the 1972 to 2017 evidence base relevant to HEMS.

Microplitis demolitor Bracovirus Proviral Loci and Clustered Replication Genes Exhibit Distinct DNA Amplification Patterns during Replication.

UNLABELLED: Polydnaviruses are large, double-stranded DNA viruses that are beneficial symbionts of parasitoid wasps. Polydnaviruses in the genus Bracovirus (BVs) persist in wasps as proviruses, and their genomes consist of two functional components referred to as proviral segments and nudivirus-like genes. Prior studies established that the DNA domains where proviral segments reside are amplified during replication and that segments within amplified loci are circularized before packaging into nucleocapsids. One DNA domain where nudivirus-like genes are located is also amplified but never packaged into virions. We recently sequenced the genome of the braconid Microplitis demolitor, which carries M. demolitor bracovirus (MdBV). Here, we took advantage of this resource to characterize the DNAs that are amplified during MdBV replication using a combination of Illumina and Pacific Biosciences sequencing approaches. The results showed that specific nucleotide sites identify the boundaries of amplification for proviral loci. Surprisingly, however, amplification of loci 3, 4, 6, and 8 produced head-to-tail concatemeric intermediates; loci 1, 2, and 5 produced head-to-head/tail-to-tail concatemers; and locus 7 yielded no identified concatemers. Sequence differences at amplification junctions correlated with the types of amplification intermediates the loci produced, while concatemer processing gave rise to the circularized DNAs that are packaged into nucleocapsids. The MdBV nudivirus-like gene cluster was also amplified, albeit more weakly than most proviral loci and with nondiscrete boundaries. Overall, the MdBV genome exhibited three patterns of DNA amplification during replication. Our data also suggest that PacBio sequencing could be useful in studying the replication intermediates produced by other DNA viruses. IMPORTANCE: Polydnaviruses are of fundamental interest because they provide a novel example of viruses evolving into beneficial symbionts. All polydnaviruses are associated with insects called parasitoid wasps, which are of additional applied interest because many are biological control agents of pest insects. Polydnaviruses in the genus Bracovirus (BVs) evolved ~100 million years ago from an ancestor related to the baculovirus-nudivirus lineage but have also established many novelties due to their symbiotic lifestyle. These include the fact that BVs are transmitted only vertically as proviruses and produce replication-defective virions that package only a portion of the viral genome. Here, we studied Microplitis demolitor bracovirus (MdBV) and report that its genome exhibits three distinct patterns of DNA amplification during replication. We also identify several previously unknown features of BV genomes that correlate with these different amplification patterns.

Mutualistic polydnaviruses share essential replication gene functions with pathogenic ancestors.

Viruses are usually thought to form parasitic associations with hosts, but all members of the family Polydnaviridae are obligate mutualists of insects called parasitoid wasps. Phylogenetic data founded on sequence comparisons of viral genes indicate that polydnaviruses in the genus Bracovirus (BV) are closely related to pathogenic nudiviruses and baculoviruses. However, pronounced differences in the biology of BVs and baculoviruses together with high divergence of many shared genes make it unclear whether BV homologs still retain baculovirus-like functions. Here we report that virions from Microplitis demolitor bracovirus (MdBV) contain multiple baculovirus-like and nudivirus-like conserved gene products. We further show that RNA interference effectively and specifically knocks down MdBV gene expression. Coupling RNAi knockdown methods with functional assays, we examined the activity of six genes in the MdBV conserved gene set that are known to have essential roles in transcription (lef-4, lef-9), capsid assembly (vp39, vlf-1), and envelope formation (p74, pif-1) during baculovirus replication. Our results indicated that MdBV produces a baculovirus-like RNA polymerase that transcribes virus structural genes. Our results also supported a conserved role for vp39, vlf-1, p74, and pif-1 as structural components of MdBV virions. Additional experiments suggested that vlf-1 together with the nudivirus-like gene int-1 also have novel functions in regulating excision of MdBV proviral DNAs for packaging into virions. Overall, these data provide the first experimental insights into the function of BV genes in virion formation.

Control of spasticity in a multiple sclerosis model using central nervous system-excluded CB1 cannabinoid receptor agonists.

The purpose of this study was the generation of central nervous system (CNS)-excluded cannabinoid receptor agonists to test the hypothesis that inhibition of spasticity, due to CNS autoimmunity, could be controlled by affecting neurotransmission within the periphery. Procedures included identification of chemicals and modeling to predict the mode of exclusion; induction and control of spasticity in the ABH mouse model of multiple sclerosis; conditional deletion of CB1 receptor in peripheral nerves; side-effect profiling to demonstrate the mechanism of CNS-exclusion via drug pumps; genome-wide association study in N2(129×ABH) backcross to map polymorphic cannabinoid drug pump; and sequencing and detection of cannabinoid drug-pump activity in human brain endothelial cell lines. Three drugs (CT3, SAB378 and SAD448) were identified that control spasticity via action on the peripheral nerve CB1 receptor. These were peripherally restricted via drug pumps that limit the CNS side effects (hypothermia) of cannabinoids to increase the therapeutic window. A cannabinoid drug pump is polymorphic and functionally lacking in many laboratory (C57BL/6, 129, CD-1) mice used for transgenesis, pharmacology, and toxicology studies. This phenotype was mapped and controlled by 1-3 genetic loci. ABCC1 within a cluster showing linkage is a cannabinoid CNS-drug pump. Global and conditional CB1 receptor-knockout mice were used as controls. In summary, CNS-excluded CB1 receptor agonists are a novel class of therapeutic agent for spasticity.