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Human aldehyde oxidase (hAOX1) is mainly present in the liver and has an emerging role in drug metabolism, since it accepts a wide range of molecules as substrates and inhibitors. Herein, we employed an integrative approach by combining NMR, X-ray crystallography, and enzyme inhibition kinetics to understand the inhibition modes of three hAOX1 inhibitors-thioridazine, benzamidine, and raloxifene. These integrative data indicate that thioridazine is a noncompetitive inhibitor, while benzamidine presents a mixed type of inhibition. Additionally, we describe the first crystal structure of hAOX1 in complex with raloxifene. Raloxifene binds tightly at the entrance of the substrate tunnel, stabilizing the flexible entrance gates and elucidating an unusual substrate-dependent mechanism of inhibition with potential impact on drug-drug interactions. This study can be considered as a proof-of-concept for an efficient experimental screening of prospective substrates and inhibitors of hAOX1 relevant in drug discovery.

CO-releasing molecules (CORMs) have been widely studied for their anti-inflammatory, antiapoptotic, and antiproliferative effects. CORM-3 is a water-soluble Ru-based metal carbonyl complex, which metallates serum proteins and readily releases CO in biological media. In this work, we evaluated the anti-inflammatory and wound-healing effects of gold nanoparticles-CORM-3 conjugates, AuNPs@PEG@BSA·Ru(CO)

Vanadium compounds have frequently been proposed as therapeutics, but their application has been hampered by the lack of information on the different V-containing species that may form and how these interact with blood and cell proteins, and with enzymes. Herein, we report several resolved crystal structures of lysozyme with bound V

In this study we define a new sample of distant powerful radio galaxies in order to study their host-galaxy properties and provide targets for future observations of HI absorption with new radio telescopes and to understand the fuelling and feedback from such sources. We have cross-matched the Sydney University Molonglo Sky Survey (SUMSS) radio catalogue at 843 MHz with the VISTA Hemisphere Survey (VHS) near-infrared catalogue using the Likelihood Ratio technique. Photometric redshifts from the Dark Energy Survey are then used to assign redshifts to the radio source counterparts. We found a total of 249 radio sources with photometric redshifts over a 148 deg^2 region. By fitting the optical and near-infrared photometry with spectral synthesis models we determine the stellar mass and star-formation rates of the radio sources, finding typical stellar masses of 10^{11} - 10^{12}M$_{\odot}$ for the powerful high-redshift radio galaxies. We also find a population of low-mass blue galaxies. However, by comparing the derived star-formation rates to the radio luminosity, we suggest that these sources are false positives in our likelihood ratio analysis. Follow up, higher-resolution (, 11 pages, 9 figures. Accepted for publication in MNRAS

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD), a Lancefield group C streptococci (GCS), is a frequent cause of bovine mastitis. This highly prevalent disease is the costliest in dairy industry. Adherence and biofilm production are important factors in streptoccocal pathogenesis. We have previously described the adhesion and internalization of SDSD isolates in human cells and now we describe the biofilm production capability of this bacterium. In this work we integrated microbiology, imaging and computational methods to evaluate the biofilm production capability of SDSD isolates; to assess the presence of biofilm regulatory protein BrpA homolog in the biofilm producers; and to predict a structural model of BrpA-like protein and its binding to putative inhibitors. Our results show that SDSD isolates form biofilms on abiotic surface such as glass (hydrophilic) and polystyrene (hydrophobic), with the strongest biofilm formation observed in glass. This ability was mainly associated with a proteinaceous extracellular matrix, confirmed by the dispersion of the biofilms after proteinase K and trypsin treatment. The biofilm formation in SDSD isolates was also confirmed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Under SEM observation, VSD16 isolate formed cell aggregates during biofilm growth while VSD9 and VSD10 formed smooth and filmy layers. We show that brpA-like gene is present and expressed in SDSD biofilm-producing isolates and its expression levels correlated with the biofilm production capability, being more expressed in the late exponential phase of planktonic growth compared to biofilm growth. Fisetin, a known biofilm inhibitor and a putative BrpA binding molecule, dramatically inhibited biofilm formation by the SDSD isolates but did not affect planktonic growth, at the tested concentrations. Homology modeling was used to predict the 3D structure of BrpA-like protein. Using high throughput virtual screening and molecular docking, we selected five ligand molecules with strong binding affinity to the hydrophobic cleft of the protein, making them potential inhibitor candidates of the SDSD BrpA-like protein. These results warrant further investigations for developing novel strategies for SDSD anti-biofilm therapy.

We would like to thank Herminia de Lencastre (ITQB-AX NOVA), Adriano Henriques (ITQB-AX NOVA) and PaulaTamagnini (i3S) for the gift of chromosomal DNA, Paulo Tavares (I2BC-CNRS) and Adriano Henriques for the gift of flagellin and anti-sigmaA antibodies, respectively, and Diogo Pereira for carrying out some growth kinetic assays. This work was supported by the FundacAo para a Ciencia e a Tecnologia [Grant Numbers PTDC/BIA-MIC/30696/2017 to ISN and UID/Multi/04378/2019 to Unidade de Ciencias Biomoleculares Aplicadas UCIBIO; and fellowship PD/BD/105737/2014 to F.L.]. ATP-binding cassette (ABC) type I importers are widespread in bacteria and play a crucial role in its survival and pathogenesis. They share the same modular architecture comprising two intracellular nucleotide-binding domains (NBDs), two transmembrane domains (TMDs) and a substrate-binding protein. The NBDs bind and hydrolyze ATP, thereby generating conformational changes that are coupled to the TMDs and lead to substrate translocation. A group of multitask NBDs that are able to serve as the cellular motor for multiple sugar importers was recently discovered. To understand why some ABC importers share energy-coupling components, we used the MsmX ATPase from Bacillus subtilis as a model for biological and structural studies. Here we report the first examples of functional hybrid interspecies ABC type I importers in which the NBDs could be exchanged. Furthermore, the first crystal structure of an assigned multitask NBD provides a framework to understand the molecular basis of the broader specificity of interaction with the TMDs. publishersversion published

Biofilms are generally defined as communities of cells involved in a self-produced extracellular matrix adhered to a surface. In biofilms, the bacteria are less sensitive to host defense mechanisms and antimicrobial agents, due to multiple strategies, that involve modulation of gene expression, controlled metabolic rate, intercellular communication, composition, and 3D architecture of the extracellular matrix. These factors play a key role in streptococci pathogenesis, contributing to therapy failure and promoting persistent infections. The species of the pyogenic group together with Streptococcus pneumoniae are the major pathogens belonging the genus Streptococcus, and its biofilm growth has been investigated, but insights in the genetic origin of biofilm formation are limited. This review summarizes pyogenic streptococci biofilms with details on constitution, formation, and virulence factors associated with formation.

