Fluorescence Microscopy-Based Quantitation of GLUT4 Translocation

Postprandial insulin-stimulated glucose uptake into target tissue is crucial for the maintenance of normal blood glucose homeostasis. This step is rate-limited by the number of facilitative glucose transporters type 4 (GLUT4) present in the plasma membrane. Since insulin resistance and impaired GLUT4 translocation are associated with the development of metabolic disorders such as type 2 diabetes, this transporter has become an important target of antidiabetic drug research. The application of screening approaches that are based on the analysis of GLUT4 translocation to the plasma membrane to identify substances with insulinomimetic properties has gained global research interest in recent years. Here, we review methods that have been implemented to quantitate the translocation of GLUT4 to the plasma membrane. These methods can be broadly divided into two sections: microscopy-based technologies (e.g., immunoelectron, confocal or total internal reflection fluorescence microscopy) and biochemical and spectrometric approaches (e.g., membrane fractionation, photoaffinity labeling or flow cytometry). In this review, we discuss the most relevant approaches applied to GLUT4 thus far, highlighting the advantages and disadvantages of these approaches, and we provide a critical discussion and outlook into new methodological opportunities.

Evaluation of Virtual Screening Strategies for the Identification of γ-Secretase Inhibitors and Modulators

γ-Secretase is an intramembrane aspartyl protease that is important in regulating normal cell physiology via cleavage of over 100 transmembrane proteins, including Amyloid Precursor Protein (APP) and Notch family receptors. However, aberrant proteolysis of substrates has implications in the progression of disease pathologies, including Alzheimer’s disease (AD), cancers, and skin disorders. While several γ-secretase inhibitors have been identified, there has been toxicity observed in clinical trials associated with non-selective enzyme inhibition. To address this, γ-secretase modulators have been identified and pursued as more selective agents. Recent structural evidence has provided an insight into how γ-secretase inhibitors and modulators are recognized by γ-secretase, providing a platform for rational drug design targeting this protease. In this study, docking- and pharmacophore-based screening approaches were evaluated for their ability to identify, from libraries of known inhibitors and modulators with decoys with similar physicochemical properties, γ-secretase inhibitors and modulators. Using these libraries, we defined strategies for identifying both γ-secretase inhibitors and modulators incorporating an initial pharmacophore-based screen followed by a docking-based screen, with each strategy employing distinct γ-secretase structures. Furthermore, known γ-secretase inhibitors and modulators were able to be identified from an external set of bioactive molecules following application of the derived screening strategies. The approaches described herein will inform the discovery of novel small molecules targeting γ-secretase.

Drugging Fuzzy Complexes in Transcription

Transcription factors (TFs) are one of the most promising but underutilized classes of drug targets. The high degree of intrinsic disorder in both the structure and the interactions (i.e., “fuzziness”) of TFs is one of the most important challenges to be addressed in this context. Here, we discuss the impacts of fuzziness on transcription factor drug discovery, describing how disorder poses fundamental problems to the typical drug design, and screening approaches used for other classes of proteins such as receptors or enzymes. We then speculate on ways modern biophysical and chemical biology approaches could synergize to overcome many of these challenges by directly addressing the challenges imposed by TF disorder and fuzziness.

Diagnostic Criteria and Lipid Screening of Dyslipidemia in Children

Obesity is associated with significant morbidities and life-threatening conditions. Evidence shows that obesity in the pediatric population has increased by ten folds recently. This has been attributed to the remarkable recent alternations in socioeconomic factors and the overall increase in the incidence of obesity among the different populations. The pathogenesis of atherosclerosis and cardiovascular diseases is usually initiated in childhood. Previous studies indicates that early identification and proper treatment of dyslipidemia in the pediatric population can significantly reduce the risk of developing cardiovascular diseases and associated morbidities. Therefore, it is vital to screen children's lipid profiles to identify dyslipidemia and apply better interventions. This can significantly reduce the risk of premature cardiovascular diseases and accelerated atherosclerosis. The present study aims to identify the diagnostic criteria and various lipid screening approaches proposed in the literature to identify dyslipidemia in children. Two main approaches for screening dyslipidemia in children were reported. These include universal and selective screening approaches. While the latter is recommended to identify high-risk children, universal screening is also recommended to identify children missed by targeted screening (usually treated by pharmacological modalities).

