Lipid Exchange Research Articles

LIPID METABOLISM INDICATORS IN SOW BLOOD UNDER THE INFLUENCE OF IRON AND GERMANIUM NANOCOMPOUNDS

The relevance of the conducted research is determined by the lack of information in the available literature regarding the influence of iron and germanium nanoparticles on lipid exchange indicators in the plasma of sows. The aim of the study was to determine the effect of administering iron and germanium nanoparticles on lipid exchange indicators in the blood of sows before and after farrowing. The experiment was conducted on 24 large white sows, aged 2-3 years, which were divided into two groups (control and experimental). For ten days before farrowing, the sows in the experimental group were administered a complex of micronutrient compounds of iron – 3 mg/day and germanium – 0.01 mg/day. Blood plasma of the animals was used as material for the study, in which specific lipid exchange indicators were determined. The effect of farrowing on the content of triacylglycerols, non-esterified fatty acids, and total cholesterol in the blood plasma of animals was established (F= 33.0–156.0 > FU=2.9; P < 0.001). Before farrowing, there is a restructuring of lipid metabolism in sows, characterized by a decrease in the content of triacylglycerols and total cholesterol, and an increase in the content of non-esterified fatty acids in the blood plasma. It was found that within a day before farrowing, the content of triacylglycerols decreases by 30.6% (P≤0.001), total cholesterol by 18.3% (P≤0.01), and non-esterified fatty acids increase by 1.5 times (P≤0.001). Administering iron and germanium nanoparticles significantly affects the content of triacylglycerols (η²ᵪ=0.53; P≤0.05), total cholesterol (η²ᵪ=0.69; P≤0.01), and non-esterified fatty acids (η²ᵪ=0.50; P≤0.05) in the blood plasma of sows only on the day of farrowing. During this time, in the blood of sows administered metal nanoparticles, the content of triacylglycerols and total cholesterol is higher by 23.7% (P≤0.05) and 11.5% (P≤0.01), respectively, and the content of non-esterified fatty acids is lower by 9.2% (P≤0.05) compared to the indicators of animals in the control group. The prospects for further research lie in the development of modern methods to increase the productivity and resistance of sows using metal nanoparticles.

The use of hemifusion to create asymmetric giant unilamellar vesicles: Insights on induced order domains.

The natural asymmetry of the lipid bilayer in biological membranes is, in part, a testament to the complexity of the structure and function of this barrier limiting and protecting cells (or organelles). These lipid bilayers consist of two lipid leaflets with different lipid compositions, resulting in unique interactions within each leaflet. These interactions, combined with interactions between the two leaflets, determine the overall behavior of the membrane. Model membranes provide the most suitable option for investigating the fundamental interactions of lipids. This report describes a comprehensive method to make asymmetric giant unilamellar vesicles (aGUVs) using the technique of hemifusion. In this method, calcium ions induce the hemifusion of giant unilamellar vesicles (GUVs) with a supported lipid bilayer (SLB), both having different lipid compositions. During hemifusion, a stalk, or a more commonly seen hemifusion diaphragm, connects the outer leaflets of GUVs and the SLB. The lateral diffusion of lipids naturally promotes the lipid exchange between the connected outer leaflets. After calcium chelation to prevent further fusion, a mechanical shear detaches aGUVs from the SLB. A fluorescence quench assay is employed to test the extent of bilayer asymmetry. A fluorescence quenching assay tests bilayer asymmetry and verifies dye and lipid migration to a GUV's outer leaflet.

Lipid exchange at ER-trans-Golgi contact sites governs polarized cargo sorting.

Oxysterol binding protein (OSBP) extracts cholesterol from the ER to deliver it to the TGN via counter exchange and subsequent hydrolysis of the phosphoinositide PI(4)P. Here, we show that this pathway is essential in polarized epithelial cells where it contributes not only to the proper subcellular distribution of cholesterol but also to the trans-Golgi sorting and trafficking of numerous plasma membrane cargo proteins with apical or basolateral localization. Reducing the expression of OSBP, blocking its activity, or inhibiting a PI4Kinase that fuels OSBP with PI(4)P abolishes the epithelial phenotype. Waves of cargo enrichment in the TGN in phase with OSBP and PI(4)P dynamics suggest that OSBP promotes the formation of lipid gradients along the TGN, which helps cargo sorting. During their transient passage through the trans-Golgi, polarized plasma membrane proteins get close to OSBP but fail to be sorted when OSBP is silenced. Thus, OSBP lipid exchange activity is decisive for polarized cargo sorting and distribution in epithelial cells.

