The Differential Effects of HDL Subpopulations on Lipoprotein Lipase (LPL)-Mediated VLDL Catabolism

High-density lipoprotein (HDL) subpopulations functional assessment is more relevant for HDL anti-atherogenic activity than cholesterol level. The aim of the study was to assess the impact of HDL-2 and HDL-3 on lipoprotein lipase (LPL)-mediated very-low-density lipoprotein (VLDL) catabolism related to hypertriglyceridemia development. VLDL and HDLs were isolated from serum by ultracentrifugation. VLDL was incubated with LPL in the absence and presence of total HDL or HDL subpopulations. Next, VLDL remnants were separated, and their composition and electrophoretic mobility was assessed. Both HDL subpopulations increased the efficiency of triglyceride lipolysis and apolipoprotein CII and CIII removal from VLDL up to ~90%. HDL-3 exerted significantly greater impact than HDL-2 on apolipoprotein E (43% vs. 18%, p < 0.001), free cholesterol (26% vs. 18%, p < 0.05) and phospholipids (53% vs. 43%, p < 0.05) removal from VLDL and VLDL remnant electrophoretic mobility (0.18 vs. 0.20, p < 0.01). A greater release of these components was also observed in the presence of total HDL with a low HDL-2/HDL-3 cholesterol ratio. Both HDL subpopulations affect VLDL composition during lipolysis, but HDL-3 exhibited a greater effect on this process. Altered composition of HDL related to significant changes in the distribution between HDL-2 and HDL-3 can influence the VLDL remnant features, affecting atherosclerosis progression.

Electrophoretic Mobility and Electric Conductivity of Salt-Free Suspensions of Charged Soft Particles

A unit cell model is employed to analyze the electrophoresis and electric conduction in a concentrated suspension of spherical charged soft particles (each is a hard core coated with a porous polyelectrolyte layer) in a salt-free medium. The linearized Poisson–Boltzmann equation applicable to a unit cell is solved for the equilibrium electrostatic potential distribution in the liquid solution containing the counterions only surrounding a soft particle. The counterionic continuity equation and modified Stokes/Brinkman equations are solved for the ionic electrochemical potential energy and fluid velocity distributions, respectively. Closed-form formulas for the electrophoretic mobility of the soft particles and effective electric conductivity of the suspension are derived, and the effect of particle interactions on these transport characteristics is interesting and significant. Same as the case in a suspension containing added electrolytes under the Debye–Hückel approximation, the scaled electrophoretic mobility in a salt-free suspension is an increasing function of the fixed charge density of the soft particles and decreases with increases in the core-to-particle radius ratio, ratio of the particle radius to the permeation length in the porous layer, and particle volume fraction, keeping the other parameters unchanged. The normalized effective electric conductivity of the salt-free suspension also increases with an increase in the fixed charge density and with a decrease in the core-to-particle radius ratio, but is not a monotonic function of the particle volume fraction.

A possible role of gas-phase electrophoretic mobility molecular analysis (nES GEMMA) in extracellular vesicle research

The emerging role of extracellular vesicles (EVs) as biomarkers and their envisioned therapeutic use require advanced techniques for their detailed characterization. In this context, we investigated gas-phase electrophoresis on a nano electrospray gas-phase electrophoretic mobility molecular analyzer (nES GEMMA, aka nES differential mobility analyzer, nES DMA) as an alternative to standard analytical techniques. In gas-phase electrophoresis, single-charged, surface-dry, native, polydisperse, and aerosolized analytes, e.g., proteins or bio-nanoparticles, are separated according to their electrophoretic mobility diameter, i.e., globular size. Subsequently, monodisperse particles are counted after a nucleation step in a supersaturated atmosphere as they pass a focused laser beam. Hence, particle number concentrations are obtained in accordance with recommendations of the European Commission for nanoparticle characterization (2011/696/EU from October 18th, 2011). Smaller sample constituents (e.g., co-purified proteins) can be detected next to larger ones (e.g., vesicles). Focusing on platelet-derived EVs, we compared different vesicle isolation techniques. In all cases, nanoparticle tracking analysis (NTA) confirmed the presence of vesicles. However, nES GEMMA often revealed a significant co-purification of proteins from the sample matrix, precluding gas-phase electrophoresis of less-diluted samples containing higher vesicle concentrations. Therefore, mainly peaks in the protein size range were detected. Mass spectrometry revealed that these main contaminants belonged to the group of globulins and coagulation-related components. An additional size exclusion chromatography (SEC) step enabled the depletion of co-purified, proteinaceous matrix components, while a label-free quantitative proteomics approach revealed no significant differences in the detected EV core proteome. Hence, the future in-depth analysis of EVs via gas-phase electrophoresis appears feasible. Graphical abstract

