Improved adeno-associated virus empty and full capsid separation using weak partitioning multi-column AEX chromatography.

Recombinant adeno-associated virus (rAAV) empty and full capsid separation has been a topic of interest in the rAAV gene therapy community for many years and the anion exchange chromatography (AEX) step has undergone various process optimizations to improve rAAV empty capsid separation, including AEX stationary phase, mobile phase, and process parameters. Here, we present a new AEX method that employs both weak partitioning chromatography (WPC) and multi-column chromatography (MCC) to achieve improved full rAAV percentage in the AEX pool. The WPC technology allows empty rAAV to be displaced by full rAAV during loading, while the MCC technology enables parallel column processing which further increases AEX step productivity. Our results show that, compared to baseline AEX batch chromatography, the AEX-WPC-MCC method demonstrated improvements in both AEX pool full rAAV percentage (∼ 20% increase) and rAAV genome recovery (∼ 20% increase). As a result, the productivity (full capsid generated per liter of AEX column per hour of processing time) of the AEX step increased by ∼34-fold from the baseline AEX batch run to the AEX-WPC-MCC run. It is foreseeable that this AEX-WPC-MCC method could find applications in large-scale rAAV manufacturing processes to improve AEX yield and reduce the cost of goods of rAAV manufacturing.

Influence of osseous structure characteristics of unilateral alveolar cleft on outcomes of alveolar bone grafting: a retrospective study.

BACKGROUND: To enhance the success rate of alveolar bone grafting, it is crucial to identify the factors that may influence the postoperative bone formation. This study aimed to investigate the impact of various osseous structure characteristics of alveolar clefts on the survival ratio of autogenous cancellous bone particle grafts. METHODS: A retrospective study was conducted on 60 patients who underwent surgery performed by the same surgeon between 2016 and 2022. Two researchers measured and recorded the bone defect volume (DV), postoperative bone formation volume at 1 year, contact area between the graft and the bone surface within the cleft (S), cleft width (CW), osseous occlusion relationships, and presence of a cleft palate and initial bone bridge within the cleft for each patient. Pearson correlation analysis, Spearman's correlation analysis, and multiple linear regression analysis were performed. RESULTS: The analysis results revealed statistical correlations between DV, CW, ratio of S to DV, cleft palate, initial bone bridge presence, and occlusion relationships with the survival rate. Multiple linear regression analysis showed that initial bone bridge and occlusion relationships affected the graft survival rate. CONCLUSIONS: Based on the presence of initial bone bridges and occlusions, we can make a rough estimate of the postoperative bone formation outcome in patients. However, the underlying mechanisms by which these two factors influence the bone formation require further investigation. In addition, preoperative orthodontic treatment to improve occlusal relationships may improve the postoperative bone formation outcomes in alveolar bone grafting.

Correction of Asymmetry of Palpebral Folds by Adopting Interrupted and Continuous Buried Suture Techniques Respectively on Different Eyelid.

People with an obvious palpebral fold on 1 eye and a narrow or no palpebral fold on the other eye are usually more satisfied with the shape of the obvious palpebral fold and want to acquire symmetry by minimally invasive surgical methods that preserve the shape of the original folds they are more satisfied with. This study introduced a minimally invasive approach using 2 different buried suture techniques on different eyelids to acquire symmetry. The continuous suture method aimed to build palpebral folds, whereas the 3-point interrupted suture method aimed to enhance naturally formed palpebral folds. The 3-point interrupted buried suture method was used on the eye with an obvious palpebral fold, and the continuous buried suture method was performed on the eye with a narrow or no palpebral fold. Twenty patients underwent this procedure to correct the asymmetry between June 2010 and July 2022. The mean follow-up period was 18.12 months. The average swelling period was 7 days on the side using the interrupted buried suture technique and 10 days on the side using the continuous suture method. It took ~3 weeks to recover a relatively natural appearance. According to postoperative follow-up data, all patients were satisfied with the results. Our surgical approach of adopting 2 different buried suture techniques on different eyelids allowed the building of the palpebral fold on one eyelid while enhancing the original fold on the other eyelid, leading to satisfactory results of palpebral fold symmetry.

Optical Nanosensors for in vivo Physiological Chloride Detection for Monitoring Cystic Fibrosis Treatment.

