Graphene modification with gold nanoparticles using the gas aggregation technique

Gold nanoparticles (Au-NPs) were deposited on single layer graphene (SLG) and few layers graphene (FLG) by applying the gas aggregation technique, previously adapted to a 4-gun commercial magnetron sputtering system. The samples were supported on SiO2 (280 nm)/Si substrates, and the influence of the applied DC power and deposition times on the nanoparticle-graphene system was investigated by Confocal Raman Microscopy. Analysis of the G and 2D bands of the Raman spectra shows that the integrated intensity ratio (I-2D/I-G) was higher for SLG than for FLG. For the samples produced using a sputtering power of 30W, the intensity (peak height) of the G and 2D bands increased with the deposition time, whereas for those produced applying 60W the peak heights of the G and 2D bands decreased with the deposition time. This behaviour was ascribed to the formation of larger Au-NPs aggregates in the last case. A significant increase of the Full Width Half Maximum (FWHM) of the G band for SLG and FLG was also observed as a function of the DC power and deposition time. Surprisingly, the fine details of the Raman spectra revealed an unintentional doping of SLG and FLG accompanying the increase of size and aggregation of the Au-NPs. (C) 2011 Elsevier B.V. All rights reserved.

SPATIAL PATTERN OF FOUR FOREST COMMUNITIES BY RIPLEY'S K FUNCTION IN SAO PAULO STATE

Investigating tree's spatial patterns according to their size classes and according to their more abundant species can provide evidences about the structure of the vegetal community, since the spatial pattern is a key question for forestry ecology studies. The tree spatial organization patterns on the environment depend on several ecological processes and on the specific characteristics of each environment, so that the best understanding of this frame provides important elements for the knowledge on forestry formation. This paper aimed to study tree spatial patterns, according to the diameter classes and from four most abundant species in different forests, in order to provide evidences regarding to the ecology of each vegetal community. The spatial pattern description in each forestry formation was developed using Ripley's K function. The studied forestry formations were: Ombrophilous Forest, Cerradao, Seasonal Forest and Restinga Forest. In this work, a 10.24 ha plot was installed in each forestry formation, and every tree, with a circumference at breast height (CBH) larger than 15 cm were measured, georeferenced and identified. The obtained data highlights the aggregated character in tropical forests, as observed in every studied forest. The 'Cerraddo' and 'Restinga' forest trees showed close aggregate patterns. In the Ombrophilous forest, for all distance scales, the aggregate pattern was meaningful. In the seasonal forest, a random tendency was observed, although a meaningful aggregation was observed in all short distances. The spatial pattern by diameter classes was generally aggregated for trees smaller than 10 cm of diameter and between 10 and 20 cm and random for the others, proving the existence of a tendency which in young trees is more aggregated than in old ones. The spatial pattern of the dominant species is always strongly similar to the general pattern of each forestry formation. The differences between the spatial patterns of two or three coincident species, among the forestry formations, indicate that its pattern is influenced by each different environment. This stands out the importance of its self-ecology and of its ecological processes, intrinsic of each community that can explain the observed patterns.

Innate Immunity in Insects, Function and Regulation of Hemolin from Hyalophora cecropia

