Molecular interactions of phospholipid monolayers with a model phospholipase.

The intrinsic overexpression of secretory phospholipase A2 (sPLA2) in various pro-inflammatory diseases and cancers has the potential to be exploited as a therapeutic strategy for diagnostics and treatment. To explore this potential and advance our knowledge of the role of sPLA2 in related diseases, it is necessary to systematically investigate the molecular interaction of the enzyme with lipids. By employing a Langmuir trough integrated with X-ray reflectivity and grazing incidence X-ray diffraction techniques, this study examined the molecular packing structure of 1,2-palmitoyl-sn-glycero-3-phosphocholine (DPPC) films before and after enzyme adsorption and enzyme-catalyzed degradation. Molecular interaction of sPLA2 (from bee venom) with the DPPC monolayer exhibited Ca2+ dependence. DPPC molecules at the interface without Ca2+ retained a monolayer organization; upon adsorption of sPLA2 to the monolayer the packing became tighter. In contrast, sPLA2-catalyzed degradation of DPPC occurred in the presence of Ca2+, leading to disruption of the ordered monolayer structure of DPPC. The interfacial film became a mixture of highly ordered multilayer domains of palmitic acid (PA) and loosely packed monolayer phase of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (lysoPC) that potentially contained the remaining un-degraded DPPC. The redistribution of lipid degradation products into the third dimension, which produced multilayer PA domains, damaged the structural integrity of the original lipid layer and may explain the bursting of liposomes observed in other studies after a latency period of mixing liposomes with sPLA2. A quantitative understanding of the lipid packing and lipid-enzyme interaction provides an intuitive means of designing and optimizing lipid-related drug delivery systems.

Colloidal Confinement of Polyphosphate on Gold Nanoparticles Robustly Activates the Contact Pathway of Blood Coagulation.

Platelet-sized polyphosphate (polyP) was functionalized on the surface of gold nanoparticles (GNPs) via a facile conjugation scheme entailing EDAC (N-(3-(dimethylamino)propyl)-N'-ethylcarbodiimide hydrochloride)-catalyzed phosphoramidation of the terminal phosphate of polyP to cystamine. Subsequent reduction of the disulfide moiety allowed for anchoring to the colloidal surface. The ability of the synthesized polyP-GNPs to initiate the contact pathway of clotting in human pooled normal plasma (PNP) was then assayed by quantifying changes in viscous, mechanical, and optical properties upon coagulation. It is revealed that the polyP-GNPs are markedly superior contact activators compared to molecularly dissolved, platelet-sized polyP (of equivalent polymer chain length). Moreover, the particles' capacity to mobilize Factor XII (FXII) and its coactivating proteins appear to be identical to very-long-chain polyP typically found in bacteria. These data imply that nanolocalization of anionic procoagulants on colloidal surfaces, achieved through covalent anchoring, may yield a robust contact surface with the ability to sufficiently cluster active clotting factors together above their threshold concentrations to cease bleeding. The polyP-GNPs therefore serve as a promising foundation in the development of a nanoparticle hemostat to treat a range of hemorrhagic scenarios.

Artificial Dense Granules: A Procoagulant Liposomal Formulation Modeled after Platelet Polyphosphate Storage Pools.

Granular platelet-sized polyphosphate nanoparticles (polyP NPs) were encapsulated in sterically stabilized liposomes, forming a potential, targeted procoagulant nanotherapy resembling human platelet dense granules in both structure and functionality. Dynamic light scattering (DLS) measurements reveal that artificial dense granules (ADGs) are colloidally stable and that the granular polyP NPs are encapsulated at high efficiencies. High-resolution scanning transmission electron microscopy (HR-STEM) indicates that the ADGs are monodisperse particles with a 150 nm diameter dense core consisting of P, Ca, and O surrounded by a corrugated 25 nm thick shell containing P, C, and O. Further, the ADGs manifest promising procoagulant activity: Detergent solubilization by Tween 20 or digestion of the lipid envelope by phospholipase C (PLC) allows for ADGs to trigger autoactivation of Factor XII (FXII), the first proteolytic step in the activation of the contact pathway of clotting. Moreover, ADGs' ability to reduce the clotting time of human plasma in the presence of PLC further demonstrate the feasibility to develop ADGs into a potential procoagulant nanomedicine.

Size-controlled synthesis of granular polyphosphate nanoparticles at physiologic salt concentrations for blood clotting.