SFRH/BD/116515/2014 PTDC/BBB-EBB/2723/2014 UID/Multi/04378/2019 grant agreement number 810856 Reducing CO2 is a challenging chemical transformation that biology solves easily, with high efficiency and specificity. In particular, formate dehydrogenases are of great interest since they reduce CO2 to formate, a valuable chemical fuel and hydrogen storage compound. The metal-dependent formate dehydrogenases of prokaryotes can show high activity for CO2 reduction. Here, we report an expression system to produce recombinant W/Sec-FdhAB from Desulfovibrio vulgaris Hildenborough fully loaded with cofactors, its catalytic characterization and crystal structures in oxidized and reduced states. The enzyme has very high activity for CO2 reduction and displays remarkable oxygen stability. The crystal structure of the formate-reduced enzyme shows Sec still coordinating the tungsten, supporting a mechanism of stable metal coordination during catalysis. Comparison of the oxidized and reduced structures shows significant changes close to the active site. The DvFdhAB is an excellent model for studying catalytic CO2 reduction and probing the mechanism of this conversion. publishersversion published

Cytosolic glutathione S-transferase (GST) enzymes participate in several cellular processes in addition to facilitating glutathione conjugation reactions that eliminate endogenous and exogenous toxic compounds, especially electrophiles. GSTs are thought to interact with various kinases, resulting in the modulation of apoptotic processes and cellular proliferation. The present research used a combination of in silico and in vitro studies to investigate protein���protein interactions between the seven most abundant cytosolic GSTs���GST alpha-1 (GST-A1), GST alpha-2 (GST-A2), GST mu-1 (GST-M1), GST mu-2 (GST-M2), GST mu-5 (GST-M5), GST theta-1 (GST-T1) and GST pi-1 (GST-P1)���and Mitogen-activated protein kinase 8 (MAPK8) and Apoptosis signal-regulating kinase 1 (ASK1). MAPK8 and ASK1 were chosen as this study���s protein interaction partners because of their predominant role in electrophile or cytokine-induced stress-mediated apoptosis, inflammation and fibrosis. The highest degree of sequence homology or sequence similarity was observed in two GST subgroups: the GST-A1, GST-A2 and GST-P1 isoforms constituted subgroup1; the GST-M1, GST-M2 and GST-M5 isoforms constituted subgroup 2. The GST-T1 isoform diverged from these isoforms. In silico investigations revealed that GST-M1 showed a significantly higher binding affinity to MAPK8, and its complex was more structurally stable than the other isoforms, in the order GST-M1 > GST-M5 > GST-P1 > GST-A2 > GST-A1 > GST-M2 > GST-T1. Similarly, GST-A1, GST-P1 and GST-T1 actively interacted with ASK1, and their structural stability was also better, in the order GST-T1 > GST-A1 > GST-P1 > GST-A2 > GST-M5 > GST-M1 > GST-M2. To validate in silico results, we performed in vitro crosslinking and mass spectroscopy experiments. Results indicated that GST-M1 interacted with GST-T1 to form heterodimers and confirmed the predicted interaction between GST-M1 and MAPK8. Communicated by Ramaswamy H. Sarma

Aldehyde oxidases are molybdenum and flavin dependent enzymes characterized by a very wide substrate specificity and performing diverse reactions that include oxidations (e.g., aldehydes and aza-heterocycles), hydrolysis of amide bonds, and reductions (e.g., nitro, S-oxides and N-oxides). Oxidation reactions and amide hydrolysis occur at the molybdenum site while the reductions are proposed to occur at the flavin site. AOX activity affects the metabolism of different drugs and xenobiotics, some of which designed to resist other liver metabolizing enzymes (e.g., cytochrome P450 monooxygenase isoenzymes), raising its importance in drug development. This work consists of a comprehensive overview on aldehyde oxidases, concerning the genetic evolution of AOX, its diversity among the human population, the crystal structures available, the known catalytic reactions and the consequences in pre-clinical pharmacokinetic and pharmacodynamic studies. Analysis of the different animal models generally used for pre-clinical trials and comparison between the human (hAOX1), mouse homologs as well as the related xanthine oxidase (XOR) are extensively considered. The data reviewed also include a systematic analysis of representative classes of molecules that are hAOX1 substrates as well as of typical and well characterized hAOX1 inhibitors. The considerations made on the basis of a structural and functional analysis are correlated with reported kinetic and metabolic data for typical classes of drugs, searching for potential structural determinants that may dictate substrate and/or inhibitor specificities.

Funding Information: Acknowledgments: The authors acknowledge the support from FEDER funds through the POCI‐ COMPETE 2020–Operational Programme Competitiveness and Internationalisation in Axis I– Strengthening research, technological development and innovation (Project POCI‐01‐0145‐FEDER‐ 007491) and National Funds (Project UID/Multi/00709/2013), Jorge Barroca‐Ferreira’s and Sandra Rocha’s individual PhD Fellowship (SFRH/BD/130068/2017 and SFRH/BD/115693/2016, respectively), and Luís A. Passarinha’s sabbatical fellowship (SFRH/BSAB/150376/2019) from FCT– Fundação para a Ciência e Tecnologia. This work was also supported by the Applied Molecular Biosciences Unit UCIBIO (UIDB/04378/2020 and UIDP/04378/2020) and the Associate Laboratory Institute for Health and Bioeconomy–i4HB (project LA/P/0140/2020) which are financed by National Funds from FCT/MCTES. Funding Information: The authors acknowledge the support from FEDER funds through the POCI- COMPETE 2020?Operational Programme Competitiveness and Internationalisation in Axis I? Strengthening research, technological development and innovation (Project POCI-01-0145-FEDER- 007491) and National Funds (Project UID/Multi/00709/2013), Jorge Barroca-Ferreira?s and Sandra Rocha?s individual PhD Fellowship (SFRH/BD/130068/2017 and SFRH/BD/115693/2016, respectively), and Lu?s A. Passarinha?s sabbatical fellowship (SFRH/BSAB/150376/2019) from FCT? Funda??o para a Ci?ncia e Tecnologia. This work was also supported by the Applied Molecular Biosciences Unit UCIBIO (UIDB/04378/2020 and UIDP/04378/2020) and the Associate Laboratory Institute for Health and Bioeconomy?i4HB (project LA/P/0140/2020) which are financed by National Funds from FCT/MCTES. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Background: The STEAP1 is a cell-surface antigen over-expressed in prostate cancer, which contributes to tumor progression and aggressiveness. However, the molecular mechanisms underlying STEAP1 and its structural determinants remain elusive. Methods: The fraction capacity of Butyl- and Octyl-Sepharose matrices on LNCaP lysates was evaluated by manipulating the ionic strength of binding and elution phases, followed by a Co-Immunoprecipitation (Co-IP) polishing. Several potential stabilizing additives were assessed, and the melting temperature (Tm) values ranked the best/worst compounds. The secondary structure of STEAP1 was identified by circular dichroism. Results: The STEAP1 was not fully captured with 1.375 M (Butyl), in contrast with interfering heterologous proteins, which were strongly retained and mostly eluted with water. This single step demonstrated higher selectivity of Butyl-Sepharose for host impurities removal from injected crude samples. Co-IP allowed recovering a purified fraction of STEAP1 and contributed to unveil potential physiologically interacting counterparts with the target. A Tm of ~55 °C was determined, confirming STEAP1 stability in the purification buffer. A predominant α-helical structure was identified, ensuring the protein’s structural stability. Conclusions: A method for successfully isolating human STEAP1 from LNCaP cells was provided, avoiding the use of detergents to achieve stability, even outside a membrane-mimicking environment. publishersversion published

