CRISPR/Cas9 gene editing in Drosophila via visual selection in a summer classroom.

CRISPR/Cas9 methods are a powerful in vivo approach to edit the genome of Drosophila melanogaster. To convert existing Drosophila GAL4 lines to LexA driver lines in a secondary school classroom setting, we applied the CRISPR-based genetic approach to a collection of Gal4 'driver' lines. The integration of the yellow+ coat color marker into homology-assisted CRISPR knock-in (HACK) enabled visual selection of Gal4-to-LexA conversions using brightfield stereo-microscopy available in a broader set of standard classrooms. Here, we report the successful conversion of eleven Gal4 lines with expression in neuropeptide-expressing cells into corresponding, novel LexA drivers. The conversion was confirmed by LexA- and Gal4-specific GFP reporter gene expression. This curriculum was successfully implemented in a summer course running 16 hours/week for seven weeks. The modularity, flexibility, and compactness of this course should enable development of similar classes in secondary schools and undergraduate curricula, to provide opportunities for experience-based science instruction, and university-secondary school collaborations that simultaneously fulfill research needs in the community of science.

Emulating VIEW 1 and VIEW 2 Clinical Trial Outcome Data Using the American Academy of Ophthalmology IRIS Registry.

BACKGROUND AND OBJECTIVE: A retrospective, noninterventional cohort study of the American Academy of Ophthalmology IRIS Registry, an electronic health record (EHR)-based comprehensive eye disease and condition registry, intended to assess whether the IRIS® Registry (Intelligent Research in Sight) could emulate the VIEW randomized clinical trials (VIEW RCTs) eligibility criteria, treatment protocol regimen, and primary endpoint. PATIENTS AND METHODS: Deidentified patients having an anti-VEGF injection of aflibercept or ranibizumab between January 1, 2013, and December 31, 2018, from the IRIS Registry. Patients were treated in accordance with one of three treatment regimens from the VIEW RCT: monthly intravitreal aflibercept injection (IAI 2Q4), intravitreal aflibercept every 2 months after 3 initial monthly doses (IAI 2Q8), or monthly ranibizumab (RQ4) injection. The main outcome measures are the number and proportion of patients meeting VIEW RCT eligibility and treatment group criteria, demographic, and clinical differences between IRIS Registry treatment groups, mean change in best documented visual acuity at one year, and evaluation of the primary endpoint of the VIEW RCT: difference in the proportion of patients maintaining vision. RESULTS: Among the 90,900 patients who met VIEW RCT eligibility criteria, 4,457 (4.85%) met treatment group criteria. The percentage of patients maintaining vision at one year was over 90%. No statistically significant difference was observed when comparing the proportion of patients maintaining vision among the RQ4 treatment group to the IAI 2Q4 or IAI 2Q8 treatment group. CONCLUSIONS: A small percentage of real-world patients met VIEW RCT study eligibility criteria and treatment protocol regimen. Among patients meeting all available criteria, the primary endpoint interpretation yielded by an observational EHR-based dataset suggested comparable results to the VIEW RCT. [Ophthalmic Surg Lasers Imaging Retina 2023;54:6-14.].

Hospital-level factors associated with death during pneumonia-associated hospitalization among adults-New York City, 2010-2014.

BACKGROUND: In New York City (NYC), pneumonia is a leading cause of death and most pneumonia deaths occur in hospitals. Whether the pneumonia death rate in NYC reflects reporting artifact or is associated with factors during pneumonia-associated hospitalization (PAH) is unknown. We aimed to identify hospital-level factors associated with higher than expected in-hospital pneumonia death rates among adults in NYC. METHODS: Data from January 1, 2010-December 31, 2014 were obtained from the New York Statewide Planning and Research Cooperative System and the American Hospital Association Database. In-hospital pneumonia standardized mortality ratio (SMR) was calculated for each hospital as observed PAH death rate divided by expected PAH death rate. To determine hospital-level factors associated with higher in-hospital pneumonia SMR, we fit a hospital-level multivariable negative binomial regression model. RESULTS: Of 148,172 PAH among adult NYC residents in 39 hospitals during 2010-2014, 20,820 (14.06%) resulted in in-hospital death. In-hospital pneumonia SMRs varied across NYC hospitals (0.77-1.23) after controlling for patient-level factors. An increase in average daily occupancy and membership in the Council of Teaching Hospitals were associated with increased in-hospital pneumonia SMR. CONCLUSIONS: Differences in in-hospital pneumonia SMRs between hospitals might reflect differences in disease severity, quality of care, or coding practices. More research is needed to understand the association between average daily occupancy and in-hospital pneumonia SMR. Additional pneumonia-specific training at teaching hospitals can be considered to address higher in-hospital pneumonia SMR in teaching hospitals.

Diabetes prevalence in rural Indigenous Guatemala: A geographic-randomized cross-sectional analysis of risk.

BACKGROUND: Developing countries and Indigenous populations are disproportionately affected by global trends in diabetes (T2DM), but inconsistent data are available to corroborate this pattern in Guatemala and indigenous communities in Central America. Historic estimates of T2DM, using a variety of sampling techniques and diagnostic methods, in Guatemala include a T2DM prevalence of: 4·2% (1970) and 8·4% (2003). Objectives of this geographically randomized, cross-sectional analysis of risk include: (1) use HbA1c to determine prevalence of T2DM and prediabetes in rural Indigenous community of Atitlán (2) identify risk factors for T2DM including age, BMI and gender. METHODS: A spatially random sampling method was used to identify 400 subjects. Prevalence was compared using the confidence interval method, and logistic regression and linear regression were used to assess association between diabetes and risk factors. FINDINGS: The overall prevalence of T2DM using HbA1c was 13·81% and prediabetes was also 13·81% in Atitlán, representing a tripling in diabetes from historic estimates and a large population with pre-diabetes. The probability of diabetes increased dramatically with increasing age, however no significant overall relationship existed with gender or BMI. CONCLUSIONS: Diabetes is a larger epidemic than previously expected and appears to be related to ageing rather than BMI. Our proposed explanations for these findings include: possible Indigenous unique genetic susceptibility to T2DM, shortcomings in BMI as a metric for adiposity in assessing risk, changes in lifestyle and diet, and an overall aging population. The conclusion of this study suggest that (1) T2DM in rural regions of Guatemala may be of epidemic proportion. With pre-diabetes, more than 25% of the population will be diabetic in the very near future; (2) Age is a significant risk factor in the Indigenous population but BMI is not. This suggests that in some populations diabetes may be a disease of ageing.