A novel flow cytometry assay using dihydroethidium as redox-sensitive probe reveals NADPH oxidase-dependent generation of superoxide anion in human platelets exposed to amyloid peptide β

This is the author accepted manuscript. The final version is available from Taylor & Francis via the DOI in this recordReactive oxygen species (ROS) generation is critical in the regulation of platelets, which has important implications in the modulation of hemostasis and thrombosis. Nonetheless, despite several assays have been described and successfully utilized in the past, the analysis of ROS generation in human platelets remains challenging. Here we show that dihydroethidium (DHE) allows the characterization of redox responses upon platelet activation by physiological and pathological stimuli. In particular, the flow cytometry assay that we describe here allowed us to confirm that thrombin, collagen-related peptide (CRP) and arachidonic acid but not adenosine diphosphate (ADP) stimulate superoxide anion formation in a concentration-dependent manner. 0.1unit/ml thrombin, 3 μg/ml CRP and 30 μM arachidonic acid are commonly used to stimulate platelets in vitro and here were shown to stimulate a significant increase in superoxide anion formation. The ROS scavenger N-acetylcysteine (NAC) abolished superoxide anion generation in response to all tested stimuli, but the pan-NADPH oxidase (NOX) inhibitor VAS2870 only inhibited superoxide anion formation in response to thrombin and CRP. The involvement of NOXs in thrombin and CRP-dependent responses was confirmed by the inhibition of platelet aggregation induced by these stimuli by VAS2870, while platelet aggregation in response to arachidonic acid was insensitive to this inhibitor. In addition, the pathological platelet stimulus amyloid β (Aβ) 1–42 peptide induced superoxide anion formation in a concentration-dependent manner. Aβ peptide stimulated superoxide anion formation in a NOX-dependent manner, as proved by the use of VAS2870. Aβ 1–42 peptide displayed only moderate activity as an aggregation stimulus, but was able to significantly potentiate platelet aggregation in response to submaximal agonists concentrations, such as 0.03 unit/ml thrombin and 10 μM arachidonic acid. The inhibition of NOXs by 10 μM VAS2870 abolished Aβ-dependent potentiation of platelet aggregation in response to 10 μM arachidonic acid, suggesting that the pro-thrombotic activity of Aβ peptides depends on NOX activity. Similar experiments could not be performed with thrombin or collagen, as NOXs are required for the signaling induced by these stimuli. These findings shed some new light on the pro-thrombotic activity of Aβ peptides. In summary, here we describe a novel and reliable assay for the detection of superoxide anion in human platelets. This is particularly important for the investigation of the pathophysiological role of redox stress in platelets, a field of research of increasing importance, but hindered by the absence of a reliable and easily accessible ROS detection methodology applicable to platelets

A novel flow cytometry assay using dihydroethidium as redox-sensitive probe reveals NADPH oxidase-dependent generation of superoxide anion in human platelets exposed to amyloid peptide β

Reactive oxygen species (ROS) generation is critical in the regulation of platelets, which has important implications in the modulation of hemostasis and thrombosis. Nonetheless, despite several assays have been described and successfully utilized in the past, the analysis of ROS generation in human platelets remains challenging. Here we show that dihydroethidium (DHE) allows the characterization of redox responses upon platelet activation by physiological and pathological stimuli. In particular, the flow cytometry assay that we describe here allowed us to confirm that thrombin, collagen-related peptide (CRP) and arachidonic acid but not adenosine diphosphate (ADP) stimulate superoxide anion formation in a concentration-dependent manner. 0.1unit/ml thrombin, 3 μg/ml CRP and 30 μM arachidonic acid are commonly used to stimulate platelets in vitro and here were shown to stimulate a significant increase in superoxide anion formation. The ROS scavenger N-acetylcysteine (NAC) abolished superoxide anion generation in response to all tested stimuli, but the pan-NADPH oxidase (NOX) inhibitor VAS2870 only inhibited superoxide anion formation in response to thrombin and CRP. The involvement of NOXs in thrombin and CRP-dependent responses was confirmed by the inhibition of platelet aggregation induced by these stimuli by VAS2870, while platelet aggregation in response to arachidonic acid was insensitive to this inhibitor. In addition, the pathological platelet stimulus amyloid β (Aβ) 1–42 peptide induced superoxide anion formation in a concentration-dependent manner. Aβ peptide stimulated superoxide anion formation in a NOX-dependent manner, as proved by the use of VAS2870. Aβ 1–42 peptide displayed only moderate activity as an aggregation stimulus, but was able to significantly potentiate platelet aggregation in response to submaximal agonists concentrations, such as 0.03 unit/ml thrombin and 10 μM arachidonic acid. The inhibition of NOXs by 10 μM VAS2870 abolished Aβ-dependent potentiation of platelet aggregation in response to 10 μM arachidonic acid, suggesting that the pro-thrombotic activity of Aβ peptides depends on NOX activity. Similar experiments could not be performed with thrombin or collagen, as NOXs are required for the signaling induced by these stimuli. These findings shed some new light on the pro-thrombotic activity of Aβ peptides. In summary, here we describe a novel and reliable assay for the detection of superoxide anion in human platelets. This is particularly important for the investigation of the pathophysiological role of redox stress in platelets, a field of research of increasing importance, but hindered by the absence of a reliable and easily accessible ROS detection methodology applicable to platelets

