Myosin XXI is the only myosin expressed in Leishmania parasites. Although it is assumed that it performs a variety of motile functions, the motor's oligomerization states, cargo-binding, and motility are unknown. Here we show that binding of a single calmodulin causes the motor to adopt a monomeric state and to move actin filaments. In the absence of calmodulin, nonmotile dimers that cross-linked actin filaments were formed. Unexpectedly, structural analysis revealed that the dimerization domains include the calmodulin-binding neck region, essential for the generation of force and movement in myosins. Furthermore, monomeric myosin XXI bound to mixed liposomes, whereas the dimers did not. Lipid-binding sections overlapped with the dimerization domains, but also included a phox-homology domain in the converter region. We propose a mechanism of myosin regulation where dimerization, motility, and lipid binding are regulated by calmodulin. Although myosin-XXI dimers might act as nonmotile actin cross-linkers, the calmodulin-binding monomers might transport lipid cargo in the parasite.unconventional myosin | motor properties
Backgrounded membrane imaging (BMI) is a novel automated, 96-well plate-based microscopic approach for subvisible particle analysis. We scientifically evaluated BMI with respect to sizing and counting accuracy, working range, impact of refractive index, and interferences by silicone oil droplets, and compared BMI to state-of-the-art dynamic image analysis (DIA). Image quality was found to be comparable to current DIA methodologies. However, with the first versions of BMI image analysis software, an undersizing of polystyrene beads was observed. BMI linear concentration range was found to reach an upper limit (7.1 Â 10 5 particles/mL) similar to DIA. In the absence of silicone oil droplets, BMI and DIA showed good agreement in total particle concentrations (particle diameter !2 mm) but differences in size distributions for particle sizes !4 mm. Analyses of prefilled syringe products and silicone oil emulsions demonstrated the removal of silicone oil in BMI sample processing. In contrast to DIA, particle counting by BMI remained unaffected by changes in refractive index. Overall, we demonstrated BMI to be a promising orthogonal method for subvisible particle characterization. Aspects like low required sample volume, high throughput, and ease of handling can make BMI a valuable alternative or complement to DIA in particular for formulation screening.
Particles in biopharmaceutical formulations remain a hot topic in drug product development. With new product classes emerging it is crucial to discriminate particulate active pharmaceutical ingredients from particulate impurities. Technical improvements, new analytical developments and emerging tools (e.g., machine learning tools) increase the amount of information generated for particles. For a proper interpretation and judgment of the generated data a thorough understanding of the measurement principle, suitable application fields and potential limitations and pitfalls is required. Our review provides a comprehensive overview of novel particle analysis techniques emerging in the last decade for particulate impurities in therapeutic protein formulations (protein-related, excipient-related and primary packaging material-related), as well as particulate biopharmaceutical formulations (virus particles, virus-like particles, lipid nanoparticles and cellbased medicinal products). In addition, we review the literature on applications, describe specific analytical approaches and illustrate advantages and drawbacks of currently available techniques for particulate biopharmaceutical formulations.
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