Bioengineered Model of Human LGMD2B Skeletal Muscle Reveals Roles of Intracellular Calcium Overload in Contractile and Metabolic Dysfunction in Dysferlinopathy.

Dysferlin is a multi-functional protein that regulates membrane resealing, calcium homeostasis, and lipid metabolism in skeletal muscle. Genetic loss of dysferlin results in limb girdle muscular dystrophy 2B/2R (LGMD2B/2R) and other dysferlinopathies - rare untreatable muscle diseases that lead to permanent loss of ambulation in humans. The mild disease severity in dysferlin-deficient mice and diverse genotype-phenotype relationships in LGMD2B patients have prompted the development of new in vitro models for personalized studies of dysferlinopathy. Here the first 3-D tissue-engineered hiPSC-derived skeletal muscle ("myobundle") model of LGMD2B is described that exhibits compromised contractile function, calcium-handling, and membrane repair, and transcriptomic changes indicative of impaired oxidative metabolism and mitochondrial dysfunction. In response to the fatty acid (FA) challenge, LGMD2B myobundles display mitochondrial deficits and intracellular lipid droplet (LD) accumulation. Treatment with the ryanodine receptor (RyR) inhibitor dantrolene or the dissociative glucocorticoid vamorolone restores LGMD2B contractility, improves membrane repair, and reduces LD accumulation. Lastly, it is demonstrated that chemically induced chronic RyR leak in healthy myobundles phenocopies LGMD2B contractile and metabolic deficit, but not the loss of membrane repair capacity. Together, these results implicate intramyocellular Ca2+ leak as a critical driver of dysferlinopathic phenotype and validate the myobundle system as a platform to study LGMD2B pathogenesis.

Application of Size Exclusion Chromatography with Multiangle Light Scattering in the Analytical Development of a Preclinical Stage Gene Therapy Program.

To provide safe recombinant adeno-associated viruses (rAAV) to patients, scalable manufacturing processes are required. However, these processes may introduce impurities that impact the performance and quality of the final drug product. Empty rAAV capsids are product-related impurities. Regulatory guidance requires that accurate analytical methods be implemented early in product development to measure the level of empty capsids. A process confirmation vector, produced from 200 L production, was used to develop and optimize a size exclusion chromatography with UV and multiangle light scattering (SEC-MALS) method. Vector produced from a 500 L production was used to assess the full-to-empty ratio using the following analytical methods: sedimentation velocity analytical ultracentrifugation (SV-AUC), droplet digital PCR (ddPCR) with capsid enzyme-linked immunosorbent assay (ELISA), bulk absorbance at 260/280 nm, cryogenic electron microscopy, and SEC-MALS. This test article was used for a 30-day, non-Good Laboratory Practices animal study that assessed biodistribution of the product (STRX-330). SEC-MALS outperformed the other methods and correlated well with SV-AUC values of full-to-empty particles. In addition, SEC-MALS agreed with ddPCR and ELISA measurements for vector genomes/mL and capsid particles/mL, respectively. SEC-MALS was linear, accurate, and precise while achieving chromatography quality control (QC) recommendations. Compared to other stability-indicating assays, SEC-MALS performed similarly to ddPCR, capsid ELISA, and infectivity assays in accelerated stress studies. In response to alkaline, but not acidic stress, SEC-MALS indicated distinct changes in the DNA content of the monomer Adeno-associated viruses (AAV) peak for STRX-330, which was supported by ddPCR data. Conversely, acidic treatment resulted in more aggregated vector, but did not impact the DNA content. This work indicates that SEC-MALS is a valuable analytical tool in the analytical development and QC testing of AAV. In addition, this work suggests that SEC-MALS can provide fundamental understanding of AAV in response to environmental stress. This may impact steps of the manufacturing process to minimize conditions that reduce performance.

Enhanced Recovery Pathway Improves Colorectal Surgery Outcomes in Private and Safety-Net Settings.