We investigated the binding of the cephalosporin-class drug cefoperazone (CFP) with bovine serum albumin (BSA) using spectroscopic techniques and in silico methods. The aim of this study was to (i) emphasize the importance of correcting for the inner filter effect in this type of study and (ii) understand the binding mechanism of CFP with BSA by addressing protein conformation and plausible binding sites. Formation of the complex was confirmed by UV–visible spectroscopy. Quenching of BSA fluorescence in the presence of CFP was also observed. Because of the high absorption of CFP in the fluorescence emission range of BSA, the fluorescence emission spectra were corrected for the inner filter effect. Fluorescence emission was studied at excitation wavelengths of 280 and 295 nm. The uncorrected data showed a significant contribution of tyrosine at the excitation wavelength of 280 nm; however, after correction, this contribution became negligible. The static-type mechanism was found to be involved in quenching, with almost 1:1 binding between BSA and CFP. Hydrogen bonding and hydrophobic forces were found to dominate the protein–ligand interactions with a slight decrease in the α-helical contents. Synchronous fluorescence spectral data (at Δλ = 15 and 60 nm) were also corrected for the inner filter effect, with the results being similar to those of excitation at 280 and 295 nm. Molecular docking and molecular dynamics (MD) simulation results suggest that, apart from the two known drug binding sites (drug site I and II), one putative binding site (binding site III) located between BSA domains 1 and 3 was also possible for CFP. MD simulations of the previously reported drug binding sites (drug site I and II) and putative binding site III revealed that binding site III showed excellent binding profiles and could be a target for future research related to BSA-drug binding.

Aldehyde Oxidase (hAOX1) is a cytosolic enzyme involved in the metabolism of drugs and xenobiotic compounds. The enzyme belongs to the xanthine oxidase (XO) family of Mo containing enzyme and is a homo‐dimer of two 150 kDa monomers. Nonsynonymous Single Nucleotide Polymorphisms (nsSNPs) of hAOX1 have been reported as affecting the ability of the enzyme to metabolize different substrates. Some of these nsSNPs have been biochemically and structurally characterized but the lack of a systematic and comprehensive study regarding all described and validated nsSNPs is urgent, due to the increasing importance of the enzyme in drug development, personalized medicine and therapy, as well as in pharmacogenetic studies. The objective of the present work was to collect all described nsSNPs of hAOX1 and utilize a series of bioinformatics tools to predict their effect on protein structure stability with putative implications on phenotypic functional consequences. Of 526 nsSNPs reported in NCBI‐dbSNP, 119 are identified as deleterious whereas 92 are identified as nondeleterious variants. The stability analysis was performed for 119 deleterious variants and the results suggest that 104 nsSNPs may be responsible for destabilizing the protein structure, whereas five variants may increase the protein stability. Four nsSNPs do not have any impact on protein structure (neutral nsSNPs) of hAOX1. The prediction results of the remaining six nsSNPs are nonconclusive. The in silico results were compared with available experimental data. This methodology can also be used to identify and prioritize the stabilizing and destabilizing variants in other enzymes involved in drug metabolism.

The Southern Photometric Local Universe Survey (S-PLUS) is imaging similar to 9300 deg(2) of the celestial sphere in 12 optical bands using a dedicated 0.8mrobotic telescope, the T80-South, at the Cerro Tololo Inter-american Observatory, Chile. The telescope is equipped with a 9.2k x 9.2k e2v detector with 10 mu m pixels, resulting in a field of view of 2 deg(2) with a plate scale of 0.55 arcsec pixel-1. The survey consists of four main subfields, which include two non-contiguous fields at high Galactic latitudes (vertical bar b vertical bar > 30 degrees, 8000 deg(2)) and two areas of the Galactic Disc and Bulge (for an additional 1300 deg(2)). S-PLUS uses the Javalambre 12-band magnitude system, which includes the 5 ugriz broad-band filters and 7 narrow-band filters centred on prominent stellar spectral features: the Balmer jump/[OII], Ca H + K, Hd, G band, Mg b triplet, H alpha, and the Ca triplet. S-PLUS delivers accurate photometric redshifts (dz /(1 + z) = 0.02 or better) for galaxies with r < 19.7 AB mag and z < 0.4, thus producing a 3D map of the local Universe over a volume of more than 1 (Gpc/h)(3). The final S-PLUS catalogue will also enable the study of star formation and stellar populations in and around the Milky Way and nearby galaxies, as well as searches for quasars, variable sources, and low-metallicity stars. In this paper we introduce the main characteristics of the survey, illustrated with science verification data highlighting the unique capabilities of S-PLUS. We also present the first public data release of similar to 336 deg(2) of the Stripe 82 area, in 12 bands, to a limiting magnitude of r = 21, available at datalab.noao.edu/splus.© 2019 The Author(s).Published by Oxford University Press on behalf of the Royal Astronomical Society, The S-PLUS project, including the T80S robotic telescope and the S-PLUS scientific survey, was founded as a partnership between the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), the Observatorio Nacional (ON), the Federal University of Sergipe (UFS), and the Federal University of Santa Catarina (UFSC), with important financial and practical contributions from other collaborating institutes in Brazil, Chile (Universidad de La Serena), and Spain (Centro de Estudios de Fisica del Cosmos de Aragon, CEFCA). The members of the collaboration are grateful for the support received from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq; grants 312333/2014-5, 306968/2014-2, 142436/2014-3, 459553/2014-3, 400738/2014-7, 302037/2015-2, 312307/2015-2, 300336/2016-0, 304184/2016-0, 304971/2016-2, 401669/2016-5, 308968/2016-6, 309456/2016-9, 421687/2016-9, 150237/2017-0, 311331/2017-3, 304819/2017-4, and 200289/2017-9), FAPESP (grants 2009/54202-8, 2011/51680-6, 2014/07684-5, 2014/11806-9, 2014/13723-3, 2014/18632-6, 2016/17119-9, 2016/12331-0, 2016/21532-9, 2016/21664-2, 2016/23567-4, 2017/01461-2, 2017/23766-0, 2018/02444-7, and 2018/21661-9), the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES; grants 88881.030413/2013-01 and 88881.156185/2017-01), the Fundacao de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ; grants 202.876/2015, 202.835/2016, and 203.186/2016), the Financiadora de Estudos e Projetos (FINEP; grants 1217/13-01.13.0279.00 and 0859/10-01.10.0663.00), the Direccion de Investigacion y Desarrollo de la Universidad de La Serena (DIDULS/ULS; projects PR16143 and PTE16146 and the Programa de Investigadores Asociados), and the Direccion de Postgrado y Postitulo. TCB, VMP, and DDW acknowledge the support from the Physics Frontier Center for the Evolution of the Elements (JINA-CEE) through the US National Science Foundation (grant PHY 14-30152). JLNC is grateful for financial support received from the Southern Office of Aerospace Research and development (SOARD; grants FA9550-15-1-0167 and FA9550-18-1-0018) of the Air Force Office of the Scientific Research International Office of the United States (AFOSR/IO). YJT and RAD acknowledge support from the Spanish National Research Council (CSIC) I-COOP + 2016 program (grant COOPB20263), and the Spanish Ministry of Economy, Industry, and Competitiveness (MINECO; grants AYA2013-48623-C2-1-P and AYA2016-81065-C2-1-P). RAOM acknowledges support from the Direccion General de Asuntos del Personal Academico of the Universidad Nacional Autonoma de Mexico (DGAPA-UNAM) through a post-doctoral fellowship from the Programa de Becas Posdoctorales en la UNAM. This work has made use of data from the Sloan Digital Sky Survey. Funding for the SDSS and SDSS-II has been provided by the Alfred P. Sloan Foundation, the Participating Institutions, the National Science Foundation, the U.S. Department of Enenergy, the National Aeronautics and Space Administration, the Japanese Monbukagakusho, the Max Planck Society, and the Higher Education Funding Council for England. The SDSS Web Site is http://www.sdss.org/.The SDSS is managed by the Astrophysical Research Consortium for the Participating Institutions. The Participating Institutions are the American Museum of Natural History, Astrophysical Institute Potsdam, University of Basel, University of Cambridge, Case Western Reserve University, University of Chicago, Drexel University, Fermilab, the Institute for Advanced Study, the Japan Participation Group, Johns Hopkins University, the Joint Institute for Nuclear Astrophysics, the Kavli Institute for Particle Astrophysics and Cosmology, the Korean Scientist Group, the Chinese Academy of Sciences (LAMOST), Los Alamos National Laboratory, the Max-Planck-Institute for Astronomy (MPIA), the Max-Planck-Institute for Astrophysics (MPA), the New Mexico State University, Ohio State University, University of Pittsburgh, University of Portsmouth, Princeton University, the United States Naval Observatory, and the University of Washington. This publication makes use of data products from the Widefield Infrared Survey Explorer, which is a joint project of the University of California, Los Angeles, and the Jet Propulsion Laboratory/California Institute of Technology, funded by the National Aeronautics and Space Administration. We are grateful for the contributions of CTIO staff in helping in the construction, commissioning, and maintenance of the telescope and camera and we are particularly thankful to the CTIO director, Steve Heathcote, for his support at every phase, without which this project would not have been completed. We thank Cesar Iniguez for making the 2D measurements of the filter transmissions at CEFCA. We warmly thank David Cristobal-Hornillos and his group for helping us to install and run the reduction package JYPE version 0.9.9 in the S-PLUS computer system in Chile. We warmly thank Mariano Moles, Javier Cenarro, Tamara Civera, Sergio Chueca, Javier Hernandez Fuertes, Antonio Marin Franch, Jesus Varella, and Hector Vazquez Ramio -the success of the S-PLUS project relies on the dedication of these and other CEFCA staff members in building OAJ and running J-PLUS and J-PAS. We deeply thank Rene Laporte and INPE, as well as Keith Taylor, for their contributions to the T80S camera.