Zika virus prM protein contains cholesterol binding motifs required for virus entry and assembly

For successful infection of host cells and virion production, enveloped viruses, including Zika virus (ZIKV), extensively rely on cellular lipids. However, how virus protein–lipid interactions contribute to the viral life cycle remains unclear. Here, we employ a chemo-proteomics approach with a bifunctional cholesterol probe and show that cholesterol is closely associated with the ZIKV structural protein prM. Bioinformatic analyses, reverse genetics alongside with photoaffinity labeling assays, and atomistic molecular dynamics simulations identified two functional cholesterol binding motifs within the prM transmembrane domain. Loss of prM–cholesterol association has a bipartite effect reducing ZIKV entry and leading to assembly defects. We propose a model in which membrane-resident M facilitates cholesterol-supported lipid exchange during endosomal entry and, together with cholesterol, creates a platform promoting virion assembly. In summary, we identify a bifunctional role of prM in the ZIKV life cycle by mediating viral entry and virus assembly in a cholesterol-dependent manner.

Experimentally determined leaflet-leaflet phase diagram of an asymmetric lipid bilayer.

We have determined the partial leaflet-leaflet phase diagram of an asymmetric lipid bilayer at ambient temperature using asymmetric giant unilamellar vesicles (aGUVs). Symmetric GUVs with varying amounts of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) were hemifused to a supported lipid bilayer (SLB) composed of DOPC, resulting in lipid exchange between their outer leaflets. The GUVs and SLB contained a red and green lipid fluorophore, respectively, thus enabling the use of confocal fluorescence imaging to determine both the extent of lipid exchange (quantified for individual vesicles by the loss of red intensity and gain of green intensity) and the presence or absence of phase separation in aGUVs. Consistent with previous reports, we found that hemifusion results in large variation in outer leaflet exchange for individual GUVs, which allowed us to interrogate the phase behavior at multiple points within the asymmetric composition space of the binary mixture. When initially symmetric GUVs showed coexisting gel and fluid domains, aGUVs with less than ~50% outer leaflet exchange were also phase-separated. In contrast, aGUVs with greater than 50% outer leaflet exchange were uniform and fluid. In some cases, we also observed three coexisting bilayer-spanning phases: two registered phases and an anti-registered phase. These results suggest that a relatively large unfavorable midplane interaction between ordered and disordered phases in opposing leaflets (i.e., a midplane surface tension) can overwhelm the driving force for lateral phase separation within one of the leaflets, resulting in an asymmetric bilayer with two uniformly mixed leaflets that is poised to phase-separate upon leaflet scrambling.

The state of lipid exchange in persons living in the territory of environmental illusion

Introduction. Currently, it is important to consider the issues of diagnosing nonspecific disorders occurred due to exposure of residents to chemicals in cities with chemical industry enterprises on the territory.
 Materials and methods. Working 30–60 year men, permanently residing downwind from the industrial site of LLC “Usolekhimprom” in the territory exposed to mercury in the city of Usolye-Sibirskoye, were examined.
 Results. The maximum concentration of total cholesterol (TCH) was 8.7 mmol/L, cholesterol (CH) of low density lipoproteins (LDL) – 6.0 mmol/L, triglycerides (TG) – 2.3 mmol/L, phospholipids – 5.5 mmol/L. The minimum content of high-density lipoprotein cholesterol (HDL cholesterol) was 0.5 mmol/L. In terms of the prevalence of disorders, hypercholesterolemia held the first place – 63% of cases, the second – a high level of LDL cholesterol – 59%, the third – an increase in ApoB – 45%. Hyperphospholipidemia occurred in 35%, hypertriglyceridemia – in 23%. Reduced HDL cholesterol was recorded in 5.3%. Low concentration of Apo A1 was registered in 20% of cases. A high level of atherogenic index was common among 16%. The prevalence of high LDL-CH was consistent with that of hypercholesterolemia, as evidenced by the correlation coefficient between the indices (R=0.77) and between the level of TCH and Apo B (R=0.80). A HDL cholesterol high level does not always indicate the antiatherogenic ability of lipoproteins, and therefore the revealed low prevalence of hypoalphacholesterolemia in the examined individuals (lower than in Russia - 16%) does not indicate antiatherogenic changes. The triglyceridemia level was associated with the ApoB concentration, which indicates its contribution to the development of proatherogenic disorders. A possible mechanism of hyperphospholipidemia is the formation of a compensatory-adaptive stimulus to the phospholipids synthesis in response to hypercholesterolemia and hypertriglyceridemia.
 Limitations. Male persons. Age 30–60 years.
 Conclusion. In persons living in the ecological trouble zone, a high frequency of lipid metabolism disorders was revealed. This fact can be considered as one of the main predisposing factors for the development of cardiovascular diseases in this cohort.

