Leukocyte Inclusion within a Platelet Rich Plasma-Derived Fibrin Scaffold Stimulates a More Pro-Inflammatory Environment and Alters Fibrin Properties

One of the main differences among platelet-rich plasma (PRP) products is the inclusion of leukocytes that may affect the biological efficacy of these autologous preparations. The purpose of this study was to evaluate whether the addition of leukocytes modified the morphological, biomechanical and biological properties of PRP under normal and inflammatory conditions. The release of pro-inflammatory cytokines from plasma rich in growth factors (PRGF) and leukocyte-platelet rich plasma (L-PRP) scaffolds was determined by enzyme-linked immunosorbent assay (ELISA) and was significantly increased under an inflammatory condition when leukocytes were included in the PRP. Fibroblasts and osteoblasts treated with L-PRP, under an inflammatory situation, underwent a greater activation of NFĸB pathway, proliferated significantly less and secreted a higher concentration of pro-inflammatory cytokines. These cellular events were assessed through Western blot and fluorimetric and ELISA methods, respectively. Therefore, the inclusion of leukocytes induced significantly higher pro-inflammatory conditions.     

A methodology for numerically analysing the hepatic artery haemodynamics during B-TACE: a proof of concept

Balloon-occluded transarterial chemoembolisation (B-TACE) is an intraarterial transcatheter treatment for liver cancer. In B-TACE, an artery-occluding microballoon catheter occludes an artery and promotes collateral circulation for drug delivery to tumours. This paper presents a methodology for analysing the haemodynamics during B-TACE, by combining zero-dimensional and three-dimensional modelling tools. As a proof of concept, we apply the methodology to a patient-specific hepatic artery geometry and analyse two catheter locations. Results show that the blood flow redistribution can be predicted in this proof-of-concept study, suggesting that this approach could potentially be used to optimise catheter location.     