Insects are useful models for the study of innate immune reactions and development. The distinction between recognition mechanisms preceding the breakdown of apoptotic cells during metamorphosis, and the breakdown of cells in response to infections, is unclear. Hemolin, a Lepidopteran member of the immunoglobulin superfamily, is a candidate molecule in self/nonself recognition. This thesis investigates hemolin function and hemolin gene regulation at a molecular level. We investigated the binding and cell adhesion properties of hemolin from H. cecropia and demonstrated that the proteins could homodimerize in presence of calcium. Moreover, a higher molecular weight membrane form of hemolin was present on hemocytes. These results, taken together with an earlier finding that soluble hemolin inhibits hemocyte adhesion, indicated that the secreted hemolin could modulate hemocyte aggregation in a competitive manner in the blood. In addition, hemolin was expressed in different tissues and at different developmental stages. Since hemolin is expressed both during development and during the immune response, its different regulatory factors must act in concert. We found that the third intron contains an enhancer, through which Dif, C/EBP and HMGI synergistically activate a reporter construct in vitro. We concluded that the enhancer is used during infection, since the κB-site is crucial for an immune response. Interestingly, we also found that the active form of the steroid hormone, ecdysone, induces the hemolin gene transcription in vivo, and in addition, acts synergistically during bacterial infection. Preliminary in vivo results indicate a secondary effect of ecdysone and the importance of hormone receptor elements in the upstream promoter region of hemolin. To explore the use of Drosophila as a genetic tool for understanding hemolin function and regulation, we sought to isolate the functional homologue in this species. A fly cDNA library in yeast was screened using H. cecropia hemolin as bait. The screen was not successful. However, it did lead to the discovery of a Drosophila protein with true binding specificity for hemolin. Subsequent characterization revealed a new, highly conserved gene, which we named yippee. Yippee is distantly related to zinc finger proteins and represents a novel family of proteins present in numerous eukaryotes, including fungi, plants and humans. Notably, when the Drosophila genome sequence was revealed, no hemolin orthologue could be detected. Finally, an extensive Drosophila genome chip analysis was initiated. The goal was to investigate the Drosophila immune response, and, in contrast to earlier studies of artificially injected flies, to examine a set of natural microbes, orally and externally applied. In parallel experiments viruses, bacteria, fungi and parasites were compared to unchallenged controls. We obtained a unique set of genes that were up-regulated in the response to the parasite Octosporea muscadomesticae and to the fungus Beauveria bassiana. We expect both down-regulated and up-regulated genes to serve as a source for the discovery of new effector molecules, in particular those that are active against parasites and fungi.

A randomised determination of the effect of fluvastatin and atorvastatin on top of dual antiplatelet treatment on platelet aggregation after implantation of coronary drug-eluting stents. The EFA-Trial

Drug-drug interaction between statins metabolised by cytochrome P450 3A4 and clopidogrel have been claimed to attenuate the inhibitory effect of clopidogrel. However, published data regarding this drug-drug interaction are controversial. We aimed to determine the effect of fluvastatin and atorvastatin on the inhibitory effect of dual antiplatelet therapy with acetylsalicylic acid (ASA) and clopidogrel. One hundred one patients with symptomatic stable coronary artery disease undergoing percutaneous coronary intervention and drug-eluting stent implantation were enrolled in this prospective randomised study. After an interval of two weeks under dual antiplatelet therapy with ASA and clopidogrel, without any lipid-lowering drug, 87 patients were randomised to receive a treatment with either fluvastatin 80 mg daily or atorvastatin 40 mg daily in addition to the dual antiplatelet therapy for one month. Platelet aggregation was assessed using light transmission aggregometry and whole blood impedance platelet aggregometry prior to randomisation and after one month of receiving assigned statin and dual antiplatelet treatment. Platelet function assessment after one month of statin and dual antiplatelet therapy did not show a significant change in platelet aggregation from 1st to 2nd assessment for either statin group. There was also no difference between atorvastatin and fluvastatin treatment arms. In conclusion, neither atorvastatin 40 mg daily nor fluvastatin 80 mg daily administered in combination with standard dual antiplatelet therapy following coronary drug-eluting stent implantation significantly interfere with the antiaggregatory effect of ASA and clopidogrel.

European Working Group on Clinical Cell Analysis: Consensus protocol for the flow cytometric characterisation of platelet function

An increased or disturbed activation and aggregation of platelets plays a major role in the pathophysiology of thrombosis and haemostasis and is related to cardiovascular disease processes. In addition to qualitative disturbances of platelet function, changes in thrombopoiesis or an increased elimination of platelets, (e. g., in autoimmune thrombocytopenia), are also of major clinical relevance. Flow cytometry is increasingly used for the specific characterisation of phenotypic alterations of platelets which are related to cellular activation, haemostatic function and to maturation of precursor cells. These new techniques also allow the study of the in vitro response of platelets to stimuli and the modification thereof under platelet-targeted therapy as well as the characterisation of platelet-specific antibodies. In this protocol, specific flow cytometric techniques for platelet analysis are recommended based on a description of the current state of flow cytometric methodology. These recommendations are an attempt to promote the use of these new techniques which are at present broadly evaluated for diagnostic purposes. Furthermore, the definition of the still open questions primarily related to the technical details of the method should help to promote the multi-center evaluation of procedures with the goal to finally develop standardized operation procedures as the basis of interlaboratory reproducibility when applied to diagnostic testing.