Lateral Transport of Domains in Anionic Lipid Bilayer Membranes under DC Electric Fields: A Coarse-Grained Molecular Dynamics Study.

Dynamic lateral transport of lipids, proteins, and self-assembled structures in biomembranes plays a crucial role in diverse cellular processes. In this study, we perform coarse-grained molecular dynamics simulations on a vesicle composed of a binary mixture of neutral and anionic lipids to investigate the lateral transport of individual lipid molecules and the self-assembled lipid domains upon an applied direct current (DC) electric field. Under the potential force of the electric field, a phase-separated domain rich in anionic lipids is trapped in the opposite direction of the electric field. The subsequent reversal of the electric field induces unidirectional domain motion. During the domain motion, the domain size remains constant, but a considerable amount of the anionic lipids is exchanged between the anionic-lipid-rich domain and the surrounding bulk. While the speed of the domain motion (collective lipid motion) shows a significant positive correlation with the electric field strength, the exchange of anionic lipids between the domain and bulk (individual lipid motion) exhibits no clear correlation with the field strength. The mean velocity field of the lipids surrounding the domain displays a two-dimensional (2D) source dipole. We revealed that the balance between the potential force of the applied electric field and the quasi-2D hydrodynamic frictional force well explains the dependence of the domain motions on the electric field strengths. The present results provide insight into the hierarchical dynamic responses of self-assembled lipid domains to the applied electric field and contribute to controlling the lateral transportation of lipids and membrane inclusions.

Obesity as a risk factor for chronic kidney disease: a contemporary view on the problem. Review

The review article is devoted to an actual clinical problem of participation of obesity in development and progression of chronic kidney disease (CKD). Some pathogenic links due to which a negative renal effect of obesity is mediated including in particular, dyslipidemia (DLP), endothelial dysfunction (ED) of renal vessels, activation of renin‑angiotensin‑aldosterone system (RAAS), arterial hypertension (AH), a production of adipose tissue mediators with renal injuring properties such as leptin, tumor necrosis factor‑α, interleukines 1,6,8 are discussed. The data about the main factors of progression of renal damage associated with obesity included insulin resistance (IR), hyperinsulinemia, DLP, systemic and renal hemodynamic disorders caused by activation of RAAS (AH, intraglomerular hypertension) are given. The role of metabolic syndrome and its components in pathogenesis of CKD as well as a role of diabetes mellitus (DM) frequently combined with obesity in development of diabetic kidney disease (diabetic nephropathy) is generalized. Among adipokines a special attention in the present review is paid for pathogenic role of leptin and adiponektin in obesity with its conditions promoted renal damage. The participation of resistance to leptin in the pathogenesis of CKD through such links as IR, ED, oxidative stress and inflammation is discussed. The role of ED and glomerular hyperfiltration in pathogenesis of microalbuminuria is elucidated. The significance of injury of kidney tubular cell structures so called podocytes in pathogenesis of CKD in obesity is uncovered. The clinical significance of early revealing of renal pathology as well as body weight, glycemia level, arterial blood pressure control and following of healthy mode of life in reduction of risk development of CKD associated with obesity, DM and other metabolic disorders is generalized. It’s emphasized that evaluation of lipid and carbohydrate exchange parameters, serum leptin level, IR indices is considered to be perspective for diagnostics of renal damage in obesity.

The association of rs1799983 eNOS gene polymorphism with dyslipidemia and efficacy of atorvastatin in correction of lipid exchange disorders in patients with diabetic nephropathy and essential hypertension

