Rational design of 4-aryl-1,2,3-triazoles for indoleamine 2,3-dioxygenase 1 inhibition.

Indoleamine 2,3-dioxygenase 1 (IDO1) is an important therapeutic target for the treatment of diseases such as cancer that involve pathological immune escape. Starting from the scaffold of our previously discovered IDO1 inhibitor 4-phenyl-1,2,3-triazole, we used computational structure-based methods to design more potent ligands. This approach yielded highly efficient low molecular weight inhibitors, the most active being of nanomolar potency both in an enzymatic and in a cellular assay, while showing no cellular toxicity and a high selectivity for IDO1 over tryptophan 2,3-dioxygenase (TDO). A quantitative structure-activity relationship based on the electrostatic ligand-protein interactions in the docked binding modes and on the quantum chemically derived charges of the triazole ring demonstrated a good explanatory power for the observed activities.

EADock: docking of small molecules into protein active sites with a multiobjective evolutionary optimization.

In recent years, protein-ligand docking has become a powerful tool for drug development. Although several approaches suitable for high throughput screening are available, there is a need for methods able to identify binding modes with high accuracy. This accuracy is essential to reliably compute the binding free energy of the ligand. Such methods are needed when the binding mode of lead compounds is not determined experimentally but is needed for structure-based lead optimization. We present here a new docking software, called EADock, that aims at this goal. It uses an hybrid evolutionary algorithm with two fitness functions, in combination with a sophisticated management of the diversity. EADock is interfaced with the CHARMM package for energy calculations and coordinate handling. A validation was carried out on 37 crystallized protein-ligand complexes featuring 11 different proteins. The search space was defined as a sphere of 15 A around the center of mass of the ligand position in the crystal structure, and on the contrary to other benchmarks, our algorithm was fed with optimized ligand positions up to 10 A root mean square deviation (RMSD) from the crystal structure, excluding the latter. This validation illustrates the efficiency of our sampling strategy, as correct binding modes, defined by a RMSD to the crystal structure lower than 2 A, were identified and ranked first for 68% of the complexes. The success rate increases to 78% when considering the five best ranked clusters, and 92% when all clusters present in the last generation are taken into account. Most failures could be explained by the presence of crystal contacts in the experimental structure. Finally, the ability of EADock to accurately predict binding modes on a real application was illustrated by the successful docking of the RGD cyclic pentapeptide on the alphaVbeta3 integrin, starting far away from the binding pocket.

Detailed analysis and follow-up studies of a high-throughput screening for indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a key regulator of immune responses and therefore an important therapeutic target for the treatment of diseases that involve pathological immune escape, such as cancer. Here, we describe a robust and sensitive high-throughput screen (HTS) for IDO1 inhibitors using the Prestwick Chemical Library of 1200 FDA-approved drugs and the Maybridge HitFinder Collection of 14,000 small molecules. Of the 60 hits selected for follow-up studies, 14 displayed IC50 values below 20 μM under the secondary assay conditions, and 4 showed an activity in cellular tests. In view of the high attrition rate we used both experimental and computational techniques to identify and to characterize compounds inhibiting IDO1 through unspecific inhibition mechanisms such as chemical reactivity, redox cycling, or aggregation. One specific IDO1 inhibitor scaffold, the imidazole antifungal agents, was chosen for rational structure-based lead optimization, which led to more soluble and smaller compounds with micromolar activity.

The Implied Book and the Narrative Text. On a Blind Spot in Narratological Theory - from a Media Studies Perspective

The article is concerned with the formal definition of a largely unnoticed factor in narrative structure. Based on the assumptions that (1) the semantics of a written text depend, among other factors, directly on its visual alignment in space, that (2) the formal structure of a text has to meet that of its spatial presentation and that (3) these assumptions hold true also for narrative texts (which, however, in modern times typically conceal their spatial dimensions by a low-key linear layout), it is argued that, how ever low-key, the expected material shape of a given narrative determines the configuration of its plot by its author. The ,implied book' thus denotes an author's historically assumable, not necessarily conscious idea of how his text, which is still in the process of creation, will be dimensionally presented and under these circumstances visually absorbed. Assuming that an author's knowledge of this later (potentially) substantiated material form influences the composition, the implied book is to be understood as a text-genetically determined, structuring moment of the text. Historically reconstructed, it thus serves the methodical analysis of structural characteristics of a completed text.

