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Blocking the interaction between the apoptosis-inducing factor (AIF) and cyclophilin A (CypA) by the AIF fragment AIF(370-394) is protective against glutamate-induced neuronal cell death and brain injury in mice. Starting from AIF(370-394), we report the generation of the disulfide-bridged and shorter variant AIF(381-389) and its structural characterization by nuclear magnetic resonance (NMR) in the free and CypA-bound state. AIF(381-389) in both the free and bound states assumes a β-hairpin conformation similar to that of the fragment in the AIF protein and shows a highly reduced conformational flexibility. This peptide displays a similar in vitro affinity for CypA, an improved antiapoptotic activity in cells and an enhanced proteolytic stability compared to the parent peptide. The NMR-based 3D model of the AIF(381-389)/CypA complex provides a better understanding of the binding hot spots on both the peptide and the protein and can be exploited to design AIF/CypA inhibitors with improved pharmacokinetic and pharmacodynamics features.

The noncondensable gases in most geothermal resources include CO2 and smaller amounts of other gases. Currently, the worldwide geothermal power is a small sector within the energy industry, and CO2 emissions related to the utilisation of geothermal resources are consequently small. In some countries, however, such as Turkey and Iceland, geothermal energy production contributes significantly to their energy budget, and their CO2 emissions are relatively significant. SUCCEED is a targeted innovation and research project, which aims to investigate the reinjection of CO2 produced at geothermal power production sites and develop, test, and demonstrate at field scale innovative measurement, monitoring and verification (MMV) technologies that can be used in most CO2 geological storage projects. The project is carried out at two operating geothermal energy production sites, the Kızıldere geothermal field in Turkey and the CarbFix project site at the Hellisheiði geothermal field in Iceland. Together with a brief description of the project, this paper presents the details of the two field sites and the progress made in seismic velocity characterisation and modelling relevant to the Kızıldere geothermal field in Turkey.

This paper deals with the design, acquisition and processing of vertical seismic profiling data collected in 2016 at the directional Thonex‐01 geothermal well, drilled in 1993 in the Geneva Canton (Switzerland). The aim of the study was to obtain additional information from existing‐abandoned well for downhole geophysical characterization of potential geothermal reservoirs in the Geneva area. The main results obtained by this study allowed an improved subsurface image by multi‐offset acquisitions near and around the deviated well. Specifically we were able to (1) improve the velocity model of the Geneva Basin, which, at present, is only constrained by one unique deep exploration well, located at the south‐west of the basin, far away from the study area; (2) image the transition between the Tertiary Molasse siliciclastic sediments and the Mesozoic carbonates; (3) define an acquisition strategy for future vertical seismic profiling surveys in support of the geothermal exploration campaigns in the area. This study has demonstrated the value of vertical seismic profiling method for the refinement of the subsurface geology at local scale, and its usefulness to assist the planning of second well associated with geothermal doublets.

Seven families of double-stranded DNA viruses have been identified, among which the Fuselloviridae and Rudiviridae are the most well-studied specimens and therefore represent model systems for detailed studies of archaeal virus biology. Two distinct genetic elements, SSV2 and pSSVx, belong to Fuselloviridae and coexist in the same Sulfolobus islandicus REY15/4 host, thus representing one of the few known two-virus systems in Archaea (1). pSSVx is a satellite virus that generates virus particles with the help of SSV2-associated packaging mechanisms. The transcriptional pattern of pSSVx undergoes a temporal variation of gene expression during its own life cycle, thus providing a good model for studying regulation of gene expression in Archaea (2). This genetic element encodes four TFs possibly implicated in the regulation of gene expression, i.e. ORF-c68, ORF51, ORF91 and ORF76. Among these, ORF76, here named Stf76 (Sulfolobus transcription factor 76 aminoacid protein), has homologs in almost all conjugative and cryptic plasmids from Sulfolobus (3), thus suggesting a relevant role for this protein in replication and/or maintenance of the plasmid. In this study, we have performed a detailed structural and functional characterization of Stf76. The corresponding gene has been cloned, expressed in Escherichia coli and the recombinant protein purified to homogeneity. To elucidate its interaction with the identified DNA operator sequence, analyses regarding its DNA binding capabilities by means of EMSA, circular dichroism, spectrofluorimetric and isothermal titration calorimetry experiments have been performed. Moreover, a structural study has been undertaken by Nuclear Magnetic Resonance spectroscopy leading to:(i) the solution structure of Stf76 based on CS-Rosetta approach, (ii) the characterization of the Stf76-DNA interaction by chemical shift perturbation analysis, (iii) a structural model describing the interaction of a single Stf76 monomer with its DNA operator. Altogether these results contribute to elucidate the regulatory mechanism underpinning the role of this protein (4). REFERENCES: 1. ARNOLD, H.P., ET AL.(1999) THE GENETIC ELEMENT PSSVX OF THE EXTREMELY THERMOPHILIC CRENARCHAEON SULFOLOBUS IS A HYBRID BETWEEN A PLASMID AND A VIRUS. MOL MICROBIOL, 34, 217-226. 2. CONTURSI, P., ET.AL. (2010) TRANSCRIPTION TERMINATION IN THE PLASMID/VIRUS HYBRID PSSVX FROM SULFOLOBUS ISLANDICUS. EXTREMOPHILES, 14, 453-463. 3. LIPPS, G. (2006) PLASMIDS AND VIRUSES OF THE THERMOACIDOPHILIC CRENARCHAEOTE SULFOLOBUS. EXTREMOPHILES, 10, 17-28. 4. CONTURSI P., FARINA B., PIRONE L. ET AL. NUCLEIC ACIDS RES. ACCEPTED 28 FEB. 2014.

