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Kinetoplastid organisms, including Trypanosoma brucei, are a significant health burden in many tropical and semitropical countries. Much of their metabolism is poorly understood. To better study kinetoplastid metabolism, chemical probes that inhibit kinetoplastid enzymes are needed. To discover chemical probes, we have developed a high-throughput flow cytometry screening assay that simultaneously measures multiple glycolysis-relevant metabolites in live T. brucei bloodstream form parasites. We transfected parasites with biosensors that measure glucose, ATP, or glycosomal pH. The glucose and ATP sensors were FRET biosensors, while the pH sensor was a GFP-based biosensor. The pH sensor exhibited a different fluorescent profile from the FRET sensors, allowing us to simultaneously measure pH and either glucose or ATP. Cell viability was measured in tandem with the biosensors using thiazole red. We pooled sensor cell lines, loaded them onto plates containing a compound library, and then analyzed them by flow cytometry. The library was analyzed twice, once with the pooled pH and glucose sensor cell lines and once with the pH and ATP sensor cell lines. Multiplexing sensors provided some internal validation of active compounds and gave potential clues for each compound's target(s). We demonstrated this using the glycolytic inhibitor 2-deoxyglucose and the alternative oxidase inhibitor salicylhydroxamic acid. Individual biosensor-based assays exhibited a Z'-factor value acceptable for high-throughput screening, including when multiplexed. We tested assay performance in a pilot screen of 14,976 compounds from the Life Chemicals Compound Library. We obtained hit rates from 0.2 to 0.4% depending on the biosensor, with many compounds impacting multiple sensors. We rescreened 44 hits, and 28 (64%) showed repeatable activity for one or more sensors. One compound exhibited EC 50 values in the low micromolar range against two sensors. We expect this method will enable the discovery of glycolytic chemical probes to improve metabolic studies in kinetoplastid parasites., Competing Interests: Declaration of competing interest All authors declare that they have no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Hypermobile Ehlers-Danlos syndrome (hEDS) is a common heritable connective tissue disorder that lacks a known genetic etiology. To identify genetic contributions to hEDS, whole exome sequencing was performed on families and a cohort of sporadic hEDS patients. A missense variant in Kallikrein-15 (KLK15 p. Gly226Asp), segregated with disease in two families and genetic burden analyses of 197 sporadic hEDS patients revealed enrichment of variants within the Kallikrein gene family. To validate pathogenicity, the variant identified in familial studies was used to generate knock-in mice. Consistent with our clinical cohort, Klk15 G224D/+ mice displayed structural and functional connective tissue defects within multiple organ systems. These findings support Kallikrein gene variants in the pathogenesis of hEDS and represent an important step towards earlier diagnosis and better clinical outcomes.

The rising costs of healthcare, increased chronic illnesses, and healthcare provider burnout has led to an environment desperate for scalable solutions to ease practice burdens. With a projected shortage in the number of primary healthcare providers available to provide team-based care, community-based pharmacy practitioners are accessible and eager to assist. In order to provide enhanced patient care services to aid their clinician colleagues, community-based pharmacists will have to transform their practices to support the provision of enhanced services and medication optimization in value-based payment models. The purpose of this article is to define how multiple factors in pharmacy, healthcare, technology and payment models aligned to create an opportunity for the Community Pharmacy Foundation and CPESN® USA to implement a nationwide community pharmacy practice model called 'Flip the Pharmacy'. This new model aims to scale community pharmacy practice transformation and move beyond filling prescriptions at a moment-in-time to caring for patients over time through a 24-month step-wise program paired with in-person pharmacist coaching. Preliminary observations from the first six months of the program highlight community pharmacy as a site of care with community-based pharmacist practitioners providing and documenting targeted patient care interventions., (© Individual authors.)