The Promise and Illusion of Continuous, Cuffless Blood Pressure Monitoring.

PURPOSE OF REVIEW: Blood pressure (BP) fluctuations outside of clinic are increasingly recognized for their role in the development of cardiovascular disease, syncope, and premature death and as a promising target for tailored hypertension treatment. However, current cuff-based BP devices, including home and ambulatory devices, are unable to capture the breadth of BP variability across human activities, experiences, and contexts. RECENT FINDINGS: Cuffless, wearable BP devices offer the promise of beat-to-beat, continuous, noninvasive measurement of BP during both awake and sleep periods with minimal patient inconvenience. Importantly, cuffless BP devices can characterize BP variability, allowing for the identification of patient-specific triggers of BP surges in the home environment. Unfortunately, the pace of evidence, regulation, and validation testing has lagged behind the pace of innovation and direct consumer marketing. We provide an overview of the available technologies and devices for cuffless BP monitoring, considerations for the calibration and validation of these devices, and the promise and pitfalls of the cuffless BP paradigm.

TMEM16E regulates endothelial cell procoagulant activity and thrombosis.

Endothelial cells (ECs) normally form an anticoagulant surface under physiological conditions, but switch to support coagulation following pathogenic stimuli. This switch promotes thrombotic cardiovascular disease. To generate thrombin at physiologic rates, coagulation proteins assemble on a membrane containing anionic phospholipid, most notably phosphatidylserine (PS). PS can be rapidly externalized to the outer cell membrane leaflet by phospholipid "scramblases," such as TMEM16F. TMEM16F-dependent PS externalization is well characterized in platelets. In contrast, how ECs externalize phospholipids to support coagulation is not understood. We employed a focused genetic screen to evaluate the contribution of transmembrane phospholipid transport on EC procoagulant activity. We identified 2 TMEM16 family members, TMEM16F and its closest paralog, TMEM16E, which were both required to support coagulation on ECs via PS externalization. Applying an intravital laser-injury model of thrombosis, we observed, unexpectedly, that PS externalization was concentrated at the vessel wall, not on platelets. TMEM16E-null mice demonstrated reduced vessel-wall-dependent fibrin formation. The TMEM16 inhibitor benzbromarone prevented PS externalization and EC procoagulant activity and protected mice from thrombosis without increasing bleeding following tail transection. These findings indicate the activated endothelial surface is a source of procoagulant phospholipid contributing to thrombus formation. TMEM16 phospholipid scramblases may be a therapeutic target for thrombotic cardiovascular disease.

Leclercia Adecarboxylata Causing Necrotizing Fasciitis in an Immunocompetent Athlete Injecting Illicit Testosterone Supplements.

Leclercia adecarboxylata (L. adecarboxylata) is an uncommon and often misdiagnosed cause of multiple infection types including skin and soft tissue, cholecystitis, and septicemia. It commonly afflicts immunocompromised hosts or individuals who experience trauma in aquatic environments. We present a case where this bacteria causes necrotizing fasciitis as a consequence of injecting street bought testosterone supplements. This patient was treated successfully with excisional debridement of the wound as well as a one week course of Linezolid and Bactrim.

High-Efficiency, Selection-free Gene Repair in Airway Stem Cells from Cystic Fibrosis Patients Rescues CFTR Function in Differentiated Epithelia.

Cystic fibrosis (CF) is a monogenic disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Mortality in CF patients is mostly due to respiratory sequelae. Challenges with gene delivery have limited attempts to treat CF using in vivo gene therapy, and low correction levels have hindered ex vivo gene therapy efforts. We have used Cas9 and adeno-associated virus 6 to correct the ΔF508 mutation in readily accessible upper-airway basal stem cells (UABCs) obtained from CF patients. On average, we achieved 30%-50% allelic correction in UABCs and bronchial epithelial cells (HBECs) from 10 CF patients and observed 20%-50% CFTR function relative to non-CF controls in differentiated epithelia. Furthermore, we successfully embedded the corrected UABCs on an FDA-approved porcine small intestinal submucosal membrane (pSIS), and they retained differentiation capacity. This study supports further development of genetically corrected autologous airway stem cell transplant as a treatment for CF.

