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.

Acute Effects of Coffee Consumption on Health among Ambulatory Adults.

BACKGROUND: Coffee is one of the most commonly consumed beverages in the world, but the acute health effects of coffee consumption remain uncertain. METHODS: We conducted a prospective, randomized, case-crossover trial to examine the effects of caffeinated coffee on cardiac ectopy and arrhythmias, daily step counts, sleep minutes, and serum glucose levels. A total of 100 adults were fitted with a continuously recording electrocardiogram device, a wrist-worn accelerometer, and a continuous glucose monitor. Participants downloaded a smartphone application to collect geolocation data. We used daily text messages, sent over a period of 14 days, to randomly instruct participants to consume caffeinated coffee or avoid caffeine. The primary outcome was the mean number of daily premature atrial contractions. Adherence to the randomization assignment was assessed with the use of real-time indicators recorded by the participants, daily surveys, reimbursements for date-stamped receipts for coffee purchases, and virtual monitoring (geofencing) of coffee-shop visits. RESULTS: The mean (±SD) age of the participants was 39±13 years; 51% were women, and 51% were non-Hispanic White. Adherence to the random assignments was assessed to be high. The consumption of caffeinated coffee was associated with 58 daily premature atrial contractions as compared with 53 daily events on days when caffeine was avoided (rate ratio, 1.09; 95% confidence interval [CI], 0.98 to 1.20; P = 0.10). The consumption of caffeinated coffee as compared with no caffeine consumption was associated with 154 and 102 daily premature ventricular contractions, respectively (rate ratio, 1.51; 95% CI, 1.18 to 1.94); 10,646 and 9665 daily steps (mean difference, 1058; 95% CI, 441 to 1675); 397 and 432 minutes of nightly sleep (mean difference, 36; 95% CI, 25 to 47); and serum glucose levels of 95 mg per deciliter and 96 mg per deciliter (mean difference, -0.41; 95% CI, -5.42 to 4.60). CONCLUSIONS: In this randomized trial, the consumption of caffeinated coffee did not result in significantly more daily premature atrial contractions than the avoidance of caffeine. (Funded by the University of California, San Francisco, and the National Institutes of Health; CRAVE ClinicalTrials.gov number, NCT03671759.).

Randomized Comparison of a Radiofrequency Wire Versus a Radiofrequency Needle System for Transseptal Puncture.

BACKGROUND: Transseptal puncture is a necessary component of many electrophysiology and structural heart procedures. Improving this technique has broad ramifications for the overall efficiency and safety of these interventions. A new technology uses a specialized introducer wire to cross the septum with radiofrequency (RF) energy, eliminating the need for a transseptal needle and wire/needle exchanges. OBJECTIVES: This study sought to compare the efficacy and safety of an RF needle versus RF wire approach for transseptal puncture. METHODS: Individuals ≥18 years of age undergoing double transseptal puncture for atrial fibrillation or left atrial flutter ablation were randomized to a transseptal approach with either an RF needle or RF wire. The primary outcome was time to achieve first transseptal puncture. Secondary outcomes included second and combined transseptal puncture time, fluoroscopy time, number of equipment exchanges, and complications. RESULTS: A total of 75 participants were enrolled (36 RF needle, 39 RF wire). No crossovers occurred. Randomization to the RF wire resulted in a significant reduction in first transseptal time compared with the RF needle (median 9.2 [IQR: 5.7-11.2] minutes vs 6.9 [IQR: 5.2-8.4] minutes, P = 0.03). Second and combined transseptal times, and number of equipment exchanges, were also reduced with the RF wire. One participant in the RF needle group experienced transient atrioventricular block due to mechanical trauma from the sheath/dilator assembly. There were no complications in the RF wire group. CONCLUSIONS: The RF wire technique resulted in faster time to transseptal puncture and fewer equipment exchanges compared with an RF needle with no difference in complications.

A genetic strategy to measure insulin signaling regulation and physiology in Drosophila.

Insulin regulation is a hallmark of health, and impaired insulin signaling promotes metabolic diseases like diabetes mellitus. However, current assays for measuring insulin signaling in all animals remain semi-quantitative and lack the sensitivity, tissue-specificity or temporal resolution needed to quantify in vivo physiological signaling dynamics. Insulin signal transduction is remarkably conserved across metazoans, including insulin-dependent phosphorylation and regulation of Akt/Protein kinase B. Here, we generated transgenic fruit flies permitting tissue-specific expression of an immunoepitope-labelled Akt (AktHF). We developed enzyme-linked immunosorption assays (ELISA) to quantify picomolar levels of phosphorylated (pAktHF) and total AktHF in single flies, revealing dynamic tissue-specific physiological regulation of pAktHF in response to fasting and re-feeding, exogenous insulin, or targeted genetic suppression of established insulin signaling regulators. Genetic screening revealed Pp1-87B as an unrecognized regulator of Akt and insulin signaling. Tools and concepts here provide opportunities to discover tissue-specific regulators of in vivo insulin signaling responses.