Amyloid peptide β1-42 induces integrin αIIbβ3 activation, platelet adhesion and thrombus formation in a NADPH oxidase-dependent manner

This is the final version. Available on open access from Hindawi Publishing Corporation via the DOI in this recordThe progression of Alzheimer’s dementia is associated with neurovasculature impairment, which includes inflammation, microthromboses, and reduced cerebral blood flow. Here, we investigate the effects of β amyloid peptides on the function of platelets, the cells driving haemostasis. Amyloid peptide β1-42 (Aβ1-42), Aβ1-40, and Aβ25-35 were tested in static adhesion experiments, and it was found that platelets preferentially adhere to Aβ1-42 compared to other Aβ peptides. In addition, significant platelet spreading was observed over Aβ1-42, while Aβ1-40, Aβ25-35, and the scAβ1-42 control did not seem to induce any platelet spreading, which suggested that only Aβ1-42 activates platelet signalling in our experimental conditions. Aβ1-42 also induced significant platelet adhesion and thrombus formation in whole blood under venous flow condition, while other Aβ peptides did not. The molecular mechanism of Aβ1-42 was investigated by flow cytometry, which revealed that this peptide induces a significant activation of integrin αIIbβ3, but does not induce platelet degranulation (as measured by P-selectin membrane translocation). Finally, Aβ1-42 treatment of human platelets led to detectable levels of protein kinase C (PKC) activation and tyrosine phosphorylation, which are hallmarks of platelet signalling. Interestingly, the NADPH oxidase (NOX) inhibitor VAS2870 completely abolished Aβ1-42-dependent platelet adhesion in static conditions, thrombus formation in physiological flow conditions, integrin αIIbβ3 activation, and tyrosine- and PKC-dependent platelet signalling. In summary, this study highlights the importance of NOXs in the activation of platelets in response to amyloid peptide β1-42. The molecular mechanisms described in this manuscript may play an important role in the neurovascular impairment observed in Alzheimer’s patients.Alzheimer´s Research UKBritish Heart FoundationNational Institute for Health Research (NIHR

Amyloid peptide β 1-42 induces integrin α IIb β 3 activation, platelet adhesion, and thrombus formation in a NADPH Oxidase-Dependent Manner

The progression of Alzheimer's dementia is associated with neurovasculature impairment, which includes inflammation, microthromboses, and reduced cerebral blood flow. Here, we investigate the effects of β amyloid peptides on the function of platelets, the cells driving haemostasis. Amyloid peptide β1-42 (Aβ1-42), Aβ1-40, and Aβ25-35 were tested in static adhesion experiments, and it was found that platelets preferentially adhere to Aβ1-42 compared to other Aβ peptides. In addition, significant platelet spreading was observed over Aβ1-42, while Aβ1-40, Aβ25-35, and the scAβ1-42 control did not seem to induce any platelet spreading, which suggested that only Aβ1-42 activates platelet signalling in our experimental conditions. Aβ1-42 also induced significant platelet adhesion and thrombus formation in whole blood under venous flow condition, while other Aβ peptides did not. The molecular mechanism of Aβ1-42 was investigated by flow cytometry, which revealed that this peptide induces a significant activation of integrin αIIbβ3, but does not induce platelet degranulation (as measured by P-selectin membrane translocation). Finally, Aβ1-42 treatment of human platelets led to detectable levels of protein kinase C (PKC) activation and tyrosine phosphorylation, which are hallmarks of platelet signalling. Interestingly, the NADPH oxidase (NOX) inhibitor VAS2870 completely abolished Aβ1-42-dependent platelet adhesion in static conditions, thrombus formation in physiological flow conditions, integrin αIIbβ3 activation, and tyrosine- and PKC-dependent platelet signalling. In summary, this study highlights the importance of NOXs in the activation of platelets in response to amyloid peptide β1-42. The molecular mechanisms described in this manuscript may play an important role in the neurovascular impairment observed in Alzheimer's patients

