Rates and correlates of medicine disposal program implementation at pharmacies in North Carolina: A longitudinal study, 2016-2021.

BACKGROUND: Unused prescription opioids from family and friends continue to be the primary access point to prescription opioids for nonmedical use among youth. Implementation of medicine disposal boxes at pharmacies is one approach to facilitate removal of unused prescription opioids from the home to prevent diversion. OBJECTIVES: We sought to examine the implementation rates of disposal boxes at pharmacies in North Carolina from 2016 to 2021 and place-based health disparities in availability. METHODS: We identified pharmacies with a disposal box in 2016, 2018, and 2021 among licensed pharmacies in North Carolina in 2018 (N = 2587). We computed descriptive statistics to describe disposal box implementation rates over time and used geographic information systems to identify spatial trends. We used separate logistic regression models in 2018 and 2021 to assess the relationship between neighborhood characteristics and the likelihood of a pharmacy implementing a disposal box. RESULTS: We found an increase in disposal boxes over time with 43 pharmacies (1.7%) in 2016, 144 (5.6%) in 2018, and 350 (13.5%) in 2021 implementing a disposal box. In 2018, independent pharmacies were more likely than chains to have a disposal box. In 2021, medical-affiliated and pharmacies defined as "other" were less likely than chains to have a disposal box. In both 2018 and 2021, pharmacies in census tracts with a higher percentage of the population below the federal poverty line were more likely to have a disposal box. In 2021, pharmacies in tracts with a higher percentage of the population unemployed were less likely to have a disposal box. In 2018, pharmacies located in counties with a greater number of opioid overdose deaths were more likely to have a disposal box. CONCLUSION: Our findings highlight the growth of disposal boxes in North Carolina over time and the potential for continued expansion to provide opportunities to prevent prescription opioid diversion.

Characterizing the molecular regulation of inhibitory immune checkpoints with multimodal single-cell screens.

The expression of inhibitory immune checkpoint molecules, such as programmed death-ligand (PD-L)1, is frequently observed in human cancers and can lead to the suppression of T cell-mediated immune responses. Here, we apply expanded CRISPR-compatible (EC)CITE-seq, a technology that combines pooled CRISPR screens with single-cell mRNA and surface protein measurements, to explore the molecular networks that regulate PD-L1 expression. We also develop a computational framework, mixscape, that substantially improves the signal-to-noise ratio in single-cell perturbation screens by identifying and removing confounding sources of variation. Applying these tools, we identify and validate regulators of PD-L1 and leverage our multimodal data to identify both transcriptional and post-transcriptional modes of regulation. Specifically, we discover that the Kelch-like protein KEAP1 and the transcriptional activator NRF2 mediate the upregulation of PD-L1 after interferon (IFN)-γ stimulation. Our results identify a new mechanism for the regulation of immune checkpoints and present a powerful analytical framework for the analysis of multimodal single-cell perturbation screens.

Modifiable Risk Factors Associated With Disposal of Unused Prescription Drugs by Parents of Adolescents.

The safe disposal of unused medications is one primary prevention strategy to reduce nonmedical prescription drug use among adolescents. We sought to identify modifiable risk factors associated with disposal of unused prescription drugs by parents of adolescents residing in ten south central Kentucky counties with disposal programs. In the fall of 2017, 4148 parents of adolescents participated in an anonymous, paper-based survey. We conducted generalized logit mixed models adjusted for within-school clustering to assess the relationship between disposal behaviors and modifiable risk factors while controlling for respondents' sociodemographic characteristics. The analytic sample consisted of parents in households in which someone had been prescribed an opioid medication within the past 12 months (N = 627). Our findings indicated that almost 42% of parents reported disposing of unused prescription medication within the past 12 months, and the majority disposed of medications at home rather than using a disposal program. Parents who perceived that any, compared to none, of their child's close friends engaged in nonmedical prescription opioid use had higher odds of reporting use of a disposal program. Parents who were aware of disposal programs, compared to those who were not aware, had greater odds of using them, rather than not disposing at all or disposing unused prescription medications at home. Compared to parents who perceived prescription drugs to be hard for adolescents to obtain for nonmedical use, parents who believed that prescription drugs were easily accessible to adolescents for nonmedical use had lower odds of using disposal programs than disposing of medications at home. Collectively, our findings suggest that enhancing awareness of disposal programs, while addressing parents' perceptions of their children's peers' use of nonmedical prescription opioids, should be considered to facilitate the disposal of unused medications and optimize current public health prevention efforts related to adolescent nonmedical use of these drugs.

Naloxone Availability in Retail Pharmacies and Neighborhood Inequities in Access.