The origin of the arc-shaped Sh2-296 nebula is still unclear. Mainly due to its morphology, the nebula has been suggested to be a 0.5 Myr-old supernova remnant (SNR) that could be inducing star formation in the CMa OB1 association. We aim to show, for the first time, that the nebula is part of a large, shell-like structure, which we have designated the ``CMa shell'', enclosing a bubble created by successive supernova (SN) explosions. We identified three runaway stars, associated with bow-shock structures, in the direction of the CMa shell and we investigate the possibility that they have originated in the center of the shell. By analyzing images of the CMa OB1 association at several wavelengths, we clearly see that the Sh2-296 nebula is in fact part of a large structure, which can be approximated by a large (with a diameter of ~60 pc) elliptical shell. Using the recent Gaia-DR2 astrometric data, we trace back the path of the three runaway stars, in order to find their original position in the past, with relation to the CMa shell. We also revise the heating and ionization of the Sh2-296 nebula, by comparing the photon budget provided by the O stars in the region with results from radio observations. We find that the runaway stars have likely been ejected from a Trapezium-like progenitor cluster on three successive SN explosions having taken place ~6, ~2 and ~1 Myr ago. We also show that the few late-type O stars in the region cannot explain the ionization of the Sh~2-296 nebula and other mechanisms need to be at work. We argue that, though we now have evidence for several SNe events in the CMa OB1 association, the SNe probably played a minor role in triggering star formation in these clouds. In contrast, the CMa OB1 association, as it is now, likely testifies to the last stages of a star-forming region., 15 pages, 10 figures, accepted by A&A

Plastic Optical Fibers (POFs) are becoming popular for the development of sensors with multiple applications. In this work, we report a preliminary study related with the coating of modified POFs with proteins. Samples of POF were immersed in buffer and in proteins’ solutions under different experimental conditions: modification of the POF sample, immersion time, temperature, buffer solution and concentration of protein. A simple and easy methodology using protein staining solution was performed to confirm the coating of the POFs with proteins and to evaluate the dependency with the experimental conditions. Further developments will be focused on the coating of POFs with modified proteins for the selective detection of contaminants in water contributing to the development of low-cost POF chemical sensors and biosensors.

This work was supported by the Applied Molecular Biosciences Unit-UCIBIO which is financed by national funds from FCT/MCTES (UID/Multi/04378/2019), Project PTDC/BBB-BEP/1185/2014 and Grant SFRH/BPD/ 84581/2012 (CC). SL thanks the Deutsche Forschungsge-meinschaft (DFG) for the support with Grant LE1171/8-3. The ESRF staff are also acknowledged, in particular ID30B beamline staff (ESRF, Grenoble). Human aldehyde oxidase (hAOX1) is a molybdenum enzyme with high toxicological importance, but its physiological role is still unknown. hAOX1 metabolizes different classes of xenobiotics and is one of the main drug-metabolizing enzymes in the liver, along with cytochrome P450. hAOX1 oxidizes and inactivates a large number of drug molecules and has been responsible for the failure of several phase I clinical trials. The interindividual variability of drug-metabolizing enzymes caused by single nucleotide polymorphisms (SNPs) is highly relevant in pharmaceutical treatments. In this study, we present the crystal structure of the inactive variant G1269R, revealing the first structure of a molybdenum cofactor (Moco)-free form of hAOX1. These data allowed to model, for the first time, the flexible Gate 1 that controls access to the active site. Furthermore, we inspected the thermostability of wild-type hAOX1 and hAOX1 with various SNPs (L438V, R1231H, G1269R or S1271L) by CD spectroscopy and ThermoFAD, revealing that amino acid exchanges close to the Moco site can impact protein stability up to 10 °C. These results correlated with biochemical and structural data and enhance our understanding of hAOX1 and the effect of SNPs in the gene encoding this enzyme in the human population. Enzymes: Aldehyde oxidase (EC1.2.3.1); xanthine dehydrogenase (EC1.17.1.4); xanthine oxidase (EC1.1.3.2). Databases: Structural data are available in the Protein Data Bank under the accession number 6Q6Q. publishersversion published

International journal of molecular sciences 20(4), 860 - (2019). doi:10.3390/ijms20040860, Bcl-2 protein is involved in cell apoptosis and is considered an interesting target for anti-cancer therapy. The present study aims to understand the stability and conformational changes of Bcl-2 upon interaction with the inhibitor venetoclax, and to explore other drug-target regions. We combined biophysical and in silico approaches to understand the mechanism of ligand binding to Bcl-2. Thermal shift assay (TSA) and urea electrophoresis showed a significant increase in protein stability upon venetoclax incubation, which is corroborated by molecular docking and molecular dynamics simulations. An 18 °C shift in Bcl-2 melting temperature was observed in the TSA, corresponding to a binding affinity multiple times higher than that of any other reported Bcl-2 inhibitor. This protein-ligand interaction does not implicate alternations in protein conformation, as suggested by SAXS. Additionally, bioinformatics approaches were used to identify deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) of Bcl-2 and their impact on venetoclax binding, suggesting that venetoclax interaction is generally favored against these deleterious nsSNPs. Apart from the BH3 binding groove of Bcl-2, the flexible loop domain (FLD) also plays an important role in regulating the apoptotic process. High-throughput virtual screening (HTVS) identified 5 putative FLD inhibitors from the Zinc database, showing nanomolar affinity toward the FLD of Bcl-2, Published by Molecular Diversity Preservation International, Basel

