Understanding the two-component sensing System of virulent bacteria

Report for the scientific sojourn carried out at the l’ Institute for Computational Molecular Science of the Temple University, United States, from 2010 to 2012. Two-component systems (TCS) are used by pathogenic bacteria to sense the environment within a host and activate mechanisms related to virulence and antimicrobial resistance. A prototypical example is the PhoQ/PhoP system, which is the major regulator of virulence in Salmonella. Hence, PhoQ is an attractive target for the design of new antibiotics against foodborne diseases. Inhibition of the PhoQ-mediated bacterial virulence does not result in growth inhibition, presenting less selective pressure for the generation of antibiotic resistance. Moreover, PhoQ is a histidine kinase (HK) and it is absent in animals. Nevertheless, the design of satisfactory HK inhibitors has been proven to be a challenge. To compete with the intracellular ATP concentrations, the affinity of a HK inhibidor must be in the micromolar-nanomolar range, whereas the current lead compounds have at best millimolar affinities. Moreover, the drug selectivity depends on the conformation of a highly variable loop, referred to as the “ATP-lid, which is difficult to study by X-Ray crystallography due to its flexibility. I have investigated the binding of different HK inhibitors to PhoQ. In particular, all-atom molecular dynamics simulations have been combined with enhanced sampling techniques in order to provide structural and dynamic information of the conformation of the ATP-lid. Transient interactions between these drugs and the ATP-lid have been identified and the free energy of the different binding modes has been estimated. The results obtained pinpoint the importance of protein flexibility in the HK-inhibitor binding, and constitute a first step in developing more potent and selective drugs. The computational resources of the hosting institution as well as the experience of the members of the group in drug binding and free energy methods have been crucial to carry out this work.

Distributed all-atom simulations of intrinsically unstructured proteins

The Computational Biophysics Group at the Universitat Pompeu Fabra (GRIB-UPF) hosts two unique computational resources dedicated to the execution of large scale molecular dynamics (MD) simulations: (a) the ACMD molecular-dynamics software, used on standard personal computers with graphical processing units (GPUs); and (b) the GPUGRID. net computing network, supported by users distributed worldwide that volunteer GPUs for biomedical research. We leveraged these resources and developed studies, protocols and open-source software to elucidate energetics and pathways of a number of biomolecular systems, with a special focus on flexible proteins with many degrees of freedom. First, we characterized ion permeation through the bactericidal model protein Gramicidin A conducting one of the largest studies to date with the steered MD biasing methodology. Next, we addressed an open problem in structural biology, the determination of drug-protein association kinetics; we reconstructed the binding free energy, association, and dissaciociation rates of a drug like model system through a spatial decomposition and a Makov-chain analysis. The work was published in the Proceedings of the National Academy of Sciences and become one of the few landmark papers elucidating a ligand-binding pathway. Furthermore, we investigated the unstructured Kinase Inducible Domain (KID), a 28-peptide central to signalling and transcriptional response; the kinetics of this challenging system was modelled with a Markovian approach in collaboration with Frank Noe’s group at the Freie University of Berlin. The impact of the funding includes three peer-reviewed publication on high-impact journals; three more papers under review; four MD analysis components, released as open-source software; MD protocols; didactic material, and code for the hosting group.

Human-Computer Interaction with older people through ethnographical lenses: everyday interactions with ICT