A LexAop > UAS > QUAS trimeric plasmid to generate inducible and interconvertible Drosophila overexpression transgenes.

The existence of three independent binary systems for conditional gene expression (Gal4/UAS; LexA/LexAop; QF/QUAS) has greatly expanded versatile genetic analyses in the Drosophila melanogaster; however, the experimental application of these tools is limited by the need to generate multiple collections of noninterchangeable transgenic fly strains for each inducible gene expression system. To address this practical limitation, we developed a modular vector that contains the regulatory elements from all three binary systems, enabling Gal4-, LexA- or QF-dependent expression of transgenes. Our methods also incorporate DNA elements that facilitate independent site-specific recombination and elimination of regulatory UAS, LexAop or QUAS modules with spatial and temporal control, thus offering unprecedented possibilities and logistical advantages for in vivo genetic modulation and efficient interconversion of overexpression transgenic fly lines.

Transgenic Drosophila lines for LexA-dependent gene and growth regulation.

Conditional expression of short hairpin RNAs with binary genetic systems is an indispensable tool for studying gene function. Addressing mechanisms underlying cell-cell communication in vivo benefits from simultaneous use of 2 independent gene expression systems. To complement the abundance of existing Gal4/UAS-based resources in Drosophila, we and others have developed LexA/LexAop-based genetic tools. Here, we describe experimental and pedagogical advances that promote the efficient conversion of Drosophila Gal4 lines to LexA lines, and the generation of LexAop-short hairpin RNA lines to suppress gene function. We developed a CRISPR/Cas9-based knock-in system to replace Gal4 coding sequences with LexA, and a LexAop-based short hairpin RNA expression vector to achieve short hairpin RNA-mediated gene silencing. We demonstrate the use of these approaches to achieve targeted genetic loss-of-function in multiple tissues. We also detail our development of secondary school curricula that enable students to create transgenic flies, thereby magnifying the production of well-characterized LexA/LexAop lines for the scientific community. The genetic tools and teaching methods presented here provide LexA/LexAop resources that complement existing resources to study intercellular communication coordinating metazoan physiology and development.

Issues in Recruiting and Retaining Asian American Breast Cancer Survivors in a Technology-Based Intervention Study.

BACKGROUND: Difficulties in recruiting and retaining Asian Americans in traditional research have been well documented. Despite an increasing number of technology-based cancer studies among racial/ethnic minorities, little is still known about potential issues in recruiting and retaining racial/ethnic minority cancer survivors for technology-based intervention research. OBJECTIVE: This discussion article aims to examine issues in recruiting and retaining a group of racial/ethnic minorities-Asian American breast cancer survivors-for a technology-based intervention study. METHODS: The parent study is an ongoing large-scale, national-scope, technology-based intervention study among a target number of 330 Asian American breast cancer survivors. During the recruitment and retention process, research diaries were written by research team members, and the written records of weekly research team meetings were kept. The written records were analyzed using a content analysis. Then, the themes were used to support the discussion points made in the article. RESULTS: There existed subethnic differences in research participation; it was easier to recruit Chinese participants compared with other subethnic groups. The use of culturally matched research team members and multiple languages was essential. Gatekeepers were also elemental for recruitment and retention. Various motivation strategies were needed to retain the participants. Each subethnic group used different communication apps. Finally, trust building was essential to retain the participants in the intervention. CONCLUSIONS: Researchers need to consider these practical issues in future technology-based intervention research. IMPLICATIONS FOR PRACTICE: These issues need to be considered in future program/intervention development and implementation among racial/ethnic minority cancer survivors.

Teaching Hand Surgery in the Developing World: Utilizing Educational Resources in Global Health.

The development of surgical capacity in the developing world is essential to address the global burden of surgical disease. Training local surgeons in low-income and middle-income countries is critical in this endeavor. The challenges to teaching hand surgery in the developing world include a shortage of local faculty, absence of a defined curriculum, no competency-based evaluation systems, few subspecialty training opportunities, and lack of financial support. To teach hand surgery in the developing world effectively, the authors suggest principles and components of a global training curriculum.