Identification of novel molecules that can selectively inhibit the growth of tumor cells, avoid causing side effects to patients and/or intrinsic or acquired resistance, usually associated with common chemotherapeutic agents, is of utmost importance. Organometallic compounds have gained importance in oncologic chemotherapy, such as organotin(iv) complexes. In this study, we assessed the anti-tumor activity of the cyclic trinuclear organotin(iv) complex with an aromatic oximehydroxamic acid group [nBu2Sn(L)]3(H2L = N,2-dihydroxy-5-[N-hydroxyethanimidoyl]benzamide) - MG85 - and provided further characterization of its biological targets. We have previously shown the high anti-proliferative activity of this complex against human colorectal and hepatocellular carcinoma cell lines and lower cytotoxicity in neonatal non-tumor fibroblasts. MG85 induces tumor cell apoptosis and down-regulation of proteins related to tubulin dynamics (TCTP and COF1). Further characterization included the: (i) evaluation of interference in the cell cycle progression, including the expression of critical genes; (ii) affinity to DNA and the corresponding mode of binding; (iii) genotoxic potential in cells with deficient DNA repair pathways; and (iv) in vivo tumor reduction efficiency using mouse colorectal carcinoma xenografts.

The authors thank the Oxford Protein Production Facility, Research Complex (Harwell, Didcot, UK) for access to high-throughput facilities and assistance during the experiments; beamline staff at I02 and I04 for assistance during data collection at Diamond Light Source (Didcot, UK). DVV, TAF, FL and MS were supported by fellowships SFRH/BD/62415/2009, SFRH/BD/36843/2007, info:eu-repo/grantAgreement/FCT/5876/147258/PT PD/BD/105737/2014 and PD/BD/128202/2016 from Fundacao para a Ciencia e Tecnologia (FCT-MCTES), Portugal. This project was supported by Project PTDC/BIA-MIC/3195/2012 from FCT-MCTES, Portugal, Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER - COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI), Pest-OE/BIA/UI0457/2001 (CREM) and Unidade de Ciencias Biomoleculares Aplicadas-UCIBIO which is financed by national funds from FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728). The NMR spectrometers are part of the National NMR Network (PTNMR) and are supported by Infrastructure Project No 022161 (co-financed by FEDER through COMPETE 2020, POCI, and PORL and FCT through PIDDAC). Gram-positive bacteria homeostasis and antibiotic resistance mechanisms are dependent on the intricate architecture of the cell wall, where amidated peptidoglycan plays an important role. The amidation reaction is carried out by the bi-enzymatic complex MurT-GatD, for which biochemical and structural information is very scarce. In this work, we report the first crystal structure of the glutamine amidotransferase member of this complex, GatD from Staphylococcus aureus, at 1.85 Å resolution. A glutamine molecule is found close to the active site funnel, hydrogen-bonded to the conserved R128. In vitro functional studies using 1H-NMR spectroscopy showed that S. aureus MurT-GatD complex has glutaminase activity even in the absence of lipid II, the MurT substrate. In addition, we produced R128A, C94A and H189A mutants, which were totally inactive for glutamine deamidation, revealing their essential role in substrate sequestration and catalytic reaction. GatD from S. aureus and other pathogenic bacteria share high identity to enzymes involved in cobalamin biosynthesis, which can be grouped in a new sub-family of glutamine amidotransferases. Given the ubiquitous presence of GatD, these results provide significant insights into the molecular basis of the so far undisclosed amidation mechanism, contributing to the development of alternative therapeutics to fight infections. publishersversion published

Selective base pairing is the foundation of DNA recognition. Here, we elucidate the molecular and structural details of a FRET-based two-component molecular beacon relying on steady-state fluorescence spectroscopy, small-angle X-ray scattering (SAXS), microscale thermophoresis (MST), and differential electrophoretic mobility. This molecular beacon was designed to detect the most common fusion sequences causing chronic myeloid leukemia, e14a2 and e13a2. The emission spectra indicate that the self-assembly of the different components of the biosensor occurs sequentially, triggered by the fully complementary target. We further assessed the structural alterations leading to the specific fluorescence FRET signature by SAXS, MST, and the differential electrophoretic mobility, where the size range observed is consistent with hybridization and formation of a 1:1:1 complex for the probe in the presence of the complementary target and revelator. These results highlight the importance of different techniques to explore conformational DNA changes in solution and its potential to design and characterize molecular biosensors for genetic disease diagnosis.

Vanadium is an element ubiquitously present in our planet's crust and thus there are several organisms that use vanadium for activity or function of proteins. Examples are the vanadium-dependent haloperoxidases and the vanadium-containing nitrogenases. Some organisms that use vanadium have extremely efficient and selective protein-dependent systems for uptake and transport of vanadium and are able to accumulate high levels of vanadium from seawater, vanabins being a unique family of vanadium binding proteins found in ascidians involved in this process. For all of the systems a discussion regarding the role of the V-containing proteins is provided, mostly centered on structural aspects of the vanadium site and, when possible or relevant, relating this to the mechanisms operating. Phosphate is very important in biological systems and is involved in an extensive number of biological recognition and bio-catalytic systems. Vanadate(V) is able to inhibit many of the enzymes involved in these processes, such as ATPases, phosphatases, ribonucleases, phosphodiesterases, phosphoglucomutase and glucose-6-phosphatase, and it appears clear that this is closely related to the analogous physicochemical properties of vanadate and phosphate. The ability of vanadium to interfere with the metabolic processes involving Ca2+ and Mg2+, connected with its versatility to undergo changes in coordination geometry, allow V to influence the function of a large variety of phosphate-metabolizing enzymes and vanadate(V) salts and compounds have been frequently used either as inhibitors of these enzymes, or as probes to study the mechanisms of their reactions and catalytic cycle. In this review we give an overview of the many examples so far reported, also disclosing that vanadate(IV) may also have an equally efficient inhibiting effect. The prospective application of vanadium compounds as therapeutics has also been an important topic of research. How vanadium may be transported in blood and up-taken by cells are particularly relevant issues, this being mainly dependent on transferrin (and albumin) present in blood plasma. The thousands of studies reported on the effects of vanadium compounds reflect the complexity of the interactions occurring. Although it is not easy to anticipate/determine if a particular effect observed in a test tube or in vitro is also going to take place in vivo, it is clear that vanadium ions may interfere with many metabolic processes at many distinct levels. Emphasis is given on structural and functional aspects of vanadium–protein interactions relevant for vanadium binding and/or for clarification of role of the metal center in the reaction mechanisms. The additional knowledge that the presence of vanadium can change the action of a protein, other than simply inhibiting it, may also be important to understand how vanadium affects biological systems. This possibility, together with the vanadate–phosphate analogy further potentiates the belief that vanadium probably has relevant functions in living beings, which may involve interaction or incorporation of the metal ion and/or its compounds with several proteins.