Personalized approaches for continuous monitoring of chloride levels are potentially valuable for evaluating the efficacy of new treatments of genetic disorders such as cystic fibrosis. In this report, we validated optode-based nanosensors for real-time chloride monitoring in the interstitial fluid of living animals. These nanosensors take advantage of a ratiometric sensing scheme which demonstrates reversible and selective chloride detection in the physiological range. We further investigate how skin pigmentation affects the sensor performance during in vivo fluorescence imaging. We successfully monitored endogenous chloride changes using nanosensors during pharmacological treatment in a cystic fibrosis mouse model. We believe this platform is a valuable tool for chloride detection which could assess the efficacy of new treatments for cystic fibrosis.

Real-time particle-by-particle detection of erythrocyte-camouflaged microsensor with extended circulation time in the bloodstream.

Personalized medicine offers great potential benefits for disease management but requires continuous monitoring of drugs and drug targets. For instance, the therapeutic window for lithium therapy of bipolar disorder is very narrow, and more frequent monitoring of sodium levels could avoid toxicity. In this work, we developed and validated a platform for long-term, continuous monitoring of systemic analyte concentrations in vivo. First, we developed sodium microsensors that circulate directly in the bloodstream. We used "red blood cell mimicry" to achieve long sensor circulation times of up to 2 wk, while being stable, reversible, and sensitive to sodium over physiologically relevant concentration ranges. Second, we developed an external optical reader to detect and quantify the fluorescence activity of the sensors directly in circulation without having to draw blood samples and correlate the measurement with a phantom calibration curve to measure in vivo sodium. The reader design is inherently scalable to larger limbs, species, and potentially even humans. In combination, this platform represents a paradigm for in vivo drug monitoring that we anticipate will have many applications in the future.

Imaging Sodium Flux during Action Potentials in Neurons with Fluorescent Nanosensors and Transparent Microelectrodes.

Sodium flux plays a pivotal role in neurobiological processes including initiation of action potentials and regulation of neuronal cell excitability. However, unlike the wide range of fluorescent calcium indicators used extensively for cellular studies, the choice of sodium probes remains limited. We have previously demonstrated optode-based nanosensors (OBNs) for detecting sodium ions with advantageous modular properties such as tunable physiological sensing range, full reversibility, and superb selectivity against key physiological interfering ion potassium. (1) Motivated by bridging the gap between the great interest in sodium imaging of neuronal cell activity as an alternative to patch clamp and limited choices of optical sodium indicators, in this Letter we report the application of nanosensors capable of detecting intracellular sodium flux in isolated rat dorsal root ganglion neurons during electrical stimulation using transparent microelectrodes. Taking advantage of the ratiometric detection scheme offered by this fluorescent modular sensing platform, we performed dual color imaging of the sensor to monitor the intracellular sodium currents underlying trains of action potentials in real time. The combination of nanosensors and microelectrodes for monitoring neuronal sodium dynamics is a novel tool for investigating the regulatory role of sodium ions involved during neural activities.

Comparative Study of Poly (ε-Caprolactone) and Poly(Lactic-co-Glycolic Acid) -Based Nanofiber Scaffolds for pH-Sensing.

PURPOSE: This study aims to develop biodegradable and biocompatible polymer-based nanofibers that continuously monitor pH within microenvironments of cultured cells in real-time. In the future, these fibers will provide a scaffold for tissue growth while simultaneously monitoring the extracellular environment. METHODS: Sensors to monitor pH were created by directly electrospinning the sensor components within a polymeric matrix. Specifically, the entire fiber structure is composed of the optical equivalent of an electrode, a pH-sensitive fluorophore, an ionic additive, a plasticizer, and a polymer to impart mechanical stability. The resulting poly(ε-caprolactone) (PCL) and poly(lactic-co-glycolic acid) (PLGA) based sensors were characterized by morphology, dynamic range, reversibility and stability. Since PCL-based nanofibers delivered the most desirable analytical response, this matrix was used for cellular studies. RESULTS: Electrospun nanofiber scaffolds (NFSs) were created directly out of optode material. The resulting NFS sensors respond to pH changes with a dynamic range centered at 7.8 ± 0.1 and 9.6 ± 0.2, for PCL and PLGA respectively. NFSs exhibited multiple cycles of reversibility with a lifetime of at least 15 days with preservation of response characteristics. By comparing the two NFSs, we found PCL-NFSs are more suitable for pH sensing due to their dynamic range and superior reversibility. CONCLUSION: The proposed sensing platform successfully exhibits a response to pH and compatibility with cultured cells. NSFs will be a useful tool for creating 3D cellular scaffolds that can monitor the cellular environment with applications in fields such as drug discovery and tissue engineering.