Integrins are heterodimeric cell-surface proteins that play important roles during developmental and pathological processes. Diverse human pathologies involve integrin adhesion including thrombotic diseases, inflammation, tumour progression, fibrosis, and infectious diseases. Although in the past decade, novel integrin-inhibitor drugs have been developed for integrin-based medical applications, the structural determinants modulating integrin-ligands recognition mechanisms are still poorly understood, reducing the number of integrin subtype exclusive antagonists. In this scenario, we have very recently showed, by means of chemical and biological assays, that a chimeric peptide (named RGDechi), containing a cyclic RGD motif linked to an echistatin C-terminal fragment, is able to interact with the components of integrin family with variable affinities, the highest for αv β3. Here, in order to understand the mechanistic details driving the molecular recognition mechanism of αv β3 by RGDechi, we have performed a detailed structural and dynamics characterization of the free peptide by natural abundance nuclear magnetic resonance (NMR) spectroscopy. Our data indicate that RGDechi presents in solution an heterogeneous conformational ensemble characterized by a more constrained and rigid pentacyclic ring and a largely unstructured acyclic region. Moreover, we propose that the molecular recognition of αv β3 integrin by RGDechi occurs by a combination of conformational selection and induced fit mechanisms. Finally, our study indicates that a detailed NMR characterization, by means of natural abundance 15 N and 13 C, of a mostly unstructured bioactive peptide may provide the molecular basis to get essential structural insights into the binding mechanism to the biological partner.

Background The peptide VLL-28, identified in the sequence of an archaeal protein, the transcription factor Stf76 from Sulfolobus islandicus, was previously identified and characterized as an antimicrobial peptide, possessing a broad-spectrum antibacterial activity. Methods Through a combined approach of NMR and Circular Dichroism spectroscopy, Dynamic Light Scattering, confocal microscopy and cell viability assays, the interaction of VLL-28 with the membranes of both parental and malignant cell lines has been characterized and peptide mechanism of action has been studied. Results It is here demonstrated that VLL-28 selectively exerts cytotoxic activity against murine and human tumor cells. By means of structural methodologies, VLL-28 interaction with the membranes has been proven and the binding residues have been identified. Confocal microscopy data show that VLL-28 is internalized only into tumor cells. Finally, it is shown that cell death is mainly caused by a time-dependent activation of apoptotic pathways. Conclusions VLL-28, deriving from the archaeal kingdom, is here found to be endowed with selective cytotoxic activity towards both murine and human cancer cells and consequently can be classified as an ACP. General significance VLL-28 represents the first ACP identified in an archaeal microorganism, exerting a trans-kingdom activity.

In the present work we performed a combined experimental and computational study on the interaction of the natural antimalarial endoperoxide plakortin and its synthetic analogue 4a with heme. Obtained results indicate that the studied compounds produce reactive carbon radical species after being reductively activated by heme. In particular, similarly to artemisinin, the formation of radicals prone to inter-molecular reactions should represent the key event responsible for Plasmodium death. To our knowledge this is the first experimental investigation on the reductive activation of simple antimalarial endoperoxides (1,2-dioxanes) by heme and results were compared to the ones previously obtained from the reaction with FeCl2. The obtained experimental data and the calculated molecular interaction models represent crucial tools for the rational optimization of our promising class of low-cost synthetic antimalarial endoperoxides.