Report for the scientific sojourn carried out at the School of Computing of the University of Dundee, United Kingdom, from 2010 to 2012. This document is a scientific report of the work done, main results, publications and accomplishment of the objectives of the 2-year post-doctoral research project with reference number BP-A 00239. The project has addressed the topic of older people (60+) and Information and Communication Technologies (ICT), which is a topic of growing social and research interest, from a Human-Computer Interaction perspective. Over a 2-year period (June 2010-June 2012), we have conducted classical ethnography of ICT use in a computer clubhouse in Scotland, addressing interaction barriers and strategies, social sharing practices in Social Network Sites, and ICT learning, and carried out rapid ethnographical studies related to geo-enabled ICT and e-government services towards supporting independent living and active ageing. The main results have provided a much deeper understanding of (i) the everyday use of Computer-Mediated Communication tools, such as video-chats and blogs, and its evolution as older people’s experience with ICT increases over time, (ii) cross-cultural aspects of ICT use in the north and south of Europe, (iii) the relevance of cognition over vision in interacting with geographical information and a wide range of ICT tools, despite common stereotypes (e.g. make things bigger), (iv) the important relationship offline-online to provide older people with socially inclusive and meaningful eservices for independent living and active ageing, (v) how older people carry out social sharing practices in the popular YouTube, (vi) their user experiences and (vii) the challenges they face in ICT learning and the strategies they use to become successful ICT learners over time. The research conducted in this project has been published in 17 papers, 4 in journals – two of which in JCR, 5 in conferences, 4 in workshops and 4 in magazines. Other public output consists of 10 invited talks and seminars.

Prediction of enzyme function by combining sequence similarity and protein interactions

Background: A number of studies have used protein interaction data alone for protein function prediction. Here, we introduce a computational approach for annotation of enzymes, based on the observation that similar protein sequences are more likely to perform the same function if they share similar interacting partners. Results: The method has been tested against the PSI-BLAST program using a set of 3,890 protein sequences from which interaction data was available. For protein sequences that align with at least 40% sequence identity to a known enzyme, the specificity of our method in predicting the first three EC digits increased from 80% to 90% at 80% coverage when compared to PSI-BLAST. Conclusion: Our method can also be used in proteins for which homologous sequences with known interacting partners can be detected. Thus, our method could increase 10% the specificity of genome-wide enzyme predictions based on sequence matching by PSI-BLAST alone.

Biana: a software framework for compiling biological interactions and analyzing networks

Background: The analysis and usage of biological data is hindered by the spread of information across multiple repositories and the difficulties posed by different nomenclature systems and storage formats. In particular, there is an important need for data unification in the study and use of protein-protein interactions. Without good integration strategies, it is difficult to analyze the whole set of available data and its properties.Results: We introduce BIANA (Biologic Interactions and Network Analysis), a tool for biological information integration and network management. BIANA is a Python framework designed to achieve two major goals: i) the integration of multiple sources of biological information, including biological entities and their relationships, and ii) the management of biological information as a network where entities are nodes and relationships are edges. Moreover, BIANA uses properties of proteins and genes to infer latent biomolecular relationships by transferring edges to entities sharing similar properties. BIANA is also provided as a plugin for Cytoscape, which allows users to visualize and interactively manage the data. A web interface to BIANA providing basic functionalities is also available. The software can be downloaded under GNU GPL license from http://sbi.imim.es/web/BIANA.php.Conclusions: BIANA's approach to data unification solves many of the nomenclature issues common to systems dealing with biological data. BIANA can easily be extended to handle new specific data repositories and new specific data types. The unification protocol allows BIANA to be a flexible tool suitable for different user requirements: non-expert users can use a suggested unification protocol while expert users can define their own specific unification rules.

On optimal monetary and fiscal policy interactions in open economies

This paper studies monetary and fiscal policy interactions in a two country model, where taxes on firms sales are optimally chosen and the monetary policy is set cooperatively.It turns out that in a two country setting non-cooperative fiscal policy makers have an incentive to change taxes on sales depending on shocks realizations in order to reduce output production. Therefore whether the fiscal policy is set cooperatively or not matters for optimal monetary policy decisions. Indeed, as already shown in the literature, the cooperative monetary policy maker implements the flexible price allocation only when special conditions on the value of the distortions underlying the economy are met. However, if non-cooperative fiscal policy makers set the taxes on firms sales depending on shocks realizations, these conditions cannot be satisfied; conversely, when fiscal policy is cooperative, these conditions are fulfilled. We conclude that whether implementing the flexible price allocation is optimal or not depends on the fiscal policy regime.

