Palmitoylated Proteins in Plasmodium falciparum-Infected Erythrocytes: Investigation with Click Chemistry and Metabolic Labeling.

The examination of the complex cell biology of the human malaria parasite Plasmodium falciparum usually relies on the time-consuming generation of transgenic parasites. Here, metabolic labeling and click chemistry are employed as a fast transfection-independent method for the microscopic examination of protein S-palmitoylation, an important post-translational modification during the asexual intraerythrocytic replication of P. falciparum. Applying various microscopy approaches such as confocal, single-molecule switching, and electron microscopy, differences in the extent of labeling within the different asexual developmental stages of P. falciparum and the host erythrocytes over time are observed.

At-home hemoglobin A1c testing during COVID-19 improved glycemic control.

OBJECTIVES: COVID-19 has exacerbated barriers to routine testing for chronic disease management. This study investigates whether a home hemoglobin A1c (HbA1c) test kit intervention increases frequency of HbA1c testing and leads to changes in HbA1c 6 months post testing and whether self-reinforcement education improves maintenance of HbA1c testing. STUDY DESIGN: Retrospective analysis of a randomized, controlled quality improvement intervention among members with type 2 diabetes (T2D) in a large commercial health plan. METHODS: Participants were 41,214 commercial fully insured members with T2D without an HbA1c test in the past 6 months or with only 1 HbA1c test in the last 12 months. Members were randomly assigned to either a control group or an at-home HbA1c testing intervention group consisting of either an opt-in test or a direct-to-member opt-out HbA1c test kit shipment. A third cohort of members was assigned to a self-reinforcement group to encourage continued testing twice per year. Main outcomes were HbA1c testing rates and HbA1c levels (in %). RESULTS: A total of 11.1% (508 of 4590) at-home HbA1c kits were completed. At-home HbA1c test kits increased testing rates by 4.9% compared with controls (P < .001). Members with an HbA1c level of at least 7% who requested and completed at-home HbA1c testing had a 0.38% reduction in HbA1c in the 6 months post intervention when controlling for baseline HbA1c (P < .001). Members who received self-reinforcement messaging had a 0.37% HbA1c reduction post intervention (P = .015). CONCLUSIONS: This novel, at-home approach to test HbA1c is an effective intervention to increase testing rates and facilitate HbA1c reduction over time in patients with T2D.

Biallelic NAA60 variants with impaired n-terminal acetylation capacity cause autosomal recessive primary familial brain calcifications.

Primary familial brain calcification (PFBC) is characterized by calcium deposition in the brain, causing progressive movement disorders, psychiatric symptoms, and cognitive decline. PFBC is a heterogeneous disorder currently linked to variants in six different genes, but most patients remain genetically undiagnosed. Here, we identify biallelic NAA60 variants in ten individuals from seven families with autosomal recessive PFBC. The NAA60 variants lead to loss-of-function with lack of protein N-terminal (Nt)-acetylation activity. We show that the phosphate importer SLC20A2 is a substrate of NAA60 in vitro. In cells, loss of NAA60 caused reduced surface levels of SLC20A2 and a reduction in extracellular phosphate uptake. This study establishes NAA60 as a causal gene for PFBC, provides a possible biochemical explanation of its disease-causing mechanisms and underscores NAA60-mediated Nt-acetylation of transmembrane proteins as a fundamental process for healthy neurobiological functioning.

Cascades of diabetes and hypertension care in Samoa: Identifying gaps in the diagnosis, treatment, and control continuum - a cross-sectional study.

