The application of L-PRP in AIDS patients with crural chronic ulcers: A pilot study

Purpose

Nonhealing wounds or skin ulcerations are the result of insufficient repair and destruction of a local healing potential. Opportunistic infections which cause a lot of ulcer complications influence the worsening general condition of patients with AIDS, ultimately leading to death. The chronicity of the condition and poor results of conventional therapy have prompted the search for new methods of treatment.

Platelet-rich plasma combined with naringin induces osteogenic differentiation of human bone marrow mesenchymal stem cells In vitro北大核心

BACKGROUND: Platelet-rich plasma (PRP) and naringin can both promote proliferation and induce osteogenic differentiation of mesenchymal stem cells. However, their combined use is rarely reported. OBJECTIVE: To observe the effect of PRP combined with naringin on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) in vitro. METHODS: BMSCs at passage 3 were divided into four groups: (1) blank control group, cells were cultured in α-MEM; (2) PRP group, cells were cultured in α-MEM containing PRP; (3) naringin group, cells were cultured in α-MEM containing naringin; and (4) combined group, cells were cultured in a-MEM containing PRP and naringin. The contents oO used PRP and naringin were 12.5% and 50 µg/L respectively. Cell proliferation was detected by MTT assay. Expression oO related genes in hBMSCs was detected by RT-PCR. Alkaline phosphatase staining, collagen type I immunohistochemical staining, and alizarin red staining were used to analyze the osteogenic differentiation of hBMSCs. RESULTS AND CONCLUSION: The proliferation of hBMSCs was increased in each group, especially in the combined group. Cells in all the groups oxcept the blank control group were positive for alkaline phosphatase staining, collagen type I immunohistochemical staining, and alizarin red staining, and the positive effect was more obvious in the combined group. However, negative or weakly positive response was found in the blank control group. At 7 and 14 days, the expression of alkaline phosphatase and collagen type I was significantly higher in the PRP, naringin and combined groups than the blank control group (P < 0.05); at 14 days, the expression of alkaline phosphatase and collagen type I was significantly higher in the combined group than the PRP and naringin groups (P < 0.05). To conclude, PRP combined with naringin can promote the proliferation of hBMSCs and induce the osteogenic differentiation of hBMSCs. Moreover, there is a synergistic effect between PRP and naringin. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.     

Formation of the Legionella-containing vacuole: phosphoinositide conversion,GTPase modulation and ER dynamics

The environmental bacterium Legionella pneumophila replicates in free-living amoeba as well as in alveolar macrophages upon inhalation of bacteria-laden aerosols. Resistance of the opportunistic pathogen to macrophages is a prerequisite to cause a severe pneumonia called Legionnaires’ disease. L. pneumophila grows intracellularly in a unique, ER-associated compartment, the Legionella-containing vacuole (LCV). The bacterial Icm/Dot type IV secretion system represents an essential virulence factor, which translocates approximately 300 “effector proteins” into protozoan or mammalian host cells. Some of these effectors contribute to the formation of the LCV by targeting conserved host factors implicated in membrane dynamics, such as phosphoinositide lipids and small GTPases. Here we review recent findings on the role of phosphoinositides, small and large GTPases as well as ER dynamics for pathogen vacuole formation and intracellular replication of L. pneumophila.     

Subclinical cardiovascular disease and Systemic Sclerosis: A comparison between risk charts,quantification of coronary calcium and carotid ultrasonography

Background and objectives

Recently published population-based cohort studies have shown a high prevalence of cardiovascular disease in Systemic Sclerosis (SSc) patients. The aim of this study is to compare three different methods to measure cardiovascular risk in patients with scleroderma.

Remodeling of intermediate metabolism in the diatom Phaeodactylum tricornutum under nitrogen stress