Objective — to search an association of rs1799983 endothelial nitric oxide synthase (eNOS) gene polymorphism with dyslipidemia (DLP) and the efficacy of atorvastatin used in correction of lipid exchange disorders in patients with diabetic nephropathy (DN) and essential hypertension (EH).
 Materials and methods. The study had been conducted in the clinical Department of Arterial Hypertension and Kidney Disease at L. T. Mala National Therapy Institute of NAMS of Ukraine (Kharkiv). Clinical examinations involved 89 patients (pts) (43(48.3%) females and 46(51.7%) males) with DN of II—IV stages and EH of II—III stages aged 34 to 78 years old (an average age is 57.11±1.89 years old). They were also genotyped for rs1799983 eNOS gene polymorphism (G894T) with a use of polymerase chain reaction. GG genotype was revealed in 29(32.6%) of pts, GT genotype was found in 33(37.1%) of cases and 27(30.3%) participants of the study had TT genotype. With the use of immune enzyme method, serum levels of total cholesterol (TC), high‑density lipoprotein cholesterol (HDL‑C) and triglycerides (TG) were measured in all included pts. Very low density lipoprotein cholesterol (VLDL‑C), low‑density lipoprotein cholesterol (LDL‑C), non‑HDL‑C and coefficient of atherogenicity were calculated by known formulas. Atorvastatin was prescribed to all the pts included in study for correction of lipid exchange disorders in daily doses of 10—20 mg (an average daily dose was 16.7±0.91 mg) for 24 weeks.
 Results. Rs1799983 eNOS gene polymorphism in pts with DN and EH was found to be associated with lipid parameters characterized a phenotype of diabetic DLP with its peculiar disorders in the systems of reverse cholesterol transport and lipoprotein lipolysis of TG‑containing VLDL. In this case a dominant G allele was associated with less significant lipid disorders than a recessive T allele. A homozygous state with allele G (genotype GG) in pts with DN and EH was associated with abnormalities in the system of TG‑containing VLDL lipolysis and an appearance of T allele in genotype (genotypes GT and TT) made an additional negative contribution to the state of lipid exchange and transport. In pts with genotype GT abnormalities in reverse cholesterol transport added to disorders in the system of TG‑containing VLDL lipolysis and in pts with genotype TT there was a combination of disorders in the systems of direct, reverse cholesterol transport and lipoprotein lipolysis. The study of hypolipidemic effects of atorvastatin depended on genotype of rs1799983 eNOS gene polymorphism showed that in GG genotype atorvastatin demonstrated mainly a hypocholesterolemic action while in genotypes contained T allele (GT and TT) we observed a reduction of hypocholesterolemic effect against the background of significant hypotriglyceridemic action and elevation of HDL‑C.
 Conclusions. In pts with DN and EH rs1799983 eNOS gene polymorphism was associated with diabetic DLP. A recessive allele T was associated with significant lipid disorders compared with a dominant G allele. A significant hypocholesterolemic effect of atorvastatin was observed in case of a presence of G allele in pts genotype while a presence of T allele in pts genotype (GT and TT) was associated with a reduction of atorvastatin cholesterol lowering action.

Sec22b regulates phagosome maturation by promoting ORP8-mediated lipid exchange at endoplasmic reticulum-phagosome contact sites

Phagosome maturation is critical for immune defense, defining whether ingested material is destroyed or converted into antigens. Sec22b regulates phagosome maturation, yet how has remained unclear. Here we show Sec22b tethers endoplasmic reticulum-phagosome membrane contact sites (MCS) independently of the known tether STIM1. Sec22b knockdown increases calcium signaling, phagolysosome fusion and antigen degradation and alters phagosomal phospholipids PI(3)P, PS and PI(4)P. Levels of PI(4)P, a lysosome docking lipid, are rescued by Sec22b re-expression and by expression of the artificial tether MAPPER but not the MCS-disrupting mutant Sec22b-P33. Moreover, Sec22b co-precipitates with the PS/PI(4)P exchange protein ORP8. Wild-type, but not mutant ORP8 rescues phagosomal PI(4)P and reduces antigen degradation. Sec22b, MAPPER and ORP8 but not P33 or mutant-ORP8 restores phagolysosome fusion in knockdown cells. These findings clarify an alternative mechanism through which Sec22b controls phagosome maturation and beg a reassessment of the relative contribution of Sec22b-mediated fusion versus tethering to phagosome biology.

Lipid exchange and inflammatory markers in patients with coronary artery disease and atrial fibrillation