An Inexpensive Bismuth-Petrolatum Dressing for Treatment of Burns.

BACKGROUND: Xeroform remains the current standard for treating superficial partial-thickness burns but can be prohibitively expensive in developing countries with prevalent burn injuries. This study (1) describes the production of an alternative low-cost dressing and (2) compares the alternative dressing and Xeroform using the metrics of cost-effectiveness, antimicrobial activity, and biocompatibility in vitro, and wound healing in vivo. METHODS: To produce the alternative dressing, 3% bismuth tribromophenate powder was combined with petroleum jelly by hand and applied to Kerlix gauze. To assess cost-effectiveness, the unit costs of Xeroform and components of the alternative dressing were compared. To assess antimicrobial properties, the dressings were placed on agar plated with Escherichia coli and the Kirby-Bauer assay performed. To assess biocompatibility, the dressings were incubated with human dermal fibroblasts and cells stained with methylene blue. To assess in vivo wound healing, dressings were applied to excisional wounds on rats and the rate of re-epithelialization calculated. RESULTS: The alternative dressing costs 34% of the least expensive brand of Xeroform. Antimicrobial assays showed that both dressings had similar bacteriostatic effects. Biocompatibility assays showed that there was no statistical difference (P < 0.05) in the cytotoxicity of Xeroform, alternative dressing, and Kerlix gauze. Finally, the in vivo healing model showed no statistical difference (P < 0.05) in mean re-epithelialization time between Xeroform (13.0 ± 1.6 days) and alternative dressing (13.5 ± 1.0 days). CONCLUSIONS: Xeroform is biocompatible, reduces infection, and enhances healing of burn wounds by preventing desiccation and mechanical trauma. Handmade petrolatum gauze may be a low-cost replacement for Xeroform. Future studies will focus on clinical trials in burn units.

Smoking and rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease caused by both genetic and environmental factors. Smoking has been implicated as one of the most important extrinsic risk factors for its development and severity. Recent developments have shed light on the pathophysiology of RA in smokers, including oxidative stress, inflammation, autoantibody formation and epigenetic changes. The association of smoking and the development of RA have been demonstrated through epidemiologic studies, as well as through in vivo and animal models of RA. With increased use of biological agents in addition to standard disease-modifying antirheumatic drugs (DMARDs), there has been interest in how smoking affects drug response in RA treatment. Recent evidence suggests the response and drug survival in people treated with anti-tumour necrosis factor (anti-TNF) therapy is poorer in heavy smokers, and possible immunological mechanisms for this effect are presented in the current paper.

Assessment of intrapartum antibiotic prophylaxis for the prevention of early-onset group B Streptococcal disease.

BACKGROUND: Most early-onset group B streptococcal (GBS) disease in recent years has occurred in newborns of prenatally GBS-negative mothers who missed intrapartum antibiotic prophylaxis (IAP). We aimed to assess the accuracy of prenatal culture in predicting GBS carriage during labor, the IAP use, and occurrence of early-onset GBS disease. METHODS: We obtained vaginal-rectal swabs at labor for GBS culture from 5497 women of ≥ 32 weeks' gestation and surface cultures at birth from newborns between February 5, 2008 and February 4, 2009 at 3 hospitals in Houston, TX and Oakland, CA. Prenatal cultures were performed by a healthcare provider during routine care, and culture results were obtained from medical records. The accuracy of prenatal culture in predicting intrapartum GBS carriage was assessed by positive and negative predictive values. Mother-to-newborn transmission of GBS was assessed. Newborns were monitored for early-onset GBS disease. RESULTS: GBS carriage was 24.5% by prenatal and 18.8% by labor cultures. Comparing prenatal with labor GBS cultures of 4696 women, the positive predictive value was 50.5% and negative predictive value was 91.7%. IAP, administered to 93.3% of prenatally GBS-positive women, was 83.7% effective in preventing newborn's GBS colonization. Mother-to-newborn transmission of GBS occurred in 2.6% of elective cesarean deliveries. Two newborns developed early-onset GBS disease (0.36/1000 births); the prenatal GBS culture of one was negative, the other's was unknown. CONCLUSIONS: IAP was effective in interrupting mother-to-newborn transmission of GBS. However, approximately 10% of prenatally GBS-negative women were positive during labor and missed IAP, whereas approximately 50% of prenatally GBS-positive women were negative during labor and received IAP. These findings emphasize the need for rapid diagnostics during labor.