Use of the KlADH3 promoter for the quantitative production of the murine PDE5A isoforms in the yeast Kluyveromyces lactis

Background: Phosphodiesterases (PDE) are a superfamily of enzymes that hydrolyse cyclic nucleotides (cAMP/ cGMP), signal molecules in transduction pathways regulating crucial aspects of cell life. PDEs regulate the intensity and duration of the cyclic nucleotides signal modulating the downstream biological efect. Due to this critical role associated with the extensive distribution and multiplicity of isozymes, the 11 mammalian families (PDE1 to PDE11) constitute key therapeutic targets. PDE5, one of these cGMP-specifc hydrolysing families, is the molecular target of several well known drugs used to treat erectile dysfunction and pulmonary hypertension. Kluyveromyces lactis, one of the few yeasts capable of utilizing lactose, is an attractive host alternative to Saccharomyces cerevisiae for heterologous protein production. Here we established K. lactis as a powerful host for the quantitative production of the murine PDE5 isoforms. Results: Using the promoter of the highly expressed KlADH3 gene, multicopy plasmids were engineered to produce the native and recombinant Mus musculus PDE5 in K. lactis. Yeast cells produced large amounts of the purifed A1, A2 and A3 isoforms displaying Km, Vmax and Sildenafl inhibition values similar to those of the native murine enzymes. PDE5 whose yield was nearly 1 mg/g wet weight biomass for all three isozymes (30 mg/L culture), is well tolerated by K. lactis cells without major growth defciencies and interferences with the endogenous cAMP/cGMP signal transduction pathways. Conclusions: To our knowledge, this is the frst time that the entire PDE5 isozymes family containing both regulatory and catalytic domains has been produced at high levels in a heterologous eukaryotic organism. K. lactis has been shown to be a very promising host platform for large scale production of mammalian PDEs for biochemical and structural studies and for the development of new specifc PDE inhibitors for therapeutic applications in many pathologies

High-Efficiency Digital Readout Systems for Fast Pixel-based Vertex Detectors

Particle physics is one of the science branches which heavily relies on most advanced technologies due to the increasing complexity of the problems it has to face. In future colliders, luminosities and beam energies are scaling upwards. These are necessary conditions for the discovery of new physics which both result in a larger amount of data that need to be brought out of the detector. That’s why one of the crucial points for new experiments is the evolution of data acquisition systems. Data acquisition systems employed in particle physics experiments followed the global technology trend and moved towards digital electronics and transmission lines, in this chapter we will describe how the effort of our work has been applied in this direction trying to extend digital processing on the very front-end of the detector. We will show how digital elaboration on the very front-end can help coping with new stringent requirements

New trends in platinum and palladium complexes as antineoplastic agents

The discovery of cisplatin (cis-Pt(NH3)2Cl2) as an antineoplastic agent has focused attention on the rational design of metal complexes that can be potentially used in cancer chemotherapy. Today, the pharmaceutical industry invests more than $1 billion each year in the development of new metal-based drugs to improve biological activities, in terms of cellular selectivity, therapeutic efficiency and minimization of side effects. Chemotherapies based on transition metals play a key role in cancer treatment, and among them platinum and palladium are the most fruitful. This article reviews the main recent advances in the design and synthesis of platinum- and palladium-based drugs, their structural features and biological studies of them. The rationale for the choice of the ligand, related to leaving groups, the geometry of the complex and the oxidation state of the metal ion, is discussed. An overview of the main biological techniques and approaches for testing the interaction of these molecules with the biological environment, mainly DNA, to validate the effect is also provided

Immunohistochemical Localisation of PDE5 in Rat Lung during Pre- and Postnatal Development

