Simplified homology-assisted CRISPR for gene editing in Drosophila.

In vivo genome editing with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 generates powerful tools to study gene regulation and function. We revised the homology-assisted CRISPR knock-in method to convert Drosophila GAL4 lines to LexA lines using a new universal knock-in donor strain. A balancer chromosome-linked donor strain with both body color (yellow) and eye red fluorescent protein (RFP) expression markers simplified the identification of LexA knock-in using light or fluorescence microscopy. A second balancer chromosome-linked donor strain readily converted the second chromosome-linked GAL4 lines regardless of target location in the cis-chromosome but showed limited success for the third chromosome-linked GAL4 lines. We observed a consistent and robust expression of the yellow transgene in progeny harboring a LexA knock-in at diverse genomic locations. Unexpectedly, the expression of the 3xP3-RFP transgene in the "dual transgene" cassette was significantly increased compared with that of the original single 3xP3-RFP transgene cassette in all tested genomic locations. Using this improved screening approach, we generated 16 novel LexA lines; tissue expression by the derived LexA and originating GAL4 lines was similar or indistinguishable. In collaboration with 2 secondary school classes, we also established a systematic workflow to generate a collection of LexA lines from frequently used GAL4 lines.

Testing an insurance-based monetary incentive program for exercise: RCT design and rationale.

Participation in regular physical activity (PA) has numerous health benefits; however, as few as 10% of U.S. adults meet guidelines when device-based assessments of PA are used. The present paper presents the design and rationale for an RCT examining the efficacy of an exercise incentive program currently offered by at least three major US insurance companies, in which participants must attend a YMCA fitness facility at least 50 times within 6 months to receive an incentive. In a factorial design, incentive amount ($200, $100, $0) is crossed with a comparison of the standard gain-framed incentive program and a loss-framed incentive condition in which participants are told their membership fee is being held and will be returned or forfeited depending on their fitness facility attendance. Participants (N = 330) are randomized to gain-framed $100 incentive (n = 55), gain-framed $200 incentive (n = 55), loss-framed $100 incentive (n = 55), loss-framed $200 incentive (n = 55), or control (n = 110). Each participant is enrolled in the same condition for two consecutive 6-month periods for a total of 12 months per participant. The primary outcome is number of visits to the fitness facility over each 6-month period, verified by objective swipe-card data. Secondary outcomes include total moderate-to-vigorous PA (MVPA) over 7-day periods assessed at 3-month intervals through accelerometers (Actigraph wGT3x-BT) and self-report. Habit formation and anticipated regret are putative mediators and household income is a putative moderator of the incentive-based programs. A payer-perspective, within-trial cost-utility analysis will quantify the incremental costs per (a) quality-adjusted life year gained, (b) YMCA attendance, and (c) change in MVPA.

Generation of LexA enhancer-trap lines in Drosophila by an international scholastic network.

Conditional gene regulation in Drosophila through binary expression systems like the LexA-LexAop system provides a superb tool for investigating gene and tissue function. To increase the availability of defined LexA enhancer trap insertions, we present molecular, genetic, and tissue expression studies of 301 novel Stan-X LexA enhancer traps derived from mobilization of the index SX4 line. This includes insertions into distinct loci on the X, II, and III chromosomes that were not previously associated with enhancer traps or targeted LexA constructs, an insertion into ptc, and seventeen insertions into natural transposons. A subset of enhancer traps was expressed in CNS neurons known to produce and secrete insulin, an essential regulator of growth, development, and metabolism. Fly lines described here were generated and characterized through studies by students and teachers in an international network of genetics classes at public, independent high schools, and universities serving a diversity of students, including those underrepresented in science. Thus, a unique partnership between secondary schools and university-based programs has produced and characterized novel resources in Drosophila, establishing instructional paradigms devoted to unscripted experimental science.

Comparing a recommendation for self-paced versus moderate intensity physical activity for midlife adults: Rationale and design.

