A flexible method for the fabrication of gold nanostructures using oligonucleotide derivatives

Several linear and branched DNA structures from 80-200 nm with a biotine molecule in the middle have been prepared. These structures have been decorated by addition of positively charged gold nanoparticles carrying 4-(dimethylamino)pyridine ligands. Streptavidin binds to the central biotine molecule introducing a 20 nm gap in the structure in which a biotinylated nanoparticle can be introduced. The simplest structure (80 nm, linear) is formed by 4 oligonucleotides. By changing some of these components changes on length, shape, and recognition system easily can be introduced.     

Improvement of platelet aggregation abnormalities in thrombocytosis after thrombocytopheresis

Platelet function tests were performed in three patients with thrombocytosis in myeloproliferative disorders before and after a swift reduction of platelet count by thrombopheresis. The decrease of platelet count obtained after the procedure was reversed in six days. In two patients with platelet aggregation defects, the normalization of aggregation abnormalities was observed after pheresis, followed by a progressive decrease of platelet response until the pre-pheresis values on 6th day. In the third patient with normal platelet aggregation, a progressive increase of platelet aggregation response was noted on the days following thrombopheresis with ischaemic symptoms of a foot toe. In all three patients, the changes of platelet aggregation were accompanied by a related increase of megathrombocytes. In the two patients with platelet aggregation abnormalities, plasma and platelet beta-thromboglobulin levels were related to changes in platelet count and aggregation.     

Sources of Variation in a Two-Step Monitoring Protocol for Species Clustered in Conspicuous Points: Dolichotis patagonum as a Case Study

In species showing distributions attached to particular features of the landscape or conspicuous signs, counts are commonly made by making focal observations where animals concentrate. However, to obtain density estimates for a given area, independent searching for signs and occupancy rates of suitable sites is needed. In both cases, it is important to estimate detection probability and other possible sources of variation to avoid confounding effects on measurements of abundance variation. Our objective was to assess possible bias and sources of variation in a two-step protocol in which random designs were applied to search for signs while continuously recording video cameras were used to perform abundance counts where animals are concentrated, using mara (Dolichotis patagonum) as a case study. The protocol was successfully applied to maras within the Península Valdés protected area, given that the protocol was logistically suitable, allowed warrens to be found, the associated adults to be counted, and the detection probability to be estimated. Variability was documented in both components of the two-step protocol. These sources of variation should be taken into account when applying this protocol. Warren detectability was approximately 80% with little variation. Factors related to false positive detection were more important than imperfect detection. The detectability for individuals was approximately 90% using the entire day of observations. The shortest sampling period with a similar detection capacity than a day was approximately 10 hours, and during this period, the visiting dynamic did not show trends. For individual mara, the detection capacity of the camera was not significantly different from the observer during fieldwork. The presence of the camera did not affect the visiting behavior of adults to the warren. Application of this protocol will allow monitoring of the near-threatened mara providing a minimum local population size and a baseline for measuring long-term trends.     

Some amino acids of the Pseudomonas aeruginosa MutL D(Q/M)HA(X)(2)E(X)(4)E conserved motif are essential for the in vivo function of the protein but not for the in vitro endonuclease activity

Human and Saccharomyces cerevisiae MutLα, and some bacterial MutL proteins, possess a metal ion-dependent endonuclease activity which is important for the in vivo function of these proteins. Conserved amino acids of the C-terminal region of human PMS2, S. cerevisiae PMS1 and of some bacterial MutL proteins have been implicated in the metal-binding/endonuclease activity. However, the contribution of individual amino acids to these activities has not yet been fully elucidated. In this work we show that Pseudomonas aeruginosa MutL protein possess an in vitro metal ion-dependent endonuclease activity. In agreement with previous published results, we observed that mutation of the aspartic acid, the first histidine or the first glutamic acid of the conserved C-terminal DMHAAHERITYE region results in nonfunctional in vivo proteins. We also determined that the arginine residue is essential for the in vivo function of this protein. However, we unexpectedly observed that although the first glutamic acid mutant derivative is not functional in vivo, its in vitro endonuclease activity is even higher than that of the wild-type protein.     

Composition-driven Surface Domain Structuring Mediated by Sphingolipids and Membrane-active Proteins

Biomembranes contain a wide variety of lipids and proteins within an essentially two-dimensional structure. The coexistence of such a large number of molecular species causes local tensions that frequently relax into a phase or compositional immiscibility along the lateral and transverse planes of the interface. As a consequence, a substantial microheterogeneity of the surface topography develops and that depends not only on the lipid-protein composition, but also on the lateral and transverse tensions generated as a consequence of molecular interactions. The presence of proteins, and immiscibility among lipids, constitute major perturbing factors for the membrane sculpturing both in terms of its surface topography and dynamics. In this work, we will summarize some recent evidences for the involvement of membrane-associated, both extrinsic and amphitropic, proteins as well as membrane-active phosphohydrolytic enzymes and sphingolipids in driving lateral segregation of phase domains thus determining long-range surface topography.     

Free fatty acid receptor 3 is a key target of short chain fatty acid: What is the impact on the sympathetic nervous system?