INTRODUCTION: This study examines the implementation of North Carolina's statewide naloxone standing order and identifies community characteristics associated with pharmacy stocking and willingness to sell naloxone under the standing order. METHODS: In April-June 2019, a mystery caller protocol was completed to assess if (1) North Carolina pharmacies had naloxone available and were willing to dispense it without a prescription, (2) pharmacy characteristics associated with availability, and (3) there were neighborhood differences (e.g., Census tract population size, density, racial composition, SES, rates of opioid overdoses, and rates of opioid prescriptions dispensed) in availability. Using random sampling stratified by inclusion on North Carolina's public list of pharmacies participating in the standing order, chain, independent, and health department pharmacies in North Carolina were sampled (n=161 of 2,044). In June 2019, the data were analyzed. Survey weights were utilized to calculate the prevalence of availability, and regression models were conducted to examine associations. RESULTS: An estimated 61.7% (95% CI=54.3, 68.5) of North Carolina retail pharmacies have naloxone available without a prescription. The odds of naloxone availability were lower for independent pharmacies than chains (OR=0.12, 95% CI=0.06, 0.25). Inclusion on North Carolina's public list of pharmacies had greater odds of naloxone availability (OR=2.32, 95% CI=1.22, 4.43). Naloxone availability was lower in communities with higher percentages of residents with public health insurance (OR=0.97, 95% CI=0.95, 0.999). CONCLUSIONS: Though more than half of the pharmacies in North Carolina participate in the standing order for naloxone, efforts to identify the best practices for ensuring widespread implementation of statewide standing orders for naloxone are warranted.

Virtual Reality for Pediatric Needle Procedural Pain: Two Randomized Clinical Trials.

OBJECTIVE: To assess the efficacy and safety of a virtual reality distraction for needle pain in 2 common hospital settings: the emergency department (ED) and outpatient pathology (ie, outpatient laboratory). The control was standard of care (SOC) practice. STUDY DESIGN: In 2 clinical trials, we randomized children aged 4-11 years undergoing venous needle procedures to virtual reality or SOC at 2 tertiary Australian hospitals. In the first study, we enrolled children in the ED requiring intravenous cannulation or venipuncture. In the second, we enrolled children in outpatient pathology requiring venipuncture. In the ED, 64 children were assigned to virtual reality and 59 to SOC. In pathology, 63 children were assigned to virtual reality and 68 to SOC; 2 children withdrew assent in the SOC arm, leaving 66. The primary endpoint was change from baseline pain between virtual reality and SOC on child-rated Faces Pain Scale-Revised. RESULTS: In the ED, there was no change in pain from baseline with SOC, whereas virtual reality produced a significant reduction in pain (between-group difference, -1.78; 95% CI, -3.24 to -0.317; P = .018). In pathology, both groups experienced an increase in pain from baseline, but this was significantly less in the virtual reality group (between-group difference, -1.39; 95% CI, -2.68 to -0.11; P = .034). Across both studies, 10 participants experienced minor adverse events, equally distributed between virtual reality/SOC; none required pharmacotherapy. CONCLUSIONS: In children aged 4-11 years of age undergoing intravenous cannulation or venipuncture, virtual reality was efficacious in decreasing pain and was safe. TRIAL REGISTRATION: Australia and New Zealand Clinical Trial Registry: ACTRN12617000285358p.

A mixing heteroduplex mobility assay (mHMA) to genotype homozygous mutants with small indels generated by CRISPR-Cas9 nucleases.

The development of gene editing technologies, especially the CRISPR-Cas9 system, has been pivotal for understanding the functional role of proteins. Rapid and efficient genotyping methods are necessary to screen for generated mutations and streamline the isolation of homozygotes. CRISPR-Cas9 system targeting a single site in the gene typically results in small indels. Many genotyping methods utilize the heteroduplex that is formed when wild-type and mutant amplicons with small indels anneal during PCR creating a bubble due to mismatched strands. These methods include T7 endonuclease/Cel-I assay, high resolution melting (HRM) analysis, and heteroduplex mobility assay (HMA). Our protocol explains a simple, two step method of a mixing HMA (mHMA) to identify homozygous mutants, a modification of the previously published HMA. We have utilized the mHMA for screening and genotyping numerous CRISPR generated models. The mHMA method to differentiate homozygous wild type from homozygous mutant animals eliminates - •DNA sequencing, even with small indels that can be difficult to discern on a gel.•additional enzymatic reaction steps, such as with the T7EI/Cel-I assay.•specialized equipment and analysis tools, such as with HRM analysis.

Analysis of novel domain-specific mutations in the zebrafish ndr2/cyclops gene generated using CRISPR-Cas9 RNPs.

Nodal-related protein (ndr2) is amember of the transforming growth factor type ß superfamily of factors and is required for ventral midline patterning of the embryonic central nervous system in zebrafish. In humans, mutations in the gene encoding nodal cause holoprosencephaly and heterotaxy. Mutations in the ndr2 gene in the zebrafish (Danio rerio) lead to similar phenotypes, including loss of the medial floor plate, severe deficits in ventral forebrain development and cyclopia. Alleles of the ndr2 gene have been useful in studying patterning of ventral structures of the central nervous system. Fifteen different ndr2 alleles have been reported in zebrafish, of which eight were generated using chemical mutagenesis, four were radiation-induced and the remaining alleles were obtained via random insertion, gene targeting (TALEN) or unknown methods. Therefore, most mutation sites were random and could not be predicted a priori. Using the CRISPR-Cas9 system from Streptococcus pyogenes, we targeted distinct regions in all three exons of zebrafish ndr2 and observed cyclopia in the injected (G0) embryos.We show that the use of sgRNA-Cas9 ribonucleoprotein (RNP) complexes can cause penetrant cyclopic phenotypes in injected (G0) embryos. Targeted polymerase chain reaction amplicon analysis using Sanger sequencing showed that most of the alleles had small indels resulting in frameshifts. The sequence information correlates with the loss of ndr2 activity. In this study, we validate multiple CRISPR targets using an in vitro nuclease assay and in vivo analysis using embryos. We describe one specific mutant allele resulting in the loss of conserved terminal cysteine-coding sequences. This study is another demonstration of the utility of the CRISPR-Cas9 system in generating domain-specific mutations and provides further insights into the structure-function of the ndr2 gene.