In mammalian lung, at the transition to extrauterine life, NO/cGMP signal transduction system is known to play crucial roles in the regulation of vascular resistance and is supposed to act in angiogenesis. PDE5, which is the most abundant cGMP metabolizing enzyme within the lung, is highly expressed in the perinatal period, but its localisation in the different pulmonary cells is still poorly known. In our research, PDE5 immunohistochemical distribution was investigated in foetal and neonatal rat lung. The highest expression of PDE5 was found in cells randomly located in the stroma; in newborns, in particular, many cells in the intersaccular walls were heavily labelled, while much lower staining levels were shown by smooth myocytes belonging to vessels and airways. On the basis of their immunoreactivity for α-SM actin and/or desmin, most of the heavily PDE5-positive cells were identified as interstitial myofibroblasts and transitional pericytes, while only a few were interpreted as interstitial lipofibroblasts

Fast nonlinear Froude–Krylov force calculation for prismatic floating platforms: a wave energy conversion application case

AbstractComputationally fast and accurate mathematical models are essential for effective design, optimization, and control of wave energy converters. However, the energy-maximising control strategy, essential for reaching economic viability, inevitably leads to the violation of linearising assumptions, so the common linear models become unreliable and potentially unrealistic. Partially nonlinear models based on the computation of Froude–Krylov forces with respect to the instantaneous wetted surface are promising and popular alternatives, but they are still too slow when floaters of arbitrary complexity are considered; in fact, mesh-based spatial discretisation, required by such geometries, becomes the computational bottle-neck, leading to simulations 2 orders of magnitude slower than real-time, unaffordable for extensive iterative optimizations. This paper proposes an alternative analytical approach for the subset of prismatic floating platforms, common in the wave energy field, ensuring computations 2 orders of magnitude faster than real-time, hence 4 orders of magnitude faster than state-of-the-art mesh-based approaches. The nonlinear Froude–Krylov model is used to investigate the nonlinear hydrodynamics of the floater of a pitching wave energy converter, extracting energy either from pitch or from an inertially coupled internal degree of freedom, especially highlighting the impact of state constraints, controlled/uncontrolled conditions, and impact on control parameters' optimization, sensitivity and effectiveness

Expression of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in mouse tissues and cell lines using an antibody against the enzyme amino-terminal domain

We have produced a polyclonal antibody that specifically recognizes cGMP-binding cGMP-specific phosphodiesterase (PDES). The antibody was raised in rabbit using as immunogen a fusion protein, in which glutathione S-transferase was coupled to a 171 amino acid polypeptide of the N-terminal region of bovine PDE5. The antibody is able to immunoprecipitate PDES activity from mouse tissues and neuroblastoma extracts while it has no effect on all other PDE isoforms present in the extracts. PDES activity recovered in the immunoprecipitates retains its sensitivity to specific inhibitors such as zaprinast (IC50 = 0.6 muM) and sildenafil (IC50 = 3.5 nM), Bands of the expected molecular mass were revealed when solubilized immunoprecipitates were analysed in Western blots. The antibody selectively stained cerebellar Purkinje neurones, which are known to express high levels of PDES mRNA. Western blot analysis of mouse tissues revealed the highest expression signal in mouse lung, followed by heart and cerebellum, while a lower signal was evident in brain, kidney and a very low signal was present in the liver. In the hybrid neuroblastoma-glioma NG108-15 cells the antibody revealed a high PDE5 induction after dibutyryl-cAMP treatment. (C) 2001 Elsevier Science B,V, All rights reserved

Investigation of the Role of the Environment on the Photoluminescence and the Exciton Relaxation of CsPbBr3 Nanocrystals Thin Films

In this work, we present a detailed optical investigation of the effects of the environment on the photoluminescence (PL) spectra and the relaxation dynamics of pristine and aged CsPbBr 3 nanocrystal (NC) thin films. We demonstrate that, contrary to previous results on similar NCs, the PL intensity of pristine NCs is higher when the sample is in wet air than in vacuum, due to the passivation of defects reducing the free exciton trapping and the bound excitons non-radiative relaxation. The aged NCs show a PL intensity increase in wet air nine times stronger than the pristine ones, due to an interplay between static and dynamic effects, increasing the number of emitting NCs and reducing the non-radiative recombination rate of free excitons. These results improve the understanding of the possible interactions between perovskite NCs and the environment, which could be relevant for the development of optical gas sensors exploiting perovskite NCs