Diatoms are unicellular algae that accumulate significant amounts of triacylglycerols as storage lipids when their growth is limited by nutrients. Using biochemical, physiological, bioinformatics, and reverse genetic approaches, we analyzed how the flux of carbon into lipids is influenced by nitrogen stress in a model diatom, Phaeodactylum tricornutum. Our results reveal that the accumulation of lipids is a consequence of remodeling of intermediate metabolism, especially reactions in the tricarboxylic acid and the urea cycles. Specifically, approximately one-half of the cellular proteins are cannibalized; whereas the nitrogen is scavenged by the urea and glutamine synthetase/glutamine 2-oxoglutarate aminotransferase pathways and redirected to the de novo synthesis of nitrogen assimilation machinery, simultaneously, the photobiological flux of carbon and reductants is used to synthesize lipids. To further examine how nitrogen stress triggers the remodeling process, we knocked down the gene encoding for nitrate reductase, a key enzyme required for the assimilation of nitrate. The strain exhibits 40–50% of the mRNA copy numbers, protein content, and enzymatic activity of the wild type, concomitant with a 43% increase in cellular lipid content. We suggest a negative feedback sensor that couples photosynthetic carbon fixation to lipid biosynthesis and is regulated by the nitrogen assimilation pathway. This metabolic feedback enables diatoms to rapidly respond to fluctuations in environmental nitrogen availability.In plants, carbon and nitrogen are directed to specific tissues or structures in accordance with developmental programs. In contrast, unicellular algae flexibly direct carbon and nitrogen to various macromolecules associated with specific intracellular compartments to optimize growth under varying environmental conditions. The signals responsible for this optimization strategy are poorly understood. They clearly are not driven by a developmental program but rather, responses to environmental cues. For example, under optimal growth conditions, ∼40% of the photosynthetically fixed carbon in typical eukaryotic microalga is directed toward the synthesis of amino acids that ultimately are incorporated into proteins (1–3). Over 50 y ago, however, it was recognized that, when nitrogen limits growth, intermediate metabolism is altered, and many microalgae can accumulate storage lipids, mainly in the form of triacylglycerols (TAGs) (4–6). This phenomenon is especially pronounced in diatoms.Diatoms, a highly successful class of eukaryotic algae that rose to ecological prominence during the past 30 My (7), often form massive blooms under turbulent conditions when nutrient supplies are highly variable (8). The ability of these organisms to optimize their growth under such conditions requires coordination of intermediate metabolism of carbon and nitrogen (9, 10). To optimize their growth, the first priority of the cells is to assimilate nitrogen into proteins, which also requires reducing equivalents and carbon skeletons that are primarily supplied by the tricarboxylic acid (TCA) cycle. However, when nitrogen availability decreases, the sink for TCA cycle metabolites declines, and acetyl-CoA, the source of carbon for the cycle, can be shunted toward fatty acid (FA) biosynthesis. Therefore, under nitrogen stress, cellular protein content decreases, whereas storage lipids increase (11, 12). This phenomenon has led to the hypothesis that overexpression of genes involved in lipid biosynthesis may increase the flux of carbon toward lipids (13, 14). Although this phenomenon is well-known, the signals that trigger the process remain unresolved. Genetic manipulations of lipid production in the model diatom, Phaeodactylum tricornutum, are ambiguous. Although there is one report showing that an overexpression of a type II diacylglycerol acyltransferase (DGAT; ProtID 49462) involved in TAG biosynthesis increases the accumulation of natural lipids in P. tricornutum (15), there are several reports indicating that manipulating FA biosynthesis does not significantly affect rates of lipid production (13, 14, 16).Using biochemical, physiological, bioinformatic, and reverse genetic approaches, we examine here how a diatom remodels intermediate metabolism to rapidly respond to nitrogen stress and its resupply. Our results reveal how carbon is redirected toward lipid biosynthesis under nitrogen stress in P. tricornutum.     