BACKGROUND: Samoa is a Pacific Island country facing one of the highest burdens of non-communicable disease globally. METHODS: In this study, we apply a cascade-of-care approach to understand gaps in the awareness, treatment, and control cascade of diabetes and hypertension in a cross-sectional, convenience sample of 703 young, high-risk Samoan adults (29.5-50.9 years). FINDINGS: Non-communicable diseases were prevalent in the study sample: 19.5% (95% CI: 16.6%-22.7%) of participants had diabetes; 47.6% (95% CI: 43.7%-51.4%) presented with pre-diabetes or diabetes; 31.0% (95% CI: 27.5%-34.6%) had hypertension; and nearly 90% (95% CI: 86.7%-91.5%) had overweight or obesity. Among those with diabetes and hypertension, only 20.5% (95% CI: 13.9%-28.4%) and 11.8% (95% CI: 7.8%-16.9%) of participants were aware of their condition, respectively. Only 0.8% (95% CI: 0.0%-4.2%) of all participants with diabetes had achieved glycemic control; only 2.8% (95% CI: 1.1%-6.1%) of those with hypertension achieved control. INTERPRETATION: We found a significant burden of diabetes and hypertension in Samoa, exceeding the recent prevalence estimates of other low- to middle-income countries by nearly two-fold. A severe unmet need in both detection and subsequent control and monitoring of these chronic conditions exists. Our results suggest that the initial diagnosis and surveillance stage in the cascade of care for chronic conditions should be a major focus of primary care efforts; national screening campaigns and programs that leverage village and district nurses to deliver community-based primary care may significantly impact gap closure in the NCD cascade. FUNDING: This study was supported by the U.S. National Institutes of Health R01HL140570 (PIs: McGarvey and DeLany); AR was supported by NIH FIC D43TW010540; HK and AR-C were supported by the Minority Health and Health Disparities International Research Training (MHIRT) Program at Brown University, NIH Grant # 5T37MD008655.

Early Conversion to Aflibercept for Persistent Diabetic Macular Edema Results in Better Visual Outcomes and Lower Treatment Costs.

PURPOSE: To evaluate the functional and anatomic outcomes, as well as cost-effectiveness, of the timing of conversion to intravitreal aflibercept (IVA) in patients with treatment-resistant diabetic macular edema (DME). METHODS: Thirty consecutive eyes (25 patients) were identified that were treated with ≥3 intravitreal bevacizumab (IVB) and/or ranibizumab (IVR) injections prior to treatment with ≥3 IVA injections. Eyes that received ≤6 IVB and/or IVR injections (early-switch) were compared to those that received ≥7 injections (late-switch) prior to conversion to IVA. Treatment effectiveness was measured in quality-adjusted life years (QALYs). A micro-simulation model examined the impact of treatment duration on outcomes. RESULTS: Early- (n=18) and late- (n=12) switch eyes had similar vision prior to conversion to IVA. Despite improvements in retinal thickness, only the early-switch eyes maintained vision gains after conversion to IVA through the end of follow-up (p=0.027). Early switch saved $22,884/eye and produced an additional 0.027 QALYs. CONCLUSION: Early conversion to IVA optimizes vision outcomes and results in lower overall treatment expenditures.

Mass variability in drops of multidose eyedrops: a quality and reliability study.

PURPOSE: This quality and reliability study aimed to identify the mass variability of multidose eyedrops and to verify the existence of a reference pattern for the drop volume of eyedrops using standard lubricant eyedrops available on the Brazilian market. METHODS: Five brands of lubricant eyedrops were evaluated. An ideal standard 20 µL drop of eyedrops from each manufacturer was captured using an adjustable micropipette. The eyedrop bottles were randomly selected, and five measurements of the samples' masses were collected using calibrated precision scales. RESULTS: The mass of the 20 µL samples varied significantly (p<0.001) among the different manufacturers. However, among eyedrops of the same brand, mass variation was not statistically different. The global mean mass of all weighed drops was 18.24 mg, and non-uniformity was identified across all eyedrop brands. CONCLUSION: Significant variations in the drop masses of common lubricant eyedrops were identified using standard laboratory equipment. Heterogeneity in the drop volume of standard eyedrop medications suggest that potential dosage discrepancies exist, possibly altering treatment efficacy. A pre-established reference measure may lead to the production of more appropriately sized eyedrops for use in human eyes.

Remote Screening for Optic Nerve Cupping Using Smartphone-based Nonmydriatic Fundus Photography.