Arquitectura, integració i implementació d'un projecte modular J2EE

L'objectiu principal del TFC consisteix en la creació d'una complexa estructura modular J2EE basada i mantinguda per Maven, amb la utilització com frameworks Spring, Hibernate i Flex principalment. Aquesta estructura permet reprendre el desenvolupament inicial, nous desenvolupaments i manteniments d'una aplicació, amb un cost temporal mínim per la part de l'equip de desenvolupament.

Using time and stochastic models for software reliability forecasting and analysis

Tämä työ luo katsauksen ajallisiin ja stokastisiin ohjelmien luotettavuus malleihin sekä tutkii muutamia malleja käytännössä. Työn teoriaosuus sisältää ohjelmien luotettavuuden kuvauksessa ja arvioinnissa käytetyt keskeiset määritelmät ja metriikan sekä varsinaiset mallien kuvaukset. Työssä esitellään kaksi ohjelmien luotettavuusryhmää. Ensimmäinen ryhmä ovat riskiin perustuvat mallit. Toinen ryhmä käsittää virheiden ”kylvöön” ja merkitsevyyteen perustuvat mallit. Työn empiirinen osa sisältää kokeiden kuvaukset ja tulokset. Kokeet suoritettiin käyttämällä kolmea ensimmäiseen ryhmään kuuluvaa mallia: Jelinski-Moranda mallia, ensimmäistä geometrista mallia sekä yksinkertaista eksponenttimallia. Kokeiden tarkoituksena oli tutkia, kuinka syötetyn datan distribuutio vaikuttaa mallien toimivuuteen sekä kuinka herkkiä mallit ovat syötetyn datan määrän muutoksille. Jelinski-Moranda malli osoittautui herkimmäksi distribuutiolle konvergaatio-ongelmien vuoksi, ensimmäinen geometrinen malli herkimmäksi datan määrän muutoksille.

Congenital hypogonadotropic hypogonadism with split hand/foot malformation: a clinical entity with a high frequency of FGFR1 mutations.

PURPOSE: Congenital hypogonadotropic hypogonadism (CHH) and split hand/foot malformation (SHFM) are two rare genetic conditions. Here we report a clinical entity comprising the two. METHODS: We identified patients with CHH and SHFM through international collaboration. Probands and available family members underwent phenotyping and screening for FGFR1 mutations. The impact of identified mutations was assessed by sequence- and structure-based predictions and/or functional assays. RESULTS: We identified eight probands with CHH with (n = 3; Kallmann syndrome) or without anosmia (n = 5) and SHFM, seven of whom (88%) harbor FGFR1 mutations. Of these seven, one individual is homozygous for p.V429E and six individuals are heterozygous for p.G348R, p.G485R, p.Q594*, p.E670A, p.V688L, or p.L712P. All mutations were predicted by in silico analysis to cause loss of function. Probands with FGFR1 mutations have severe gonadotropin-releasing hormone deficiency (absent puberty and/or cryptorchidism and/or micropenis). SHFM in both hands and feet was observed only in the patient with the homozygous p.V429E mutation; V429 maps to the fibroblast growth factor receptor substrate 2α binding domain of FGFR1, and functional studies of the p.V429E mutation demonstrated that it decreased recruitment and phosphorylation of fibroblast growth factor receptor substrate 2α to FGFR1, thereby resulting in reduced mitogen-activated protein kinase signaling. CONCLUSION: FGFR1 should be prioritized for genetic testing in patients with CHH and SHFM because the likelihood of a mutation increases from 10% in the general CHH population to 88% in these patients.Genet Med 17 8, 651-659.