Cytoskeletal Components Define Protein Location to Membrane Microdomains

The plasma membrane is an important compartment that undergoes dynamic changes in composition upon external or internal stimuli. The dynamic subcompartmentation of proteins in ordered low-density (DRM) and disordered high-density (DSM) membrane phases is hypothesized to require interactions with cytoskeletal components. Here, we systematically analyzed the effects of actin or tubulin disruption on the distribution of proteins between membrane density phases. We used a proteomic screen to identify candidate proteins with altered submembrane location, followed by biochemical or cell biological characterization in Arabidopsis thaliana. We found that several proteins, such as plasma membrane ATPases, receptor kinases, or remorins resulted in a differential distribution between membrane density phases upon cytoskeletal disruption. Moreover, in most cases, contrasting effects were observed: Disruption of actin filaments largely led to a redistribution of proteins from DRM to DSM membrane fractions while disruption of tubulins resulted in general depletion of proteins from the membranes. We conclude that actin filaments are necessary for dynamic movement of proteins between different membrane phases and that microtubules are not necessarily important for formation of microdomains as such, but rather they may control the protein amount present in the membrane phases.Living cells need borders and molecular compartments for biochemical reactions and storage of metabolites. The plasma membrane therefore is a prerequisite for the evolution of different life forms. It consists of a phospholipid bilayer into which proteins and special lipid species such as sterols, sphingolipids, and glycolipids are inserted. The first complex model of plasma membrane was proposed in 1972 by Jonathan Singer and Garth Nicolson (1), replacing the concept of the plasma membrane as a strict protein–lipid–protein sandwich that was generally accepted until then. In Singer and Nicolson''s model, the cell membrane is a two-dimensionally oriented viscous solution in which the membrane constituents are orientated in the most thermodynamically favorable manner, hiding hydrophobic hydrocarbon chains inside the lipid bilayer and exposing polar and ionic groups to the aqueous phase. This fluid mosaic model also implied that membrane proteins as well as lipid components are distributed in a homogeneous lipid bilayer at long range, but they can form specific aggregates and phases at short range, which were also termed “lipid rafts” or membrane microdomains.Over the past 30 years, it has become evident that the plasma membrane is not such a homogeneous structure as it was initially proposed. We now know that the lipid bilayer is asymmetric (2) and that the free diffusion of membrane proteins is restricted by their interactions with intracellular and extracellular components (3). More recently, Simons and Ikonen suggested that large ordered phases, enriched with cholesterol and sphingolipids, emerge within the plasma membrane and that they function as platforms for enrichment of certain proteins while excluding others (4). This current membrane model suggests that the mixture of sterols and polar lipids within the plasma membrane can appear in two distinct phases: liquid disordered (L_d) and liquid ordered (L_o) phase (5). In this view, the so-called membrane microdomains are considered to be part of the L_o phase. Based on work on model membranes, it is suggested that lateral segregation of components into L_d and L_o phases occurs spontaneously (6) with the self-associating properties between sterols and highly saturated hydrocarbon chains of phopsho- and sphingolipids as the main driving force (7). Additionally, it is suggested that also specific lipid-protein and protein-protein interactions are essential for the formations of membrane domains as well as for stabilization of smaller nanodomains which subsequently may cause formation of larger platforms. In contrast to the animal cells, in plants these membrane microdomains seem to be rather immobile (8), possibly due to their attachment to the outer cell wall. More recently, it became obvious that membrane microdomains within a single cell are highly diverse and of different compositions (9). Generally, in the plant model, organisms'' plasma membrane microdomains turned out to be important in plant defense (10, 11), cell polarity (12, 13), and general signaling properties of the plasma membrane (14, 15).The cytoskeleton was identified as an essential cellular component with important roles in membrane topography, bordering, trafficking, and organelle movement (16). Single particle tracking in mammalian cells revealed that the transferrin receptor and macroglobulin receptor demonstrate normal Brownian diffusion but only within a specific membrane compartment (17). Two hypothetical models were proposed in order to explain this phenomenon (supplemental Fig. 1). Direct interactions between transmembrane proteins and cytoskeleton are suggested to creates a barrier, called “fence,” where cytosolic parts of transmembrane proteins collides with cytoskeletal components, limiting their diffusion to certain areas. These molecules can jump over the “fence” to a neighboring compartment, possibly due to the dynamic nature of the interaction of membrane proteins and cytoskeleton, where they are again temporally trapped (17). This phenomenon was recently described also in A. thaliana where the interplay between membrane microdomains and microtubules plays a role in secondary cell wall formation (reviewed in (18)). The second model assumes, additionally, that particular transmembrane proteins are anchored to and lined up along cytoskeleton and act as “pickets” to arrest free diffusion of other membrane components, including nontransmembrane proteins, within the enclosed compartment (19).For plants, the composition of these sterol-rich membranes phases was analyzed in several biochemical studies (14, 20–22). Thereby, low-density preparations of plasma membrane fractions after treatment with nonionic detergents (DRM¹ fractions) were considered as a biochemical representation enriched in cellular membrane ordered phases or microdomains. Proteomic studies in mammalian cells consistently reported that the DRM fraction is highly enriched with several cytoskeletal proteins such as actin, tubulin, myosin, dynamin, actinin, and supervillin (23–25). Additionally, the level of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a lipid connecting the plasma membrane to actin filaments, was also significantly elevated in DRM preparations (26). Treatment with microtubule and actin depolymerizing agent results in drastic loss of many signaling proteins from these DRM fractions prepared from adult rat cardiac myocytes (27) or human embryonic retinal cells (28).Based on this knowledge, we propose two hypothetical models for the relationship between cytoskeleton and membrane microdomains for plant cells: (i) Actin filaments and microtubules could be important in the membrane phase separation or formation of the membrane microdomains themselves. In this case, disruption of the cytoskeleton would cause a lack of phase segregation in the plasma membrane. (ii) The cytoskeleton is only important for the incorporation of specific protein into the sterol-enriched regions but not for the general formation of these phase separations. This view implies that phase separations or membrane microdomains would still be present after cytoskeleton disruption but their protein composition can be different. Another possible scenario is (iii) that cytoskeletal elements serve as anchors for membrane microdomains at particular position in the plasma membrane, so the absence of these anchors would cause the increased mobility of microdomains (supplemental Fig. 1).The primary aim of this study was to characterize the interplay between cytoskeletal components and different membrane phases (microdomains) in A. thaliana suspension cell cultures. To reach this goal, biochemical and proteomic approaches were combined with confocal microscopy and activity assays measuring the influence of actin or tubulin disruption on the composition, localization, and biochemical properties of the sterol-enriched membrane microdomains. Thereby, for biochemical analyses, low-density detergent-resistant membrane fractions are analyzed as containing cellular sterol-rich membrane compartments.     

