Photochromic conversion in a red/green cyanobacteriochrome from Synechocystis PCC6803: quantum yields in solution and photoswitching dynamics in living E. coli cells.

The protein encoded by the gene slr1393 from the cyanobacterium Synechocystis sp. PCC6803 (Slr1393) is composed of three GAF domains, a PAS domain, and a histidine kinase motif. The third GAF domain (referred to as GAF3) was previously characterized as the sole domain in this protein, being able to carry phycocyanobilin (PCB) as the chromophore and to accomplish photochemistry. GAF3 shows photochromicity, and is able to switch between a red-absorbing parental state (GAF3R, λmax = 649 nm) and a green-absorbing photoproduct state (GAF3G, λmax = 536 nm) upon appropriate irradiation. In this study we have determined the photochemical quantum yields for the interconversion between both forms using two methods: an "absolute" method and a reference-based control. The latter is a comparative procedure which exploits a well-characterized blue-light photoreceptor, YtvA from Bacillus subtilis, and the cyanobacterial phytochrome Cph1 as actinometers. The former is an ad hoc developed, four laser-based setup where two cw lasers provide the pump beams to induce photoswitching (red to green and green to red, respectively) and two cw lasers simultaneously monitor the appearance and disappearance of the two species. Interestingly, fit analysis of the recorded transient absorbance changes provided a quantum yield for the green → red conversion (≈0.3) at least three times larger than for the red → green conversion (≈0.08). These data are in agreement with the results from the comparative method documenting the usefulness of the 'direct' method developed here for quantum yields' determination. The light-induced switching capability of this photochromic protein allowed measuring the kinetics of GAF3 immobilized on a glass plate, and within living, overexpressing Escherichia coli cells.

Clinical and Peripheral Biomarkers in Female Patients Affected by Anorexia: Does the Neutrophil/Lymphocyte Ratio (NLR) Affect Severity?

Anorexia Nervosa (AN) is a disabling disorder characterized by extreme weight loss and frequent chronicization, especially in its most severe forms. This condition is associated with a pro-inflammatory state; however, the role of immunity in symptom severity remains unclear. Total cholesterol, white blood cells, neutrophils, lymphocytes, platelets, iron, folate, vitamin D and B12 were dosed in 84 female AN outpatients. Mildly severe (Body Mass Index-BMI ≥ 17) versus severe (BMI < 17) patients were compared using one-way ANOVAs or χ2 tests. A binary logistic regression model was run to investigate the potential association between demographic/clinical variables or biochemical markers and the severity of AN. Patients with severe anorexia (compared to mild forms) were older (F = 5.33; p = 0.02), engaged in more frequent substance misuse (χ2 = 3.75; OR = 3.86; p = 0.05) and had a lower NLR (F = 4.12; p = 0.05). Only a lower NLR was predictive of severe manifestations of AN (OR = 0.007; p = 0.031). Overall, our study suggests that immune alterations may be predictive of AN severity. In more severe forms of AN, the response of the adaptive immunity is preserved, while the activation of the innate immunity may be reduced. Further studies with larger samples and a wider panel of biochemical markers are needed to confirm the present results.

A new FRAP/FRAPa method for three-dimensional diffusion measurements based on multiphoton excitation microscopy.

We present a new convenient method for quantitative three-dimensionally resolved diffusion measurements based on the photobleaching (FRAP) or photoactivation (FRAPa) of a disk-shaped area by the scanning laser beam of a multiphoton microscope. Contrary to previously reported spot-photobleaching protocols, this method has the advantage of full scalability of the size of the photobleached area and thus the range of diffusion coefficients, which can be measured conveniently. The method is compatible with low as well as high numerical aperture objective lenses, allowing us to perform quantitative diffusion measurements in three-dimensional extended samples as well as in very small volumes, such as cell nuclei. Furthermore, by photobleaching/photoactivating a large area, diffusion along the optical axis can be measured separately, which is convenient when studying anisotropic diffusion. First, we show the rigorous mathematical derivation of the model, leading to a closed-form formula describing the fluorescence recovery/redistribution phase. Next, the ability of the multiphoton FRAP method to correctly measure absolute diffusion coefficients is tested thoroughly on many test solutions of FITC-dextrans covering a wide range of diffusion coefficients. The same is done for the FRAPa method on a series of photoactivatable green fluorescent protein solutions with different viscosities. Finally, we apply the method to photoactivatable green fluorescent protein diffusing freely in the nucleus of living NIH-3T3 mouse embryo fibroblasts.

Role of three-dimensional bleach distribution in confocal and two-photon fluorescence recovery after photobleaching experiments.

The quantitative analysis of fluorescence perturbation experiments such as fluorescence recovery after photobleaching (FRAP) requires suitable analytical models to be developed. When diffusion in 3D environments is considered, the description of the initial condition produced by the perturbation (i.e., the photobleaching of a selected region in FRAP) represents a crucial aspect. Though it is widely known that bleaching profiles approximations can lead to errors in quantitative FRAP measurements, a detailed analysis of the sources and the effects of these approximations has never been conducted until now. In this study, we measured the experimental 3D bleaching distributions obtained in conventional and two-photon excitation schemes and analyzed the deviations from the ideal cases usually adopted in FRAP experiments. In addition, we considered the non-first-order effects generated by the high energy pulses usually delivered in FRAP experiments. These data have been used for finite-element simulations mimicking FRAP experiments on free diffusing molecules and compared with FRAP model curves based on the ideal bleach distributions. The results show that two-photon excitation more closely fits ideal bleaching patterns even in the event of fluorescence saturation, achieving estimations of diffusion coefficients within 20% accuracy of the correct value.