Clinical efficacy of virtual reality for acute procedural pain management: A systematic review and meta-analysis.

BACKGROUND: Acutely painful procedures are commonplace. Current approaches to pain most often involve pharmacotherapy, however, there is interest in virtual reality (VR) as a non-pharmacological alternative. A methodologically rigorous systematic review and meta-analysis is lacking. METHODS: Following PRISMA guidelines, we searched the Cochrane Library, Ovid MEDLINE, Embase, CINAHL, ERIC, NIHR Centre for Review and Dissemination, Proquest, the System for Information on Grey Literature in Europe and the WHO International Clinical Trials Registry Platform from inception to 5 November 2017. Included studies were randomised with an experimental trial design, included a non-VR control group and examined the efficacy of VR with regards to an acutely painful clinical intervention. Bias was assessed along Cochrane guidelines, with performance bias not assessed due to the non-blindable nature of VR. We extracted summary data for maximal pain score and used standard mean difference DerSimonian-Laird random-effects meta-analysis (RevMan 5.3). This review was prospectively registered (PROSPERO CRD42017058204). FINDINGS: Of the 12,450 studies identified, 20 studies were eligible for the systematic review. No trials reported in sufficient detail to judge their risk of bias, and 10 studies were at high risk of bias in at least one domain. 16 studies (9 randomised controlled trials, 7 crossover studies) examining 656 individuals were included in quantitative synthesis. Pain scales were heterogenous, but mostly employed 100-point scales. Across all trials, meta-analysis was suggestive of a -0.49 (95%CI -0.83 to -0.41, p = 0.006) standardised mean difference reduction in pain score with VR. However there was a high degree of statistical heterogeneity (χ2 p<0.001, I2 81%, 95%CI for I2 70-88%), driven by randomised studies, with substantial clinical heterogeneity. CONCLUSION: These data suggest that VR may have a role in acutely painful procedures, however included studies were clinically and statistically heterogenous. Further research is required to validate findings, establish cost efficacy and optimal clinical settings for usage. Future trials should report in accordance with established guidelines.

Widespread natural variation in murine natural killer T-cell number and function.

Natural killer T (NKT) cells comprise a novel T-lymphocyte subset that can influence a wide variety of immune responses through their ability to secrete large amounts of a variety of cytokines. Although variation in NKT-cell number and function has been extensively studied in autoimmune disease-prone mice, in which it has been linked to disease susceptibility, relatively little is known of the natural variation of NKT-cell number and function among normal inbred mouse strains. Here, we demonstrate strain-dependent variation in the susceptibility of C57BL/6J and BALB/cJ mice to NKT-mediated airway hyperreactivity, which correlated with significant increases in serum interleukin-4 (IL-4) and IL-13 elicited by the synthetic glycosphingolipid alpha-galactosylceramide. Examination of NKT-cell function revealed a significantly greater frequency of cytokine-producing NKT cells in C57BL/6J versus BALB/cJ mice as well as significant differences in the kinetics of NKT-cell cytokine production. Extension of this analysis to a panel of inbred mouse strains indicated that variability in NKT-cell cytokine production was widespread. Similarly, an examination of NKT-cell frequency revealed a significantly greater number of liver NKT cells in the C57BL/6J mice versus BALB/cJ mouse livers. Again, examination of a panel of inbred mouse strains revealed that liver NKT-cell numbers were quite variable, spanning over a 100-fold range. Taken together, these results demonstrate the presence of widespread natural variation in NKT-cell number and function among common inbred mouse strains, which may have implications for the examination of the influence of NKT cells in immune responses and disease pathogenesis among different genetic backgrounds.

False reports of childhood events in appropriate interviews.

The present study employed the "parental misinformation" paradigm to examine whether individuals report false events from their childhood even when they are interviewed in an appropriate manner by a trained interviewer. Each participant was interviewed on three occasions. By the final interview, one participant produced a "full" report, and six participants produced "partial" reports, of childhood events that did not occur. Although participants reported perceiving greater pressure to report the false events than the real events, independent judges' ratings of social pressure in the interviews did not differ as a function of what type of event participants were being asked about. Participants also reported higher confidence in their parents', compared to their own, recall of events from their childhood. False reports were also positively correlated with scores on both the full and the revised versions of the Dissociative Experiences Scale, and negatively correlated with score on the Self-Monitoring scale. These results indicate that, despite being interviewed in an appropriate manner by a trained interviewer, some participants will falsely report events from their childhoods.