Preemptive analgesia by lornoxicam--an NSAID--significantly inhibits perioperative platelet aggregation

BACKGROUND AND OBJECTIVE: To investigate whether preemptive administered lornoxicam changes perioperative platelet function during thoracic surgery. METHODS: A total of 20 patients scheduled for elective thoracic surgery were randomly assigned to receive either lornoxicam (16 mg, i.v.; n = 10) or placebo (n = 10) preoperatively. All patients underwent treatment of solitary lung metastasis and denied any antiplatelet medication within the past 2 weeks. Blood samples were drawn via an arterial catheter directly into silicone-coated Vacutainer tubes containing 0.5 mL of 0.129 M buffered sodium citrate 3.8% before, 15 min, 4 h and 8 h after the study medication was administered. Platelet aggregation curves were obtained by whole blood electrical impedance aggregometry (Chrono Log). RESULTS: Platelet aggregation was significantly reduced 15 min, 4 h and 8 h after lornoxicam administration compared to placebo (P < 0.05) for collagen, adenosine diphosphate and arachidonic acid as trigger substances. Adenosine diphosphate-induced platelet aggregation decreased by 85% 15 min after lornoxicam administration, and remained impaired for 8 h. CONCLUSION: Platelet aggregation assays are impaired for at least 8 h after lornoxicam application. Therefore perioperative analgesia by use of lornoxicam should be carefully administered under consideration of subsequent platelet dysfunction.

The effect of desmopressin on platelet function: a selective enhancement of procoagulant COAT platelets in patients with primary platelet function defects.

1-deamino-8-d-arginine vasopressin (desmopressin [DDAVP]) is clinically efficacious in patients with mild platelet function disorders but it is not known which mechanisms mediate this effect. Our aim was to evaluate the impact of in vivo DDAVP administration in these patients. We assessed von Willebrand factor (VWF), factor VIII, platelet activation and aggregation, platelet-dependent thrombin generation, and platelet intracellular Na(+)/Ca(2+) fluxes, before and 2 and 4 hours after DDAVP (0.3 µg/kg). We found (1) no significant changes for P-selectin expression, PAC-1 binding, δ-granule content and secretion, and platelet-aggregation; (2) significant decreases of secretion of α-granules and GPIIb-IIIa activation induced by adenosine 5'-diphosphate, convulxin, and thrombin; (3) significant increases of procoagulant platelets induced by convulxin/thrombin and platelet-dependent thrombin generation; and (4) significant increases of intracellular Na(+)/Ca(2+) concentrations. We show that in vivo DDAVP selectively and markedly enhances the ability to form procoagulant platelets and increases platelet-dependent thrombin generation by enhancing Na(+)/Ca(2+) mobilization. This report indicates that the beneficial hemostatic effect of DDAVP is not limited to an increase in large VWF multimers. An enhancement of platelet procoagulant activity appears to be an additional and (at least in platelet disorders) -possibly clinically relevant mechanism of DDAVP's action.

Defective pituitary function and gamete interactions in $\beta$1,4-galactosyltransferase null mice