Chlorite dismutase (Cld) is a b-type hemecontaining enzymethat catalyzes the reduction of chlorite into chloride plus dioxygen. This enzyme has gained attention because it can be used in the development of bioremediation processes, biosensors, and controlled dioxygen production. In the present work, Cld was purified from Magnetospirillum sp. cells cultured anaerobically with acetate/perchlorate until stationary phase. Biochemical, spectroscopic and X-ray crystallography methods showed that Cld from Magnetospirillum sp. is a ~140 kDa homopentamer comprising ~27.8 kDa monomers. Preliminary X-ray crystallography studies confirmed the quaternary structure and the presence of one b-type heme per monomer. The EPR spectroscopic signature of the as-purified Cld samples is affected by the buffer composition used during the purification. Potassium phosphate buffer is the only buffer that affected neither the spectral nor the kinetic properties of Cld. Kinetic studies in solution revealed that Cld from Magnetospirillum sp. decomposes chlorite at high turnover rates with optimal pH 6.0. A temperature below 10 °C is required to avoid enzyme inactivation due to cofactor bleaching during turnover, and to achieve full substrate consumption. Cld kinetic parameters were not affected when kinetic assays were performed in the presence of air or under argon atmosphere, but chloride is a weak mixed inhibitor that modifies the EPR signal of as-prepared samples. Fil: Freire, Diana M.. Universidade Nova de Lisboa. Faculdade de Ciências e Tecnologia. Departamento de Química; Portugal Fil: Rivas, Maria Gabriela. Universidade Nova de Lisboa. Faculdade de Ciências e Tecnologia. Departamento de Química; Portugal. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina Fil: Dias, André M.. Universidade Nova de Lisboa. Faculdade de Ciências e Tecnologia. Departamento de Química; Portugal Fil: Lopes, Ana T.. Universidade Nova de Lisboa. Faculdade de Ciências e Tecnologia. Departamento de Química; Portugal Fil: Costa, Cristina. Universidade Nova de Lisboa. Faculdade de Ciências e Tecnologia. Departamento de Química; Portugal Fil: Santos Silva, Teresa. Universidade Nova de Lisboa. Faculdade de Ciências e Tecnologia. Departamento de Química; Portugal Fil: Van Doorslaer, Sabine. University of Antwerp. Department of Physics, ; Bélgica Fil: González, Pablo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidade Nova de Lisboa. Faculdade de Ciências e Tecnologia. Departamento de Química; Portugal

Aldehyde oxidase (AOX) is a xanthine oxidase (XO)-related enzyme with emerging importance due to its role in the metabolism of drugs and xenobiotics. We report the first crystal structures of human AOX1, substrate free (2.6-angstrom resolution) and in complex with the substrate phthalazine and the inhibitor thioridazine (2.7-angstrom resolution). Analysis of the protein active site combined with steady-state kinetic studies highlight the unique features, including binding and substrate orientation at the active site, that characterize human AOX1 as an important drug-metabolizing enzyme. Structural analysis of the complex with the noncompetitive inhibitor thioridazine revealed a new, unexpected and fully occupied inhibitor-binding site that is structurally conserved among mammalian AOXs and XO. The new structural insights into the catalytic and inhibition mechanisms of human AOX that we now report will be of great value for the rational analysis of clinical drug interactions involving inhibition of AOX1 and for the prediction and design of AOX-stable putative drugs.

A few ruthenium based metal carbonyl complexes, e.g. CORM-2 and CORM-3, have therapeutic activity attributed to their ability to deliver CO to biological targets. In this work, a series of related complexes with the formula [Ru(CO)3Cl2L] (L = DMSO (3), L-H3CSO(CH2)2CH(NH2)CO2H) (6a); D,L-H3CSO(CH2)2CH(NH2)CO2H (6b); 3-NC5H4(CH2)2SO3Na (7); 4-NC5H4(CH2)2SO3Na (8); PTA (9); DAPTA (10); H3CS(CH2)2CH(OH)CO2H (11); CNCMe2CO2Me (12); CNCMeEtCO2Me (13); CN(c-C3H4)CO2Et) (14)) were designed, synthesized and studied. The effects of L on their stability, CO release profile, cytotoxicity and anti-inflammatory properties are described. The stability in aqueous solution depends on the nature of L as shown using HPLC and LC-MS studies. The isocyanide derivatives are the least stable complexes, and the S-bound methionine oxide derivative is the more stable one. The complexes do not release CO gas to the headspace, but release CO2 instead. X-ray diffraction of crystals of the model protein Hen Egg White Lysozyme soaked with 6b (4UWN) and 8 (4UWN) shows the addition of Ru(II)(CO)(H2O)4 at the His15 binding site. Soakings with 7(4UWN) produced the metallacarboxylate [Ru(COOH)(CO)(H2O)3](+) bound to the His15 site. The aqueous chemistry of these complexes is governed by the water-gas shift reaction initiated with the nucleophilic attack of HO(-) on coordinated CO. DFT calculations show this addition to be essentially barrierless. The complexes have low cytotoxicity and low hemolytic indices. Following i.v. administration of CORM-3, the in vivo bio-distribution of CO differs from that obtained with CO inhalation or with heme oxygenase stimulation. A mechanism for CO transport and delivery from these complexes is proposed.

Previous studies generally agree that in the blood serum vanadium is transported mainly by human serum transferrin (hTF). In this work through the combined use of electrochemical techniques, matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry and small-angle X-ray scattering (SAXS) data it is confirmed that both VIV and VV bind to apo-hTF and holo-hTF. The electrochemical behavior of solutions containing vanadate(V) solutions at pH=7.0, analyzed by using two different voltammetric techniques, with different time windows, at a mercury electrode, Differential Pulse Polarography (DPP) and Cyclic Voltammetry (CV), is consistent with a stepwise reduction of VV→VIV and VIV→VII. Globally the voltammetric data are consistent with the formation of 2:1 complexes in the case of the system VV-apo-hTF and both 1:1 and 2:1 complexes in the case of VV-holo-hTF; the corresponding conditional formation constants were estimated. MALDI-TOF mass spectrometric data carried out with samples of VIVOSO4 and apo-hTF and of NH4VVO3 with both apo-hTF and holo-hTF with V:hTF ratios of 3:1 are consistent with the binding of vanadium to the proteins. Additionally the SAXS data suggest that both VIVOSO4 and NaVVO3 can effectively interact with human apo-transferrin, but for holo-hTF no clear evidence was obtained supporting the existence or the absence of protein-ligand interactions. This latter data suggest that the conformation of holo-hTF does not change in the presence of either VIVOSO4 or NH4VVO3. Therefore, it is anticipated that VIV or VV bound to holo-hTF may be efficiently up-taken by the cells through receptor-mediated endocytosis of hTF.