The earth's crust presents two dissimilar rheological behaviors depending on the in situ stress-temperature conditions. The upper, cooler part is brittle, while deeper zones are ductile. Seismic waves may reveal the presence of the transition but a proper characterization is required. We first obtain a stress–strain relation, including the effects of shear seismic attenuation and ductility due to shear deformations and plastic flow. The anelastic behavior is based on the Burgers mechanical model to describe the effects of seismic attenuation and steady-state creep flow. The shear Lamé constant of the brittle and ductile media depends on the in situ stress and temperature through the shear viscosity, which is obtained by the Arrhenius equation and the octahedral stress criterion. The P and S wave velocities decrease as depth and temperature increase due to the geothermal gradient, an effect which is more pronounced for shear waves. We then obtain the P−S and SH equations of motion recast in the velocity-stress formulation, including memory variables to avoid the computation of time convolutions. The equations correspond to isotropic anelastic and inhomogeneous media and are solved by a direct grid method based on the Runge–Kutta time stepping technique and the Fourier pseudospectral method. The algorithm is tested with success against known analytical solutions for different shear viscosities. A realistic example illustrates the computation of surface and reverse-VSP synthetic seismograms in the presence of an abrupt brittle–ductile transition.

The critical role of integrins in tumor progression and metastasis has stimulated intense efforts to identify pharmacological agents that can modulate integrin function. In recent years, alpha(v)beta(3) and alpha(v)beta(5) integrin antagonists were demonstrated to be effective in blocking tumor progression. RGDechi-hCit, a chimeric peptide containing a cyclic RGD motif linked to an echistatin C-terminal fragment, is able to recognize selectively alpha(v)beta(3) integrin both in vitro and in vivo. High-resolution molecular details of the selective alpha(v)beta(3) recognition of the peptide are certainly required, nonetheless RGDechi-hCit internalization limited the use of classical in cell NMR experiments. To overcome such limitations, we used WM266 isolated cellular membranes to accomplish a detailed NMR interaction study that, combined with a computational analysis, provides significant structural insights into alpha(v)beta(3) molecular recognition by RGDechi-hCit. Remarkably, on the basis of the identified molecular determinants, we design a RGDechi-hCit mutant that is selective for alpha(v)beta(5) integrin.

In this work, a sensitive and convenient protease-based fluorimetric high-throughput screening (HTS) assay for determining peptidyl-prolyl cis-trans isomerase activity was developed. The assay was based on a new intramolecularly quenched substrate, whose fluorescence and structural properties were examined together with kinetic constants and the effects of solvents on its isomerization process. Pilot screens performed using the Library of Pharmacologically Active Compounds (LOPAC) and cyclophilin A (CypA), as isomerase model enzyme, indicated that the assay was robust for HTS, and that comparable results were obtained with a CypA inhibitor tested both manually and automatically. Moreover, a new compound that inhibits CypA activity with an IC50 in the low micromolar range was identified. Molecular docking studies revealed that the molecule shows a notable shape complementarity with the catalytic pocket confirming the experimental observations. Due to its simplicity and precision in the determination of extent of inhibition and reaction rates required for kinetic analysis, this assay offers many advantages over other commonly used assays.

The zinc coordination sphere in prokaryotic zinc finger domain is extremely versatile and influences the stability and the folding property of the domain. Of a particular interest is the fourth zinc coordinating position, which is frequently occupied by two successive histidines, both able to coordinate the metal ion. To clarify their structural and functional role we report the NMR solution structure and the dynamics behavior of Ros87 H42A, which is a functional mutant of Ros87, the DNA binding domain of the Ros protein containing a prokaryotic Cys(2)His(2) zinc finger domain. The structural analysis indicates that reducing the spacer among the two coordinating histidines from 4 (among His37 and His42) amino acids to 3 (among His37 and His41) increases the helicity of the first a-helix. At the same time, the second helix appears more mobile in the mu s-ms timescale and the hydrophobic core is reduced. These data explain the high frequency of three-residue His spacers in the eukaryotic zinc finger domain and their absence in the prokaryotic counterpart. Furthermore, the structural comparison shows that the second coordination position is more sensitive to H42A mutation with respect to the first and the third position, providing the rationale of the high variability of the second and the fourth zinc coordinating position in Ros homologs, which adopt different metal coordination but preserve similar tertiary structures and DNA binding activities. Finally, H/D exchange measurements and NMR thermal unfolding analysis indicate that this mutant likely unfolds via a different mechanism with respect to the wild-type. (C) 2013 Published by Elsevier Inc.

