Purity and DNA content of AAV capsids assessed by analytical ultracentrifugation and orthogonal biophysical techniques.

Development and manufacturing adeno-associated virus (AAV)-based vectors for gene therapy requires suitable analytical methods to assess the quality of the formulations during development, as well as the quality of different batches and the consistency of the processes. Here, we compare biophysical methods to characterize purity and DNA content of viral capsids from five different serotypes (AAV2, AAV5, AAV6, AAV8, and AAV9). For this purpose, we apply multiwavelength sedimentation velocity analytical ultracentrifugation (SV-AUC) to obtain the species' contents and to derive the wavelength-specific correction factors for the respective insert-size. In an orthogonal manner we perform anion exchange chromatography (AEX) and UV-spectroscopy and the three methods yield comparable results on empty/filled capsid contents with these correction factors. Whereas AEX and UV-spectroscopy can quantify empty and filled AAVs, only SV-AUC could identify the low amounts of partially filled capsids present in the samples used in this study. Finally, we employ negative-staining transmission electron microscopy and mass photometry to support the empty/filled ratios with methods that classify individual capsids. The obtained ratios are consistent throughout the orthogonal approaches as long as no other impurities and aggregates are present. Our results show that the combination of selected orthogonal methods can deliver consistent empty/filled contents on non-standard genome sizes, as well as information on other relevant critical quality attributes, such as AAV capsid concentration, genome concentration, insert size length and sample purity to characterize and compare AAV preparations.

Global intercompany assessment of ICIEF platform comparability for the characterization of therapeutic proteins.

An international team spanning 19 sites across 18 biopharmaceutical and in vitro diagnostics companies in the United States, Europe, and China, along with one regulatory agency, was formed to compare the precision and robustness of imaged CIEF (ICIEF) for the charge heterogeneity analysis of the National Institute of Standards and Technology (NIST) mAb and a rhPD-L1-Fc fusion protein on the iCE3 and the Maurice instruments. This information has been requested to help companies better understand how these instruments compare and how to transition ICIEF methods from iCE3 to the Maurice instrument. The different laboratories performed ICIEF on the NIST mAb and rhPD-L1-Fc with both the iCE3 and Maurice using analytical methods specifically developed for each of the molecules. After processing the electropherograms, statistical evaluation of the data was performed to determine consistencies within and between laboratory and outlying information. The apparent isoelectric point (pI) data generated, based on two-point calibration, for the main isoform of the NIST mAb showed high precision between laboratories, with RSD values of less than 0.3% on both instruments. The SDs for the NIST mAb and the rhPD-L1-Fc charged variants percent peak area values for both instruments are less than 1.02% across different laboratories. These results validate the appropriate use of both the iCE3 and Maurice for ICIEF in the biopharmaceutical industry in support of process development and regulatory submissions of biotherapeutic molecules. Further, the data comparability between the iCE3 and Maurice illustrates that the Maurice platform is a next-generation replacement for the iCE3 that provides comparable data.

Viscoelasticity of cross-linked actin networks: experimental tests, mechanical modeling and finite-element analysis.

Filamentous actin is one of the main constituents of the eukaryotic cytoskeleton. The actin cortex, a densely cross-linked network, resides underneath the lipid bilayer. In the present work we propose a continuum mechanical formulation for describing the viscoelastic properties of in vitro actin networks, which serve as model systems for the cortex, by including the microstructure, i.e. the behavior of a single filament and its spatial arrangement. The modeling of the viscoelastic response in terms of physically interpretable parameters is conducted using a multiscale approach consisting of two steps: modeling of the single filament response of F-actin by a worm-like chain model including the extensibility of the filament, and assembling the three-dimensional biopolymer network by using the microsphere model which accounts for filaments equally distributed in space. The viscoelastic effects of the network are taken into account using a generalized Maxwell model. The Cauchy stress and elasticity tensors are obtained within a continuum mechanics framework and implemented into a finite-element program. The model is validated on the network level using large strain experiments on reconstituted actin gels. Comparisons of the proposed model with rheological experiments recover reasonable values for the material parameters. Finite-element simulations of the indentation of a sphere on a network slab and the aspiration of a droplet in a micropipette allow for further insights of the viscoelastic behavior of actin networks.

Engaging community-based veterans' organizations in health promotion programs.

Community organizations, such as churches, clubs, and senior centers, can be important locations for health programs. However, little is known about the organizational factors that influence participation and engagement in health programs. To learn more, we evaluated a community-based program designed to help US military veterans better manage their high blood pressure. The program involved training a pair of veterans to deliver health-related presentations at their local units. We found that factors such as larger meeting attendance size, rural location, age diversity, and member enthusiasm were positively associated with both a willingness to participate and a high level of engagement in program activities.

Fragmentation is crucial for the steady-state dynamics of actin filaments.

Despite the recognition that actin filaments are important for numerous cellular processes, and decades of investigation, the dynamics of in vitro actin filaments are still not completely understood. Here, we follow the time evolution of the length distribution of labeled actin reporter filaments in an unlabeled F-actin solution via fluorescence microscopy. Whereas treadmilling and diffusive length fluctuations cannot account for the observed dynamics, our results suggest that at low salt conditions, spontaneous fragmentation is crucial.

Preliminary description of the feasibility of using peer leaders to encourage hypertension self-management.

BACKGROUND: Despite consensus that effective treatment of hypertension reduces morbidity and mortality, control rates remain relatively low. This report describes key features of a peer support program designed to motivate individuals to improve self-management of hypertension. METHODS: We recruited Veterans of Foreign Wars posts in southeastern Wisconsin and trained members of these posts to be peer health leaders over a period of 18 months. The curriculum covered information important to blood pressure control, as well as peer educator skills. During this time, the peer leaders presented educational materials and encouraged self-monitoring of blood pressure at post meetings. Surveys and focus groups were conducted to evaluate the adoption of the program at the posts. RESULTS: After a series of informational mailings and visits to veteran posts, 15 posts and 27 peer leaders volunteered to participate. Fourteen posts (93%) continued active participation throughout the study period, as did 24 peer leaders. Peer leaders reported that they gained health knowledge, skills, and confidence to perform as informational resources at their posts, resulting in greater levels of health support among post members. CONCLUSION: The partnership of health care professional, medical school, and veteran service organization successfully organized and maintained a community-based, peer-led program to promote healthy behaviors among Wisconsin's armed services veterans. Community physicians should be familiar with programs of this type as chronic disease self-management grows in appeal in our communities and increasing numbers of veterans return from armed service duty.