Aim. To investigate lipid exchange violations, inflammatory markers levels, their connections in coronary artery disease (CAD) patients with atrial fibrillation (AF) and their role in AF paroxysm development and duration.
 Materials and methods. 300 patients were divided into three groups: 27 patients without CAD and arrhythmias formed the control group (CG), 149 patients with CAD but without arrhythmias were included in the main group, and 124 patients with CAD and AF paroxysm were in the comparison group. The average duration of AF paroxysm in the studied group was 2 [1; 3] days (mean [95 % CI]). Such indexes were examined: total cholesterol (TC), triglycerides (TG), low-density lipoproteins (LDL), high-density lipoprotein (HDL), lipoprotein (α) (Lp(α)), apolipoprotein A1 (ApoA1), apoprotein B (ApoB), C-reactive protein (CRP) and interleukin 6 (IL-6).
 Results. According to our data, CAD is characterized by significant lipid exchange violations (increasing TC, LDL, TG, Lpα, ApoB and decreasing HDL; p < 0.05) and increasing inflammatory markers (CRP, IL-6). The presence of AF paroxysm in CAD patients deepened such changes (increasing ApoB, IL-6, CRP; p < 0.05) and ApoB/ApoA1 ratio was increased (p < 0.05). Significant strong and middle strength correlations between inflammatory markers (CRP, IL-6) and lipid exchange indexes (LDL, TG, ApoA1, ApoB, ApoB/ApoA1) were found (p < 0.05). For validation the role of lipid exchange and inflammation in AF pathogenesis ROC curve was performed: LDL + 1.6 × CRP, the area under ROC curve 0.8519 (p < 0.05). This formula can help us to predict the development of AF paroxysm in CAD patients. Moreover, a linear regression equation was created: AF Paroxysm Duration (days) = 0.91 × IL-6 – 0.95 (p < 0.05), which will help to predict AF paroxysm duration in CAD patients, too.
 Conclusions. AF paroxysm occurrence in CAD patients is based on dyslipidemia and inflammation. It is connected with increasing IL-6, CRP, ApoB/ApoA1 levels. The duration of AF paroxysm in CAD patients directly depends on the IL-6 level.

Protein–lipid charge interactions control the folding of outer membrane proteins into asymmetric membranes

Biological membranes consist of two leaflets of phospholipid molecules that form a bilayer, each leaflet comprising a distinct lipid composition. This asymmetry is created and maintained in vivo by dedicated biochemical pathways, but difficulties in creating stable asymmetric membranes in vitro have restricted our understanding of how bilayer asymmetry modulates the folding, stability and function of membrane proteins. In this study, we used cyclodextrin-mediated lipid exchange to generate liposomes with asymmetric bilayers and characterize the stability and folding kinetics of two bacterial outer membrane proteins (OMPs), OmpA and BamA. We found that excess negative charge in the outer leaflet of a liposome impedes their insertion and folding, while excess negative charge in the inner leaflet accelerates their folding relative to symmetric liposomes with the same membrane composition. Using molecular dynamics, mutational analysis and bioinformatics, we identified a positively charged patch critical for folding and stability. These results rationalize the well-known ‘positive-outside’ rule of OMPs and suggest insights into the mechanisms that drive OMP folding and assembly in vitro and in vivo.

Orchestrating vesicular and nonvesicular membrane dynamics by intrinsically disordered proteins.

Compartmentalization by membranes is a common feature of eukaryotic cells and serves to spatiotemporally confine biochemical reactions to control physiology. Membrane-bound organelles such as the endoplasmic reticulum (ER), the Golgi complex, endosomes and lysosomes, and the plasma membrane, continuously exchange material via vesicular carriers. In addition to vesicular trafficking entailing budding, fission, and fusion processes, organelles can form membrane contact sites (MCSs) that enable the nonvesicular exchange of lipids, ions, and metabolites, or the secretion of neurotransmitters via subsequent membrane fusion. Recent data suggest that biomolecule and information transfer via vesicular carriers and via MCSs share common organizational principles and are often mediated by proteins with intrinsically disordered regions (IDRs). Intrinsically disordered proteins (IDPs) can assemble via low-affinity, multivalent interactions to facilitate membrane tethering, deformation, fission, or fusion. Here, we review our current understanding of how IDPs drive the formation of multivalent protein assemblies and protein condensates to orchestrate vesicular and nonvesicular transport with a special focus on presynaptic neurotransmission. We further discuss how dysfunction of IDPs causes disease and outline perspectives for future research.

Atomic force microscopy of erythrocytes of patients with different severity of pancreatitis

Aim of the study was to prove the possibility of using red blood cell probe microscopy as a predictor of the development of pancreonecrosis.Material and methods. Atomic force microscopy was performed in 143 people (82 were male and 61 were female). The study was performed on an Integra Aura probe microscope (ZAO NT-MDT, Russia). Each erythrocyte was scanned for cell membrane and cell as a whole. A total of 940 red blood cells were examined. According to the severity of acute pancreatitis, patients are distributed in three groups. As a control group, 37 people without somatic pathology were examined. Diagnosis and treatment of patients was carried out in the public health institution “Design Bureau” Russian Railways-Medicine “Krasnoyarsk” from 2015–2019.Results. The study showed that the shape of erythrocyte does not depend on the severity of pancreatitis and does not change, however, structural changes of the membrane occur in the form of the formation of multiple erosions on its surface. The membrane is deformed, and its adhesion increases. These changes reduce the transport capacity of the blood, which, in our opinion, leads to an increase in the general intoxication of the body and to a deterioration in the condition of the patient.Conclusions. Apparently, pancreatic enzymes entering the free bloodstream in the first phase of pancreatitis cause not only oxidative stress, but also the reorganization and destruction of the erythrocyte membrane. The increase in membrane rigidity and deformation of the erythrocyte surface cytoskeleton against the background of increased adhesion seems to disrupt the gas transmission function of erythrocyte and microrheological properties of blood, which in turn disrupts the exchange of amino acids, lipids and detoxification capabilities of blood. The appearance of erythrocytes in the free bloodstream with the presence of erosion on the surface of the erythrocyte membrane can serve as a prediction of a non-favorable course of acute pancreatitis and a predictor of a possible transition of edema pancreatitis to pancreonecrosis.