Global Transcriptional Response to CRISPR/Cas9-AAV6-Based Genome Editing in CD34+ Hematopoietic Stem and Progenitor Cells.

Genome-editing technologies are currently being translated to the clinic. However, cellular effects of the editing machinery have yet to be fully elucidated. Here, we performed global microarray-based gene expression measurements on human CD34+ hematopoietic stem and progenitor cells that underwent editing. We probed effects of the entire editing process as well as each component individually, including electroporation, Cas9 (mRNA or protein) with chemically modified sgRNA, and AAV6 transduction. We identified differentially expressed genes relative to control treatments, which displayed enrichment for particular biological processes. All editing machinery components elicited immune, stress, and apoptotic responses. Cas9 mRNA invoked the greatest amount of transcriptional change, eliciting a distinct viral response and global transcriptional downregulation, particularly of metabolic and cell cycle processes. Electroporation also induced significant transcriptional change, with notable downregulation of metabolic processes. Surprisingly, AAV6 evoked no detectable viral response. We also found Cas9/sgRNA ribonucleoprotein treatment to be well tolerated, in spite of eliciting a DNA damage signature. Overall, this data establishes a benchmark for cellular tolerance of CRISPR/Cas9-AAV6-based genome editing, ensuring that the clinical protocol is as safe and efficient as possible.

Increase in Dye:Dendrimer Ratio Decreases Cellular Uptake of Neutral Dendrimers in RAW Cells.

Neutral generation 3 poly(amidoamine) dendrimers were labeled with Oregon Green 488 (G3-OGn) to obtain materials with controlled fluorophore:dendrimer ratios (n = 1-2), a mixture containing mostly 3 dyes per dendrimer, a mixture containing primarily 4 or more dyes per dendrimer (n = 4+), and a stochastic mixture (n = 4avg). The UV absorbance of the dye conjugates increased linearly as n increased and the fluorescence emission decreased linearly as n increased. Cellular uptake was studied in RAW cells and HEK 293A cells as a function of the fluorophore:dendrimer ratio (n). The cellular uptake of G3-OG n (n = 3, 4+, 4avg) into RAW cells was significantly lower than G3-OG n (n = 1, 2). The uptake of G3-OG n (n = 3, 4+, 4avg) into HEK 293A cells was not significantly different from G3-OG1. Thus, the fluorophore:dendrimer ratio was observed to change the extent of uptake in the macrophage uptake mechanism but not in the HEK 293A cell. This difference in endocytosis indicates the presence of a pathway in the macrophage that is sensitive to hydrophobicity of the particle.

Quantitative Measurement of Cationic Polymer Vector and Polymer-pDNA Polyplex Intercalation into the Cell Plasma Membrane.

Cationic gene delivery agents (vectors) are important for delivering nucleotides, but are also responsible for cytotoxicity. Cationic polymers (L-PEI, jetPEI, and G5 PAMAM) at 1× to 100× the concentrations required for translational activity (protein expression) induced the same increase in plasma membrane current of HEK 293A cells (30-50 nA) as measured by whole cell patch-clamp. This indicates saturation of the cell membrane by the cationic polymers. The increased currents induced by the polymers are not reversible for over 15 min. Irreversibility on this time scale is consistent with a polymer-supported pore or carpet model and indicates that the cell is unable to clear the polymer from the membrane. For polyplexes, although the charge concentration was the same (at N/P ratio of 10:1), G5 PAMAM and jetPEI polyplexes induced a much larger current increase (40-50 nA) than L-PEI polyplexes (<20 nA). Both free cationic lipid and lipid polyplexes induced a lower increase in current than cationic polymers (<20 nA). To quantify the membrane bound material, partition constants were measured for both free vectors and polyplexes into the HEK 293A cell membrane using a dye influx assay. The partition constants of free vectors increased with charge density of the vectors. Polyplex partition constants did not show such a trend. The long lasting cell plasma permeability induced by exposure to the polymer vectors or the polyplexes provides a plausible mechanism for the toxicity and inflammatory response induced by exposure to these materials.