Highly Sensitive Determination of Hydrogen Peroxide and Glucose by Fluorescence Correlation Spectroscopy

BACKGROUND: Because H(2)O(2) is generated by various oxidase-catalyzed reactions, a highly sensitive determination method of H(2)O(2) is applicable to measurements of low levels of various oxidases and their substrates such as glucose, lactate, glutamate, urate, xanthine, choline, cholesterol and NADPH. We propose herein a new, highly sensitive method for the measurement of H(2)O(2) and glucose using fluorescence correlation spectroscopy (FCS). METHODOLOGY/PRINCIPAL FINDINGS: FCS has the advantage of allowing us to determine the number of fluorescent molecules. FCS measures the fluctuations in fluorescence intensity caused by fluorescent probe movement in a small light cavity with a defined volume generated by confocal illumination. We thus developed a highly sensitive determination system of H(2)O(2) by FCS, where horseradish peroxidase (HRP) catalyzes the formation of a covalent bond between fluorescent molecules and proteins in the presence of H(2)O(2). Our developed system gave a linear calibration curve for H(2)O(2) in the range of 28 to 300 nM with the detection limit of 8 nM. In addition, by coupling with glucose oxidase (GOD)-catalyzed reaction, the method allows to measure glucose in the range of 80 nM to 1.5 µM with detection limit of 24 nM. The method was applicable to the assay of glucose in blood plasma. The mean concentration of glucose in normal human blood plasma was determined to be 4.9 mM. CONCLUSIONS/SIGNIFICANCE: In comparison with commercial available methods, the detection limit and the minimum value of determination for glucose are at least 2 orders of magnitude more sensitive in our system. Such a highly sensitive method leads the fact that only a very small amount of plasma (20 nL) is needed for the determination of glucose concentration in blood plasma

Illuminating the life of GPCRs

The investigation of biological systems highly depends on the possibilities that allow scientists to visualize and quantify biomolecules and their related activities in real-time and non-invasively. G-protein coupled receptors represent a family of very dynamic and highly regulated transmembrane proteins that are involved in various important physiological processes. Since their localization is not confined to the cell surface they have been a very attractive "moving target" and the understanding of their intracellular pathways as well as the identified protein-protein-interactions has had implications for therapeutic interventions. Recent and ongoing advances in both the establishment of a variety of labeling methods and the improvement of measuring and analyzing instrumentation, have made fluorescence techniques to an indispensable tool for GPCR imaging. The illumination of their complex life cycle, which includes receptor biosynthesis, membrane targeting, ligand binding, signaling, internalization, recycling and degradation, will provide new insights into the relationship between spatial receptor distribution and function. This review covers the existing technologies to track GPCRs in living cells. Fluorescent ligands, antibodies, auto-fluorescent proteins as well as the evolving technologies for chemical labeling with peptide- and protein-tags are described and their major applications concerning the GPCR life cycle are presented

Aligning evidence generation and use across health, development, and environment

© 2019 The Authors Although health, development, and environment challenges are interconnected, evidence remains fractured across sectors due to methodological and conceptual differences in research and practice. Aligned methods are needed to support Sustainable Development Goal advances and similar agendas. The Bridge Collaborative, an emergent research-practice collaboration, presents principles and recommendations that help harmonize methods for evidence generation and use. Recommendations were generated in the context of designing and evaluating evidence of impact for interventions related to five global challenges (stabilizing the global climate, making food production sustainable, decreasing air pollution and respiratory disease, improving sanitation and water security, and solving hunger and malnutrition) and serve as a starting point for further iteration and testing in a broader set of contexts and disciplines. We adopted six principles and emphasize three methodological recommendations: (1) creation of compatible results chains, (2) consideration of all relevant types of evidence, and (3) evaluation of strength of evidence using a unified rubric. We provide detailed suggestions for how these recommendations can be applied in practice, streamlining efforts to apply multi-objective approaches and/or synthesize evidence in multidisciplinary or transdisciplinary teams. These recommendations advance the necessary process of reconciling existing evidence standards in health, development, and environment, and initiate a common basis for integrated evidence generation and use in research, practice, and policy design

Molecular network analysis using reverse phase protein microarrays for patient tailored therapy

The practice of medicine has always aimed at individualized treatment of disease. The relationship between patient and physician has always been a personal one, and the physician's choice of treatment has been intended to be the best fit for the patient's needs. The necessary pooling/grouping of disease families and their assignment to a number of drugs or treatment methods has, consequently, led to an increase in the number of effective therapies. However, given the heterogeneity of most human diseases, and cancer specifically, it is currently impossible for the treating clinician to effectively predict a patient's response and outcome based on current technologies, much less the idiosyncratic resistances and adverse effects associated with the limited therapeutic options