Despite much attention, the function of oligosaccharide chains of glycoproteins remains largely unknown. Our understanding of oligosaccharide function in vivo has been limited to the use of reagents and targeted mutations that eliminate entire oligosaccharide chains. However, most, if not all biological functions for oligosaccharides have been attributed to specific terminal sequences on these oligosaccharides, yet there have been few studies to examine the consequences of modifying terminal oligosaccharide structures in vivo. To address this issue, mice were created bearing a targeted mutation in $\beta$1,4-galactosyltransferase, an enzyme responsible for elaboration of many of the proposed biologically-active carbohydrate epitopes. Most galactosyltransferase-null mice died within the first few weeks after birth and were characterized by stunted growth, thin skin, sparse hair, and dehydration. In addition, the adrenal cortices were poorly stratified and spermatogenesis was delayed. The few surviving adults had puffy skin (myxedema), difficulty delivering pups at birth (dystocia), and failed to lactate (agalactosis). All of these defects are consistant with endocrine insufficiency, which was confirmed by markedly decreased levels of serum thyroxine. The anterior pituitary gland appeared functionally delayed in newborn mutant mice, since the constituent cells were quiescent and nonsecretory, unlike that of control littermates. However, the anterior pituitary acquired a normal secretory phenotype during neonatal development, although it remained abnormally small and its glycoprotein hormones were devoid of $\beta$1,4-galactosyl residues. These results support in vitro studies suggesting that incomplete glycosylation of pituitary hormones leads to the creation of hormone antagonists that down regulate subsequent endocrine function producing polyglandular endocrine insufficiency. More surprisingly, the fact that some mice survive this neonatal period indicates the presence of a previously unrecognized compensatory pathway for glycoprotein hormone glycosylation and/or action.^ In addition to its well-studied biosynthetic function in the Golgi complex, a GalTase isoform is also expressed on the sperm surface where it functions as a gamete receptor during fertilization by binding to its oligosaccharide ligand on the egg coat glycoprotein, ZP3. Aggregation of GalTase by multivalent ZP3 oligosaccharides activates a G-protein cascade leading to the acrosome reaction. Although GalTase-null males are fertile, the mutant sperm bind less ZP3 than wild-type sperm, and are unable to undergo the acrosome reaction in response to either zona pellucida glycoproteins or to anti-GalTase anti-serum, as do wild-type sperm. However, mutant and wild-type sperm undergo the acrosome reaction normally in response to calcium ionophore which bypasses the requirement for ZP3 binding. Interestingly, the phenotype of the GalTase-null sperm is reciprocal to that of sperm that overexpress surface GalTAse and which bind more ZP3 leading to precocious acrosome reactions. These results confirm that GalTase functions as at least one of the sperm receptors for ZP3, and that GalTase participates in the ZP3-induced signal transduction pathway during zona pellucida-induced acrosome reactions. ^

Integrins in human T cell function: Transdominant regulation, conformational constraints, and intracellular effectors

Integrin adhesion molecules have both positive and negative potential in the regulation of peripheral blood T cell (PB T cell) activation, yet their mechanism of action in the mediation of human T lymphocyte function remains largely undefined. The goals of this study then were to elucidate integrin signaling mechanisms in PB T cells.^ By ligating $\beta$1 integrins with mAb 18D3, it was demonstrated that costimulation of PB T cell proliferation induced by coimmobilizing antibodies specific for $\beta$1, $\beta$2, and $\beta$7 integrin subfamilies in conjunction with the anti-CD3 mAb OKT3 was inhibited. Costimulation of T cell proliferation induced by non-integrins CD4, CD26, CD28, CD44, CD45RA, or CD45RO was unaffected. Inhibition of costimulation correlated with diminished IL-2 production. In his manner, $\beta$1 integrins could regulate heterologous integrins of the $\beta$2 and $\beta$7 subfamilies in a transdominant fashion. It was also demonstrated that integrin costimulation of T cell activation was acutely sensitive to the structural conformation of $\beta$1 integrins. Using the cyclic hexapeptide CWLDVC (TBC772, which is based on the $\alpha4\beta1$ integrin binding site in fibronectin) in soluble form, it was shown that integrins locked into a conformation displaying a neo-epitope called the ligand induced binding site (LIBS) recognized by mAb 15/7 were inhibited from sending mitogenic signals to T cells. When BSA-conjugated TBC772 was coimmobilized with anti-CD3 mAb OKT3, costimulation of proliferation occurred. This suggested that temporally uncoupling integrin receptor occupancy from receptor crosslinking inhibited $\beta$1 integrin signaling mechanisms. When subsets of PB T cells were examined to determine those initially activated by integrins within 6 hours of activation, costimulation induced intracellular accumulation of IL-2 predominantly in the CD4$\sp+$ and CD45RO$\sp+$ T cell subsets. This was similar to a number of PB T cell costimulatory molecules including CD26, CD43, CD44. Only CD28 costimulated IL-2 production from both CD45RA$\sp+$ and CD45RO$\sp+$ subpopulations.^ The GTPase Rho has been implicated in regulating integrin mediated stress fiber formation and anchorage dependent growth in fibroblasts, so studies were initiated to determine if Rho played a role in integrin dependent T cell function. In order to perform this, a technique based on scrape-loading was developed to incorporate macromolecules into PB T cells that maintained their functional activity. With this technique, C3 exoenzyme from Clostridium botulinum was incorporated into PB T cells. C3 ADP-ribosylates Rho proteins on Asn$\sp{41},$ which is in close proximity to the Rho effector domain, rendering it inactive. It was demonstrated that functional Rho is not required for basal or upregulated PB T cell adhesion to $\beta$1 integrin substrates, however PB T cell homotypic aggregation induced by PMA, which is an event mediated predominantly by the integrin $\rm\alpha L\beta2,$ was delayed. PB T cells lacking Rho function displayed altered cell morphology on $\beta$1 integrin ligands, producing stellate, dendritic-like pseudopodia. Rho activity was also found to be required for integrin dependent costimulation of proliferation. When intracellular accumulation of IL-2 was measured, inactivation of Rho prevented both integrin and CD28 costimulatory activity. Rho was identified to lie upstream of signals mediating PKC activation and Ca$\sp{++}$ fluxes, as PMA and ionomycin activation of PB T cells was unaffected by the inactivation of Rho. ^

