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This study aims to develop a 4-limb canine gait analysis system using wireless inertial measurement units (IMUs). 3D printed sensor holders were designed to ensure quick and consistent sensor mounting. Signal analysis algorithms were developed to automatically determine the timing of swing start and end in a stride. To evaluate the accuracy of the new system, a synchronized study was conducted in which stride parameters in four dogs were measured simultaneously using the 4-limb IMU system and a pressure-sensor based walkway gait system. The results showed that stride parameters measured in both systems were highly correlated. Bland-Altman analyses revealed a nominal mean measurement bias between the two systems in both forelimbs and hindlimbs. Overall, the disagreement between the two systems was less than 10% of the mean value in over 92% of the data points acquired from forelimbs. The same performance was observed in hindlimbs except for one parameter due to small mean values. We demonstrated that this 4-limb system could successfully visualize the overall gait types and identify rapid gait changes in dogs. This method provides an effective, low-cost tool for gait studies in veterinary applications or in translational studies using dog models of neuromuscular diseases., (© 2022. The Author(s).)

Amorphous solid dispersion (ASD) is a widely employed formulation technique for drugs with poor aqueous solubility. Polymers are integral components of ASDs, but mechanisms by which polymers lead to the generation and maintenance of supersaturated solutions, which enhance oral absorption in vivo, are poorly understood. Herein, a diverse group of newly synthesized cellulose derivatives was evaluated for their ability to inhibit crystallization of enzalutamide, a poorly soluble compound used to treat prostate cancer. ASDs were prepared from selected polymers, specifically a somewhat hydrophobic polymer that was extremely effective at inhibiting drug crystallization, and a less effective, but more hydrophilic, crystallization inhibitor, that might afford better release. Drug membrane transport rate was evaluated in vitro and compared to in vivo performance, following oral dosing in rats. Good correlation was noted between the in vitro diffusion cell studies and the in vivo data. The ASD formulated with the less effective crystallization inhibitor outperformed the ASD prepared with the highly effective crystallization inhibitor in terms of the amount and rate of drug absorbed in vivo. This study provides valuable insight into key factors impacting oral absorption from enabling ASD formulations, and how best to evaluate such formulations using in vitro approaches.

Multicellular tumor spheroids have been increasingly used by researchers to produce more physiologically relevant experimental environments. However, tracking of spheroid growth and treatment-induced volume reduction has not been readily adopted. Here, squamous carcinoma cells were seeded at different starting cell numbers with growth and reduction kinetics monitored using live cell imaging. Following the initial growth phase, spheroids were treated with auristatin as small molecule (MMAE) or as antibody-drug conjugate containing non-cleavable auristatin drug payload (033-F). Compared to cells in monolayers, 033-F had notably weaker potency against spheroids despite potency levels of MMAE being similar against monolayers and spheroids. Accumulation of released payload from 033-F was reduced in higher volume spheroids, likely contributing to the potency differences. Despite lowered potency towards spheroids with 033-F, spheroid volume was still readily reduced by 033-F in a dose-dependent fashion, with >85% volume reductions at the highest concentrations for all spheroid sizes. Additionally, the core of the larger spheroids showed more resiliency towards microtubule inhibition. Overall, this work highlights how various in-vivo 'features' such as tumor penetration, cell interactions, and increased resistance to therapeutics can be integrated into a spheroid model and tracked over time by automated imaging technology.

Dysfunction in the contractile properties of the diaphragm muscle contributes to the morbidity and mortality in many neuromuscular and respiratory diseases. Methods that can accurately quantify diaphragm function in mouse models are essential for preclinical studies. Diaphragm function is usually measured using the diaphragm strip. Two methods have been used to attach the diaphragm strip to the force transducer. The suture method is easy to adopt but it cannot maintain the physiological orientation of the muscle fibers. Hence, results may not accurately reflect diaphragm contractility. The clamp method can better maintain diaphragm muscle fiber orientation but is used less often because detailed information on clamp fabrication and application has never been published. Importantly, a side-by-side comparison of the two methods is lacking. To address these questions, we engineered diaphragm clamps using mechanically highly durable material. Here, we present a detailed and ready-to-use protocol on the design and manufacture of diaphragm clamps. Also, we present a step by step protocol on how to mount the diaphragm strip to the clamp and then to the muscle force measurement system. We compared the diaphragm force from the same mouse with both suture and clamp methods. We found the clamp method yielded a significantly higher muscle force. Finally, we validated the utility of the clamp method in the mdx model of Duchenne muscular dystrophy. In summary, the clamp method described in this paper yields reliable and consistent diaphragm force data. This method will be useful to any laboratory interested in performing mouse diaphragm function assay.

The Phosphatidylinositol glycan class A (PIG-A) gene mutation assay phenotypically measures erythrocyte mutations, assessed here for their correlation to neoplastic progression in the gastro-oesophageal reflux disease (GORD)-Barrett's metaplasia (BM)-oesophageal adenocarcinoma (OAC) model. Endoscopy patients underwent venipuncture and erythrocytes fluorescently stained for glycosyl phosphatidylinositol (GPI)-anchored proteins; CD55 and CD59. Using flow cytometry, GPI-anchor negative erythrocytes (mutants) were scored and compared amongst groups. The study enlisted 200 patients and 137 healthy volunteers. OAC patients had a three-fold increase in erythrocyte mutant frequency (EMF) compared to GORD patients (p < 0.001) and healthy volunteers (p < 0.001). In OAC patients, higher EMF was associated with worsening tumour staging (p = 0.014), nodal involvement (p = 0.019) and metastatic disease (p = 0.008). Chemotherapy patients demonstrated EMF's over 19-times higher than GORD patients. Patients were further classified into groups containing those with non-neoplastic disease and those with high-grade dysplasia/cancer with 72.1% of cases correctly classified by high EMF. Within the non-neoplastic group, aspirin users had lower EMF (p = 0.001) and there was a positive correlation between body mass index (p = 0.03) and age (p < 0.001) and EMF. Smokers had EMF's over double that of non-smokers (p = 0.011). Results suggest this test could help detect OAC and may be a useful predictor of disease progression.