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Mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene have been identified as one of the most common genetic causes of Parkinson's disease (PD). The LRRK2 PD-associated mutations LRRK2G2019S and LRRK2R1441C, located in the kinase domain and in the ROC-COR domain, respectively, have been demonstrated to impair mitochondrial function. Here, we sought to further our understanding of mitochondrial health and mitophagy by integrating data from LRRK2R1441C rat primary cortical and human induced pluripotent stem cell-derived dopamine (iPSC-DA) neuronal cultures as models of PD. We found that LRRK2R1441C neurons exhibit decreased mitochondrial membrane potential, impaired mitochondrial function and decreased basal mitophagy levels. Mitochondrial morphology was altered in LRRK2R1441C iPSC-DA but not in cortical neuronal cultures or aged striatal tissue, indicating a cell-type-specific phenotype. Additionally, LRRK2R1441C but not LRRK2G2019S neurons demonstrated decreased levels of the mitophagy marker pS65Ub in response to mitochondrial damage, which could disrupt degradation of damaged mitochondria. This impaired mitophagy activation and mitochondrial function were not corrected by the LRRK2 inhibitor MLi-2 in LRRK2R1441C iPSC-DA neuronal cultures. Furthermore, we demonstrate LRRK2 interaction with MIRO1, a protein necessary to stabilize and to anchor mitochondria for transport, occurs at mitochondria, in a genotype-independent manner. Despite this, we found that degradation of MIRO1 was impaired in LRRK2R1441C cultures upon induced mitochondrial damage, suggesting a divergent mechanism from the LRRK2G2019S mutation., (© The Author(s) 2023. Published by Oxford University Press.)

Variants at the GBA locus, encoding glucocerebrosidase, are the strongest common genetic risk factor for Parkinson's disease (PD). To understand GBA-related disease mechanisms, we use a multi-part-enrichment proteomics and post-translational modification (PTM) workflow, identifying large numbers of dysregulated proteins and PTMs in heterozygous GBA-N370S PD patient induced pluripotent stem cell (iPSC) dopamine neurons. Alterations in glycosylation status show disturbances in the autophagy-lysosomal pathway, which concur with upstream perturbations in mammalian target of rapamycin (mTOR) activation in GBA-PD neurons. Several native and modified proteins encoded by PD-associated genes are dysregulated in GBA-PD neurons. Integrated pathway analysis reveals impaired neuritogenesis in GBA-PD neurons and identify tau as a key pathway mediator. Functional assays confirm neurite outgrowth deficits and identify impaired mitochondrial movement in GBA-PD neurons. Furthermore, pharmacological rescue of glucocerebrosidase activity in GBA-PD neurons improves the neurite outgrowth deficit. Overall, this study demonstrates the potential of PTMomics to elucidate neurodegeneration-associated pathways and potential drug targets in complex disease models., Competing Interests: Declaration of interests The authors’ current additional affiliations, unrelated to this work, are as follows: D.L.E.V., Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands; L.N.K., Nature Reviews Neurology, London, UK; U.C., School of Medicine, Cardiff University, Cardiff, UK; F.S.B., School of Biological Sciences, University of Edinburgh, Edinburgh, UK; J.B., Clinical Neurosciences, University of Cambridge, Cambridge, UK; J.P.C., Astbury Center for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds, Leeds, UK; and T.M.C., Mend the Gap, University of British Columbia, Vancouver, BC, Canada., (Copyright © 2023. Published by Elsevier Inc.)