Current U.S. guidelines recommend that adults obtain 150 min per week of moderate intensity physical activity (PA), 75 min of vigorous intensity PA, or some equivalent combination. However, less than half of U.S. adults reach this goal, with the proportion even smaller among adults with overweight or obesity. Moreover, regular PA declines after age 45-50. Previous research suggests a shift in national guidelines to emphasize PA of a self-selected intensity (i.e., self-paced), instead of prescribed moderate intensity PA, may result in better adherence to PA programs, particularly among midlife adults with overweight or obesity. The present paper presents the protocol for a field-based RCT testing the hypothesis that adherence to PA programs is improved when PA is explicitly recommended to be self-paced rather than prescribed at moderate intensity among midlife (ages 50-64) adults (N = 240) with overweight or obesity. All participants receive a 12-month intervention designed to help them overcome barriers to regular PA and are randomly assigned to either self-paced or prescribed moderate intensity PA. The primary outcome is total volume of PA (minutes by intensity) as measured by accelerometry. Secondary outcomes include self-reported min/week of PA and changes in bodyweight. Additionally, using ecological momentary assessment, we examine putative mediators of treatment effects. We hypothesize self-paced PA will lead to a more positive affective response to PA, more perceived autonomy, and lower perceived exertion during PA, and thus greater increases in PA behavior. Findings will have direct implications for PA intensity recommendations among midlife adults with overweight or obesity.

Syringes must be prioritized globally to ensure equitable access to COVID-19 and other essential vaccines and to sustain safe injection practices.

Supply of autodisable (AD) syringes has been a key component of global COVID-19 vaccination campaigns, and it is critical to maintaining safe injection practices for routine immunization as well as pandemic response. AD syringe production increased significantly in response to demand, but distribution challenges have included the need to coordinate syringes to meet the specific delivery requirements of various COVID-19 vaccines, shipping bottlenecks, and syringe export restrictions. Stockpiling syringes, ensuring standardization of future vaccine dose volumes, and geographical diversification of syringe production would improve syringe logistics in the future. Balancing syringe supply and demand and stabilizing the market over the long term is essential to ensure that the world is prepared for possible new variants of COVID-19 or a new global outbreak. This will require concerted action on the part of public, nonprofit, and private partners.

Testing the Feasibility and Acceptability of an Acceptance and Commitment Therapy Intervention to Increase Physical Activity among Individuals with Depression: A Protocol Paper.

Background: Engaging in regular physical activity (PA) is particularly important among individuals with depression, who are at heightened risk for a host of negative health outcomes. However, people with depression are 50% less likely to meet national guidelines for PA and face unique barriers to PA adherence, including lower distress tolerance and motivation for exercise. Acceptance and Commitment Therapy (ACT) may offer promise for increasing PA among adults with depressive symptoms due to its effects on distress tolerance and motivation. Therefore, we developed ACTivity, an ACT-based intervention designed to promote PA among low-active adults with elevated depressive symptoms. Prior to testing the efficacy of ACTivity in an RCT, an important first step is to conduct a preliminary trial to establish feasibility of study procedures for the ACTivity and comparison intervention programs, as well as to establish the credibility/acceptability of the intervention. The purpose of this paper is to describe the ACTivity intervention and the design of this feasibility trial. Method/Design: We will conduct a feasibility RCT with two parallel groups and a 1:1 allocation ratio comparing ACTivity to a comparison intervention (relaxation training + PA promotion) among 60 low-active adults with elevated depressive symptoms. All study procedures will be conducted remotely. Discussion: Results of this feasibility study will inform a subsequent RCT designed to test the efficacy of ACTivity. If shown to be efficacious, ACTivity will provide a treatment that can be widely disseminated to increase PA among adults with depressive symptoms and thereby decrease their risk for chronic disease.

Cu, Fe, and Zn isotope ratios in murine Alzheimer's disease models suggest specific signatures of amyloidogenesis and tauopathy.

