Oxygen diffusion pathways in mutated forms of a LOV photoreceptor from Methylobacterium radiotolerans: a molecular dynamics study.

Mr4511 LOV (Light, Oxygen and Voltage) protein is a blue light sensing photoreceptor from Methylobacterium radiotolerans, binding flavin mononucleotide (FMN) as chromophore. Blue light activation of LOV domains triggers the reversible formation of a FMN-cysteine adduct by a photocycle that goes through the FMN excited triplet state. LOV domains can be engineered as fluorescent sensors and actuators for optogenetics and photomedicine [1]. First experimental data on Mr4511 LOV protein [2] indicate its high potential as a photosensitizer for singlet oxygen (SO) the cytotoxic reactive excited state of molecular oxygen, produced by diffusion limited energy transfer from the FMN triplet state. This feature is obtained after the single mutation of reactive cysteine C71, a change that prevents formation of the photoproduct. In addition, the lack of a tryptophan, conserved in ca. 75% of LOV domains and shown to strongly quench the FMN triplet lifetime (T) in LOV proteins, allows for Mr4511 LOV a longer T than for other LOV domains in C71S and C71G variants [2]. After an homology modeling of Mr4511 LOV, that has lead to a dimeric protein stabilized by the presence of a strong leucine zipper in the C-terminal helices, a mutation of the photocycle substrate cysteine into serine (C71S) has been introduced in silico to make it a SO photosensitizer, and the mutated form stability was tested by MD simulations. Afterwards, both transient and persistent oxygen channels were detected and analysed both in the wt and in the mutated protein. Molecular oxygen was then placed both outer and into the chromophore cavity and potential diffusion pathways were explored with MD simulations, showing a high accessibility of the binding cavity and a high persistence of oxygen inside. Mutations that might favor SO generation were designed based on their position with respect to the FMN and the oxygen channels, taking into account the ability of certain amino acids to quench FMN triplet state and SO. Therefore, C71S/Y61T and C71S/Y61S double mutants were generated in silico and their stability was checked. The analysis of their oxygen diffusion pathways showed an increased diffusion and persistence of oxygen molecules inside the binding cavity, indicating a promising model for SO photosensing and its biomedical and biophysical applications. [1] A. Losi, et al., Chem. Rev. 118 (2018): 10659-10709. [2] E. Consiglieri, et al., Photochem. Photobiol. Sci. 18 (2019): 2657–2660

Simulation and evaluation of optimization problem solutions in distributed energy management systems

Deregulation in electricity markets requires fast and robust optimization tools for a secure and efficient operation of the electric power system. In addition, there is the need of integrating and coordinating operational decisions taken by different utilities acting in the same market. Distributed Energy Management Systems (DEMS) may help to fulfill these requirements. The design of a DEMS requires detailed simulation results for the evaluation of its performance. To simulate the operation of a DEMS from the optimization standpoint, a general purpose distributed optimization software tool, DistOpt, is used, and its capabilities are extended to handle power system problems. The application to the optimal power flow problem is presented.published_or_final_versio

Submicrometer-Channel Organic Transistors with MHz Operation Range on Flexible Substrates by a Low-Resolution Fabrication Technique

In this paper, the development of a simple and reproducible approach for the fabrication of n-type organic field-effect transistors with a 350 nm-long channel on flexible substrates is reported. The critical feature of the device, the channel length, is obtained using a self-alignment process that exploits the vertical step of a plasma-etched thin Parylene C layer, according to the so-called step-edge architecture. The fabricated devices can operate in continuous mode and show an average and maximum transition frequency of 2.5 MHz and 5.5 MHz, respectively. The possibility of easily obtaining high-performing, short channel organic transistors on flexible substrates, without the use of expensive and high-resolution techniques, represents an interesting step toward the miniaturization of flexible circuits in the field of large-area organic electronics

Mapping the role of aromatic amino acids within a blue-light sensing LOV domain

Photosensing LOV (Light, Oxygen, Voltage) domains detect and respond to UVA/Blue (BL) light by forming a covalent adduct between the flavin chromophore and a nearby cysteine, via the decay of the flavin triplet excited state. LOV domains where the reactive cysteine has been mutated are valuable fluorescent tools for microscopy and as genetically encoded photosensitisers for reactive oxygen species. Besides being convenient tools for applications, LOV domains without the reactive cysteine (naturally occurring or engineered) can still be functionally photoactivated via formation of a neutral flavin radical. Tryptophans and tyrosines are held as the main partners as potential electron donors to the flavin excited states. In this work, we explore the relevance of aromatic amino acids in determining the photophysical features of the LOV protein Mr4511 from Methylobacterium radiotolerans by introducing point mutations into the C71S variant that does not form the covalent adduct. By using an array of spectroscopic techniques we measured the fluorescence quantum yields and lifetimes, the triplet yields and lifetimes, and the efficiency of singlet oxygen (SO) formation for eleven Mr4511 variants. Insertion of Trp residues at distances between 0.6 and 1.5 nm from the flavin chromophore results in strong quenching of the flavin excited triplet state and, at the shorter distances even of the singlet excited state. The mutation F130W (ca. 0.6 nm) completely quenches the singlet excited state, preventing triplet formation: in this case, even if the cysteine is present, the photo-adduct is not formed. Tyrosines are also quenchers for the flavin excited states, although not as efficient as Trp residues, as demonstrated with their substitution with the inert phenylalanine. For one of these variants, C71S/Y116F, we found that the quantum yield of formation for singlet oxygen is 0.44 in aqueous aerobic solution, vs 0.17 for C71S. Based on our study with Mr4511 and on literature data for other LOV domains we suggest that Trp and Tyr residues too close to the flavin chromophore (at distances less than 0.9 nm) reduce the yield of photoproduct formation and that introduction of inert Phe residues in key positions can help in developing efficient, LOV-based photosensitisers. This journal i

Expression of μ-protocadherin is negatively regulated by the activation of the β-catenin signaling pathway in normal and cancer colorectal enterocytes.