Size-controlled granular polyphosphate (PolyP) nanoparticles were synthesized by precipitation in aqueous solutions containing physiological concentrations of calcium and magnesium. We demonstrate using dynamic light scattering (DLS) that the solubility is correlated inversely with PolyP chain length, with very long chain PolyP (PolyP1000+, more than 1000 repeating units) normally found in prokaryotes precipitating much more robustly than shorter chains like those found in human platelet dense granules (PolyP80, range 76-84 repeating units). It is believed that the precipitation of PolyP is a reversible process involving calcium coordination to phosphate monomers in the polymer chain. The particles are stable in aqueous buffer and albumin suspensions on time scales roughly equivalent to catastrophic bleeding events. Transmission electron microscopy images demonstrate that the PolyP nanoparticles are spherical and uniformly electron dense, with a particle diameter of 200-250 nm, closely resembling the content of acidocalcisomes. X-ray elemental analysis further reveals that the P/Ca ratio is 67:32. The granular nanoparticles also manifest promising procoagulant effects, as measured by in vitro clotting tests assaying contact pathway activity.

Long QT2 mutation on the Kv11.1 ion channel inhibits current activity by ablating a protein kinase Cα consensus site.

Mutations that inhibit Kv11.1 ion channel activity contribute to abnormalities of cardiac repolarization that can lead to long QT2 (LQT2) cardiac arrhythmias and sudden death. However, for most of these mutations, nothing is known about the molecular mechanism linking Kv11.1 malfunction to cardiac death. We have previously demonstrated that disease-related mutations that create consensus sites for kinases on ion channels can dramatically change ion channel activity. Here, we show that a LQT2-associated mutation can inhibit Kv11.1 ion channel activity by perturbing a consensus site for the Ser/Thr protein kinase C α (PKCα). We first reveal by mass spectrometry analysis that Ser890 of the Kv11.1 ion channel is phosphorylated. Then, we demonstrate by a phospho-detection immunoassay combined with genetic manipulation that PKCα phosphorylates Ser890. Furthermore, we show that Ser890 phosphorylation is associated with an increase in Kv11.1 membrane density with alteration of recovery from inactivation. In addition, a newly discovered and as yet uncharacterized LQT2-associated nonsynonymous single nucleotide polymorphism 2660 G→A within the human ether-á-go-go-related gene 1 coding sequence, which replaces arginine 887 with a histidine residue (R887H), strongly inhibits PKCα-dependent phosphorylation of residue Ser890 on Kv11.1, and ultimately inhibits surface expression and current density. Taken together, our data provide a functional link between this channel mutation and LQT2.

Voluntary Age Regression Entering "Headspace" in a Child With Post-traumatic Stress Disorder.

In patients with post-traumatic stress disorder (PTSD), the use of coping mechanisms seems to correlate with higher levels of resiliency; however, in the age of the internet, patients may find it easier to discover new unhealthy skills that can hinder their treatment and further progress their symptoms. This report describes the case of a 12-year-old female with PTSD who was admitted for suicidal ideation and who presented with age regression that was voluntary in nature, characterized by reverting to the age of a six-year-old girl while her boyfriend took on a parental role for her. These behaviors were learned through her use of social media. This case demonstrates the use of maladaptive behaviors to cope with their trauma and the need for parental supervision on the use of the internet and social media by the younger population.

Core-shell Structure and Aggregation Number of Micelles Composed of Amphiphilic Block Copolymers and Amphiphilic Heterografted Polymer Brushes Determined by Small-Angle X-ray Scattering.

A large group of functional nanomaterials employed in biomedical applications, including targeted drug delivery, relies on amphiphilic polymers to encapsulate therapeutic payloads via self-assembly processes. Knowledge of the micelle structures will provide critical insights into design of polymeric drug delivery systems. Core-shell micelles composed of linear diblock copolymers poly(ethylene glycol)-b-poly(caprolactone) (PEG-b-PCL), poly(ethylene oxide)-b-poly(lactic acid) (PEG-b-PLA), as well as a heterografted brush consisting of a poly(glycidyl methacrylate) backbone with PEG and PLA branches (PGMA-g-PEG/PLA) were characterized by dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) measurements to gain structural information regarding the particle morphology, core-shell size, and aggregation number. The structural information at this quasi-equilibrium state can also be used as a reference when studying the kinetics of polymer micellization.

Localization of Short-Chain Polyphosphate Enhances its Ability to Clot Flowing Blood Plasma.

Short-chain polyphosphate (polyP) is released from platelets upon platelet activation, but it is not clear if it contributes to thrombosis. PolyP has increased propensity to clot blood with increased polymer length and when localized onto particles, but it is unknown whether spatial localization of short-chain polyP can accelerate clotting of flowing blood. Here, numerical simulations predicted the effect of localization of polyP on clotting under flow, and this was tested in vitro using microfluidics. Synthetic polyP was more effective at triggering clotting of flowing blood plasma when localized on a surface than when solubilized in solution or when localized as nanoparticles, accelerating clotting at 10-200 fold lower concentrations, particularly at low to sub-physiological shear rates typical of where thrombosis occurs in large veins or valves. Thus, sub-micromolar concentrations of short-chain polyP can accelerate clotting of flowing blood plasma under flow at low to sub-physiological shear rates. However, a physiological mechanism for the localization of polyP to platelet or vascular surfaces remains unknown.