Alzheimer's disease (AD) is characterized by accumulation of tau and amyloid-beta in the brain, and recent evidence suggests a correlation between associated protein aggregates and trace elements, such as copper, iron, and zinc. In AD, a distorted brain redox homeostasis and complexation by amyloid-beta and hyperphosphorylated tau may alter the isotopic composition of essential mineral elements. Therefore, high-precision isotopic analysis may reveal changes in the homeostasis of these elements. We used inductively coupled plasma-mass spectrometry (ICP-MS)-based techniques to determine the total Cu, Fe, and Zn contents in the brain, as well as their isotopic compositions in both mouse brain and serum. Results for male transgenic tau (Line 66, L66) and amyloid/presenilin (5xFAD) mice were compared with those for the corresponding age- and sex-matched wild-type control mice (WT). Our data show that L66 brains showed significantly higher Fe levels than did those from the corresponding WT. Significantly less Cu, but more Zn was found in 5xFAD brains. We observed significantly lighter isotopic compositions of Fe (enrichment in the lighter isotopes) in the brain and serum of L66 mice compared with WT. For 5xFAD mice, Zn exhibited a trend toward a lighter isotopic composition in the brain and a heavier isotopic composition in serum compared with WT. Neither mouse model yielded differences in the isotopic composition of Cu. Our findings indicate significant pathology-specific alterations of Fe and Zn brain homeostasis in mouse models of AD. The associated changes in isotopic composition may serve as a marker for proteinopathies underlying AD and other types of dementia.

Helping individuals with firearm injuries: A cluster randomized trial.

BACKGROUND: Patients with firearm injuries are at high risk of subsequent arrest and injury following hospital discharge. We sought to evaluate the effect of a 6-month joint hospital- and community-based low-intensity intervention on risk of arrest and injury among patients with firearm injuries. METHODS: We conducted a cluster randomized controlled trial, enrolling patients with firearm injuries who received treatment at Harborview Medical Center, the level 1 trauma center in Seattle, Washington, were 18 years or older at the time of injury, spoke English, were able to provide consent and a method of contact, and lived in one of the five study counties. The intervention consisted of hospital-based motivational interviewing, followed by a 6-month community-based intervention, and multiagency support. The primary outcome was the risk of subsequent arrest. The main secondary outcome was the risk of death or subsequent injury requiring treatment in the emergency department or hospitalization. RESULTS: Neither assignment to or engagement with the intervention, defined as having at least 1 contact point with the support specialist, was associated with risk of arrest at 2 years post-hospital discharge (relative risk for intervention assignment, 1.15; 95% confidence interval, 0.90-1.48; relative risk for intervention engagement, 1.07; 95% confidence interval, 0.74-2.19). There was similarly no association observed for subsequent injury. CONCLUSIONS: This study represents one of the first randomized controlled trials of a joint hospital- and community-based intervention delivered exclusively among patients with firearm injuries. The intervention was not associated with changes in risk of arrest or injury, a finding most likely due to the low intensity of the program. LEVEL OF EVIDENCE: Care management, level II.

Fidelity Assessment of a Social Work-Led Intervention Among Patients with Firearm Injuries.

PURPOSE: To support future development and refinement of social work-led intervention programs among patients with firearm injuries and to demonstrate how a fidelity assessment can be used to adjust and refine intervention delivery in an ongoing trial. METHODS: We conducted a fidelity assessment of a randomized controlled trial of a social work-led intervention among patients with a firearm injury. RESULTS: We found that our study intervention was well implemented, meeting 70% of the fidelity assessment score items, however noted lower fidelity with client-based items. DISCUSSION: As a result of fidelity assessment findings, we refined intervention delivery to improve implementation fidelity including beginning to review cases of all patients each month, rather than focusing on patients in crisis. Our fidelity assessment process and findings offer insight into the challenges of implementing an intervention among patients with firearm injuries and highlights the value of monitoring intervention fidelity during an ongoing trial.