(Invited) Nanosensor Coupling to Human and Plant Interfaces for Real Time Chemical Information Transfer

Our laboratory at MIT has been interested over the past few years in new techniques to facilitate the transfer of chemical information from living organisms, specifically plants, animals and humans, for applications ranging from precision agriculture to precision medicine. This presentation will discuss recent advances on this topic. As tool towards this end, fluorescent nanosensors hold the potential to revolutionize life sciences and medicine. However, their adaptation and translation into the in vivo environment is fundamentally hampered by unfavourable tissue scattering and intrinsic autofluorescence. Here we develop wavelength-induced frequency filtering (WIFF) whereby the fluorescence excitation wavelength is modulated across the absorption peak of a nanosensor, allowing the emission signal to be separated from the autofluorescence background, increasing the desired signal relative to noise, and internally referencing it to protect against artefacts. Using highly scattering phantom tissues, an SKH1-E mouse model and other complex tissue types, we show that WIFF improves the nanosensor signal-to-noise ratio across the visible and near-infrared spectra up to 52-fold. This improvement enables the ability to track fluorescent carbon nanotube sensor responses to riboflavin, ascorbic acid, hydrogen peroxide and a chemotherapeutic drug metabolite for depths up to 5.5 ± 0.1 cm when excited at 730 nm and emitting between 1,100 and 1,300 nm, even allowing the monitoring of riboflavin diffusion in thick tissue. As an application, nanosensors aided by WIFF detect the chemotherapeutic activity of temozolomide transcranially at 2.4 ± 0.1 cm through the porcine brain without the use of fibre optic or cranial window insertion. The ability of nanosensors to monitor previously inaccessible in vivo environments will be important for life-sciences research, therapeutics and medical diagnostics. Also towards this overall objective, our laboratory at MIT has been interested in exploring the relatively new interface between living plants and non-biological nanostructures to impart the former with new and enhanced functions, which we call Plant Nanobionics. We have developed a theory of subcellular uptake and kinetic trapping of a wide range of nanoparticles, validated in-vivo in living plants. Confocal visible and near infrared fluorescent microscopy and single particle tracking of Gold-Cystein-AF405 (GNP-Cys-AF405), Streptavidin-Quantum Dot (SA-QD), Dextran and Poly(acrylic acid) nanoceria, and various polymer-wrapped SWCNT, including lipid-PEG-SWCNT, chitosan-SWCNT and (AT)15-SWCNT, were used to demonstrate that particle size and the magnitude, but not the sign, of the zeta potential are key in determining whether a particle is spontaneously and kinetically trapped within chloroplasts or the cytosol. We develop a mathematical model of this Lipid Exchange Envelope Penetration (LEEP) mechanism, which agrees well with observations of this size and zeta potential dependence. As an application, we rationally designed a chitosan-complexed single-walled carbon nanotube (SWNT) as nanocarriers to selectively deliver plasmid DNA (pDNA) to chloroplasts of different plant species without external biolistic or chemical aid. We demonstrate chloroplast-targeted transgene delivery and expression in living mature arugula (Eruca sativa) and watercress (Nasturitium officinale) plants in planta and in isolated Arabidopsis thaliana mesophyll protoplasts. Another application of nanoparticles and nanotechnology to plant sciences is in the form of biochemical sensors that operate in planta and across diverse species. Using non-destructive optical nanosensors, we find that the spatial and temporal H2O2 concentration immediately post-wounding follows a simple logistic waveform for six dicot plant species: lettuce (Lactuca sativa), arugula (Eruca sativa), spinach (Spinacia oleracea), strawberry blite (Blitum capitatum), sorrel (Rumex acetosa), and Arabidopsis thaliana, ranked in order of wave speed from 0.44 to 3.10 cm/min. The H2O2 wave tracks the concomitant surface potential wave measured electrochemically for the series of plants. We show that the plant NADPH oxidase RbohD, glutamate receptor-like channels (GLR3.3 and GLR3.6) are all critical to the propagation of the H2O2 waveform upon wounding. Our findings highlight the utility of a new type of nanosensor probe that is species-independent and capable of real-time, spatial and temporal biochemical measurements in planta.