BACKGROUND: Enhanced recovery pathways (ERPs) can decrease length of stay (LOS) and improve colorectal surgery outcomes in private health care; however, their efficacy in the public realm, comprised largely of underserved and uninsured patients, remains uncertain. MATERIALS AND METHODS: An ERP without social interventions was implemented at a private hospital (PH) and a safety-net hospital (SNH) within a large academic medical center in 2014. Process and outcome metrics from 100 patients in the 18 mo before ERP implementation at each institution were retrospectively compared with a similar group after ERP implementation. Primary outcomes were LOS, 30-d readmission, and reoperation. RESULTS: Post-ERP groups were older than pre-ERP (P = 0.047, 0.034), with no difference in sex or body mass index. Rate of open versus minimally invasive was similar at the SNH (P = 0.067), whereas more post-ERP patients at PH underwent open surgery (P = 0.002). Ninety six percentage of PH patients were funded through private insurance or Medicare, verses 6% at the SNH. LOS at PH decreased from 8.1 to 5.9 d (P = 0.028) and at SNH from 7.0 to 5.1 d (P = 0.004). There was no change in 30-d all-cause readmission (PH P = 0.634; SNH P = 1) or reoperation (PH P = 0.610; SNH P = 0.066). CONCLUSIONS: ERP reduced LOS in both private and safety-net settings without addressing social determinants of health. Readmission and reoperation rates were unchanged. As health care moves toward a bundled payment model, ERP can help optimize outcomes and control costs in the public arena.

Decreasing Inappropriate Use of Rasburicase to Promote Cost-Effective Care.

BACKGROUND: Rasburicase is a recommended treatment of tumor lysis syndrome and patients at high-risk for developing tumor lysis syndrome. Unfortunately, it is expensive, and unnecessary use raises costs of care. METHODS: Plan, Do, Study, Act methodology was used to decrease the inappropriate use of rasburicase. In the Plan phase, a multidisciplinary quality improvement team reviewed the rasburicase ordering process and its prescription patterns at Parkland Health and Hospital System between October 2015 and September 2017 to determine appropriate interventions for improvement. In the Do phase, interventions were deployed to improve rasburicase prescriptions. In the Study phase, the team reviewed the rasburicase orders and appropriateness from February 2018 to October 2018. During the Act phase, the interventions were found to be successful, and the process changes were solidified. RESULTS: At baseline, 65 doses of rasburicase were administered during the 2-year baseline period, 21 of these (32.3%) were inappropriate. Review of the ordering process identified pitfalls: one-click ready-to-sign order, fixed default dose, no hard-stop alert requiring physicians to review and confirm appropriate indications, and lack of secondary pharmacy review. We aimed to reduce the percentage of inappropriate rasburicase orders from a baseline of 32.3% to 10% over 3 months. In February 2018, we implemented the interventions, which resulted in reduction in inappropriate rasburicase use, with only a single inappropriate order placed in 7 months postintervention. CONCLUSION: A multidisciplinary approach and classic quality improvement methodology enabled us to reduce inappropriate rasburicase use. Straightforward electronic medical record interventions and secondary pharmacy review are effective in addressing overuse.

The impact of dispositional cynicism on job-specific affect and work intentions.

Working from the Employee Work Passion Appraisal (EWPA) model, this article examines the relationship between employee dispositional cynicism, job-specific affect (i.e. positive and negative) and work intentions including intent to use discretionary effort, intent to perform, intent to endorse, intent to stay and intent to be an organisational citizen. An online survey generated participation from 747 current and potential clients of an international consulting company. To evaluate the fit of the data in accordance with the EWPA framework, structural equation modeling was conducted to test the overall fit of the proposed model and to examine the hypothesised relationships between constructs. Analyses confirmed correlations between dispositional cynicism and job-specific affect, supported notable relationships between positive job-specific affect and all work intentions, provided evidence for job-specific affect's mediation of cynicism and work intentions and uncovered a direct negative relationship between cynicism and intent to use organisational citizenship behaviour. Results suggest that state-specific workplace emotions are important for understanding the degree to which employee dispositional cynicism will ultimately influence most performance-related work intentions. However, independent of affect, employee cynicism may directly result in somewhat lower intentions to help others at work. Study limitations and practical implications for employee selection and training are considered.