PRECIS: Evaluation of nonmydriatic fundus photographs captured with a low-cost, smartphone-based camera facilitated remote screening of patients for enlarged optic nerve cup-to-disc ratio in the Independent Nation of Samoa, an underserved setting with one full-time ophthalmologist in the entire country. PURPOSE: To investigate factors that impact inter-rater agreement of glaucoma suspect optic disc status using a low-cost, handheld nonmydriatic fundus camera. METHODS: Color fundus photographs were obtained using the PanOptic iExaminer attached to an iPhone 6S by a lay examiner on 206 participants in the Independent Nation of Samoa. Images were remotely graded by an ophthalmologist and optometrist, and images from participants identified as at-risk for glaucoma were escalated to a glaucoma subspecialist for review. Fundus photo brightness, contrast, and focus were measured using the cup, rim, and temporal regions of the disc. Stereoscopic image pairs were subsequently generated from a subset of individual nonmydriatic photographs. RESULTS: Features suggestive of glaucoma based on optic disc cupping were identified in 16.0% (33/206) of participants. There was moderately strong agreement between graders (90.3%) with κ=0.53 [95% confidence interval (CI)=0.33-0.73]. The intraclass correlation coefficients for the cup-to-disc ratio (CDR) and its difference were 0.84 (95% CI=0.81-0.87) and 0.68 (95% CI=0.59-0.75). Of the 33 participants identified, 94% had clinical risk criteria for potential glaucoma when reviewed by a subspecialist. Color fundus photograph cup brightness was significantly associated with cup-to-disc (CDR) grade, R2=0.36 (P<0.001), in which a brighter disc yielded a higher CDR. CONCLUSIONS: Smartphone-based screening is a simple, low-cost method capable of measuring the CDR of the optic nerve. When combined with testing for other glaucoma risk factors such as intraocular pressure, this method of measuring CDR may help identify those patients who should be referred for further ophthalmologic assessment. We are currently conducting studies to assess the sensitivity and specificity of smart phone-based remote screening.

Microbial Cross-contamination in Multidose Eyedrops: The Impact of Instillation Angle and Bottle Geometry.

Purpose: To evaluate the impact of instillation angle and nozzle tip geometry on cross-contamination risk of multidose ocular solution bottles. Methods: Pseudomonas aeruginosa solution was passed exclusively on the outside of the nozzle to simulate contamination on the exterior of topical agents. Three drops were administered from angles of 90° and 45° from bottles with either a round or sharp tip geometry, and the cultures were examined for growth. Two-hundred sixteen cultures from nine lubricant eyedrop brands currently existing in the Brazilian market were assessed for bacterial growth. Results: After seven days, bacterial contamination was detected in 53.7% of cultures when drops were administered at 90° and in 70.4% of cultures at 45°. Eyedrops collected from a rounded nozzle tip and an instillation angle of 90° transmitted bacteria in 69.4% of cases, whereas those administered from a sharp tip transmitted bacteria in only 22.2% of cases (P = 0.001). At an instillation angle of 45°, contamination was identified in 83.3% of bottles with a rounded tip geometry and in only eight of 18 bottles (44.4%) from those with a sharp nozzle geometry (P = 0.005). Conclusions: Adjusting the instillation angle of eyedrop solutions to 90°, as well as using a nozzle geometry that prevents flow of the solution to the side of the bottle, significantly reduced contamination rates. Translational Relevance: Standardizing drop bottles and adjusting delivery angle shows promise in reducing contamination rates and may critically impact the quality of care for patients requiring topical therapeutic agents.

Liquid-liquid phase separation of the Golgi matrix protein GM130.

Golgins are an abundant class of peripheral membrane proteins of the Golgi. These very long (50-400 nm) rod-like proteins initially capture cognate transport vesicles, thus enabling subsequent SNARE-mediated membrane fusion. Here, we explore the hypothesis that in addition to serving as vesicle tethers, Golgins may also possess the capacity to phase separate and, thereby, contribute to the internal organization of the Golgi. GM130 is the most abundant Golgin at the cis Golgi. Remarkably, overexpressed GM130 forms liquid droplets in cells analogous to those described for numerous intrinsically disordered proteins with low complexity sequences, even though GM130 is neither low in complexity nor intrinsically disordered. Virtually pure recombinant GM130 also phase-separates into dynamic, liquid-like droplets in close to physiological buffers and at concentrations similar to its estimated local concentration at the cis Golgi.

Exploring the Paradoxical Relationship of a Creb 3 Regulatory Factor Missense Variant With Body Mass Index and Diabetes Among Samoans: Protocol for the Soifua Manuia (Good Health) Observational Cohort Study.