The specificity and role of microglia in epileptogenesis in mouse models of tuberous sclerosis complex.

OBJECTIVE: Microglial abnormalities have been reported in pathologic specimens from patients with tuberous sclerosis complex (TSC), a genetic disorder characterized by epilepsy, intellectual disability, and autism. However, the pathogenic role of microglia in epilepsy in TSC is poorly understood, particularly whether microglia defects may be a primary contributor to epileptogenesis or are secondary to seizures or simply epiphenomena. In this study, we tested the hypothesis that Tsc1 gene inactivation in microglia is sufficient to cause epilepsy in mouse models of TSC. METHODS: Using a chemokine receptor, Cx3cr1, to target microglia, conventional Tsc1Cx3cr1-Cre CKO (conditional knockout) mice and postnatal-inducible Tsc1Cx3cr1-CreER CKO mice were generated and assessed for molecular and histopathologic evidence of microglial abnormalities, mechanistic target of rapamycin 1 (mTORC1) pathway activation, and epilepsy. RESULTS: Tsc1Cx3cr1-Cre CKO mice exhibited a high efficiency of microglia Tsc1 inactivation, mTORC1 activation, increased microglial size and number, and robust epilepsy, which were rapamycin-dependent. However, Cre reporter studies demonstrated that constitutive Cx3cr1 expression affected not only microglia, but also a large percentage of cortical neurons, confounding the role of microglia in epileptogenesis in Tsc1 Cx3cr1-Cre CKO mice. In contrast, postnatal inactivation of Tsc1 utilizing a tamoxifen-inducible Cx3cr1-CreER resulted in a more-selective microglia Tsc1 inactivation with high efficiency, mTORC1 activation, and increased microglial size and number, but no documented epilepsy. SIGNIFICANCE: Microglia abnormalities may contribute to epileptogenesis in the context of neuronal involvement in TSC mouse models, but selective Tsc1 gene inactivation in microglia alone may not be sufficient to cause epilepsy, suggesting that microglia have more supportive roles in the pathogenesis of seizures in TSC.

Posttraumatic Growth in Relationally Betrayed Women.

The research literature on relational betrayal in a committed relationship has focused on the resulting trauma from the betrayal; however, few studies investigated the potential for posttraumatic growth following a relational betrayal. This study investigated the presence of posttraumatic growth in relationally betrayed women. The research focused on women's perceptions of the relational betrayal, and factors that facilitated posttraumatic growth. Results indicated relationally betrayed women perceived the betrayal as a traumatic event, to the extent that some met criteria for PTSD diagnosis. The passage of time was significant corollary to posttraumatic growth when moderated by a PTSD diagnosis. Finally, certain resources were reported to be more helpful than others in the development of posttraumatic growth. Clinical implications are presented.

Efficacy of SERD/SERM Hybrid-CDK4/6 Inhibitor Combinations in Models of Endocrine Therapy-Resistant Breast Cancer.

PURPOSE: Endocrine therapy, using tamoxifen or an aromatase inhibitor, remains first-line therapy for the management of estrogen receptor (ESR1)-positive breast cancer. However, ESR1 mutations or other ligand-independent ESR1 activation mechanisms limit the duration of response. The clinical efficacy of fulvestrant, a selective estrogen receptor downregulator (SERD) that competitively inhibits agonist binding to ESR1 and triggers receptor downregulation, has confirmed that ESR1 frequently remains engaged in endocrine therapy-resistant cancers. We evaluated the activity of a new class of selective estrogen receptor modulators (SERM)/SERD hybrids (SSH) that downregulate ESR1 in relevant models of endocrine-resistant breast cancer. Building on the observation that concurrent inhibition of ESR1 and the cyclin-dependent kinases 4 and 6 (CDK4/6) significantly increased progression-free survival in advanced patients, we explored the activity of different SERD- or SSH-CDK4/6 inhibitor combinations in models of endocrine therapy-resistant ESR1(+) breast cancer. EXPERIMENTAL DESIGN: SERDs, SSHs, and the CDK4/6 inhibitor palbociclib were evaluated as single agents or in combination in established cellular and animal models of endocrine therapy-resistant ESR1(+) breast cancer. RESULTS: The combination of palbociclib with a SERD or an SSH was shown to effectively inhibit the growth of MCF7 cell or ESR1-mutant patient-derived tumor xenografts. In tamoxifen-resistant MCF7 xenografts, the palbociclib/SERD or SSH combination resulted in an increased duration of response as compared with either drug alone. CONCLUSIONS: A SERD- or SSH-palbociclib combination has therapeutic potential in breast tumors resistant to endocrine therapies or those expressing ESR1 mutations. See related commentary by DeMichele and Chodosh, p. 4999.