Nutritional Status of Patients with Chronic Critical Illness

Material and methods. We examined 23 patients with chronic critical illness who were in a minimally conscious state (MCS) with 10.9±2.5 scores on the FOUR (Full Outline of Unresponsiveness) Score Coma Scale. Indicators of carbohydrate, lipid, protein and energy exchange metabolism were evaluated using specimens sampled in the morning hours. Nutritional support was provided by isocaloric isonitrogenic mixtures.Results. Blood biochemistry showed decreases in total protein and albumin levels in 86.9% and 91.3% of patients, respectively. The tests also revealed decreased concentrations of several amino acids, including essential amino acids: histidine (38.3±13.07 µmol/l), methionine (12.68±3.81 µmol/l), threonine (61.6 [58.5;87.7] µmol/l), tryptophan (33.06±15.95 µmol/l), and non-essential amino acids: arginine (40.50 [22.2; 46.9] µmol/l), glutamic acid (124.5±39.29 µmol/l), tyrosine (37.97±10.12 µmol/l). Some correlations between the concentrations of individual amino acids and other indicators were revealed, such as histidine and CRP (r=–0.68, P=0.043), tryptophan and CRP (r=–0.86, P=0.002), histidine and leukocyte count (r=–0.76, P=0.015), methionine and lysine (r=0.88, P=0.008), methionine and patient's weight (r=–0.68, P=0.042). A relationship between threonine concentration and the level of consciousness on the FOUR scale (r=–0.73, P=0.037) was also found. All patients demonstrated significant alterations of carbohydrate and lipid metabolism.Conclusion. Alteration of adequate protein metabolism seems to be the most affected constituent in the nutritional status of patients with chronic critical illness. It is manifested by a decrease in the concentration of total protein and a number of essential and non-essential amino acids, which implies the importance of highprotein nutritional support and correction of the amino acid profile.

PtdSer as a signaling lipid determined by privileged localization of ORP5 and ORP8 at ER/PM junctional foci to determine PM and ER PtdSer/PI(4)P ratio and cell function

The membrane contact site ER/PM junctions are hubs for signaling pathways, including Ca2+ signaling. Phosphatidylserine (PtdSer) mediates various physiological functions; however, junctional PtdSer composition and the role of PtdSer in Ca2+ signaling and Ca2+-dependent gene regulation are not understood. Here, we show that STIM1-formed junctions are required for PI(4)P/PtdSer exchange by ORP5 and ORP8, which have reciprocal lipid exchange modes and function as a rheostat that sets the junctional PtdSer/PI(4)P ratio. Targeting the ORP5 and ORP8 and their lipid transfer ORD domains to PM subdomains revealed that ORP5 sets low and ORP8 high junctional PI(4)P/PtdSer ratio that controls STIM1-STIM1 and STIM1-Orai1 interaction and the activity of the SERCA pump to determine the pattern of receptor-evoked Ca2+ oscillations, and consequently translocation of NFAT to the nucleus. Significantly, targeting the ORP5 and ORP8 ORDs to the STIM1 ER subdomain reversed their function. Notably, changing PI(4)P/PtdSer ratio by hydrolysis of PM or ER PtdSer with targeted PtdSer-specific PLA1a1 reproduced the ORPs function. The function of the ORPs is determined both by their differential lipid exchange modes and by privileged localization at the ER/PM subdomains. These findings reveal a role of PtdSer as a signaling lipid that controls the available PM PI(4)P, the unappreciated role of ER PtdSer in cell function, and the diversity of the ER/PM junctions. The effect of PtdSer on the junctional PI(4)P level should have multiple implications in cellular signaling and functions.