BACKGROUND: The prevalence of obesity and diabetes in Samoa, like many other Pacific Island nations, has reached epidemic proportions. Although the etiology of these conditions can be largely attributed to the rapidly changing economic and nutritional environment, a recently identified genetic variant, rs373863828 (CREB 3 regulatory factor, CREBRF: c.1370G>A p.[R457Q]) is associated with increased odds of obesity, but paradoxically, decreased odds of diabetes. OBJECTIVE: The overarching goal of the Soifua Manuia (Good Health) study was to precisely characterize the association of the CREBRF variant with metabolic (body composition and glucose homeostasis) and behavioral traits (dietary intake, physical activity, sleep, and weight control behaviors) that influence energy homeostasis in 500 adults. METHODS: A cohort of adult Samoans who participated in a genome-wide association study of adiposity in Samoa in 2010 was followed up, based on the presence or absence of the CREBRF variant, between August 2017 and March 2019. Over a period of 7-10 days, each participant completed the main study protocol, which consisted of anthropometric measurements (weight, height, circumferences, and skinfolds), body composition assessment (bioelectrical impedance and dual-energy x-ray absorptiometry), point-of-care glycated hemoglobin measurement, a fasting blood draw and oral glucose tolerance test, urine collection, blood pressure measurement, hand grip strength measurement, objective physical activity and sleep apnea monitoring, and questionnaire measures (eg, health interview, cigarette and alcohol use, food frequency questionnaire, socioeconomic position, stress, social support, food and water insecurity, sleep, body image, and dietary preferences). In January 2019, a subsample of the study participants (n=118) completed a buttock fat biopsy procedure to collect subcutaneous adipose tissue samples. RESULTS: Enrollment of 519 participants was completed in March 2019. Data analyses are ongoing, with results expected in 2020 and 2021. CONCLUSIONS: While the genetic variant rs373863828, in CREBRF, has the largest known effect size of any identified common obesity gene, very little is currently understood about the mechanisms by which it confers increased odds of obesity but paradoxically lowered odds of type 2 diabetes. The results of this study will provide insights into how the gene functions on a whole-body level, which could provide novel targets to prevent or treat obesity, diabetes, and associated metabolic disorders. This study represents the human arm of a comprehensive and integrated approach involving humans as well as preclinical models that will provide novel insights into metabolic disease. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR1-10.2196/17329.

Mass variability in drops of multidose eyedrops: a quality and reliability study / Variabilidade da massa das gotas dos colírios: um estudo de qualidade e confiabilidade

ABSTRACT Purpose: This quality and reliability study aimed to identify the mass variability of multidose eyedrops and to verify the existence of a reference pattern for the drop volume of eyedrops using standard lubricant eyedrops available on the Brazilian market. Methods: Five brands of lubricant eyedrops were evaluated. An ideal standard 20 µL drop of eyedrops from each manufacturer was captured using an adjustable micropipette. The eyedrop bottles were randomly selected, and five measurements of the samples' masses were collected using calibrated precision scales. Results: The mass of the 20 µL samples varied significantly (p<0.001) among the different manufacturers. However, among eyedrops of the same brand, mass variation was not statistically different. The global mean mass of all weighed drops was 18.24 mg, and non-uniformity was identified across all eyedrop brands. Conclusion: Significant variations in the drop masses of common lubricant eyedrops were identified using standard laboratory equipment. Heterogeneity in the drop volume of standard eyedrop medications suggest that potential dosage discrepancies exist, possibly altering treatment efficacy. A pre-established reference measure may lead to the production of more appropriately sized eyedrops for use in human eyes.

RESUMO Objetivo: Este estudo de qualidade e confiabilidade teve como objetivo identificar a variabilidade da massa do volume da gota de colírios multidose e verificar a existência de um padrão de referência para o volume da gota dos colírios usando lágrimas artificiais do mercado brasileiro. Métodos: Cinco marcas de colírios lubrificantes foram avaliadas quanto ao volume da gota. Uma gota padrão ideal de 20 µL de cada fabricante foi coletada usando uma micropipeta ajustável. Os frascos dos colírios foram selecionados aleatoriamente e cinco medidas das massas das amostras foram coletadas usando escalas de precisão calibradas. Resultados: A massa das amostras de 20 µL variou significativamente (p<0,001) entre os diferentes fabricantes. No entanto, entre os colírios da mesma marca, a variação da massa não foi estatisticamente diferente. A massa média global de todas as gotas pesadas foi de 18,24 mg e foi observada uma não-uniformidade entre todas as marcas de colírios. Conclusão: Identificou-se uma variação significativa nas massas do volume das gotas dos colírios lubrificantes, usando equipamento padrão de laboratório. A heterogeneidade no volume da gota dos colírios testados sugere a existência de discrepâncias potenciais em suas posologias, possivelmente alterando a eficácia do tratamento. Uma medida de referência pré-estabelecida pode levar à produção de colírios com gotas de tamanho mais apropriado para uso em olhos humanos.