Imaging caspase-3 activation as a marker of apoptosis-targeted treatment response in cancer.

PURPOSE: We tested whether positron emission tomography (PET) with the caspase-3-targeted isatin analog [(18)F]WC-4-116 could image caspase-3 activation in response to an apoptosis-inducing anticancer therapy. PROCEDURES: [(18)F]WC-4-116 uptake was determined in etoposide-treated EL4 cells. Biodistribution studies with [(18)F]WC-4-116 and [(18)F]ICMT-18, a non-caspase-3-targeted tracer, as well as [(18)F]WC-4-116 microPET imaging assessed responses in Colo205 tumor-bearing mice treated with death receptor 5 (DR5)-targeted agonist antibodies. Immunohistochemical staining and enzyme assays confirmed caspase-3 activation. Two-way analysis of variance or Student's t test assessed for treatment-related changes in tracer uptake. RESULTS: [(18)F]WC-4-116 increased 8 ± 2 fold in etoposide-treated cells. The [(18)F]WC-4-116 % ID/g also increased significantly in tumors with high caspase-3 enzyme activity (p < 0.05). [(18)F]ICMT-18 tumor uptake did not differ in tumors with high or low caspase-3 enzyme activity. CONCLUSIONS: [(18)F]WC-4-116 uptake in vivo reflects increased caspase-3 activation and may be useful for detecting caspase-3-mediated apoptosis treatment responses in cancer.

Best practices for improving flow and care of pediatric patients in the emergency department.

This report provides a summary of best practices for improving flow, reducing waiting times, and improving the quality of care of pediatric patients in the emergency department.

Imaging pulmonary inducible nitric oxide synthase expression with PET.

UNLABELLED: Inducible nitric oxide synthase (iNOS) activity increases in acute and chronic inflammatory lung diseases. Imaging iNOS expression may be useful as an inflammation biomarker for monitoring lung disease activity. We developed a novel tracer for PET that binds to iNOS in vivo, (18)F-NOS. In this study, we tested whether (18)F-NOS could quantify iNOS expression from endotoxin-induced lung inflammation in healthy volunteers. METHODS: Healthy volunteers were screened to exclude cardiopulmonary disease. Qualifying volunteers underwent a baseline, 1-h dynamic (18)F-NOS PET/CT scan. Endotoxin (4 ng/kg) was then instilled bronchoscopically in the right middle lobe. (18)F-NOS imaging was performed again approximately 16 h after endotoxin instillation. Radiolabeled metabolites were determined from blood samples. Cells recovered by bronchoalveolar lavage (BAL) after imaging were stained immunohistochemically for iNOS. (18)F-NOS uptake was quantified as the distribution volume ratio (DVR) determined by Logan plot graphical analysis in volumes of interest placed over the area of endotoxin instillation and in an equivalent lung region on the left. The mean Hounsfield units (HUs) were also computed using the same volumes of interest to measure density changes. RESULTS: Seven healthy volunteers with normal pulmonary function completed the study with evaluable data. The DVR increased by approximately 30%, from a baseline mean of 0.42 ± 0.07 to 0.54 ± 0.12, and the mean HUs by 11% after endotoxin in 6 volunteers who had positive iNOS staining in BAL cells. The DVR did not change in the left lung after endotoxin. In 1 volunteer with low-level iNOS staining in BAL cells, the mean HUs increased by 7% without an increase in DVR. Metabolism was rapid, with approximately 50% of the parent compound at 5 min and 17% at 60 min after injection. CONCLUSION: (18)F-NOS can be used to image iNOS activity in acute lung inflammation in humans and may be a useful PET tracer for imaging iNOS expression in inflammatory lung disease.

