A DNA-based fluorescent reporter maps HOCl production in the maturing phagosome.

Phagocytes destroy pathogens by trapping them in a transient organelle called the phagosome, where they are bombarded with reactive oxygen species (ROS) and reactive nitrogen species (RNS). Imaging reactive species within the phagosome would directly reveal the chemical dynamics underlying pathogen destruction. Here we introduce a fluorescent, DNA-based combination reporter, cHOClate, which simultaneously images hypochlorous acid (HOCl) and pH quantitatively. Using cHOClate targeted to phagosomes in live cells, we successfully map phagosomal production of a specific ROS, HOCl, as a function of phagosome maturation. We found that phagosomal acidification was gradual in macrophages and upon completion, HOCl was released in a burst. This revealed that phagosome-lysosome fusion was essential not only for phagosome acidification, but also for providing the chloride necessary for myeloperoxidase activity. This method can be expanded to image several kinds of ROS and RNS and be readily applied to identify how resistant pathogens evade phagosomal killing.

Determination of the pKa Values of trans-Resveratrol, a Triphenolic Stilbene, by Singular Value Decomposition. Comparison with Theory.

Several independent determinations of the pKa values of trans-resveratrol in water have led to conflicting results. Singular value decomposition analysis of UV absorption spectra of trans-resveratrol (t-Resv) in N2-outgased aqueous solutions buffered to pH values in the 7.0-13.6 range yielded the UV spectra of the three anionic forms and the corresponding pKa values: pKa1 = 9.16, pKa2 = 9.77, and pKa3 = 10.55 in very good agreement with calculated theoretical values. The analysis of the absorption spectra guided the assignment of the fluorescence spectrum of each anionic form. With the resolved spectra on hand, we applied the Förster equation to estimate pKa* values of 2.5 and 0, respectively, for the p- and m-OH substituents of t-Resv in S1. Theory supports a proposed mechanism for the reaction of t-Resv anions with O2.