A novel dual-plasmid platform provides scalable transfection yielding improved productivity and packaging across multiple AAV serotypes and genomes.

Transient transfection of mammalian cells using plasmid DNA is a standard method to produce adeno-associated virus (AAV) vectors allowing for flexible and scalable manufacture. Typically, three plasmids are used to encode the necessary components to facilitate vector production; however, a dual-plasmid system, termed pDG, was introduced over 2 decades ago demonstrating two components could be combined resulting in comparable productivity to triple transfection. We have developed a novel dual-plasmid system, pOXB, with an alternative arrangement of sequences that results in significantly increased AAV vector productivity and percentage of full capsids packaged in comparison to the pDG dual design and triple transfection. Here, we demonstrate the reproducibility of these findings across seven recombinant AAV genomes and multiple capsid serotypes as well as the scalability of the pOXB dual-plasmid transfection at 50-L bioreactor scale. Purified drug substance showed a consistent product quality profile in line with triple-transfected vectors, except for a substantial improvement in intact genomes packaged using the pOXB dual- transfection system. Furthermore, pOXB dual- and triple-transfection-based vectors performed consistently in vivo. The pOXB dual plasmid represents an innovation in AAV manufacturing resulting in significant process gains while maintaining the flexibility of a transient transfection platform.

Influence of embedding parameters and noise in center of pressure recurrence quantification analysis.

Recurrence quantification analysis (RQA) can extract the dynamics of postural control from center of pressure (CoP) data by quantifying the system's repeatability, complexity, and local dynamic stability through several variables. Computation of these variables requires the selection of suitable embedding parameters for state space reconstruction (i.e. time delay and embedding dimension); however, it is unclear how the parameters influence RQA variables when examining noisy CoP data. This study evaluated the sensitivity of RQA variables to embedding parameter values and noise level, and assessed methods of selecting embedding parameters for CoP data. Five healthy male subjects maintained quiet stance for 30s while the anterior-posterior CoP was measured. The effect of noise was evaluated by adding uniform white noise of increasing amplitude to the raw CoP signal. The magnitude of all RQA variables decreased with increasing noise amplitude for all subjects. A sensitivity analysis was performed by systematically altering the embedding parameters for the raw data with and without a selected level of added noise. The key result was that, for all subjects, the RQA variables were sensitive to the embedding parameter values and the level of noise in the CoP data. Finally, the performance of false nearest neighbors and average displacement algorithms for choosing embedding parameters was evaluated. Both methods gave clear and consistent results for all subjects with either raw or noisy data. The results suggest that careful selection of embedding parameters is essential when using RQA to examine postural control based on noisy CoP data.

Intranuclear rodlets in human pancreatic islet cells.

OBJECTIVES: Intranuclear rodlets (INRs) are rod-shaped intranuclear inclusions that we have described in neurons of the human brain. We recently identified these structures in pancreatic islet cells. The objectives of this study are to describe the light microscopic features and cellular pattern of distribution of INRs in human pancreatic islet cells. METHODS: Double immunofluorescence staining was performed on 5 human pancreatic tissue samples for the detection of class III beta tubulin (C3T) to detect INRs and for promyelocytic leukemia (PML) protein to examine the relationship between PML and INRs. RESULTS: Intranuclear rodlets were detected in 22.99% of pancreatic B cells compared with only 3.11%, 1.80%, and 1.60% of A, D, and PP cells, respectively. Twenty-four percent of C3T-immunoreactive INRs showed partial or complete immunoreactivity for PML. Promyelocytic leukemia staining within the nuclei of B cells was confined to INRs and was not present in the typical PML bodies present in other cell types. Spatially, PML and C3T staining of islet cell INRs appeared to be mutually exclusive within individual INRs. CONCLUSIONS: Intranuclear rodlets are present within the nuclei of pancreatic islet cells, where they reside predominantly but not exclusively in B cells. Immunoreactivity of B-cell INRs for PML suggests that the functional significance of INRs may be related to that of PML and/or PML bodies. Conversely, the exclusive localization of PML staining to INRs in B cells indicates that PML's function in B cells is selectively associated with INRs. The mutually exclusive pattern of PML and C3T staining suggests dynamic interactions between these 2 proteins in B-cell INRs. In light of evidence for the involvement of INRs and of PML bodies in disease, it will be of interest to investigate these structures in animal models of diabetes and in human diabetes.

Evaluation of time-to-contact measures for assessing postural stability.

Postural stability has traditionally been examined through spatial measures of the center of mass (CoM) or center of pressure (CoP), where larger amounts of CoM or CoP movements are considered signs of postural instability. However, for stabilization, the postural control system may utilize additional information about the CoM or CoP such as velocity, acceleration, and the temporal margin to a stability boundary. Postural time-to-contact (TtC) is a variable that can take into account this additional information about the CoM or CoP. Postural TtC is the time it would take the CoM or CoP, given its instantaneous trajectory, to contact a stability boundary. This is essentially the time the system has to reverse any perturbation before stance is threatened. Although this measure shows promise in assessing postural stability, the TtC values derived between studies are highly ambiguous due to major differences in how they are calculated. In this study, various methodologies used to assess postural TtC were compared during quiet stance and induced-sway conditions. The effects of the different methodologies on TtC values will be assessed, and issues regarding the interpretation of TtC data will also be discussed.