Dynamic label-free imaging of lipid nanodomains

Lipid rafts are submicron proteolipid domains thought to be responsible for membrane trafficking and signaling. Their small size and transient nature put an understanding of their dynamics beyond the reach of existing techniques, leading to much contention as to their exact role. Here, we exploit the differences in light scattering from lipid bilayer phases to achieve dynamic imaging of nanoscopic lipid domains without any labels. Using phase-separated droplet interface bilayers we resolve the diffusion of domains as small as 50 nm in radius and observe nanodomain formation, destruction, and dynamic coalescence with a domain lifetime of 220 ± 60 ms. Domain dynamics on this timescale suggests an important role in modulating membrane protein function.Cell membranes compartmentalize into lipid domains that enable the selective recruitment of specific proteins (1). These “lipid rafts” have been proposed to control many membrane processes including apical sorting, protein trafficking, and the clustering of proteins during signaling. The dynamic formation and destruction of lipid nanodomains are thought to provide the central mechanism to regulate this wide range of essential processes (2–4). Although many methods now provide strong evidence to support their existence in vivo (5), the combination of nanoscopic size and dynamics on millisecond timescales has placed the direct observation of their behavior beyond the scope of existing techniques. Consequently, although we know they exist, frustratingly little is known regarding their function and dynamics (6).Recent advances in fluorescence nanoscopy provide the only time-dependent information on the behavior of lipid nanodomains (7–9). Stimulated emission depletion–fluorescence correlation spectroscopy has shown cholesterol-mediated hindered nanoscale diffusion of single labeled sphingomyelin lipids that is consistent with the lipid raft hypothesis and transient binding of lipids (9). Superresolution fluorescence microscopy has also revealed protein clusters in cell membranes with 0.5-s temporal resolution (7). All of these experiments, however, are limited in temporal resolution by fluorescence, and must infer lipid nanodomains from the addition of fluorescent labels.Macroscopic phase separation in artificial lipid bilayers has been widely used to help understand the biological implications of domain formation. Different lipid phases can be visualized using fluorescence microscopy with labels that preferentially partition into a specific phase (10–12). This approach is successful for micrometer-sized domains but inevitably fails on the tens to few hundreds of nanometers scale due to limitations in phase specificity, the limited residence time of a label within a specific nanoscopic domain, and the achievable optical resolution (13). The fluorescent probe is itself an additional component that can perturb phase behavior, either directly or through photooxidation (14, 15). As a result, lipid nanodomain dynamics have not been observed directly even in artificial systems, although recent ensemble-based techniques report lipid heterogeneity on the appropriate length scales (13). In addition to fluorescence-based approaches, ellipsometry and reflection interference contrast microscopy have been used to characterize phase separation in lipid bilayers (16, 17), taking advantage of different bilayer thicknesses and refractive indices caused by varying degrees of cholesterol content and lipid packing. Given sufficient sensitivity and resolution, this approach should hold for arbitrarily small domains.We recently developed interferometric scattering microscopy (iSCAT) (18–20) and achieved sensitivity to refractive index perturbations down to the level of a single unlabeled protein molecule in solution with millisecond time resolution (21, 22). Here, we exploit the unique sensitivity of iSCAT to overcome the limitations in temporal resolution and sensitivity to image, track, and characterize lipid nanodomains without requiring any labels. We use droplet interface bilayers (DIBs) as an artificial membrane model (23, 24) with phase-separated lipid mixtures (Fig. 1A). DIBs are formed by the contact of two lipid monolayers; in this case, a monolayer formed at the interface between an aqueous droplet and a solution of phospholipids in oil, and another between a thin hydrogel film and the oil. DIBs are robust, long-lived, and defect-free, show unrestricted diffusion, form gigaohm resistance seals, and are compatible with high-resolution optical microscopy (24).Open in a separate windowFig. 1.Detection of lipid nanodomains using iSCAT. (A) Schematic of a DIB showing ordered (light gray) and disordered (black) phases. The interference between scattered and reflected fields (E_s and E_r) is detected in the far field using a digital camera. (B) The 100-ms TIRF (Top) and iSCAT (Bottom) images of a DIB containing S_o domains within a bulk L_d phase (1:1 DOPC: bSM plus 1 mol% Atto488-DPPE). The static background due to scattering from the agarose substrate can been seen in this raw iSCAT image. This background is subtracted in subsequent images. (C) Time-lapse sequence of iSCAT images of L_o nanodomains appearing from a uniform L_d phase upon cooling of a DIB below the phase transition temperature. The droplet was heated to 45 °C for 10 min. Nanodomains appeared 2–5 min after heating was stopped. Composition, 1:1:1 DPhPC:bSM:Chol. Greyscale values are of the normalized reflected intensity. (D) Trajectories corresponding to average pixel contrast within a 900 × 900-nm window centered on each nanodomain shown in C. Values before the appearance of the domain are representative of the background fluctuations at the position where the domain first becomes visible. (Scale bars: 5 μm.)     

Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site

K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) is a prenylated, membrane-associated GTPase protein that is a critical switch for the propagation of growth factor signaling pathways to diverse effector proteins, including rapidly accelerated fibrosarcoma (RAF) kinases and RAS-related protein guanine nucleotide dissociation stimulator (RALGDS) proteins. Gain-of-function KRAS mutations occur frequently in human cancers and predict poor clinical outcome, whereas germ-line mutations are associated with developmental syndromes. However, it is not known how these mutations affect K-RAS association with biological membranes or whether this impacts signal transduction. Here, we used solution NMR studies of K-RAS4B tethered to nanodiscs to investigate lipid bilayer-anchored K-RAS4B and its interactions with effector protein RAS-binding domains (RBDs). Unexpectedly, we found that the effector-binding region of activated K-RAS4B is occluded by interaction with the membrane in one of the NMR-observable, and thus highly populated, conformational states. Binding of the RAF isoform ARAF and RALGDS RBDs induced marked reorientation of K-RAS4B from the occluded state to RBD-specific effector-bound states. Importantly, we found that two Noonan syndrome-associated mutations, K5N and D153V, which do not affect the GTPase cycle, relieve the occluded orientation by directly altering the electrostatics of two membrane interaction surfaces. Similarly, the most frequent KRAS oncogenic mutation G12D also drives K-RAS4B toward an exposed configuration. Further, the D153V and G12D mutations increase the rate of association of ARAF-RBD with lipid bilayer-tethered K-RAS4B. We revealed a mechanism of K-RAS4B autoinhibition by membrane sequestration of its effector-binding site, which can be disrupted by disease-associated mutations. Stabilizing the autoinhibitory interactions between K-RAS4B and the membrane could be an attractive target for anticancer drug discovery.The K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) protein product of the KRAS gene undergoes posttranslational farnesylation and C-terminal processing, which, in conjunction with a poly-basic hypervariable region (HVR), targets K-RAS4B to anionic lipid rafts on the intracellular side of the plasma membrane (Fig. 1A) (1). This localization is essential for K-RAS4B function and enhances signaling fidelity (2). Although the significance of membrane tethering of K-RAS4B is well appreciated, a high-resolution map of how K-RAS4B interacts with the membrane is lacking. Because membrane-anchored RAS presents a major challenge to crystallization, current structural insights into the behavior of membrane-anchored RAS have come from a variety of lower-resolution techniques including in vivo FRET-based studies (3), fluorescence and infrared spectroscopic studies (4–6), and in silico models (3). These pioneering studies suggested that the K-RAS4B GTPase domain adopts certain preferred orientations on the anionic membrane and that these orientations could be influenced by the bound nucleotide. Here we present high-resolution NMR-derived models of the dynamic interactions between K-RAS4B and the lipid bilayer and how they can be impacted by disease-associated mutations or interactions with effector proteins.Open in a separate windowFig. 1.K-RAS4B signaling at the plasma membrane and the nanodisc lipid bilayer model for NMR studies. (A) Schematic illustration of K-RAS4B signaling on plasma membrane. (Inset) Schematic of a K-RAS4B:nanodisc complex. (B) Distribution of eleven isoleucine Cδ throughout the K-RAS4B GTPase-domain. (Right) ¹H-¹³C HMQC spectra of nanodisc-conjugated K-RAS4B in the GDP- (Upper) and GMPPNP- (Lower) bound forms.     

Platelet rich plasma and plasma rich in growth factors for split-thickness skin graft donor site treatment in the burn patient setting: A randomized clinical trial

IntroductionManagement of donor site morbidity in the setting of split thickness skin graft (STSG) is of crucial importance with no superior wound dressing described to date and the growing need of decreasing epithelializing time. The purpose of the study was to compare the standard of care using a hydrocolloid dressing to platelet rich plasma (PRP) and plasma rich in growth factors (PRGF) in order to determine its therapeutic potential in this setting.MethodsA randomized clinical trial was conducted in which each patient served as its own control. PRGF was obtained by means of freeze-thaw out of the PRP from the subject of the study. Patients from the study had three donor sites and each donor site received either to PRP, PRGF or the standard of care, hydrocolloid. The main variable was time to epithelialization, and secondary variables subject to study were pain, quality of the scar, complications and cost.Results20 patients were recruited with a total number of 60 donor sites to study. On the 8th post-operative day 55% and 45% of the sites treated with PRP and PRGF, respectively, complete epithelialization was observed as compared to 20% of the sites treated with hydrocolloid, statistical significance was achieved between the latter two (p = 0.036). The areas treated with PRP and PRGF received inferior values on the visual analog scale on post-op day 5 and 8 compared to hydrocolloid. Values on wound healing metrics were lower in the PRP when compared to hydrocolloid. No adverse effects were recorded.ConclusionDonor site of STSG treated with PRP in the setting of the burn patient decreased time to epithelialization. In our study a better pain control and in scar quality was observed in both, the PRP and PRGF group.