Resources Required for Implementation of SARS-CoV-2 Screening in Massachusetts K-12 Public Schools in Winter/Spring 2021

Importance CDC guidance emphasizes the importance of in-person education for students in grades kindergarten to 12 (K-12) during the COVID-19 pandemic. CDC encourages weekly SARS-CoV-2 testing of asymptomatic, unvaccinated students and staff ("screening") to reduce infection risk and provide data about in-school SARS-CoV-2 prevalence where community incidence is high. The financial costs of screening assays have been described, but the human resource requirements at the school and district level to implement a SARS-CoV-2 screening program are not well known. Objective To quantify the resources required to implement a screening program in K-12 schools. Design, Setting, and Participants A consortium of Massachusetts public K-12 schools was formed to implement and evaluate a range of SARS-CoV-2 screening approaches. Participating districts were surveyed weekly about their programs, including: type of assay used, individual vs. pooled screening, approaches to return of results and deconvolution (identification of positive individual specimens) of positive pools, number and type of personnel implementing the screening program, and hours spent on program implementation. Main Outcomes and Measures Costs, resource utilization Results In 21 participating districts, over 21 weeks from January to June 2021, the positivity rate was 0.0%-0.21% among students and 0.0%-0.13% among educators/staff, and 4 out of 21 (19%) districts had at least one classroom transition to remote learning at any point due to a positive case. The average weekly cost to implement a screening program, including assay and personnel costs, was $17.00 per person tested; this was $46.68 for individual screenings and $15.61 for pooled screenings. The total weekly costs by district ranged from $1,644-$93,486, and districts screened between 58 and 3,675 people per week. The reported number of personnel working per week ranged from 1-5 to >50, and the total number of hours worked by all personnel ranged from 5-10 to >50. Conclusion and Relevance The human resources required to implement SARS-CoV-2 screening in Massachusetts public K-12 schools were substantial. Where screening is recommended for the 2021-22 school year due to high COVID-19 incidence (e.g., where vaccination uptake is low and/or more infectious variants predominate), understanding the human resources required to implement screening will assist districts policymakers in planning.

Age and Sex Effects across the Blood Proteome after Lonizing Radiation Exposure: Consequences for Biomarker Screening and Risk Assessment

Molecular biomarkers of ionizing radiation (IR) exposure are a promising new tool in various disciplines: they can give necessary information for adaptive treatment planning in cancer radiotherapy, enable risk projection for radiation-induced survivorship diseases, or facilitate triage and intervention in radiation hazard events. However, radiation biomarker discovery has not yet resolved the most basic features of personalized medicine: age and sex. To overcome this critical bias in biomarker identification, we quantitated age and sex effects and assessed their relevance in the radiation response across the blood proteome. We used high-throughput mass spectrometry on blood plasma collected 24 hours after 0.5 Gy total body irradiation (15 MV nominal photon energy) from male and female C57BL/6N mice at juvenile (7-weeks-old) or adult (18-weeks-old) age. We also assessed sex and strain effects using juvenile male and female BALB/c nude mice. We showed that age and sex created significant effects in the proteomic response regarding both extent and functional quality of IR-induced responses. Furthermore, we found that age and sex effects appeared non-linear and were often end-point specific. Overall, age contributed more to differences in the proteomic response than sex, most notably in immune responses, oxidative stress, and apoptotic cell death. Interestingly, sex effects were pronounced for DNA damage & repair pathways and associated cellular outcome (pro-survival vs. pro-apoptotic). Only one protein (AHSP) was identified as a potential general biomarker candidate across age and sex, while GMNN, REG3B, and SNCA indicated some response similarity across age. This low yield advocated that unisex or uniage biomarker screening approaches are not feasible. In conclusion, age- and sex-specific screening approaches should be implemented as standard protocol to ensure robustness and diagnostic power of biomarker candidates. Bias-free molecular biomarkers are a necessary progression towards personalized medicine and integral for advanced adaptive cancer radiotherapy and risk assessment.