Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy

Blue Light Using Flavin (BLUF) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Förster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light- and dark-adapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule

A versatile transposon-based technology to generate loss- and gain-of-function phenotypes in the mouse liver

Background Understanding the contribution of gene function in distinct organ systems to the pathogenesis of human diseases in biomedical research requires modifying gene expression through the generation of gain- and loss-of-function phenotypes in model organisms, for instance, the mouse. However, methods to modify both germline and somatic genomes have important limitations that prevent easy, strong, and stable expression of transgenes. For instance, while the liver is remarkably easy to target, nucleic acids introduced to modify the genome of hepatocytes are rapidly lost, or the transgene expression they mediate becomes inhibited due to the action of effector pathways for the elimination of exogenous DNA. Novel methods are required to overcome these challenges, and here we develop a somatic gene delivery technology enabling long-lasting high-level transgene expression in the entire hepatocyte population of mice. Results We exploit the fumarylacetoacetate hydrolase (Fah) gene correction-induced regeneration in Fah-deficient livers, to demonstrate that such approach stabilizes luciferase expression more than 5000-fold above the level detected in WT animals, following plasmid DNA introduction complemented by transposon-mediated chromosomal gene transfer. Building on this advancement, we created a versatile technology platform for performing gene function analysis in vivo in the mouse liver. Our technology allows the tag-free expression of proteins of interest and silencing of any arbitrary gene in the mouse genome. This was achieved by applying the HADHA/B endogenous bidirectional promoter capable of driving well-balanced bidirectional expression and by optimizing in vivo intronic artificial microRNA-based gene silencing. We demonstrated the particular usefulness of the technology in cancer research by creating a p53-silenced and hRas G12V-overexpressing tumor model. Conclusions We developed a versatile technology platform for in vivo somatic genome editing in the mouse liver, which meets multiple requirements for long-lasting high-level transgene expression. We believe that this technology will contribute to the development of a more accurate new generation of tools for gene function analysis in mice.Peer reviewe

Photocycle alteration and increased enzymatic activity in genetically modified photoactivated adenylate cyclase OaPAC

Photoactivated adenylate cyclases (PACs) are light activated enzymes that combine blue light sensing capacity with the ability to convert ATP to cAMP and pyrophosphate (PPi) in a light-dependent manner. In most of the known PACs blue light regulation is provided by a blue light sensing domain using flavin which undergoes a structural reorganization after blue-light absorption. This minor structural change then is translated toward the C-terminal of the protein, inducing a larger conformational change that results in the ATP conversion to cAMP. As cAMP is a key second messenger in numerous signal transduction pathways regulating various cellular functions, PACs are of great interest in optogenetic studies. The optimal optogenetic device must be “silent” in the dark and highly responsive upon light illumination. PAC from Oscillatoria acuminata is a very good candidate as its basal activity is very small in the dark and the conversion rates increase 20-fold upon light illumination. We studied the effect of replacing D67 to N, in the blue light using flavin domain. This mutation was found to accelerate the primary electron transfer process in the photosensing domain of the protein, as has been predicted. Furthermore, it resulted in a longer lived signaling state, which was formed with a lower quantum yield. Our studies show that the overall effects of the D67N mutation lead to a slightly higher conversion of ATP to cAMP, which points in the direction that by fine tuning the kinetic properties more responsive PACs and optogenetic devices can be generated

Intensification of the photodegradation efficiency of an emergent water pollutant through process conditions optimization by means of response surface methodology

International audienceHeterogeneous photocatalysis has been increasingly investigated during the past years and has been recognized as a promising technique for clean and safe water purification. The current study exploits the advantage of this technique demonstrating that the removal of a biorefractory water pollutant named clofibric acid can be really improved by photocatalysis through a parametric comprehensive investigation and optimization study based on response surface methodology. Its novelty comes from the approach used to enhance the efficiency of the photocatalytic degradation of clofibric acid. A custom central composite design consisting of 49 trials was applied for process modeling and a quadratic robust model was derived based on the analysis of variance for the optimization of the process parameters. The effective removal of the target molecule with about 70% carbon mineralization was achieved under optimal photocatalytic conditions: 1.5 mg/L as the initial concentration of pollutant, 0.61 g/L catalyst, and an irradiation time of 190 min. Further, it was provided that nitrates play a positive role in the removal of this pollutant, while hydrogenocarbonates slow down its elimination. The ecotoxicity evaluation at different trophic levels confirmed the low toxicity of photodegradation by-products. Data analysis demonstrated that response surface methodology is a reliable approach for the optimization of the interactive effects of photocatalytic process parameters and is able to enhance their performance for the complete elimination of this hardly removed water pollutant

