Investigation of Retinal Microcirculation in Diabetic Patients Using Adaptive Optics Ophthalmoscopy and Optical Coherence Angiography.

The current research approaches the retinal microvasculature of healthy volunteers (17 subjects), patients with diabetes mellitus without retinopathy (19 subjects), and of diabetic patients with nonproliferative (17 subjects) and proliferative (21 subjects) diabetic retinopathy, by using adaptive optics ophthalmoscopy and optical coherence ophthalmoscopy angiography. For each imaging technique, several vascular parameters have been calculated in order to achieve a comparative analysis of these imaging biomarkers between the four studied groups. The results suggest that diabetic patients with or without diabetic retinopathy prove signs of retinal arteriole structural alterations, mainly showed by altered values of wall to lumen ratio, calculated for the superior or inferior temporal branch of the central retinal artery, near the optic nerve head, and significant changes of the vascular density in the retinal superficial capillary plexus. Both adaptive optics ophthalmoscopy and optical coherence ophthalmoscopy angiography are providing useful information about the retinal microvasculature from early onset of diabetic disease, having a promising diagnostic and prognostic role in the future.

Laser-induced CNV following hair removal procedure.

Objective: To describe the chronological features of choroidal neovascular membrane (CNV) development subsequent to accidental firing of diode laser into the eye of a young female during hair epilation. Methods: Descriptive case report. Results: The patient presented one week after the laser injury to a local ophthalmologist complaining of RE (right eye) blurred central vision. Snellen's visual acuity (VA) was 6/ 7.5. Optical coherence tomography (OCT) showed focal disruption of the ellipsoid and the interdigitation zones. Four weeks later, she presented with worsening symptoms and RE VA 6/ 15. Funduscopy revealed a perifoveal grayish lesion with adjacent retinal hemorrhage, which, on fluorescein angiography, was leaking, compatible with CNV. OCT showed a dome-shaped sub-retinal pigment epithelium lesion with extension into the subretinal space and little subretinal fluid. The patient was treated with one intravitreal bevacizumab injection. There was rapid regression of the CNV and improvement of VA to 6/ 7.5 at one-month visit and to 6/ 6 at 6-month visit. Conclusion: All the laser procedures should be conducted with intensive care for both the patient and the laser surgeon since inadvertent effects are constantly being reported due to lack of adherence to safety measures.

Simple discrimination of sub-cycling cells by propidium iodide flow cytometric assay in Jurkat cell samples with extensive DNA fragmentation.

Human leukemia Jurkat T cells were analyzed for apoptosis and cell cycle by flow cytometry, using the Annexin V/propidium iodide (PI) standard assay, and a simple PI staining in Triton X-100/digitonin-enriched PI/RNase buffer, respectively. Cells treated with doxorubicin or menadione displayed a very strong correlation between the apoptotic cell fraction measured by the Annexin V/PI assay, and the weight of a secondary cell population that emerged on the forward scatter (FS)/PI plot, as well as on the side scatter (SS)/PI and FL1/PI plots generated from parallel cell cycle recordings. In both cases, the Pearson correlation coefficients were >0.99. In cell cycle determinations, PI fluorescence was detected on FL3 (620/30 nm), and control samples exhibited the expected linear dependence of FL3 on FL1 (525/40 nm) signals. However, increasing doses of doxorubicin or menadione generated a growing subpopulation of cells displaying a definite right-shift on the FS/FL3, SS/FL3 and FL1/FL3 plots, as well as decreased PI fluorescence, indicative of ongoing fragmentation and loss of nuclear DNA. By gating on these events, the resulting fraction of presumably sub-cycling cells (i.e. cells with cleaved DNA, counting sub-G0/G1, sub-S and sub-G2/M cells altogether) was closely similar to the apoptotic rate assessed by Annexin V/PI labeling. Taken together, these findings suggest a possible way to recognize the entire population of cells undergoing apoptotic DNA cleavage and simultaneously determine the cell cycle distribution of non-apoptotic cells in PI-labeled cell samples with various degrees of DNA fragmentation, using a simple and reproducible multiparametric analysis of flow cytometric recordings.

Cellular determinants involving mitochondrial dysfunction, oxidative stress and apoptosis correlate with the synergic cytotoxicity of epigallocatechin-3-gallate and menadione in human leukemia Jurkat T cells.

We have investigated the growth-suppressive action of epigallocatechin-3-gallate (EGCG) on human leukemia Jurkat T cells. Results show a strong correlation between the dose-dependent reduction of clonogenic survival following acute EGCG treatments and the EGCG-induced decline of the mitochondrial level of Ca(2+). The cell killing ability of EGCG was synergistically enhanced by menadione. In addition, the cytotoxic effect of EGCG applied alone or in combination with menadione was accompanied by apoptosis induction. We also observed that in acute treatments EGCG displays strong antioxidant properties in the intracellular milieu, but concurrently triggers some oxidative stress generating mechanisms that can fully develop on a longer timescale. In parallel, EGCG dose-dependently induced mitochondrial depolarization during exposure, but this condition was subsequently reversed to a persistent hyperpolarized mitochondrial state that was dependent on the activity of respiratory Complex I. Fluorimetric measurements suggest that EGCG is a mitochondrial Complex III inhibitor and indicate that EGCG evokes a specific cellular fluorescence with emission at 400nm and two main excitation bands (at 330nm and 350nm) that may originate from a mitochondrial supercomplex containing dimeric Complex III and dimeric ATP-synthase, and therefore could provide a valuable means to characterize the functional properties of the respiratory chain.