Mu-protocadherin (MUCDHL) is an adhesion molecule predominantly expressed by colorectal epithelial cells which is markedly downregulated upon malignant transformation. Notably, treatment of colorectal cancer (CRC) cells with mesalazine lead to increased expression of MUCDHL, and is associated with sequestration of β-catenin on the plasma membrane and inhibition of its transcriptional activity. To better characterize the causal relationship between β-catenin and MUCDHL expression, we performed various experiments in which CRC cell lines and normal colonic organoids were subjected to culture conditions inhibiting (FH535 treatment, transcription factor 7-like 2 siRNA inactivation, Wnt withdrawal) or stimulating (LiCl treatment) β-catenin activity. We show here that expression of MUCDHL is negatively regulated by functional activation of the β-catenin signaling pathway. This finding was observed in cell culture systems representing conditions of physiological stimulation and upon constitutive activation of β-catenin in CRC. The ability of MUCDHL to sequester and inhibit β-catenin appears to provide a positive feedback enforcing the effect of β-catenin inhibitors rather than serving as the primary mechanism responsible for β-catenin inhibition. Moreover, MUCDHL might have a role as biomarker in the development of CRC chemoprevention drugs endowed with β-catenin inhibitory activity

Milk and butter. From the Neolithic to the current nutritional aspects

The evolution in the history of nutrition knowledge towards dairy products, is strictly related to the socio-cultural development of humans. In fact, milk and butter have accompanied humans since ancient times, which traces of the consumption of such products are dated back about the earliest times after the last (glaciation) ice age, while the application for extra nutritional uses, such as cosmetics and ceremonial rites, are reported in the writings of the Old Testament. Even in Italy, before the Roman Empire, were known rudimentary techniques of production and storage of dairy products. But only with the advent of the Etruscans, and the Romans later, that the use of milk and dairy products reach a wide diffusion in several applications. Since the advent of Christ until today, milk and its derivatives have maintained a privileged place in the human diet, but it is only with the advent of modern medicine and new findings in lipidic chemistry that emerged multiple biological and nutritional properties, very important for human health. After a short summary of the ancient history of the milk and butter, the role of dairy products in cancer, in hypercholesterolemia, and cardiovascular disease are reported. Moreover, the current opinions on saturated fatty acids, the role of polyunsaturated fatty acids and their lipid mediators obtained by the action of cyclooxygenase, lipoxygenase and the cytochrome P450 enzymes, are treated. Even if sometimes mistreated, the milk, but most of all its high fat content derivatives such as butter, is a rich source of biologically active compounds that foster a controversial action against neolplastic and cardiovascular disease. These compounds, mainly contained in the lipid fraction, for the more obvious relationships that exist between nutrition and health status, have been the subject in the last decades of intense scientific investigation in which there were expressed lights and shadows, but recognizing that not all fats are harmful and further thorough studies are necessary, in particular, on the derived lipid mediators. This will allow a significant progress based on new scientific evidences, further orienting researchers and clinicians on evidencebased nutritional science

The dark recovery rate in the photocycle of the bacterial photoreceptor YtvA is affected by the cellular environment and by hydration

We report thermal recovery kinetics of the lit state into the parental dark state, measured for the blue light-sensing photoreceptor YtvA inside overexpressing E. coli and B. subtilis bacterial cells, performed for the wild type and several mutated proteins. Recovery was followed as a recovery of the fluorescence, as this property is only found for the parental but not for the photochemically generated lit state. When cells were deposited onto a microscope glass plate, the observed thermal recovery rate in the photocycle was found ca. ten times faster in comparison to purified YtvA in solution. When the E. coli or B. subtilis colonies were soaked in an isotonic buffer, the dark relaxation became again much slower and was very similar to that observed for YtvA in solution. The observed effects show that rate constants can be tuned by the cellular environment through factors such as hydration. Copyright

The use of nematodes in assessing ecological conditions in shallow waters surrounding a Mediterranean harbour facility

The spatial distribution and structure of nematode assemblages in the area surrounding the harbour of Vado Ligure (Savona, NW Mediterranean) were studied in relation to the influence of natural and anthropogenic environmental factors. Stations were selected following an “anthropogenic gradient” from sites located near the city centre and its harbour to more pristine and distant sites. Sediment quality was determined by considering both sediment granulometric and chemical parameters (hydrocarbons, heavy metals, total organic matter, proteins, carbohydrates) as well as nematode abundance, diversity, life strategies, trophic structure and assemblage composition. A high correlation between environmental characteristics and the nematode response was found. On the basis of the comparison of these results, which identified three distinct sub-areas associated with different levels of environmental quality, a set of nematode indicator genera was selected for the future evaluation of quality status

Spiky Strings and Giant Holes

We analyse semiclassical strings in AdS in the limit of one large spin. In this limit, classical string dynamics is described by a finite number of collective coordinates corresponding to spikes or cusps of the string. The semiclassical spectrum consists of two branches of excitations corresponding to "large" and "small" spikes respectively. We propose that these states are dual to the excitations known as large and small holes in the spin chain description of N=4 SUSY Yang-Mills. The dynamics of large spikes in classical string theory can be mapped to that of a classical spin chain of fixed length. In turn, small spikes correspond to classical solitons propagating on the background formed by the large spikes. We derive the dispersion relation for these excitations directly in the finite gap formalism.Comment: 36 pages, 9 figure

role of evaluating tumor infiltrating lymphocytes programmed death ligand 1 and mismatch repair proteins expression in malignant mesothelioma

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