The role of hemorheological disorders in the pathogenesis of hemorrhagic pancreonecrosis

Введение. Вопросы патогенеза острого панкреатита и панкреонекроза до настоящего времени остаются в центре внимания исследователей и клиницистов. До сих пор до конца не выяснена роль изменений в системе гемостаза и гемореологических нарушений в развитии этого заболевания. Цель исследования: установить роль гемореологических нарушений в патогенезе геморрагического панкреонекроза и изучить специфику механизма этих расстройств. Материалы и методы. Обследовано 29 пациентов с геморрагическим панкреонекрозом (12 женщин и 17 мужчин) в возрасте от 23 до 60 лет. Исследовали вязкость крови, показатель гематокрита, количество эритроцитов и их диаметр, агрегацию, электрофоретическую подвижность, деформируемость и механическую резистентность эритроцитов, белковый состав плазмы, содержание сиаловой кислоты в плазме и в эритроцитах, параметры липидного обмена, содержание кальция и фибриногена в крови,фибринолитическую активность крови и агрегационную активность тромбоцитов, гемокоагуляционная активность исследована методом тромбоэластографии. Для определения нормальных значений исследованных показателей было обследовано 15 практически здоровых лиц (7 женщин и 8 мужчин). Результаты. У больных панкреонекрозом самым грубым нарушениям подвергаются эритроциты: их механическая резистентность снижалась в 2 раза, объем увеличивался на 18,7%, деформируемость падала на 43,8%, количество снижалось на 8,75%, показатель гематокрита при этом оставался на уровне нормальных значений по причине увеличенного объема (сферичности) клеток; в 1,8 раза возрастала агрегация эритроцитов. Вязкость крови при скорости сдвига 1 c–1 увеличивалась в 3,3 раза, а при скорости сдвига 150 c–1 — в 1,58 раза по сравнению с нормой. Причиной повышения агрегации эритроцитов являлось снижение их электрофоретической подвижности на 35,9% из-за десиализации их мембран: концентрация сиаловой кислоты в клеточных мембранах была снижена на 20,8%, а содержание конъюгированной сиаловой кислоты в плазме увеличено в 2,25 раза по сравнению с нормальными значениями. Заключение. Гемореологические расстройства, которые возникают первоначально у больных геморрагическим панкреонекрозом как результат некротических изменений поджелудочной железы, с определенного, довольно раннего этапа сами становятся фактором патогенеза данного заболевания. Доминирующим фактором прогрессивного увеличения вязкости крови у больных панкреонекрозом является нарушение морфофункциональных и физико-химических свой ств эритроцитов на фоне высокой активности протеолитических ферментов, биологически активных аминов и крайней степени токсемии. Background. The pathogenesis of acute pancreatitis and pancreonecrosis is still the focus of researchers and clinicians. The role of hemorheological disorders in these diseases remain uncertain until now. Objectives: to define the role of hemorheological disorders in the pathogenesis of hemorrhagic pancreonecrosis and to study the specifics of the mechanism of these disorders. Patients/Methods. This study included 29 patients (12 women and 17 men, age of 23 to 60 years old) with hemorrhagic pancreatic necrosis. We examined blood viscosity, hematocrit and some erythrocyte properties as count, diameter, aggregation, electrophoretic mobility, deformability and mechanical resistance; other investigated parameters were plasma protein composition, plasma and erythrocytes sialic acid concentrations, lipids, total calcium and fibrinogen concentrations, blood fibrinolytic activity, platelets aggregation activity; total hemocoagulation activity was studied with thromboelastography. Control group contained 15 practically healthy individuals (7 women and 8 men). Results. Expressed disturbances of blood rheological properties, mostly in erythrocytes were detected in patients with pancreonecrosis. Red blood cells (RBC) showed 2-times decreasing of mechanical resistance, of their volume by 18.7%, of deformability by 43.8%, of count by 8.75%. Hematocrit remained normal level due to RBC increased volume (sphericity). RBC aggregation had been increased by 1.8 times. Blood viscosity at the shear rate of 1 s–1 was increased by 3.3 times and at the shear rate of 150 s–1 by 1.58 times. Raised erythrocyte aggregation was caused by a decrease of RBC electrophoretic mobility of 35.9%. Sialic acid concentration in RBC membranes was lower of 20.8% whereas conjugated sialic acid in plasma showed increasing by 2.25 times. Conclusions. RBC morphofunctional and physicochemical disturbances cause the increase in blood viscosity in patients with pancreonecrosis. It is distinguishing feature of hemorheological disorders in hemorrhagic pancreonecrosis developing, seems, due to high activity of proteolytic enzymes and biologically active amines. Of particular importance in hemorrhagic pancreonecrosis belong to platelet involving into intravascular coagulation.

