Microchip electrophoresis with amperometric detection for the study of the generation of nitric oxide by NONOate salts

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Microchip electrophoresis (ME) with electrochemical detection was used to monitor nitric oxide (NO) production from diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO) and 1-(hydroxyl-NNO-azoxy)-l-proline disodium salt (PROLI/NO). NO was generated through acid hydrolysis of these NONOate salts. The products of acid hydrolysis were introduced into a 5-cm separation channel using gated injection. The separation was accomplished using reverse polarity and a background electrolyte consisting of 10 mM boric acid and 2 mM tetradecyltrimethylammonium bromide, pH 11. Electrochemical detection was performed using an isolated potentiostat in an in-channel configuration. Potentials applied to the working electrode, typically higher than +1.0 V vs. Ag/AgCl, allowed the direct detection of nitrite, NO, DEA/NO, and PROLI/NO. Baseline resolution was achieved for the separation of PROLI/NO and NO while resolution between DEA/NO and NO was poor (1.0 +/- 0.2). Nitrite was present in all samples tested.403823772384Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)NIH (USA) [R01 NS042929, R21 NS061202]American Heart Association (USA)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [2010/01046-6, 2008/57805-2, 2008/53868-0]NIH (USA) [R01 NS042929, R21 NS061202

An Integrated Microfluidic Device for Monitoring Changes in Nitric Oxide Production in Single T-Lymphocyte (Jurkat) Cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)A considerable amount of attention has been focused on the analysis of single cells in an effort to better understand cell heterogeneity in cancer and neurodegenerative diseases. Although microfluidic devices have several advantages for single cell analysis, few papers have actually demonstrated the ability of these devices to monitor chemical changes in perturbed biological systems. In this paper, a new microfluidic channel manifold is described that integrates cell transport, lysis, injection, electrophoretic separation, and fluorescence detection into a single device, making it possible to analyze individual cells at a rate of 10 cells/min in an automated fashion. The system was employed to measure nitric oxide (NO) production in single T-lymphocytes (Jurkat cells) using a fluorescent marker, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA). The cells were also labeled with 6-carboxyfluorescein diacetate (6-CFDA) as an internal standard. The NO production by control cells was compared to that of cells stimulated using lipopolysaccharide (LPS), which is known to cause the expression of inducible nitric oxide synthase (iNOS) in immune-type cells. Statistical analysis of the resulting electropherograms from a population of cells indicated a 2-fold increase in NO production in the induced cells. These results compare nicely to a recently published bulk cell analysis of NO.85211018810195NIH [R21NS061202]Terry Johnson Cancer Center, Kansas State UniversityDOD ASSURE [CHE-1004991]University of Catania, ItalyAmerican Heart AssociationFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)NIH [R21NS061202]DOD ASSURE [CHE-1004991]FAPESP [2010/01046-6

Monitoring intracellular nitric oxide production using microchip electrophoresis and laser-induced fluorescence detection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Nitric oxide (NO) is a biologically important short-lived reactive species that has been shown to be involved in a large number of physiological processes. The production of NO is substantially increased in immune and other cell types through the upregulation of inducible nitric oxide synthase (iNOS) caused by exposure to stimulating agents such as lipopolysaccharide (LPS). NO production in cells is most frequently measured via fluorescence microscopy using diaminofluorescein-based probes. Capillary electrophoresis with laser-induced fluorescence detection has been used previously to separate and quantitate the fluorescence derivatives of NO from potential interferences in single neurons. In this paper, microchip electrophoresis (ME) coupled to laser-induced fluorescence (LIF) detection is evaluated as a method for measurement of the NO production by Jurkat cells under control and stimulating conditions. ME is ideal for such analyses due to its fast and efficient separations, low volume requirements, and ultimate compatibility with single cell chemical cytometry systems. In these studies, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) was employed for the detection of NO, and 6-carboxyfluorescein diacetate (6-CFDA) was employed as an internal standard. Jurkat cells were stimulated using lipopolysaccharide (LPS) to produce NO, and bulk cell analysis was accomplished using ME-LIF. Stimulated cells exhibited an approximately 2.5-fold increase in intracellular NO production compared to the native cells. A NO standard prepared using diethylamine NONOate (DEA/NO) salt was used to construct a calibration curve for quantitation of NO in cell lysate. Using this calibration curve, the average intracellular NO concentrations for LPS-stimulated and native Jurkat cells were calculated to be 1.5 mM and 0.6 mM, respectively.42414420NIH [R01 NS042929, R21 NS061202]NIH NCRR [P20RR016475]Adams InstituteAmerican Heart AssociationUniversity of Catania, ItalyFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)NIH [R01 NS042929, R21 NS061202]NIH NCRR [P20RR016475]FAPESP [2010/010466

Smart University for Sustainable Governance in Smart Local Service Systems

From the Service Science perspective, our work tends to provide a conceptual and methodological contribution to affirm the role of the university as the responsible agent for the growth and development of a local area. In this sense, the purpose is to promote a harmonious growth of the whole local service system, focused on academic quality and accountability through the approach to Social Responsibility, also involving government, business and society. The University as a place for higher education and research, at the same time, represents a privileged space of convergence of different growth perspectives of the local actors. This convergence should be seen as a “place” to share and develop a common sense of value that is smart, ethically, socially, and economically sustainable, i.e. a Smart Local Service System (S-LSS)