Nervous system (NS) activity participates in metabolic homeostasis by detecting peripheral signal molecules derived from food intake and energy balance. High quality diets are thought to include fiber-rich foods like whole grain rice, breads, cereals, and grains. Several studies have associated high consumption of fiber-enriched diets with a reduced risk of diabetes, obesity, and gastrointestinal disorders. In the lower intestine, anaerobic fermentation of soluble fibers by microbiota produces short chain fatty acids (SCFAs), key energy molecules that have a recent identified leading role in the intestinal gluconeogenesis, promoting beneficial effects on glucose tolerance and insulin resistance¹. SCFAs are also signaling molecules that bind to specific G-protein coupled receptors (GPCRs) named Free Fatty Acid Receptor 3 (FFA3, GPR41) and 2 (FFA2, GPR43). However, how SCFAs impact NS activity through their GPCRs is poorly understood. Recently, studies have demonstrated the presence of FFA2 and FFA3 in the sympathetic NS of rat, mouse and human^{2, 3}. Two studies have showed that FFA3 activation by SCFAs increases firing and norepinephrine (NE) release from sympathetic neurons^{3, 4}. However, the recent study from the Ikeda Laboratory² revealed that activation of FFA3 by SCFAs impairs N-type calcium channel (NTCC) activity, which contradicts the idea of FFA3 activation leading to increased action potential evoked NE release. Here we will discuss the scope of the latter study and the putative physiological role of SCFAs and FFAs in the sympathetic NS.     

Sphingomyelinase-induced domain shape relaxation driven by out-of-equilibrium changes of composition

Sphingomyelinase (SMase)-induced ceramide (Cer)-enriched domains in a lipid monolayer are shown to result from an out-of-equilibrium situation. This is induced by a change of composition caused by the enzymatic production of Cer in a sphingomyelin (SM) monolayer that leads to a fast SM/Cer demixing into a liquid-condensed (LC), Cer-enriched and a liquid-expanded, SM-enriched phases. The morphological evolution and kinetic dependence of Cer-enriched domains is studied under continuous observation by epifluorescence microscopy. Domain shape annealing is observed from branched to rounded shapes after SMase activity quenching by EDTA, with a decay halftime of ∼10 min. An out-of-equilibrium fast domain growth is not the determinant factor for domain morphology. Domain shape rearrangement in nearly equilibrium conditions result from the counteraction of intradomain dipolar repulsion and line tension, according to McConnell's shape transition theory. Phase separation causes a transient compositional overshoot within the LC phase that implies an increased out-of-equilibrium enrichment of Cer into the LC domains. As a consequence, higher intradomain repulsion leads to transient branched structures that relax to rounded shapes by lowering the proportion of Cer in the domain to equilibrium values. The fast action of SMase can be taken as a compositional perturbation that brings about important consequences for the surface organization.     

Electromagnetic fields (EMFs) and adenosine receptors modulate prostaglandin E(2) and cytokine release in human osteoarthritic synovial fibroblasts

Synovial fibroblasts (SFs) contribute to the development of osteoarthritis (OA) by the secretion of a wide range of pro-inflammatory mediators, including cytokines and lipid mediators of inflammation. Previous studies suggest that electromagnetic fields (EMFs) may represent a potential therapeutic approach to limit cartilage degradation and control inflammation associated to OA, and that they may act through the adenosine pathway. Therefore, we investigated whether EMFs might modulate inflammatory activities of human SFs from OA patients (OASFs) treated with interleukin-1β (IL-1β), and the possible involvement of adenosine receptors (ARs) in mediating EMF effects. EMF exposure induced a selective increase in A(2A) and A(3) ARs. These increases were associated to changes in cAMP levels, indicating that ARs were functionally active also in EMF-exposed cells. Functional data obtained in the presence of selective A(2A) and A(3) adenosine agonists and antagonists showed that EMFs inhibit the release of prostaglandin E(2) (PGE(2)) and the proinflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8), while stimulating the release of interleukin-10 (IL-10), an antinflammatory cytokine. These effects seem to be mediated by the EMF-induced upregulation of A(2A) and A(3) ARs. No effects of EMFs or ARs have been observed on matrix degrading enzyme production. In conclusion, this study shows that EMFs display anti-inflammatory effects in human OASFs, and that these EMF-induced effects are in part mediated by the adenosine pathway, specifically by the A(2A) and A(3) AR activation. Taken together, these results open new clinical perspectives to the control of inflammation associated to joint diseases.     

Sphingomyelinase acts by an area-activated mechanism on the liquid-expanded phase of sphingomyelin monolayers

We describe the localization of Alexa-488-labeled SMase in SM/ceramide (Cer) lipid monolayers containing segregated liquid-condensed (LC) Cer-enriched domains surrounded by a continuous liquid-expanded (LE) SM-enriched phase. Langmuir-Schaefer films were made in order to visualize the labeled enzyme. Independently of initial conditions Alexa-SMase is preferably localized in the SM-enriched LE phase and it is not enriched at the domain boundaries. A novel mechanism is proposed for the action of SMase, which can also explain the regulatory effect of the surface topography on the enzyme activity. The homogeneous enzymatic generation of Cer in the LE phase leads to a meta-stable, kinetically trapped, supersaturated mixed monolayer. This effect acts as driving force for the segregation of the Cer-enriched domain following classical nucleation mechanisms. Accordingly, the number and size of Cer-enriched domains are determined by the extent of Cer supersaturation in the LE phase rather than by the SMase local activity. The kinetic barrier for nucleation, for which a compositional gap of at least 53 mol% of Cer is necessary to reach a thermodynamically stable LC phase, can explain the lag time to reaching full catalytic activity. Altogether, the data support an "area-activated mechanism," in which the enzyme is homogeneously active over the LE surface.