This study describes the use of methylene blue (MB) plus light (photodynamic inactivation, PDI) in the presence of hydrogen peroxide (H 2 O 2 ) to kill Staphylococcus aureus, Escherichia coli, and Candida albicans. When H 2 O 2 was added to MB plus light there was an increased antimicrobial effect, which could be due to a change in the type of ROS generated or increased microbial uptake of MB. To clarify the mechanism, the production of ROS was investigated in the presence and absence of H 2 O 2 . It was observed that ROS production was almost inhibited by the presence of H 2 O 2 when cells were not present. In addition, experiments using different sequence combinations of MB and H 2 O 2 were performed and MB optical properties inside the cell were analyzed. Spectroscopy experiments suggested that the amount of MB was higher inside the cells when H 2 O 2 was used before or simultaneously with PDI, and ROS formation inside C. albicans cells confirmed that ROS production is higher in the presence of H 2 O 2 . Moreover enzymatic reduction of MB by E. coli during photosensitizer uptake to the photochemically inactive leucoMB could be reversed by the oxidative effects of hydrogen peroxide, increasing ROS formation inside the microorganism. Therefore, the combination of a photosensitizer such as MB and H 2 O 2 is an interesting approach to improve PDI efficiency.

There are controversial reports in the literature concerning the reactivity of singlet oxygen ((1)O(2)) with the redox probe 2',7'-dichlorodihydrofluorescein (DCFH). By carefully preparing solutions in which (1)O(2) is quantitatively generated in the presence of DCFH, we were able to show that the formation rate of the fluorescent molecule derived from DCFH oxidation, which is 2',7'-dichlorofluorescein (DCF), increases in D(2)O and decreases in sodium azide, proving the direct role of (1)O(2) in this process. We have also prepared solutions in which either (1)O(2) or dication (MB(2+)) and semi-reduced (MB) radicals of the sensitizer and subsequently super-oxide radical (O(2)(-)) are generated. The absence of any effect of SOD and catalase ruled out the DCFH oxidation by O(2)(-), indicating that both (1)O(2) and MB(2+) react with DCFH. Although the formation of DCF was 1 order of magnitude larger in the presence of MB(2+) than in the presence of (1)O(2), considering the rate of spontaneous decays of these species in aqueous solution, we were able to conclude that the reactivity of (1)O(2) with DCFH is actually larger than that of MB(2+). We conclude that DCFH can continue to be used as a probe to monitor general redox misbalance induced in biologic systems by oxidizing radicals and (1)O(2).

The photodynamic effects of the cationic TMPyP (meso-tetrakis [N-methyl-4-pyridyl]porphyrin) and the anionic TPPS4 (meso-tetrakis[4-sulfonatophenyl]porphyrin) against PC/CL phosphatidylcholine/cardiolipin (85/15%) membranes were probed to address the influence of phorphyrin binding on lipid damage. Electronic absorption spectroscopy and zeta potential measurements demonstrated that only TMPyP binds to PC/CL large unilamellar vesicles (LUVs). The photodamage after irradiation with visible light was analyzed by dosages of lipid peroxides (LOOH) and thiobarbituric reactive substance and by a contrast phase image of the giant unilamellar vesicles (GUVs). Damage to LUVs and GUVs promoted by TMPyP and TPPS4 were qualitatively and quantitatively different. The cationic porphyrin promoted damage more extensive and faster. The increase in LOOH was higher in the presence of D2O, and was impaired by sodium azide and sorbic acid. The effect of D2O was higher for TPPS4 as the photosensitizer. The use of DCFH demonstrated that liposomes prevent the photobleaching of TMPyP. The results are consistent with a more stable TMPyP that generates long-lived singlet oxygen preferentially partitioned in the bilayer. Conversely, TPPS4 generates singlet oxygen in the bulk whose lifetime is increased in D2O. Therefore, the affinity of the porphyrin to the membrane modulates the rate, type and degree of lipid damage.

The aim of this work was to determine the oesophageal transit time (OTT) of a bolus using the biomagnetic technique and compare the results to those obtained by means of scintigraphy. For the biomagnetic evaluation, a test meal (yoghurt) uniformly labelled with 5 g of powder ferrite was swallowed in a single gulp by 19 normal volunteers in the upright position. One sensor (first order gradiometer) was placed at the furcula and a second one at the xiphoid process to detect the passage of the test meal and the magnetic signal output was recorded in a computer. The OTT was determined by plotting the voltage signal against time. The scintigraphic technique was used in the same volunteers: the test meal was labelled with less than 350 MBq of 99mTc-phytate and swallowed in the same way. The bolus transit was recorded at 4 frames s(-1) (100-120 frames acquisition) and the OTT was determined by drawing two regions of interest in the same areas as the sensors. The results were determined by plotting counts against time. The averages for OTTs were 3.8 +/- 0.8 s for the scintigraphic technique and 4.6 +/- 0.9 s for the biomagnetic technique. Although scintigraphic OTT was significantly shorter than magnetic OTT, there was a significant correlation between them. We conclude that the biomagnetic study may be used to evaluate OTT.

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