The functional indexes of RBCs and microcirculation in the traumatic brain injury with the action of 2-ethil-6-methil-3-hydroxypiridin succinate

Research aim To study the RBCs functional and metabolic parameters and the microcirculatory brain structure at traumatic brain injury (TBI) under the action of 2-ethyl-6-methyl-3-hydroxypyridine succinate. Methods A closed TBI was modeled by the free fall of a load on the parietooccipital regions of head. We made studies of the influence of 2-ethil-6-methil-3-hydroxipiridin succinate on aggregation and electrophoretic mobility of RBCs, catalase activity, malonic dialdehyde concentration, adenosine triphosphate and 2.3-biphosphoglycerate (2.3 – BPG) concentrations in RBCs. The state of parenchyma and microcirculatory brain mainstream in post-traumatic period of TBI have been studied on micro-preparations. Results The use of 2-ethyl-6-methyl-3-hydroxypyridine succinate under conditions of head injury leads to a decrease in MDA concentration and in aggregation of RBCs, to an increase in the 2.3—BPG concentration and RBC electrophoretic mobility compared to the control (group value). The most pronounced changes under the action of 2-ethyl-6-methyl-3-hydroxypyridine succinate were observed 3–7 days after the TBI. Significant indicators of the restoration of the microvasculature and brain tissue provoked by the use of 2-ethyl-6-methyl-3-hydroxypyridine succinate of were evident from the 7th day unlike the control group, where the restoration of structural morphological parameters was observed only on the 12th day of the post-traumatic period. Fast recovery of blood flow under the action of 2-ethyl-6-methyl-3-hydroxypyridine succinate ensured effective restoration of neurons and glia in comparison with the control group. Conclusions Early and long-term cytoprotective correction intensifies the oxygen transport function of the blood, prevents and / or reduces disorders of microvessels, neurons and glia in the post-traumatic period, thereby provides correction of hypoxic state and drives to the restoration of brain tissues homeostasis.

Ionization and structural properties of mRNA lipid nanoparticles influence expression in intramuscular and intravascular administration

Lipid Nanoparticles (LNPs) are used to deliver siRNA and COVID-19 mRNA vaccines. The main factor known to determine their delivery efficiency is the pKa of the LNP containing an ionizable lipid. Herein, we report a method that can predict the LNP pKa from the structure of the ionizable lipid. We used theoretical, NMR, fluorescent-dye binding, and electrophoretic mobility methods to comprehensively measure protonation of both the ionizable lipid and the formulated LNP. The pKa of the ionizable lipid was 2-3 units higher than the pKa of the LNP primarily due to proton solvation energy differences between the LNP and aqueous medium. We exploited these results to explain a wide range of delivery efficiencies in vitro and in vivo for intramuscular (IM) and intravascular (IV) administration of different ionizable lipids at escalating ionizable lipid-to-mRNA ratios in the LNP. In addition, we determined that more negatively charged LNPs exhibit higher off-target systemic expression of mRNA in the liver following IM administration. This undesirable systemic off-target expression of mRNA-LNP vaccines could be minimized through appropriate design of the ionizable lipid and LNP.