A matched-cohort study of pediatric head injuries: collecting data to inform an evidence-based triage assessment.

UNLABELLED: Triage nurses are the "first stop" for patients who present to the emergency department for care. The assessment of pediatric head injuries is especially challenging because signs and symptoms of head trauma in children do not correlate well with the risk of closed head injury (CHI). METHODS: A retrospective matched cohort study was conducted to compare 2 groups of patients who presented to a pediatric emergency department for evaluation of a head injury: a CHI-positive cohort and a CHI-negative cohort as identified by computed tomography scan. The purpose of the chart review was to collect specific information from both cohorts which could be used to inform a nurse-driven pediatric head injury assessment tool. RESULTS: The younger the child, the more likely they were to be asymptomatic. Scalp hematomas in infants <3 months were associated with CHI even if the infants were otherwise asymptomatic. Injuries to the temporal-parietal region were associated with CHI at every age. Frequency of caregiver report of loss of consciousness (LOC) was almost identical in both cohorts. Children in every age category sustained CHIs as the result of minor falls based on standard age-related fall criteria. DISCUSSION: The infants and children at highest risk for CHI are often the most difficult to assess. The results of this study reinforce the need for a nurse-driven, evidence-based risk scoring system that could be used to aid with early identification of infants and children who are at high risk for CHI.

Radiolabeled isatin binding to caspase-3 activation induced by anti-Fas antibody.

INTRODUCTION: Noninvasive imaging methods that can distinguish apoptosis from necrosis may be useful in furthering our understanding of diseases characterized by apoptotic dysregulation as well as aiding drug development targeting apoptotic pathways. We evaluated the ability of radiolabeled isatins to quantify caspase-3 activity induced by the activation of the extrinsic apoptotic pathway by the anti-Fas antibody in mice. METHODS: The behavior of three different radiolabeled isatins ([(18)F]WC-II-89, [(18)F]WC-IV-3 and [(11)C]WC-98) was characterized in mice with and without anti-Fas antibody treatment by microPET imaging and biodistribution studies. The activity of [(18)F]WC-II-89 was also compared with [(99m)Tc]mebrofenin. The effect of pan-caspase inhibition with quinolyl-valyl-O-methylaspartyl-[2,6-difluorophenoxy]-methyl ketone (Q-VD-OPh) on [(18)F]WC-II-89 uptake was studied. Caspase-3 activity was confirmed by a fluorometric enzyme assay. RESULTS: All three tracers behaved similarly in microPET and biodistribution studies. Increased retention of all tracers was observed in the livers of treated animals and several other organs, all of which demonstrated increased caspase-3 enzyme activity; however, impaired hepatobiliary excretion made attribution of these findings to caspase-3 activity difficult. The isatin [(18)F]WC-II-89 was retained at statistically significantly higher levels in the organs after anti-Fas antibody treatment while [(99m)Tc]mebrofenin activity cleared, suggesting specific binding to activated caspase-3, but the magnitude of increased binding was still relatively low. Caspase inhibition with Q-VD-OPh partially blocked [(18)F]WC-II-89 retention but completely blocked caspase-3 enzyme activity in the liver. CONCLUSIONS: The radiolabeled isatins appear to bind specifically to caspase-3 in vivo, but their sensitivity is limited. Further optimization is required for these tracers to be useful for clinical applications.