Effect of elevated CO2, temperature and drought on photosynthesis of nodulated alfalfa during a cutting regrowth cycle

Rising atmospheric CO₂ may increase potential net leaf photosynthesis under short-term exposure, but this response decreases under long-term exposure because plants acclimate to elevated CO₂ concentrations through a process known as downregulation. One of the main factors that may influence this phenomenon is the balance between sources and sinks in the plant. The usual method of managing a forage legume like alfalfa requires the cutting of shoots and subsequent regrowth, which alters the source/sink ratio and thus photosynthetic behaviour. The aim of this study was to determine the effect of CO₂ (ambient, around 350 vs. 700 µmol mol⁻¹), temperature (ambient vs. ambient + 4° C) and water availability (well-irrigated vs. partially irrigated) on photosynthetic behaviour in nodulated alfalfa before defoliation and after 1 month of regrowth. At the end of vegetative normal growth, plants grown under conditions of elevated CO₂ showed photosynthetic acclimation with lower photosynthetic rates, V_cmax and ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activity. This decay was probably a consequence of a specific rubisco protein reduction and/or inactivation. In contrast, high CO₂ during regrowth did not change net photosynthetic rates or yield differences in V_cmax or rubisco total activity. This absence of photosynthetic acclimation was directly associated with the new source-sink status of the plants during regrowth. After cutting, the higher root/shoot ratio in plants and remaining respiration can function as a strong sink for photosynthates, avoiding leaf sugar accumulation, the negative feed-back control of photosynthesis, and as a consequence, photosynthetic downregulation.     

Smokers with CT Detected Emphysema and No Airway Obstruction Have Decreased Plasma Levels of EGF,IL-15, IL-8 and IL-1ra

Rationale

Low-grade inflammation and emphysema have been shown to be associated with an increased risk of lung cancer. However, the systemic inflammatory response in patients with emphysema is still unknown.

Objective

To compare the plasma cytokine profiles in two groups of current or former smokers without airway obstruction: a control group of individuals without computed tomography (CT) detected emphysema vs. a study group of individuals with CT detected emphysema.

Methods

Subjects underwent a chest CT, spirometry, and determination of EGF, IL-15, IL-1ra, IL-8, MCP-1, MIP-1β, TGFα, TNFα, and VEGF levels in plasma. Cytokine levels in each group were compared adjusting for confounding factors.

Results

160 current smokers and former smokers without airway obstruction participated in the study: 80 without emphysema and 80 subjects with emphysema. Adjusted group comparisons revealed significant reductions in EGF (−0.317, p = 0.01), IL-15 (−0.21, p = 0.01), IL-8 (−0.180, p = 0.02) and IL-1ra (−0.220, p = 0.03) in subjects with emphysema and normal spirometry.

Conclusions

Current or former smokers expressing a well-defined disease characteristic such as emphysema, has a specific plasma cytokine profile. This includes a decrease of cytokines mainly implicated in activation of apoptosis or decrease of immunosurveillance. This information should be taken into account when evaluated patients with tobacco respiratory diseases.     

On the adjustment of the sex ratio and the gregarious behavior of animal populations.

Ribosome synthesis in bacteria is linked to RNA polymerase synthesis; both synthesis rates depend upon the values of six parameters: (1) fraction of total ribosomes that is functioning, (2) fraction of total RNA polymerase that is functioning, (3) fraction of functioning RNA polymerase engaged in rRNA synthesis, (4) fraction of total protein that is RNA polymerase protein, (5) peptide chain elongation rate, (6) rRNA chain elongation rate. If these parameters are constant in time, then the numbers of both ribosomes and RNA polymerase molecules increase exponentially. It is shown how the rate constant (fractional increase per unit of time) relates to these parameters and how the kinetics of ribosome and RNA polymerase synthesis respond to a change in any of these parameters.     