Identification of platelet function defects by multi-parameter assessment of thrombus formation.

Assays measuring platelet aggregation (thrombus formation) at arterial shear rate mostly use collagen as only platelet-adhesive surface. Here we report a multi-surface and multi-parameter flow assay to characterize thrombus formation in whole blood from healthy subjects and patients with platelet function deficiencies. A systematic comparison is made of 52 adhesive surfaces with components activating the main platelet-adhesive receptors, and of eight output parameters reflecting distinct stages of thrombus formation. Three types of thrombus formation can be identified with a predicted hierarchy of the following receptors: glycoprotein (GP)VI, C-type lectin-like receptor-2 (CLEC-2)>GPIb>α6β1, αIIbβ3>α2β1>CD36, α5β1, αvβ3. Application with patient blood reveals distinct abnormalities in thrombus formation in patients with severe combined immune deficiency, Glanzmann's thrombasthenia, Hermansky-Pudlak syndrome, May-Hegglin anomaly or grey platelet syndrome. We suggest this test may be useful for the diagnosis of patients with suspected bleeding disorders or a pro-thrombotic tendency.

Toxicity of eosinophil MBP is repressed by intracellular crystallization and promoted by extracellular aggregation.

Eosinophils are white blood cells that function in innate immunity and participate in the pathogenesis of various inflammatory and neoplastic disorders. Their secretory granules contain four cytotoxic proteins, including the eosinophil major basic protein (MBP-1). How MBP-1 toxicity is controlled within the eosinophil itself and activated upon extracellular release is unknown. Here we show how intragranular MBP-1 nanocrystals restrain toxicity, enabling its safe storage, and characterize them with an X-ray-free electron laser. Following eosinophil activation, MBP-1 toxicity is triggered by granule acidification, followed by extracellular aggregation, which mediates the damage to pathogens and host cells. Larger non-toxic amyloid plaques are also present in tissues of eosinophilic patients in a feedback mechanism that likely limits tissue damage under pathological conditions of MBP-1 oversecretion. Our results suggest that MBP-1 aggregation is important for innate immunity and immunopathology mediated by eosinophils and clarify how its polymorphic self-association pathways regulate toxicity intra- and extracellularly.

In vitro effects of 3 % hypertonic saline and 20 % mannitol on canine whole blood coagulation and platelet function.