New Evidence of the Enhanced Elimination of a Persistent Drug Used as a Lipid Absorption Inhibitor by Advanced Oxidation with UV-A and Nanosized Catalysts

International audienceThis work demonstrates new evidence of the efficient destruction and mineralization of an emergent organic pollutant using UV-A and titanium nanosized catalysts. The target compound considered in this work is the primary metabolite of a lipid regulator drug, clofibrate, identified in many studies as refractory during conventional wastewater treatment. The photocatalytic performance study was carried out in batch mode at laboratory scale, in aqueous suspension. Kinetic data showed that titanium dioxide P25 Aeroxide (R) exhibits the highest photocatalytic efficiency compared to the other investigated catalysts. Pollutant degradation and mineralization efficiencies strongly increased when decreasing the initial substrate concentration. Target molecules oxidized faster when the catalyst load increased, and the mineralization was enhanced under acidic conditions 92% of mineralization was achieved at pH 4 after 190 min of reaction. Radical quenching assays confirmed that HO center dot and (hvb(+)) were the reactive oxygen species involved in the photocatalytic oxidation of the considered pollutant. In addition, further results revealed that the removal efficiency decreased in real water matrices. Finally, data collected through a series of phytotoxicity tests demonstrated that the photocatalytic process considerably reduces the toxicity of the treated solutions, confirming the process's effectiveness in the removal of persistent and biorefractory emergent organic water pollutants

Lung Transplant Rehabilitation—A Review

Background: Both lung transplant recipients and candidates are characterised by reduced training capacity and low average quality of life (QoL). This review investigates the impact of training on exercise ability and QoL in patients before and after lung transplant. Methods: Searches were conducted from the beginning to 7 March 2022 using the terms “exercise,” “rehabilitation,” “lung transplant,” “exercise ability,” “survival,” “quality of life” and “telerehabilitation” in six databases, including Cochrane Central Register of Controlled Trials (CENTRAL), PubMed, CINAHL, Nursing and Allied Health, and Scopus. The inclusion criteria were studies evaluating the effects of an exercise training programme concurrent with lung transplantation as well as patients and candidates (>18 years old) through any lung diseases. The term “lung transplant rehabilitation” was used to refer to all carefully thought-out physical activities with the ultimate or intermediate objective of improving or maintaining physical health. Results: Out of 1422 articles, 10 clinical- and 3 telerehabilitation studies, candidates (n = 420) and recipients (n = 116) were related to the criteria and included in this review. The main outcome significantly improved in all studies. The 6-min walk distance, maximum exercise capacity, peak oxygen uptake, or endurance for constant load rate cycling improved measuring physical activity [aerobic exercises, breathing training, and aerobic and inspiratory muscle training sessions (IMT)]. Overall scores for dyspnoea improved after exercise training. Furthermore, health-related quality of life (HRQOL) also improved after aerobic exercise training, which was performed unsupervised or accompanied by breathing sessions. Aerobic training alone rather than combined with inspiratory muscle- (IMT) or breathing training enhanced exercise capacity. Conclusion: In conclusion, rehabilitation programmes seem to be beneficial to patients both preceding and following lung transplantation. More studies are required to determine the best training settings in terms of time scale, frequency, and work intensity in terms of improving exercise ability, dyspnoea, and HRQOL

ABCC1 polymorphisms in anthracycline-induced cardiotoxicity in childhood acute lymphoblastic leukaemia

Anthracyclines are potent cytostatic drugs the correct dosage of which is critical to aviod possible cardiac side effects. ABCC1 (MRP1) is expressed in the heart and takes part in the detoxification and protection of the cells from toxic effects of xenobiotics, including anthracyclines. Our objective was to search for associations between left ventricular function and single nucleotide polymorphisms of the ABCC1 gene in children who received anthracycline chemotherapy. We analyzed data of 235 pediatric patients with acute lymphoblastic leukemia. Patients were followed up by echocardiography (median follow up 6.3 years). Nine polymorphisms in the ABCC1 gene were genotyped. The ABCC1 rs3743527TT genotype and rs3743527TT-rs246221TC/TT genotype combination were found to be associated with lower left ventricular fractional shortening (LVFS) after chemotherapy. Our results suggest that genetic variants in the ABCC1 gene could influence anthracycline induced left ventricular dysfunction