Causes,historical development,effects and future challenges of a common environmental problem: eutrophication

The impact of the presence of us humans as well as our activities to nature has led to over-exploitation of natural resources and to dramatic changes in land-use including the use of artificial fertilizers contributes to the deterioration of the natural environment. The population density, industrial processes and the use of fertilizers are the main causes for the eutrophication of river systems, estuaries and seas. There are several ways to determine the actual nutrient levels back to the 1950s, back to 1900 or even back to 1800. Available data indicate that the natural background concentrations of nutrients (pristine conditions or the period prior to the widespread use of artificial fertilizers and detergents) were dramatically lower than today. Available time series on chemical and biological data collected from different parts of the world show the (sometimes tremendous) increase in nutrient levels and the related productivity. The same time series, however, also show the decreases in values when measures were taken to reduce the nutrient emissions. Investigations of different systems all over the world show that nearly every system responses differently to eutrophication. Especially physical boundary conditions play an important role in the manifestation of the ultimate effect of local eutrophication. Apart from the physical boundary conditions also the transformation and retention of nutrients in estuarine and coastal systems contribute to system specific responses. Depending on all these different conditions, site specific responses with even site specific problems may occur. The challenge of this millennium is to really reach a balance between nature and mankind including its population size. A beneficial step in this discussion is assessing criteria to reduce eutrophication back to a level acceptable to both humans and nature.     

Characterization of Prorocentrum elegans and Prorocentrum levis (Dinophyceae) from the southeastern Bay of Biscay by morphology and molecular phylogeny

Benthic Prorocentrum species can produce toxins that adversely affect animals and human health. They are known to co‐occur with other bloom‐forming, potentially toxic, benthic dinoflagellates of the genera Ostreopsis, Coolia, and Gambierdiscus. In this study, we report on the presence of P. elegans M.Faust and P. levis M.A.Faust, Kibler, Vandersea, P.A. Tester & Litaker from the southeastern Bay of Biscay. Sampling was carried out in the Summer‐Autumn 2010–2012 along the Atlantic coast of the Iberian Peninsula, but these two species were only found in the northeastern part of the Peninsula. Strains were isolated from macroalgae collected from rocky‐shore areas bordering accessible beaches. Morphological traits of isolated strains were analyzed by LM and SEM, whereas molecular analyses were performed using the LSU and internal transcribed spacer (ITS)1‐5.8S‐ITS2 regions of the rDNA. A bioassay with Artemia fransciscana and liquid chromatography–high‐resolution mass spectrometry analyses were used to check the toxicity of the species, whose results were negative. The strains mostly corresponded to their species original morphological characterization, which is supported by the phylogenetic analyses in the case of P. levis, whereas for P. elegans, this is the first known molecular characterization. This is also the second known report of P. elegans.     

Acute and Chronic Effects of Ethanol on Transbilayer Membrane Domains

Alcohols, including ethanol, have a specific effect on transbilayer and lateral membrane domains. Recent evidence has shown that alcohols in vitro have a greater effect on fluidity of one leaflet as compared to the other. The present study examined effects of chronic ethanol consumption on fluidity of synaptic plasma membrane (SPM) exofacial and cytofacial leaflets using trinitrobenzenesulfonic acid (TNBS) labeling and differential polarized fluorometry of 1,6-diphenyl-1,3,5-hexatriene (DPH). Mice were administered ethanol or a control liquid diet for 3 weeks. Animals were killed and SPM prepared. The exofacial leaflet of SPM was significantly more fluid than the cytofacial leaflet in both groups, as indicated by limiting anisotropy of DPH. However, differences between the two leaflets were much smaller in the ethanol-treated group. Ethanol at concentrations seen clinically had a greater effect in vitro on the more fluid exofacial leaflet. This asymmetric effect of ethanol was significantly diminished in the exofacial leaflet of the ethanol-treated mice. Chronic ethanol consumption has a specific effect on membranes. Membrane functions that may be regulated by asymmetry of fluidity and lipid distribution may be altered by chronic ethanol consumption.