Use of NIR spectroscopy and multivariate process spectra calibration methodology for pharmaceutical solid samples analysis

Accomplish high quality of final products in pharmaceutical industry is a challenge that requires the control and supervision of all the manufacturing steps. This request created the necessity of developing fast and accurate analytical methods. Near infrared spectroscopy together with chemometrics, fulfill this growing demand. The high speed providing relevant information and the versatility of its application to different types of samples lead these combined techniques as one of the most appropriated. This study is focused on the development of a calibration model able to determine amounts of API from industrial granulates using NIR, chemometrics and process spectra methodology.

Pathogenic plant-microbe interactions. What we know and how we benefit

Plants, like humans and other animals, also get sick, exhibit disease symptoms, and die. Plant diseases are caused by environmental stress, genetic or physiological disorders and infectious agents including viroids, viruses, bacteria and fungi. Plant pathology originated from the convergence of microbiology, botany and agronomy; its ultimate goal is the control of plant disease. Microbiologists have been attracted to this field of research because of the need for identification of the agents causing infectious diseases in economically important crops. In 1878—only two years after Pasteur and Koch had shown for the first time that anthrax in animals was caused by a bacteria—Burril, in the USA, discovered that the fire blight disease of apple and pear was also caused by a bacterium (nowadays known as Erwinia amylovora). In 1898, Beijerinck concluded that tobacco mosaic was caused by a “contagium vivum fluidum” which he called a virus. In 1971, Diener proved that a potato disease named potato spindle tuber was caused by infectious RNA which he called viroid

Simple and fast methods based on solid-phase extraction coupled to liquid chromatography with UV detection for the monitoring of caffeine in natural and waste waters as marker of anthropogenic impact

Two concentration methods for fast and routine determination of caffeine (using HPLC-UV detection) in surface, and wastewater are evaluated. Both methods are based on solid-phase extraction (SPE) concentration with octadecyl silica sorbents. A common “offline” SPE procedure shows that quantitative recovery of caffeine is obtained with 2 mL of an elution mixture solvent methanol-water containing at least 60% methanol. The method detection limit is 0.1 μg L−1 when percolating 1 L samples through the cartridge. The development of an “online” SPE method based on a mini-SPE column, containing 100 mg of the same sorbent, directly connected to the HPLC system allows the method detection limit to be decreased to 10 ng L−1 with a sample volume of 100 mL. The “offline” SPE method is applied to the analysis of caffeine in wastewater samples, whereas the “on-line” method is used for analysis in natural waters from streams receiving significant water intakes from local wastewater treatment plants

Impact of fisheries on activity, diet and predatory interactions between yellow-legged and Audouin's gulls breeding at the Chafarinas Is.

Interactions between yellow-legged Larus cachinnans and Audouin´s Larus audouinii gulls and fisheries operating around the Chafarinas Islands, located 4.5 km off the Moroccan Mediterranean coast, are reviewed. At the Chafarinas archipelago two distinct types of fisheries operate: trawlers and purse seines. Gulls take advantage of both fisheries. They scavenge trawler discards and congregate around shoals of fish attracted to the surface by the purse-seine lamps. When both trawlers and purse-seine boats are in operation, the diet of both gull species is similar, with epipelagic fish accounting for over 60% of the biomass, partially collected in association with the purse-seine fishery. When only trawlers operated yellow-legged gulls, but not Audouin´s gulls, augmented their diet mainly with human waste from refuse dumps, suggesting that competition for food between the two species is mainly limited to the periods when resources made available by fishery activities are abundant. Likewise, when only trawlers operated, there was an increase in the predation pressure on eggs and chicks of Audouin´s gulls. In particular, during the week of celebrations for the holy lamb festival when neither fishery operated, egg losses of Audouin´s gull increased dramatically, suggesting that severe food shortage caused by the cessation of fishing can result in an increased predation pressure by yellow-legged gulls, affecting Audouin´s gull productivity. These results suggest a novel socio-ecological link between gulls, fisheries and local feasts.

Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) Protein: Localization in Endocytic-coated Pits, Interactions with Clathrin, and the Impact of Overexpression on Clathrin-mediated Traffic

The clathrin assembly lymphoid myeloid leukemia (CALM) gene encodes a putative homologue of the clathrin assembly synaptic protein AP180. Hence the biochemical properties, the subcellular localization, and the role in endocytosis of a CALM protein were studied. In vitro binding and coimmunoprecipitation demonstrated that the clathrin heavy chain is the major binding partner of CALM. The bulk of cellular CALM was associated with the membrane fractions of the cell and localized to clathrin-coated areas of the plasma membrane. In the membrane fraction, CALM was present at near stoichiometric amounts relative to clathrin. To perform structure-function analysis of CALM, we engineered chimeric fusion proteins of CALM and its fragments with the green fluorescent protein (GFP). GFP-CALM was targeted to the plasma membrane-coated pits and also found colocalized with clathrin in the Golgi area. High levels of expression of GFP-CALM or its fragments with clathrin-binding activity inhibited the endocytosis of transferrin and epidermal growth factor receptors and altered the steady-state distribution of the mannose-6-phosphate receptor in the cell. In addition, GFP-CALM overexpression caused the loss of clathrin accumulation in the trans-Golgi network area, whereas the localization of the clathrin adaptor protein complex 1 in the trans-Golgi network remained unaffected. The ability of the GFP-tagged fragments of CALM to affect clathrin-mediated processes correlated with the targeting of the fragments to clathrin-coated areas and their clathrin-binding capacities. Clathrin-CALM interaction seems to be regulated by multiple contact interfaces. The C-terminal part of CALM binds clathrin heavy chain, although the full-length protein exhibited maximal ability for interaction. Altogether, the data suggest that CALM is an important component of coated pit internalization machinery, possibly involved in the regulation of clathrin recruitment to the membrane and/or the formation of the coated pit.

A T42A Ran Mutation: Differential Interactions with Effectors and Regulators, and Defect in Nuclear Protein Import

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran·GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran·GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin ß (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.

Reduction of symmetry in grandite solid solution

The results of a crystal structure refinement of an anisotropic grandite garnet specimen with composition Gro36-4 And63-6 are given. The structure obtained has orthorrombic symmetry (space group Fddd) and is compared with similar results obtained by other authors. In all cases the reduction of symmetry is due to the ordering of Fe3+ and Al in octahedral sites. Non cubic structures of grandites are discussed in connection with optical, morphological an grou-th features of these minerals.

New insights on the crystalline forms in binary systems of n-alkanes: Characterization of the solid ordered phases in the phase diagram tricosane + pentacosane

X-ray diffraction analyses of the pure components n-tricosane and n-pentacosane and of their binary mixed samples have enabled us to characterize the crystalline phases observed at low temperature. On the contrary to what was announced in literature on the structural behavior of mixed samples in odd-odd binary systems with D n = 2, the three domains are not all orthorhombic. This work has enabled us to show that two of the domains are, in fact, monoclinic, (Aa, Z = 4) and the other one is orthorhombic (Pca21, Z = 4). The conclusions drawn in this work can be easily transposed to other binary systems of n-alkanes.

Public health expenditure and spatial interactions in a decentralized national health system

One of the limitations of cross-country health expenditure analysis refers to the fact that the financing, the internal organization and political restraints of health care decision-making are country-specific and heterogeneous. Yet, a potential solution is to examine the influence of such effects in those countries that have undertaken decentralization processes. In such a setting, it is possible to examine potential expenditure spillovers across the geography of a country as well as the influence of the political ideology of regional incumbents on public health expenditure. This paper examines the determinants of public health expenditure within Spanish region-states (Autonomous Communities, ACs), most of them subject to similar financing structures although exhibiting significant heterogeneity as a result of the increasing decentralization, region-specific political factors along with different use of health care inputs, economic dimension and spatial interactions