The accumulation of toxic protein aggregates in multiple neurodegenerative diseases is associated with defects in the macroautophagy/autophagy-lysosome pathway. The amelioration of disease phenotypes across multiple models of neurodegeneration can be achieved through modulating the master regulator of lysosome function, TFEB (transcription factor EB). Using a novel multi-parameter high-throughput screen for cytoplasmic:nuclear translocation of endogenous TFEB and the related transcription factor TFE3, we screened the Published Kinase Inhibitor Set 2 (PKIS2) library as proof of principle and to identify kinase regulators of TFEB and TFE3. Given that TFEB and TFE3 are responsive to cellular stress we have established assays for cellular toxicity and lysosomal function, critical to ensuring the identification of hit compounds with only positive effects on lysosome activity. In addition to AKT inhibitors which regulate TFEB localization, we identified a series of quinazoline-derivative compounds that induced TFEB and TFE3 translocation. A novel series of structurally-related analogs was developed, and several compounds induced TFEB and TFE3 translocation at higher potency than previously screened compounds. KINOME scan and cell-based KiNativ kinase profiling revealed high binding for the PRKD (protein kinase D) family of kinases, suggesting good selectivity for these compounds. We describe and utilize a cellular target-validation platform using CRISPRi knockdown and orthogonal PRKD inhibitors to demonstrate that the activity of these compounds is independent of PRKD inhibition. The more potent analogs induced subsequent upregulation of the CLEAR gene network and cleared pathological HTT protein in a cellular model of proteinopathy, demonstrating their potential to alleviate neurodegeneration-relevant phenotypes. Abbreviations: AD: Alzheimer disease; AK: adenylate kinase; CLEAR: coordinated lysosomal expression and regulation; CQ: chloroquine; HD: Huntington disease; PD: Parkinson disease; PKIS2: Published Kinase Inhibitor Set 2; PRKD: protein kinase D; TFEB: transcription factor EB.

Staphylococcus aureus remains a leading cause of skin and soft tissue infections (SSTIs) globally. In the United States, many of these infections are caused by isolates classified as USA300. Our understanding of the success of USA300 as a human pathogen is due in part to data obtained from animal infection models, including rabbit SSTI models. These animal models have been used to study S. aureus virulence and pathogenesis and to gain an enhanced understanding of the host response to infection. Although significant knowledge has been gained, the need to use a relatively high inoculum of USA300 (1 × 10 8 to 5 × 10 8 CFU) is a caveat of these infection models. As a step toward addressing this issue, we created mutations in USA300 that mimic those found in S. aureus strains with naturally occurring rabbit tropism-namely, single nucleotide polymorphisms in dltB and/or deletion of rot . We then developed a rabbit SSTI model that utilizes an inoculum of 10 6 USA300 CFU to cause reproducible disease and tested whether primary SSTI protects rabbits against severe reinfection caused by the same strain. Although there was modest protection against severe reinfection, primary infection and reinfection with rabbit-tropic USA300 strains failed to increase the overall level of circulating anti-S. aureus antibodies significantly. These findings provide additional insight into the host response to S. aureus. More work is needed to further develop a low-inoculum infection model that can be used to better test the potential of new therapeutics or vaccine target antigens. IMPORTANCE Animal models of S. aureus infection are important for evaluating bacterial pathogenesis and host immune responses. These animal infection models are often used as an initial step in the testing of vaccine antigens and new therapeutics. The extent to which animal models of S. aureus infection approximate human infections remains a significant consideration for translation of results to human clinical trials. Although significant progress has been made with rabbit models of S. aureus infection, one concern is the high inoculum needed to cause reproducible disease. Here, we generated USA300 strains that have tropism for rabbits and developed a rabbit SSTI model that uses fewer CFU than previous models.

Objective: COVID-19 may have contributed to an excess of out-of-hospital cardiac arrests (OOHCAs). This observational study identified changes in OOHCA epidemiology pre- and post-COVID-19 lockdown in a single UK helicopter emergency medical service (HEMS)., Methods: A retrospective, single-center (Essex & Herts Air Ambulance), observational study was undertaken with anonymized OOHCA data (demographics, etiology, and outcomes) from March 23, 2020, to June 23, 2020, and comparative data from March 23, 2019, to June 23, 2019. Supplementary data (total OOHCAs and patient outcomes) were provided by the East of England Ambulance Service National Health Service Trust. Data were analyzed using the Mann-Whitney U test and chi-square test; P < .05 was statistically significant., Results: Of the HEMS activations during national lockdown, 33.6% were for OOHCAs compared with 25.8% during the reference time frame. The frequency of young and female OOHCAs demonstrated a statistically significant increase. Statistically significant variations in medical etiology and initial cardiac rhythm were identified., Conclusion: During the initial UK-wide lockdown, the OOHCA characteristics attended by 1 HEMS team were altered. The changes seen may be due to the pathophysiology of COVID-19 or an alteration in dispatch due to the demand placed on the wider ambulance service; this may require further consideration for any future lockdowns or pandemics., (Copyright © 2021 Air Medical Journal Associates. Published by Elsevier Inc. All rights reserved.)