Longitudinally diode-pumped Nd:YAG double-clad planar waveguide laser

We report the demonstration of a near-diffraction-limited, compact, diode-end-pumped double-clad planar waveguide Nd:YAG laser. Efficient laser operation was achieved for the three dominant Nd3+ transitions at 1.064µm 0.946µm, and 1.32µm, with TE polarised output powers of 1.33W, 0.57W, and 0.33W for the available output couplers. The output beam from the monolithic plane-plane laser cavity had measured M2 values of 1.0 and 1.8, perpendicular and parallel to the plane of the waveguide respectively

Adatom Fe(III) on the hematite surface: Observation of a key reactive surface species

The reactivity of a mineral surface is determined by the variety and population of different types of surface sites (e.g., step, kink, adatom, and defect sites). The concept of "adsorbed nutrient" has been built into crystal growth theories, and many other studies of mineral surface reactivity appeal to ill-defined "active sites." Despite their theoretical importance, there has been little direct experimental or analytical investigation of the structure and properties of such species. Here, we use ex-situ and in-situ scanning tunneling microcopy (STM) combined with calculated images based on a resonant tunneling model to show that observed nonperiodic protrusions and depressions on the hematite (001) surface can be explained as Fe in an adsorbed or adatom state occupying sites different from those that result from simple termination of the bulk mineral. The number of such sites varies with sample preparation history, consistent with their removal from the surface in low pH solutions

Multiple Sexual Partners and Condom use among 10 - 19 Year-olds in four Districts in Tanzania: What do we Learn?

Although some studies in Tanzania have addressed the question of sexuality and STIs among adolescents, mostly those aged 15 - 19 years, evidence on how multiple sexual partners influence condom use among 10 - 19 year-olds is limited. This study attempts to bridge this gap by testing a hypothesis that sexual relationships with multiple partners in the age group 10 - 19 years spurs condom use during sex in four districts in Tanzania. Secondary analysis was performed using data from the Adolescents Module of the cross-sectional household survey on Maternal, Newborn and Child Health (MNCH) that was done in Kigoma, Kilombero, Rufiji and Ulanga districts, Tanzania in 2008. A total of 612 adolescents resulting from a random sample of 1200 households participated in this study. Pearson Chi-Square was used as a test of association between multiple sexual partners and condom use. Multivariate logistic regression model was fitted to the data to assess the effect of multiple sexual partners on condom use, having adjusted for potential confounding variables. STATA (10) statistical software was used to carry out this process at 5% two-sided significance level. Of the 612 adolescents interviewed, 23.4% reported being sexually active and 42.0% of these reported having had multiple (> 1) sexual partners in the last 12 months. The overall prevalence of condom use among them was 39.2%. The proportion using a condom at the last sexual intercourse was higher among those who knew that they can get a condom if they want than those who did not. No evidence of association was found between multiple sexual partners and condom use (OR = 0.77, 95% CI = 0.35 - 1.67, P = 0.504). With younger adolescents (10 - 14 years) being a reference, condom use was associated with age group (15 - 19: OR = 3.69, 95% CI = 1.21 - 11.25, P = 0.022) and district of residence (Kigoma: OR = 7.45, 95% CI = 1.79 - 31.06, P = 0.006; Kilombero: OR = 8.89, 95% CI = 2.91 - 27.21, P < 0.001; Ulanga: OR = 5.88, 95% CI = 2.00 - 17.31, P = 0.001), Rufiji being a reference category. No evidence of association was found between multiple sexual partners and condom use among adolescents in the study area. The large proportion of adolescents who engage in sexual activity without using condoms, even those with multiple partners, perpetuates the risk of transmission of HIV infections in the community. Strategies such as sex education and easing access to and making a friendly environment for condom availability are important to address the risky sexual behaviour among adolescents

Slow relaxation in the two dimensional electron plasma under the strong magnetic field

We study slow relaxation processes in the point vortex model for the two-dimensional pure electron plasma under the strong magnetic field. By numerical simulations, it is shown that, from an initial state, the system undergoes the fast relaxation to a quasi-stationary state, and then goes through the slow relaxation to reach a final state. From analysis of simulation data, we find (i) the time scale of the slow relaxation increases linearly to the number of electrons if it is measured by the unit of the bulk rotation time, (ii) during the slow relaxation process, each electron undergoes an superdiffusive motion, and (iii) the superdiffusive motion can be regarded as the Levy flight, whose step size distribution is of the power law. The time scale that each electron diffuses over the system size turns out to be much shorter than that of the slow relaxation, which suggests that the correlation among the superdiffusive trajectories is important in the slow relaxation process.Comment: 11pages, 19 figures. Submitted to J. Phys. Soc. Jp