The RNA binding motif protein 5 (RBM5), also known as Luca15 or H37, is a component of prespliceosomal complexes that regulates the alternative splicing of several mRNAs, such as Fas and caspase-2. The RBM5 gene is located at the 2p21.3 chromosomal region, which is strongly associated with lung cancer and many other cancers. Both increased and decreased levels of RBM5 can play a role in tumor progression. In particular, downregulation of rbm5 is involved in lung cancer and other cancers upon Ras activation, and, also, represents a molecular signature associated with metastasis in various solid tumors. On the other hand, upregulation of RBM5 occurs in breast and ovarian cancer. Moreover, RBM5 was also found to be involved in the early stage of the HIV-1 viral cycle, representing a potential target for the treatment of the HIV-1 infection. While the molecular basis for RNA recognition and ubiquitin interaction has been structurally characterized, small molecules binding this zinc finger (ZF) domain that might contribute to characterizing their activity and to the development of potential therapeutic agents have not yet been reported. Using an NMR screening of a fragment library we identified several binders and the complex of the most promising one, compound 1, with the RBM5 ZF1 was structurally characterized in solution. Interestingly, the binding mechanism reveals that 1 occupies the RNA binding pocket and is therefore able to compete with the RNA to bind RBM5 RanBP2-type ZF domain, as indicated by NMR studies. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Seismic methods contribute to the exploration of geothermal areas and characterization of existing geothermal resources. Seismic velocity and attenuation depend on the pressure and temperature conditions of the geothermal systems, which are closely related to the properties of the rock frame and geothermal fluids. We calculate the seismic velocities and attenuation in terms of the subsurface distribution of the confining and pore pressures and temperature, assuming that the heat transfer from below is convective or conductive. The pore pressure is assumed hydrostatic. In hydrothermal systems the temperature is calculated assuming the boiling point condition at the specific pore pressure down to the reservoir. Beneath the reservoir it is assumed constant in convectively heated systems and following a constant temperature gradient in conductively heated systems. In Enhanced Geothermal Systems (EGS) conductive heat transfer and constant temperature gradient are assumed. We present three application examples, considering simplified subsurface models to describe the geothermal systems beneath the production wells. The seismic wave properties are calculated using the rock's mechanical Burgers model and the Arrhenius equation to take into account rock-properties-variability with temperature and the Gassmann model for fluid saturating the porous rocks.

Geophysical characterization plays a key role for the definition of the deep structures of geothermal reservoirs and the consequent assessment and validation of the geothermal conceptual model. Seismic methods may provide a valuable contribution for this purpose. This involves a deep and reliable understanding of the sensitivity of seismic-wave propagation to physical and temperature variations, with complex interactions. We present the theory and sensitivity analysis based on rock's mechanical Burgers model including Arrhenius temperature equations, integrated with Gassmann model for fluid saturated porous rocks, pressure effects for bulk and shear moduli, as well as permeability and squirt flow effects. Assuming a temperature gradient model, the analysis applied at low seismic frequencies compares the interpretation of the sensitivity effects for different typical seismic elastic quantities, showing the different performance in relation to physical effects, including melting, supercritical conditions, and observability obtained in different temperature regions. With a quantification of the physical properties, the results of the study show that in deeper zones the main expected contributions in terms of variations in seismic velocity, moduli and seismic attenuation due to temperature come from melting transition, while in shallower porous fluid-saturated formations the trends are governed by pressure effects, with minor contributions of permeability and possible effects related to soft porosity. The new calculated elastic moduli are complex-valued and frequency-dependent, and temperature dependent through the fluid properties. In this complex scenario, not always the increments in the velocity and elastic wave moduli correspond to an increment in the temperature. Moreover, with mobility decreasing as a function of depth, the analysis shows that the shear quality factor is sensitive to permeability, which introduces moderate effects for velocity and attenuation of shear waves. The analysis applies to active exploration seismic and passive seismology.