Background: Intravenous tranexamic acid (TXA) reduces bleeding deaths after injury and childbirth. It is most effective when given early. In many countries, pre-hospital care is provided by people who cannot give i.v. injections. We examined the pharmacokinetics of intramuscular TXA in bleeding trauma patients., Methods: We conducted an open-label pharmacokinetic study in two UK hospitals. Thirty bleeding trauma patients received a loading dose of TXA 1 g i.v., as per guidelines. The second TXA dose was given as two 5 ml (0·5 g each) i.m. injections. We collected blood at intervals and monitored injection sites. We measured TXA concentrations using liquid chromatography coupled to mass spectrometry. We assessed the concentration time course using non-linear mixed-effect models with age, sex, ethnicity, body weight, type of injury, signs of shock, and glomerular filtration rate as possible covariates., Results: Intramuscular TXA was well tolerated with only mild injection site reactions. A two-compartment open model with first-order absorption and elimination best described the data. For a 70-kg patient, aged 44 yr without signs of shock, the population estimates were 1.94 h -1 for i.m. absorption constant, 0.77 for i.m. bioavailability, 7.1 L h -1 for elimination clearance, 11.7 L h -1 for inter-compartmental clearance, 16.1 L volume of central compartment, and 9.4 L volume of the peripheral compartment. The time to reach therapeutic concentrations (5 or 10 mg L -1 ) after a single intramuscular TXA 1 g injection are 4 or 11 min, with the time above these concentrations being 10 or 5.6 h, respectively., Conclusions: In bleeding trauma patients, intramuscular TXA is well tolerated and rapidly absorbed., Clinical Trial Registration: 2019-000898-23 (EudraCT); NCT03875937 (ClinicalTrials.gov)., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Therapeutic strategies based on a specific oncogenic target are better justified when elimination of that particular oncogene reduces tumorigenesis in a model organism. One such oncogene, Musashi-1 ( Msi-1 ), regulates translation of target mRNAs and is implicated in promoting tumorigenesis in the colon and other tissues. Msi-1 targets include the tumor suppressor adenomatous polyposis coli ( Apc ), a Wnt pathway antagonist lost in ∼80% of all colorectal cancers. Cell culture experiments have established that Msi-1 is a Wnt target, thus positioning Msi-1 and Apc as mutual antagonists in a mutually repressive feedback loop. Here, we report that intestines from mice lacking Msi-1 display aberrant Apc and Msi-1 mutually repressive feedback, reduced Wnt and Notch signaling, decreased proliferation, and changes in stem cell populations, features predicted to suppress tumorigenesis. Indeed, mice with germline Apc mutations ( Apc Min ) or with the Apc 1322T truncation mutation have a dramatic reduction in intestinal polyp number when Msi-1 is deleted. Taken together, these results provide genetic evidence that Msi-1 contributes to intestinal tumorigenesis driven by Apc loss, and validate the pursuit of Msi-1 inhibitors as chemo-prevention agents to reduce tumor burden., (© 2017. Published by The Company of Biologists Ltd.)

We present a case of acute clenbuterol toxicity following ingestion of 20 μg of clenbuterol, resulting in symptoms of sympathetic activation, sinus tachycardia and electrolyte derangement. The patient was managed conservatively with fluid resuscitation, electrolyte replacement and monitoring, and discharged following a 5-day stay in hospital., (2016 BMJ Publishing Group Ltd.)