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Detailed Analysis of Apoptosis and Delayed Luminescence of Human Leukemia Jurkat T Cells after Proton Irradiation and Treatments with Oxidant Agents and Flavonoids

Following previous work, we investigated in more detail the relationship between apoptosis and delayed luminescence (DL) in human leukemia Jurkat T cells under a wide variety of treatments. We used menadione and hydrogen peroxide to induce oxidative stress and two flavonoids, quercetin, and epigallocatechin gallate, applied alone or in combination with menadione or H2O2. 62 MeV proton beams were used to irradiate cells under a uniform dose of 2 or 10 Gy, respectively. We assessed apoptosis, cell cycle distributions, and DL. Menadione, H2O2 and quercetin were potent inducers of apoptosis and DL inhibitors. Quercetin decreased clonogenic survival and the NAD(P)H level in a dose-dependent manner. Proton irradiation with 2 Gy but not 10 Gy increased the apoptotic rate. However, both doses induced a substantial G2/M arrest. Quercetin reduced apoptosis and prolonged the G2/M arrest induced by radiation. DL spectroscopy indicated that proton irradiation disrupted the electron flow within Complex I of the mitochondrial respiratory chain, thus explaining the massive necrosis induced by 10 Gy of protons and also suggested an equivalent action of menadione and quercetin at the level of the Fe/S center N2, which may be mediated by their binding to a common site within Complex I, probably the rotenone-binding site.

Mitochondrial respiratory complex I probed by delayed luminescence spectroscopy

Abstract. The role of mitochondrial complex I in ultraweak photon-induced delayed photon emission [delayed luminescence (DL)] of human leukemia Jurkat T cells was probed by using complex I targeting agents like rotenone, menadione, and quercetin. Rotenone, a complex I-specific inhibitor, dose-dependently increased the mitochondrial level of reduced nicotinamide adenine dinucleotide (NADH), decreased clonogenic survival, and induced apoptosis. A strong correlation was found between the mitochondrial levels of NADH and oxidized flavin mononucleotide (FMNox) in rotenone-, menadione- and quercetin-treated cells. Rotenone enhanced DL dose-dependently, whereas quercetin and menadione inhibited DL as well as NADH or FMNox. Collectively, the data suggest that DL of Jurkat cells originates mainly from mitochondrial complex I, which functions predominantly as a dimer and less frequently as a tetramer. In individual monomers, both pairs of pyridine nucleotide (NADH/reduced nicotinamide adenine dinucleotide phosphate) sites and flavin (FMN-a/FMN-b) sites appear to bind cooperatively their specific ligands. Enhancement of delayed red-light emission by rotenone suggests that the mean time for one-electron reduction of ubiquinone or FMN-a by the terminal Fe/S center (N2) is 20 or 284 μs, respectively. All these findings suggest that DL spectroscopy could be used as a reliable, sensitive, and robust technique to probe electron flow within complex I in situ.

Single Photon Avalanche Diodes: Towards the Large Bidimensional Arrays

Single photon detection is one of the most challenging goals of photonics. In recent years, the study of ultra-fast and/or low-intensity phenomena has received renewed attention from the academic and industrial communities. Intense research activity has been focused on bio-imaging applications, bio-luminescence, bio-scattering methods, and, more in general, on several applications requiring high speed operation and high timing resolution. In this paper we present design and characterization of bi-dimensional arrays of a next generation of single photon avalanche diodes (SPADs). Single photon sensitivity, dark noise, afterpulsing and timing resolution of the single SPAD have been examined in several experimental conditions. Moreover, the effects arising from their integration and the readout mode have also been deeply investigated.

A New Generation of SPAD—Single-Photon Avalanche Diodes

Design and characterization of a new generation of single-photon avalanche diodes (SPAD) array, manufactured by ST-Microelectronics in Catania, Italy, are presented. Device performances, investigated in several experimental conditions and here reported, demonstrate their suitability in many applications. SPADs are thin p-n junctions operating above the breakdown condition in Geiger mode at low voltage. In this regime a single charged carrier injected into the depleted layer can trigger a self-sustaining avalanche, originating a detectable signal. Dark counting rate at room temperature is down to 10 s-1 for devices with an active area of 10 mum in diameter, and 103 s-1 for those 50 mum wide. SPAD quantum efficiency, measured in the range 350-1050 nm, can be comparable to that of a typical silicon based detector and reaches the values of about 50% at 550 nm. Finally, the low production costs and the integration possibility are other favorable features in sight of highly dense integrated 1-D or 2-D arrays.

Spectral analysis of photo-induced delayed luminescence from human skin in vivo

The UVA induced Delayed Luminescence (DL), has been measured in vivo in the forearm skin of some healthy volunteers of different sex and age during several periods of the year. An innovative instrument able to detect, in single photon counting mode, the spectrum and the time trend of the DL emission has been used. The measured differences in the time trends of the spectral components may be related to the sex and the age. The potential development of a new analysis technique based on this phenomenon is discussed.