Glutathione S-transferases (GSTs) comprise a family of enzymes that utilizes glutathione (GSH) in many enzymatic reactions that involved in transformation of several compounds including therapeutic drug molecules and carcinogens. In addition, GSTs influence cellular survival and proliferation, by repressing apoptosis signal-regulating kinase 1 (ASK1) thus affecting the activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) in response to various intra and extracellular stresses. Molecules inhibiting the function of GSTs received attention as an adjuvant therapy to the highly toxic electrophilic agents to avoid usage of high doses and toxicity for better outcomes. There is no detailed in silico analysis exists in literature to describe the binding patterns of known inhibitors to all GST isoforms. This study is aimed at providing details of binding patterns of known and putative substrates (Busulfan, Treosulfan, SS-EBDM, SS-DEB), inhibitors (Ethacrynic acid, Sulfolane and Curcumin) with predominately-expressed seven isoforms of GST (Alpha1, Alpha2, Pi1, Mu1, Mu2, Mu5 and Theta1). In silico methodology include six steps namely (a) Retrieval of three-dimensional structure of GSTs and Ligand molecules from RCSB-PDB and NCBI-PubChem databases, (b) Protein and Ligand preparation using Auto Dock Tools (ADT), (c) Receptor grid preparation based on known binding site (Direct docking protocol) of GSTs using AutoDock/Vina plugin in PyMOL, (d) Preparation of Auto Dock Vina configuration file, (e) Running of docking calculation using Auto Dock Vina and (f) Analysis of docking results using ADT, PyMOL and LigPlus programs. Molecular docking studies of substrates/inhibitors are performed with both Apo and GSH bounded forms of GSTs. Structural parameters such as estimated free energy of binding (ΔH), estimated inhibition constant (Ki), binding orientation, intermolecular interactions were noted for all the docking interaction models. Then the parameters were compared against each substrate or inhibitor for the affinity towards a selective GST isoform. Out of the three putative or known inhibitors screened, Curcumin showed a significant high binding affinity towards all the classes of GSTs, particularly GST Alpha1 (ΔH: -9.7 kcal/mol and Ki: 0.08 µM). Ethacrynic acid also showed better binding affinity towards GST Alpha1 (ΔH: -7.6 kcal/mol and Ki: 2.7 uM). Sulfolane did not exhibited a stronger affinity towards all the seven GST isoforms. Busulfan and Treosulfan exhibited a reasonable binding affinity towards GST Alpha1 (ΔH: -5.2 and -5.3 kcal/mol) and weakened affinity for the remaining six GST isoforms. Thus, treosulfan could be a possible substrate for GST Alpha1. Manual inspection of three-dimensional structures of the docking complexes revealed that binding-sites for inhibitor and substrate are different. In an on-going study, we are evaluating the inhibitory potential of Curcumin and Ethacrynic acid against GSTs in in vitro studies. The detailed description of the binding interactions may be useful to screen new putative GST substrates and inhibitors. Presence or absence of variants in these binding pockets also can define the amount of inhibitor required and the affinity and potency of an inhibitor and or substrate. This poster is presented at " ESPT 2017 in Catania, Italy from Oct 4th-7th 2017"

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Molybdenum and tungsten are taken up by bacteria and archaea as their soluble oxyanions through high affinity transport systems belonging to the ATP-binding cassette (ABC) transporters. The component A (ModA/TupA) of these transporters is the first selection gate from which the cell differentiates between MoO4 2-, WO4 2- and other similar oxyanions. We report the biochemical characterization and the crystal structure of the apo-TupA from Desulfovibrio desulfuricans G20, at 1.4 Å resolution. Small Angle X-ray Scattering data suggests that the protein adopts a closed and more stable conformation upon ion binding. The role of the arginine 118 in the selectivity of the oxyanion was also investigated and three mutants were constructed: R118K, R118E and R118Q. Isothermal titration calorimetry clearly shows the relevance of this residue for metal discrimination and oxyanion binding. In this sense, the three variants lost the ability to coordinate molybdate and the R118K mutant keeps an extremely high affinity for tungstate. These results contribute to an understanding of the metal-protein interaction, making it a suitable candidate for a recognition element of a biosensor for tungsten detection. Fil: Otrelo Cardoso, Ana Rita. Universidade Nova de Lisboa; Portugal Fil: Nair, Rashmi R.. Universidade Nova de Lisboa; Portugal Fil: Correia, Márcia A. S.. Universidade Nova de Lisboa; Portugal Fil: Correia Cordeiro, RAquel S.. Universidade Nova de Lisboa; Portugal Fil: Panjkovich, Alejandro. European Molecular Biology Laboratory; Alemania Fil: Svergun, Dmitri I.. European Molecular Biology Laboratory; Alemania Fil: Santos Silva, Teresa. Universidade Nova de Lisboa; Portugal Fil: Rivas, Maria Gabriela. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina

The Canis Major OB1 Association has an intriguing scenario of star formation, especially in the Canis Major R1 (CMa R1) region traditionally assigned to a reflection nebula, but in reality an ionized region. This work is focused on the young stellar population associated to CMa R1, for which our previous results from ROSAT, optical and near-infrared data had revealed two stellar groups with different ages, suggesting a possible mixing of populations originated from distinct star-formation episodes. The X-ray data allow the detected sources to be characterized according to hardness ratios, light curves and spectra. Estimates of mass and age were obtained from the 2MASS catalogue, and used to define a complete subsample of stellar counterparts, for statistical purposes. A catalogue of 387 XMM-Newton sources is provided, 78% being confirmed as members or probable members of the CMa R1 association. Flares were observed for 13 sources, and the spectra of 21 bright sources could be fitted by a thermal plasma model. Mean values of fits parameters were used to estimate X-ray luminosities. We found a minimum value of log(L$_X$[erg/s]) = 29.43, indicating that our sample of low-mass stars (M$_\star$ $\leq$ 0.5 M$_\odot$), being faint X-ray emitters, is incomplete. Among the 250 objects selected as our complete subsample (defining our best sample), 171 are found to the East of the cloud, near Z CMa and dense molecular gas, 50% of them being young (< 5 Myr) and 30% being older (> 10 Myr). The opposite happens to the West, near GU CMa, in areas lacking molecular gas: among 79 objects, 30% are young and 50% are older. These findings confirm that a first episode of distributed star formation occurred in the whole studied region ~10 Myr ago and dispersed the molecular gas, while a second, localized episode (< 5 Myr) took place in the regions where molecular gas is still present., 38 pages, 21 figures, accepted for A&A

The knowledge on the binding of vanadium ions and complexes to serum proteins and how vanadium might be transported in blood and up-taken by cells has received much attention during the last decade, particularly as far as the transport of V IV O 2+ is concerned. In this work we revise and discuss some relevant aspects of previous research, namely the two main types of binding proposed for transport of V IV O(carrier) 2 complexes. New results, obtained by circular dichroism (CD), EPR and gel electrophoresis, regarding the binding of vanadium to hTF in the oxidation states +5 and +3 are also presented. Namely, evidences for the binding of V V -species to diferric-transferrin, designated by (Fe III ) 2 hTF, as well as to (Al III ) 2 hTF, are presented and discussed, the possibility of up-take of vanadate by cells through (Fe III ) 2 hTF endocytosis being suggested. It is also confirmed that V III binds strongly to hTF, forming di-vanadium(III)-transferrin, designated by (V III ) 2 hTF, and gel electrophoresis experiments indicate that (V III ) 2 hTF corresponds to a ‘closed conformation’ similar to (Fe III ) 2 hTF.

