Collection of serum albumin aggregate nanoparticles from human plasma by dielectrophoresis.

Dielectrophoresis (DEP) is a fast and reliable nanoparticle recovery method that utilizes nonuniform electric fields to manipulate particles based on their material composition and size, enabling recovery of biologically-derived nanoparticles from plasma for diagnostic applications. When applying DEP to undiluted human plasma, collection of endogenous albumin proteins was observed at electric field gradients much lower than predicted by theory to collect molecular proteins. To understand this collection, nanoparticle tracking analysis of bovine serum albumin (BSA) dissolved in 0.5× phosphate-buffered saline was performed and showed that albumin spontaneously formed aggregate nanoparticles with a mean diameter of 237 nm. These aggregates experienced a dielectrophoretic force as a function of aggregate radius rather than the diameter of individual protein molecules which contributed to their collection. In high conductance buffer (6.8 mS/cm), DEP was able to move these aggregates into regions of high electric field gradient, and in lower conductance buffer (0.68 mS/cm), these aggregates could be moved into high or low gradient regions depending on the applied frequency. Disruption of BSA aggregates using a nonionic detergent significantly decreased the particle diameter, resulting in decreased dielectrophoretic collection of albumin which increased the collection consistency of particles of interest. These results provide techniques to manipulate albumin aggregates via DEP, which impacts collection of diagnostic biomarkers.

Enhancement of dielectrophoresis-based particle collection from high conducting fluids due to partial electrode insulation.

Dielectrophoresis (DEP) is a successful method to recover nanoparticles from different types of fluid. The DEP force acting on these particles is created by an electrode microarray that produces a nonuniform electric field. To apply DEP to a highly conducting biological fluid, a protective hydrogel coating over the metal electrodes is required to create a barrier between the electrode and the fluid. This protects the electrodes, reduces the electrolysis of water, and allows the electric field to penetrate into the fluid sample. We observed that the protective hydrogel layer can separate from the electrode and form a closed domed structure and that collection of 100 nm polystyrene beads increased when this occurred. To better understand this collection increase, we used COMSOL Multiphysics software to model the electric field in the presence of the dome filled with different materials ranging from low-conducting gas to high conducting phosphate-buffered saline fluids. The results suggest that as the electrical conductivity of the material inside the dome is reduced, the whole dome acts as an insulator which increases electric field intensity at the electrode edge. This increased intensity widens the high-intensity electric field factor zone resulting in increased collection. This informs how dome formation results in increased particle collection and provides insight into how the electric field can be intensified to the increase collection of particles. These results have important applications for increasing the recovery of biologically-derived nanoparticles from undiluted physiological fluids that have high conductance, including the collection of cancer-derived extracellular vesicles from plasma for liquid biopsy applications.

A Description of Apixaban Dosing Patterns for Treatment or Prevention of Thrombotic Events in Hospitalized Patients on Dialysis.

Introduction: Apixaban is currently the only oral direct factor Xa inhibitor approved for treatment and prevention of venous thromboembolism (VTE) in patients on hemodialysis. Exclusion of dialysis patients from major clinical trials results in prescriber uncertainty regarding the optimal dose of apixaban for VTE treatment in this population. This study sought to characterize the variance in apixaban prescribing patterns for thrombotic indications other than atrial fibrillation. Methods: This retrospective, multi-center, descriptive study analyzed apixaban dosing patterns for hospitalized chronic dialysis patients with history of thrombosis. The primary outcome was incidence of deviation from manufacturer recommendations for dosing, assessed for either a new start or receipt prior to hospitalization. Secondary outcomes included observation of recurrent thrombotic and bleeding event rates during subsequent hospitalizations. Patients were analyzed into subgroups according to type of thrombotic indication for treatment. Data are reported with descriptive statistics. Results: A total of 101 patients were included. Deviations in recommended dosing were observed in 53 of 80 (66.2%) patients receiving apixaban for treatment of acute or chronic thrombosis. Of 44 patients started on apixaban during hospitalization for the indication of acute VTE, a dose deviation was observed in 79.5% of patients. Rates of rehospitalization for recurrent thrombotic events and bleeding were 11.8% and 9.9%, respectively. Conclusion: Variation in apixaban prescribing practices for the treatment of VTE in dialysis patients is common, suggesting an urgent need for prospective studies and updated dosing guidance to optimize safety with apixaban use in this population.

Multiplex Single-Cell Bioprinting for Engineering of Heterogeneous Tissue Constructs with Subcellular Spatial Resolution.

Tissue development, function, and disease are largely driven by the spatial organization of individual cells and their cell-cell interactions. Precision engineered tissues with single-cell spatial resolution, therefore, have tremendous potential for next generation disease models, drug discovery, and regenerative therapeutics. Despite significant advancements in biofabrication approaches to improve feature resolution, strategies to fabricate tissues with the exact same organization of individual cells in their native cellular microenvironment have remained virtually non-existent to date. Here we report a method to spatially pattern single cells with up to eight cell phenotypes and subcellular spatial precision. As proof-of-concept we first demonstrate the ability to systematically assess the influence of cellular microenvironments on cell behavior by controllably altering the spatial arrangement of cell types in bioprinted precision cell-cell interaction arrays. We then demonstrate, for the first time, the ability to produce high-fidelity replicas of a patient's annotated cancer biopsy with subcellular resolution. The ability to replicate native cellular microenvironments marks a significant advancement for precision biofabricated in-vitro models, where heterogenous tissues can be engineered with single-cell spatial precision to advance our understanding of complex biological systems in a controlled and systematic manner.

A quantitative systems approach to identify paracrine mechanisms that locally suppress immune response to Interleukin-12 in the B16 melanoma model.

Interleukin-12 (IL12) enhances anti-tumor immunity when delivered to the tumor microenvironment. However, local immunoregulatory elements dampen the efficacy of IL12. The identity of these local mechanisms used by tumors to suppress immunosurveillance represents a key knowledge gap for improving tumor immunotherapy. From a systems perspective, local suppression of anti-tumor immunity is a closed-loop system - where system response is determined by an unknown combination of external inputs and local cellular cross-talk. Here, we recreated this closed-loop system in vitro and combined quantitative high content assays, in silico model-based inference, and a proteomic workflow to identify the biochemical cues responsible for immunosuppression. Following an induction period, the B16 melanoma cell model, a transplantable model for spontaneous malignant melanoma, inhibited the response of a T helper cell model to IL12. This paracrine effect was not explained by induction of apoptosis or creation of a cytokine sink, despite both mechanisms present within the co-culture assay. Tumor-derived Wnt-inducible signaling protein-1 (WISP-1) was identified to exert paracrine action on immune cells by inhibiting their response to IL12. Moreover, WISP-1 was expressed in vivo following intradermal challenge with B16F10 cells and was inferred to be expressed at the tumor periphery. Collectively, the data suggest that (1) biochemical cues associated with epithelial-to-mesenchymal transition can shape anti-tumor immunity through paracrine action and (2) remnants of the immunoselective pressure associated with evolution in cancer include both sculpting of tumor antigens and expression of proteins that proactively shape anti-tumor immunity.