Obstructive Sleep Apnea-induced Endothelial Dysfunction Is Mediated by miR-210.

Rationale: Obstructive sleep apnea (OSA)-induced endothelial cell (EC) dysfunction contributes to OSA-related cardiovascular sequelae. The mechanistic basis of endothelial impairment by OSA is unclear. Objectives: The goals of this study were to identify the mechanism of OSA-induced EC dysfunction and explore the potential therapies for OSA-accelerated cardiovascular disease. Methods: The experimental methods include data mining, bioinformatics, EC functional analyses, OSA mouse models, and assessment of OSA human subjects. Measurements and Main Results: Using mined microRNA sequencing data, we found that microRNA 210 (miR-210) conferred the greatest induction by intermittent hypoxia in ECs. Consistently, the serum concentration of miR-210 was higher in individuals with OSA from two independent cohorts. Importantly, miR-210 concentration was positively correlated with the apnea-hypopnea index. RNA sequencing data collected from ECs transfected with miR-210 or treated with OSA serum showed a set of genes commonly altered by miR-210 and OSA serum, which are largely involved in mitochondrion-related pathways. ECs transfected with miR-210 or treated with OSA serum showed reduced [Formula: see text]o2 rate, mitochondrial membrane potential, and DNA abundance. Mechanistically, intermittent hypoxia-induced SREBP2 (sterol regulatory element-binding protein 2) bound to the promoter region of miR-210, which in turn inhibited the iron-sulfur cluster assembly enzyme and led to mitochondrial dysfunction. Moreover, the SREBP2 inhibitor betulin alleviated intermittent hypoxia-increased systolic blood pressure in the OSA mouse model. Conclusions: These results identify an axis involving SREBP2, miR-210, and mitochondrial dysfunction, representing a new mechanistic link between OSA and EC dysfunction that may have important implications for treating and preventing OSA-related cardiovascular sequelae.

Optimization of Microparticle Reagents to Collect and Detect Antibody.

Bead reagents are used in a large number of assays in bioscience and biotechnology to collect and purify antibodies by immobilization. Bead-based immunoassays offer high-throughput analysis of multiple antibodies in a single sample. Although a variety of antibody-binding moieties on the collection beads have been studied, the physical and material properties of collection beads have not been optimized to isolate specific antibodies over a broad range of concentrations from complex environments containing cells. We present a study of how to optimally use microparticles coated with protein G to collect low concentrations of IgG antibodies from complex solutions. We study the impact of bead material, bead size, incubation time, and protein G density to more efficiently collect antibodies and detect specific antibodies via fluorescent antigen labeling. The minimum detectable limit and the minimum incubation time for antibody collection are used as metrics to evaluate the collection parameters. We found that larger silica beads can capture more antibodies from a low concentration of sample, with a minimum incubation time of 60 min to equilibrium binding, resulting in a minimum detectable concentration of antibodies of 26 nM. We show that simple biophysical optimization of antibody collection reagents can be used to improve the collection of low concentrations of antibodies in complex environments. We demonstrate that the technology may be useful for monitoring antibody secretions from hybridoma cultures.

Heterofunctional Particles as Single Cell Sensors to Capture Secreted Immunoglobulins and Isolate Antigen-Specific Antibody Secreting Cells.

Isolating cells based on their secreted proteins remain a challenge. The authors demonstrate a capacity for high throughput single-cell protein secretion analysis and isolation based on heterofunctional particles combined with fluorescence activated cell sorting (FACS). The workflow shows that antibody secreting cells (ASCs) specific for the H1 protein from influenza virus can be isolated from B cells. The workflow consists of incubating anti-CD27 particles with the ASCs, capturing locally secreted immunoglobulins with Protein G on the particles, and identifying immunoglobulins specific to H1 via fluorescent labeled antigens followed by FACS to enrich antigen-specific ASCs. Two particles designs, Janus and mixed, are tested with hybridoma cells. Mixed particles are found to improve antibody collection, while Janus particles are found to bind target cells more effectively. Targeted hybridoma cells in coculture with non-specific hybridoma cells are identified with a sensitivity of 96% and specificity of 98%. Heterofunctional particles are used to capture ASCs that secrete antibodies specific for influenza virus from B cells from healthy adults isolated from blood after vaccination. Positive H1-tetramer sorted ASCs are validated using single ASC cultures and identify 23/56 cells specific for H1 demonstrating 164-fold enrichment from total B cells and 14.6-fold enrichment from total ASCs.