BACKGROUND: Hyperosmolar therapy, using either mannitol or hypertonic saline (HTS), is considered the treatment of choice for intracranial hypertension. However, hyperosmolar agents may impair coagulation and platelet function, limiting their use in patients at risk for hemorrhage. Despite this, studies evaluating the effects of mannitol compared to other hyperosmolar agents in dogs are largely lacking. The aim of this study was to compare the in vitro effects on global hemostasis and platelet function of 20 % mannitol and 3 % HTS on canine blood. METHODS: Citrated whole blood from 15 healthy dogs was diluted with 0.9 % saline, 20 % mannitol and 3 % HTS in ratios of 1:16 and 1:8. Rotational thromboelastometry (ROTEM) was used to assess clotting time (CT), clot formation time (CFT) and maximal clot firmness (MCF) following extrinsic activation (Ex-tem) and after platelet inhibition (Fib-tem). A platelet function analyzer (PFA-100) was used to assess closure time (CtPFA). RESULTS: No significant differences were observed between untreated whole blood and samples diluted with saline. Samples diluted with both mannitol and HTS were hypocoagulable compared to untreated whole blood samples. At a dilution of 1:16, no significant differences were found between any measured parameter in samples diluted with saline compared to mannitol or HTS. At a 1:8 dilution, CtPFA was prolonged in samples diluted with mannitol and HTS compared to saline, and CtPFA was prolonged more with mannitol than HTS. Ex-tem CT was increased at a 1:8 dilution with mannitol compared to HTS. Ex-tem CFT was prolonged at a 1:8 dilution with both agents compared to saline, and was prolonged more with mannitol than HTS. Ex-tem MCF was reduced at a 1:8 dilution with both agents compared to saline. DISCUSSION AND CONCLUSIONS: Data in this study indicate that both mannitol and HTS affect canine platelet function and whole blood coagulation in vitro in a dose-dependent fashion. The most pronounced effects were observed after high dilutions with mannitol, which impaired platelet aggregation, clot formation time, clot strength, and fibrin formation significantly more than HTS. Further in vivo studies are necessary before recommendations can be made

Sphingosine 1-Phosphate Produced by Sphingosine Kinase 2 Intrinsically Controls Platelet Aggregation In Vitro and In Vivo

RATIONALE Platelets are known to play a crucial role in hemostasis. Sphingosine kinases (Sphk) 1 and 2 catalyze the conversion of sphingosine to the bioactive metabolite sphingosine 1-phosphate (S1P). Although platelets are able to secrete S1P on activation, little is known about a potential intrinsic effect of S1P on platelet function. OBJECTIVE To investigate the role of Sphk1- and Sphk2-derived S1P in the regulation of platelet function. METHODS AND RESULTS We found a 100-fold reduction in intracellular S1P levels in platelets derived from Sphk2(-/-) mutants compared with Sphk1(-/-) or wild-type mice, as analyzed by mass spectrometry. Sphk2(-/-) platelets also failed to secrete S1P on stimulation. Blood from Sphk2-deficient mice showed decreased aggregation after protease-activated receptor 4-peptide and adenosine diphosphate stimulation in vitro, as assessed by whole blood impedance aggregometry. We revealed that S1P controls platelet aggregation via the sphingosine 1-phosphate receptor 1 through modulation of protease-activated receptor 4-peptide and adenosine diphosphate-induced platelet activation. Finally, we show by intravital microscopy that defective platelet aggregation in Sphk2-deficient mice translates into reduced arterial thrombus stability in vivo. CONCLUSIONS We demonstrate that Sphk2 is the major Sphk isoform responsible for the generation of S1P in platelets and plays a pivotal intrinsic role in the control of platelet activation. Correspondingly, Sphk2-deficient mice are protected from arterial thrombosis after vascular injury, but have normal bleeding times. Targeting this pathway could therefore present a new therapeutic strategy to prevent thrombosis.

Input Aggregation in Models of Data Envelopment Analysis: A Statistical Test with an Application to Indian Manufacturing

A problem frequently encountered in Data Envelopment Analysis (DEA) is that the total number of inputs and outputs included tend to be too many relative to the sample size. One way to counter this problem is to combine several inputs (or outputs) into (meaningful) aggregate variables reducing thereby the dimension of the input (or output) vector. A direct effect of input aggregation is to reduce the number of constraints. This, in its turn, alters the optimal value of the objective function. In this paper, we show how a statistical test proposed by Banker (1993) may be applied to test the validity of a specific way of aggregating several inputs. An empirical application using data from Indian manufacturing for the year 2002-03 is included as an example of the proposed test.

Intraoperative changes in platelet function in relation to moderate haemorrhage

Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved. Funding The study was funded by TENOVUS Scotland (G12/14). Jan Jansen is in receipt of salary support through the NHS Research Scotland (NRS) fellowship scheme. Acknowledgements The authors are extremely grateful for the expert assistance and contributions of Dr Neil Scott (Medical Statistician, University of Aberdeen), Win Culley (Research Nurse, Woodend Hospital), Dr Karen Cranfield (Consultant Anaesthetist), and Ms Sharon Wood, (Research Technician, Rowett Institute of Nutrition and Health) for fibrinogen measurement.