Novel insights into the antiproliferative effects and synergism of quercetin and menadione in human leukemia Jurkat T cells.

The flavonoid quercetin and menadione (vitamin K3) are known as potent apoptogens in human leukemia Jurkat T cells. We explored some underlying mechanisms and the potential relevance of the combination quercetin-menadione for clinical applications. In acute treatments, quercetin manifested a strong antioxidant character, but induced a transient loss of Δψm, likely mediated by opening of the mitochondrial permeability transition pore. After removal of quercetin, persistent mitochondrial hyperpolarization was generated via stimulation of respiratory Complex I. In contrast, menadione-induced Δψm dissipation was only partially and transiently reversed after menadione removal. Results indicate that Ca(2+) release is a necessary event in quercetin-induced cell death and that the survival response to quercetin is delineated within 1h from exposure. Depending on dose, the two agents exhibited either antagonistic or synergistic effects in reducing clonogenicity of Jurkat cells. 24-h combinatorial regimens at equimolar concentrations of 10-15 µM, which are compatible with a clinically achievable (and safe) scheme, reduced cell viability at efficient rates. Altogether, these findings support the idea that the combination quercetin-menadione could improve the outcome of conventional leukemia therapies, and warrant the utility of additional studies to investigate the therapeutic effects of this combination in different cellular or animal models for leukemia.

Mitochondrial respiratory complex I probed by delayed luminescence spectroscopy.

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.

Comparative analysis of Staphylococcus aureus and Escherichia coli microcalorimetric growth.

BACKGROUND: Microcalorimetric bacterial growth studies have illustrated that thermograms differ significantly with both culture media and strain. The present contribution examines the possibility of discriminating between certain bacterial strains by microcalorimetry and the qualitative and quantitative contribution of the sample volume to the observed thermograms. Growth patterns of samples of Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) were analyzed. Certain features of the thermograms that may serve to distinguish between these bacterial strains were identified. RESULTS: The thermograms of the two bacterial strains with sample volumes ranging from 0.3 to 0.7 ml and same initial bacterial concentration were analyzed. Both strains exhibit a roughly 2-peak shape that differs by peak amplitude and position along the time scale. Seven parameters corresponding to the thermogram key points related to time and heat flow values were proposed and statistically analyzed. The most relevant parameters appear to be the time to reach a heat flow of 0.05 mW (1.67 ± 0.46 h in E. coli vs. 2.99 ± 0.53 h in S. aureus, p < 0.0001), the time to reach the first peak (3.84 ± 0.5 h vs. 5.17 ± 0.49 h, p < 0.0001) and the first peak value (0.19 ± 0.02 mW vs. 0.086 ± 0.012 mW, p < 0.0001). The statistical analysis on 4 parameters of volume-normalized heat flow thermograms showed that the time to reach a volume-normalized heat flow of 0.1 mW/ml (1.75 ± 0.37 h in E. coli vs. 2.87 ± 0.65 h in S. aureus, p < 0.005), the time to reach the first volume-normalized peak (3.78 ± 0.47 h vs. 5.12 ± 0.52 h, p < 0.0001) and the first volume-normalized peak value (0.35 ± 0.05 mW/ml vs. 0.181 ± 0.040 mW/ml, p < 0.0001) seem to be the most relevant. Peakfit® decomposition and analysis of the observed thermograms complements the statistical analysis via quantitative arguments, indicating that: (1) the first peak pertains to a faster, "dissolved oxygen" bacterial growth (where the dissolved oxygen in the initial suspension acts as a limiting factor); (2) the second peak indicates a slower "diffused oxygen" growth that involves transport of oxygen contained in the unfilled part of the microcalorimetric cell; (3) a strictly fermentative growth component may slightly contribute to the observed complex thermal signal. CONCLUSION: The investigated strains of Staphylococcus aureus and Escherichia coli display, under similar experimental conditions, distinct thermal growth patterns. The two strains can be easily differentiated using a selection of the proposed parameters. The presented Peakfit analysis of the complex thermal signal provides the necessary means for establishing the optimal growth conditions of various bacterial strains. These conditions are needed for the standardization of the isothermal microcalorimetry method in view of its further use in qualitative and quantitative estimation of bacterial growth.

Signal mass and Ca²âº kinetics in local calcium events: a modeling study.

We use a detailed modeling formalism based on numerical simulations of local calcium release events where the blurring of the image, the presence of diffusional barriers provided by large organelles situated close to the release site, as well as the variable position of the scan line with respect to the release site are taken into consideration. We have investigated the effect of the fluorescence noise fluctuations on the accuracy in computing the signal mass from linescan recordings and obtained a quantitative description of both the signal mass and the local increase in the free Ca(2+) level as a function of the release current, the release duration and the orientation of the scan line, for three different levels of noise magnitudes. The model could provide a very good fit to a wide set of available experimental data regarding the signal mass of puffs visualized by fluorescence microscopy in the Xenopus oocyte loaded with 40 µM Oregon Green-1 in the absence of the calcium chelator EGTA. Numerical simulations also predict the amplitude and the kinetics of calcium signals evolving in the absence of the indicator, and indicate that sub-maximal activation of IP(3) receptors could produce in average levels of about 2 µM and 0.4 µM free Ca(2+) close to a release site located in the animal or in the vegetal hemisphere, respectively, whereas the maximal levels reached in more rare events could be 11 µM and 4 µM, respectively.