Mood Spectrum Model: Evidence reconsidered in the light of DSM-5

AIM: to investigate studies conducted with the Mood Spectrum Structured Interviews and Self-Report versions (SCI-MOODS and MOODS-SR). METHODS: We conducted a review of studies published between 1997 and August 2014. The search was performed using Pubmed and PsycINFO databases. Analysis of the papers followed the inclusion and exclusion criteria recommended by the PRISMA Guidelines, namely: (1) articles that presented a combination of at least two terms, "SCI-MOODS" [all fields] or "MOODS-SR" [all fields] or "mood spectrum" [all fields]; (2) manuscript in English; (3) original articles; and (4) prospective or retrospective original studies (analytical or descriptive), experimental or quasi-experimental studies. Exclusion criteria were: (1) other study designs (case reports, case series, and reviews); (2) non-original studies including editorials, book reviews and letters to the editor; and (3) studies not specifically designed and focused on SCI-MOODS or MOODS-SR. RESULTS: The search retrieved 43 papers, including 5 reviews of literature or methodological papers, and 1 case report. After analyzing their titles and abstracts, according to the eligibility criteria, 6 were excluded and 37 were chosen and included. The SCI-MOODS and the MOODS-SR have been tested in published studies involving 52 different samples across 4 countries (Italy, United States, Spain and Japan). The proposed mood spectrum approach has demonstrated its usefulness mainly in 3 different areas: (1) Patients with the so-called "pure" unipolar depression that might manifest hypomanic atypical and/or sub-threshold aspects systematically detectable with the mood questionnaire; (2) Spectrum features not detected by other instruments are clinically relevant, because they might manifest in waves during the lifespan, sometimes together, sometimes alone, sometimes reaching the severity for a full-blown disorder, sometimes interfering with other mental disorders or complicating the course of somatic diseases; and (3) Higher scores on the MOODS-SR factors assessing "psychomotor disturbances", "mixed instability" and "suicidality" delineate subtypes of patients characterized by the more severe forms of mood disorders, the higher risk for psychotic symptoms, and the lower quality of life after the remission of the full-blown-episode. CONCLUSION: The mood spectrum model help researchers and clinicians in the systematic assessment of those areas of psychopathology that are still neglected by the Diagnostic and Statistical Manual of Mental Disorders 5 classification

Identification of murine phosphodiesterase 5A isoforms and their functional characterization in HL-1 cardiac cell line

Phosphodiesterase 5A (PDE5A) specifically degrades the ubiquitous second messenger cGMP and experimental and clinical data highlight its important role in cardiac diseases. To address PDE5A role in cardiac physiology, three splice variants of the PDE5A were cloned for the first time from mouse cDNA library (mPde5a1, mPde5a2 and mPde5a3). The predicted amino acidic sequences of the three murine isoforms are different in the N-terminal regulatory domain. mPDE5A isoforms were transfected in HEK293T cells and they showed high affinity for cGMP and similar sensitivity to sildenafil inhibition. RT-PCR analysis showed that mPde5a1, mPde5a2 and mPde5a3 had differential tissue distribution. In the adult heart, mPde5a1 and mPde5a2 were expressed at different levels whereas mPde5a3 was undetectable. Overexpression of mPDE5As induced an increase of HL-1 number cells which progress into cell cycle. mPDE5A1 and mPDE5A3 overexpression increased the number of polyploid and binucleated cells, mPDE5A3 widened HL-1 areas and modulated hypertrophic markers more efficiently respect to the other mPDE5A isoforms. Moreover, mPDE5A isoforms had differential subcellular localization: mPDE5A1 was mainly localized in the cytoplasm, mPDE5A2 and mPDE5A3 were also nuclear localized. These results demonstrate for the first time the existence of three PDE5A isoforms in mouse and highlight their potential role in the induction of hypertrophy. This article is protected by copyright. All rights reserved