Lipid-driven interleaflet coupling of plasma membrane order regulates FcεRI signaling in mast cells

Interleaflet coupling—the influence of one leaflet on the properties of the opposing leaflet—is a fundamental plasma membrane organizational principle. This coupling is proposed to participate in maintaining steady-state biophysical properties of the plasma membrane, which in turn regulates some transmembrane signaling processes. A prominent example is antigen (Ag) stimulation of signaling by clustering transmembrane receptors for immunoglobulin E (IgE), FcεRI. This transmembrane signaling depends on the stabilization of ordered regions in the inner leaflet for sorting of intracellular signaling components. The resting inner leaflet has a lipid composition that is generally less ordered than the outer leaflet and that does not spontaneously phase separate in model membranes. We propose that interleaflet coupling can mediate ordering and disordering of the inner leaflet, which is poised in resting cells to reorganize upon stimulation. To test this in live cells, we first established a straightforward approach to evaluate induced changes in membrane order by measuring inner leaflet diffusion of lipid probes by imaging fluorescence correlation spectroscopy, by imaging fluorescence correlation spectroscopy (ImFCS), before and after methyl-α-cyclodexrin (mαCD)-catalyzed exchange of outer leaflet lipids (LEX) with exogenous order- or disorder-promoting phospholipids. We examined the functional impact of LEX by monitoring two Ag-stimulated responses: recruitment of cytoplasmic Syk kinase to the inner leaflet and exocytosis of secretory granules (degranulation). Based on the ImFCS data in resting cells, we observed global increase or decrease of inner leaflet order when outer leaflet is exchanged with order- or disorder-promoting lipids, respectively. We find that the degree of both stimulated Syk recruitment and degranulation correlates positively with LEX-mediated changes of inner leaflet order in resting cells. Overall, our results show that resting-state lipid ordering of the outer leaflet influences the ordering of the inner leaflet, likely via interleaflet coupling. This imposed lipid reorganization modulates transmembrane signaling stimulated by Ag clustering of IgE-FcεRI.

A SNARE-like protein from Solanum lycopersicum increases salt tolerance by modulating vesicular trafficking in tomato.

Intracellular vesicular trafficking ensures the exchange of lipids and proteins between endomembrane compartments. This is relevant under high salinity conditions, since both the removal of transporters and ion channels from the plasma membrane and the compartmentalization of toxic ions require the formation of vesicles, which can be maintained as multivesicular bodies or be fused to the central vacuole. SNARE proteins (Soluble N-ethylmaleimide-sensitive factor attachment receptor) participate in the vesicle fusion process and give specificity to their destination. Plant genome studies have revealed a superfamily of genes that encode for proteins called SNARE-like. These proteins appear to be participating in vesicular trafficking with similar functions to those of SNARE proteins. A SNARE-like, named SlSLSP6, in Solanum lycopersicum plants has been shown to be induced under high salinity conditions. A phylogenetic relationship of SlSLSP6 with SNARE-like proteins of salinity-tolerant plants, including Salicornia brachiata, Zostera marina and Solanum pennelli, was determined. Considering its amino acid sequence, a putative clathrin adapter complex domain and palmitoylation site was predicted. Subcellular localization analysis evidenced that SlSLSP6 is mostly localized in the plasma membrane. Using transgenic tomato plants, we identified that overexpression of SlSLSP6 increased tolerance to salt stress. This tolerance was evident when we quantified an improvement in physiological and biochemical parameters, such as higher chlorophyll content, performance index, efficiency of photosystem II and relative water content, and lower malondialdehyde content, compared to control plants. At the subcellular level, the overexpression of SlSLSP6 reduced the presence of H2O2 in roots and increased the compartmentalization of sodium in vacuoles during salt stress. These effects appear to be associated with the higher endocytic rate of FM4-64, determined in the plant root cells. Taken together, these results indicate that SlSLSP6 increases tolerance to salt stress by modulating vesicular trafficking through over-induction of the endocytic pathway. This work contributes to understanding the role of this type of SNARE-like protein during salt stress and could be a potential candidate in breeding programs for tolerance to salt stress in tomato plants.

Burst of hopping trafficking correlated reversible dynamic interactions between lipid droplets and mitochondria under starvation.

Dynamic membrane contacts between lipid droplets (LDs) and mitochondria play key roles in lipid metabolism and energy homeostasis. Understanding the dynamics of LDs under energy stimulation is thereby crucial to disclosing the metabolic mechanism. Here, the reversible interactions between LDs and mitochondria are tracked in real-time using a robust LDs-specific fluorescent probe (LDs-Tags). Through tracking the dynamics of LDs at the single-particle level, spatiotemporal heterogeneity is revealed. LDs in starved cells communicate and integrate their activities (i.e., lipid exchange) through a membrane contact site-mediated mechanism. Thus the diffusion is intermittently alternated between active and confined states. Statistical analysis shows that the translocation of LDs in response to starvation stress is non-Gaussian, and obeys nonergodic-like behavior. These results provide deep understanding of the anomalous diffusion of LDs in living cells, and also afford guidance for rationally designing efficient transporter.