The TupABC system is involved in the cellular uptake of tungsten and belongs to the ABC (ATP binding cassette)-type transporter systems. The TupA component is a periplasmic protein that binds tungstate anions, which are then transported through the membrane by the TupB component using ATP hydrolysis as the energy source (the reaction catalyzed by the ModC component). We report the heterologous expression, purification, determination of affinity binding constants and crystallization of the Desulfovibrio alaskensis G20 TupA. The tupA gene (locus tag Dde_0234) was cloned in the pET46 Enterokinase/Ligation-Independent Cloning (LIC) expression vector, and the construct was used to transform BL21 (DE3) cells. TupA expression and purification were optimized to a final yield of 10 mg of soluble pure protein per liter of culture medium. Native polyacrylamide gel electrophoresis was carried out showing that TupA binds both tungstate and molybdate ions and has no significant interaction with sulfate, phosphate or perchlorate. Quantitative analysis of metal binding by isothermal titration calorimetry was in agreement with these results, but in addition, shows that TupA has higher affinity to tungstate than molybdate. The protein crystallizes in the presence of 30% (w/v) polyethylene glycol 3350 using the hanging-drop vapor diffusion method. The crystals diffract X-rays beyond 1.4 Å resolution and belong to the P21 space group, with cell parameters a = 52.25 Å, b = 42.50 Å, c = 54.71 Å, β = 95.43°. A molecular replacement solution was found, and the structure is currently under refinement. Fil: Otrelo Cardoso, Ana Rita. Centro de Química Fina E Biotecnologia; Portugal Fil: Nair, Rashmi. Centro de Química Fina E Biotecnologia; Portugal Fil: Correia, Márcia. Centro de Química Fina E Biotecnologia; Portugal Fil: Rivas, Maria Gabriela. Centro de Química Fina E Biotecnologia; Portugal. Universidad Nacional del Litoral; Argentina Fil: Santos Silva, Teresa. Centro de Química Fina E Biotecnologia; Portugal

Aldehyde oxidases (AOXs) are molybdo-flavoenzymes characterized by broad substrate specificity, oxidizing aromatic/aliphatic aldehydes into the corresponding carboxylic acids and hydroxylating various heteroaromatic rings. Mammals are characterized by a complement of species-specific AOX isoenzymes, that varies from one in humans (AOX1) to four in rodents (AOX1, AOX2, AOX3 and AOX4). The physiological function of mammalian AOX isoenzymes is unknown, although human AOX1 is an emerging enzyme in phase-I drug metabolism. Indeed, the number of therapeutic molecules under development which act as AOX substrates is increasing. The recent crystallization and structure determination of human AOX1 as well as mouse AOX3 has brought new insights into the mechanisms underlying substrate/inhibitor binding as well as the catalytic activity of this class of enzymes.

The xanthine oxidase (XO) family comprises molybdenum-dependent enzymes that usually form homodimers (or dimers of heterodimers/trimers) organized in three domains that harbor two [2Fe-2S] clusters, one FAD, and a Mo cofactor. In this work, we crystallized an unusual member of the family, the periplasmic aldehyde oxidoreductase PaoABC from Escherichia coli. This is the first example of an E. coli protein containing a molybdopterin-cytosine-dinucleotide cofactor and is the only heterotrimer of the XO family so far structurally characterized. The crystal structure revealed the presence of an unexpected [4Fe-4S] cluster, anchored to an additional 40 residues subdomain. According to phylogenetic analysis, proteins containing this cluster are widely spread in many bacteria phyla, putatively through repeated gene transfer events. The active site of PaoABC is highly exposed to the surface with no aromatic residues and an arginine (PaoC-R440) making a direct interaction with PaoC-E692, which acts as a base catalyst. In order to understand the importance of R440, kinetic assays were carried out, and the crystal structure of the PaoC-R440H variant was also determined.

Objectives: To establish the allele and genotype frequencies of dihydrofolate reductase (DHFR) -317A>G and -680C>A variants in Indian population, to find the association of these variants with the risk of acute lymphoblastic leukemia (ALL) and to analyze the effects of non-synonymous SNPs (nsSNPs) and 3’untranslated region (3’UTR)variants of DHFR gene on its structure and function using in-silico tools. Methods: A total of 235 unrelated healthy volunteers (controls) and 127 ALL patients (cases) were recruited for the study. DNA was extracted from peripheral leucocytes. Genotyping of DHFR polymorphisms was done by realtime PCR. We investigated the deleterious effect of nsSNPs and variants in 3’UTR of DHFR gene through computational platforms. Results: In the present study, the frequency of DHFR -317G and -680 A alleles was found to be 33.3% and 59.8% respectively. The studied DHFR variants (rs408626 and rs442767) did not confer a significant risk for ALL. Insilico analysis revealed that three nsSNPs potentially affect the structure, function and activity of the DHFR protein. Four microRNA binding sites were found to be highly affected due to 3’UTR SNPs. Further, docking simulation suggested that the order of binding affinity of methotrexate towards native and all three mutant forms of DHFR is D153V (rs121913223)>native>G18R (rs61736208)>D187Y (rs200904105). Conclusion: This is the first study to report the normative genotype distribution of DHFR variants in Indian population and also to report that DHFR (-317A>G and -680C>A) variants do not confer a potential risk for development of ALL. Inter-ethnic differences exist in the distribution of DHFR variant genotypes, and this can lead to variability in therapeutic response to DHFR substrates. Protein sequence analysis revealed rs200904105 influences the phosphorylation process (post-translational modification) of DHFR and docking simulation suggested methotrexate to have a higher affinity towards rs121913223 mutant form. Therefore, studies are warranted to explore the clinical impact of these variants in the Indian population.

Complexes of the general formula fac-[Ru(CO)(3)L(3)](2+), namely CORM-2 and CORM-3, have been successfully used as experimental CO releasing molecules (CO-RMs) but their mechanism of action and delivery of CO remain unclear. The well characterized complex [Ru(CO)(3)Cl(2)(1,3-thiazole)] (1) is now studied as a potential model CO-RM of the same family of complexes using LC-MS, FTIR, and UV-vis spectroscopy, together with X-ray crystallography. The chemistry of [Ru(CO)(3)Cl(2)(1,3-thiazole)] is very similar to that of CORM-3: it only releases residual amounts of CO to the headspace of a solution in PBS7.4 and produces marginal increase of COHb after long incubation in whole blood. 1 also reacts with lysozyme to form Ru adducts. The crystallographic model of the lysozyme-Ru adducts shows only mono-carbonyl Ru species. [Ru(H(2)O)(4)(CO)] is found covalently bound to a histidine (His15) and to two aspartates (Asp18 and Asp119) at the protein surface. The CO release silence of both 1 and CORM-3 and their rapid formation of protein-Ru(CO)(x)(H(2)O)(y) (x=1,2) adducts, support our hypothesis that fac-[Ru(CO)(3)L(3)] CO-RMs deliver CO in vivo through the decay of their adducts with plasma proteins.

The biological role of carbon monoxide (CO) has completely changed in the last decade. Beyond its widely feared toxicity, CO has revealed a very important biological activity as a signaling molecule with marked protective actions namely against inflammation, apoptosis and endothelial oxidative damage. Its direct use as a therapeutic gas showed significant and consistent positive results but also intrinsic severe limitations. The possibility of replacing the gas by pro-drugs acting as CO-Releasing Molecules (CO-RMs) has clearly been demonstrated with several experimental compounds. Transition metal carbonyls complexes have proven to be the most versatile experimental CO-RMs so far. Presently, the challenge is to equip them with drug-like properties to turn them into useful pharmaceuticals. This requires studying their interactions with biological molecules namely those that control their pharmacokinetic and ADME profiles like the plasma proteins. In this account we analyze these questions and review the existing interactions between Metal Carbonyls and proteins. The recently explored case of CORM-3 is revisited to exemplify the methodologies involved and the importance of the results for the understanding of the mode of action of such pro-drugs.