BioSuper: A web tool for the superimposition of biomolecules and assemblies with rotational symmetry

Background Most of the proteins in the Protein Data Bank (PDB) are oligomeric complexes consisting of two or more subunits that associate by rotational or helical symmetries. Despite the myriad of superimposition tools in the literature, we could not find any able to account for rotational symmetry and display the graphical results in the web browser. Results BioSuper is a free web server that superimposes and calculates the root mean square deviation (RMSD) of protein complexes displaying rotational symmetry. To the best of our knowledge, BioSuper is the first tool of its kind that provides immediate interactive visualization of the graphical results in the browser, biomolecule generator capabilities, different levels of atom selection, sequence-dependent and structure-based superimposition types, and is the only web tool that takes into account the equivalence of atoms in side chains displaying symmetry ambiguity. BioSuper uses ICM program functionality as a core for the superimpositions and displays the results as text, HTML tables and 3D interactive molecular objects that can be visualized in the browser or in Android and iOS platforms with a free plugin. Conclusions BioSuper is a fast and functional tool that allows for pairwise superimposition of proteins and assemblies displaying rotational symmetry. The web server was created after our own frustration when attempting to superimpose flexible oligomers. We strongly believe that its user-friendly and functional design will be of great interest for structural and computational biologists who need to superimpose oligomeric proteins (or any protein). BioSuper web server is freely available to all users at http://ablab.ucsd.edu/BioSuper webcite.

Attracting cavities for docking. Replacing the rough energy landscape of the protein by a smooth attracting landscape.

Molecular docking is a computational approach for predicting the most probable position of ligands in the binding sites of macromolecules and constitutes the cornerstone of structure-based computer-aided drug design. Here, we present a new algorithm called Attracting Cavities that allows molecular docking to be performed by simple energy minimizations only. The approach consists in transiently replacing the rough potential energy hypersurface of the protein by a smooth attracting potential driving the ligands into protein cavities. The actual protein energy landscape is reintroduced in a second step to refine the ligand position. The scoring function of Attracting Cavities is based on the CHARMM force field and the FACTS solvation model. The approach was tested on the 85 experimental ligand-protein structures included in the Astex diverse set and achieved a success rate of 80% in reproducing the experimental binding mode starting from a completely randomized ligand conformer. The algorithm thus compares favorably with current state-of-the-art docking programs. © 2015 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.

Kestomagneettigeneraattorin staattorin mekaanisen rakenteen analysointi ja kehitys

Työssä tutkittiin kestomagneettigeneraattorin staattoriytimen aksiaalisen puristamisen ja lämpötilan vaikutuksia staattorin jännityksiin ja muodonmuutoksiin. Lisäksi tutkittiin aksiaalisen puristusjännityksen vaikutusta staattorin aksiaalisuuntaisen ominaismuodon taajuuteen. Rakenteesta tehtiin elementtimalli, jonka avulla laskettiin rakenteen siirtymät, jännitykset ja ominaistaajuudet. Rakenteen jännityksiä ja siirtymiä tutkittiin myös yksinkertaisella jousikuvauksella, jonka tuloksia verrattiin elementtimallilla saatuihin tuloksiin. Työn tavoitteena oli kehittää uusi staattorirakenne. Elementtimenetelmän tulosten perusteella staattorirakennetta voidaan yksinkertaistaa poistamalla nyt käytetty puristusrengas, jolloin rakenteesta tulee yksinkertaisempi ja kustannustehokkaampi.

Hitsatun rakenteen lujuustarkastelu kokeellisesti määritetyn kuormitushistorian perusteella

Työssä tutkittiin jätteen murskauksesta murskaimeen aiheutuvia kuormituksia vastusvenymäliuskamittauksilla. Eri jätetyyppien aiheuttamia kuormituksia tutkittiin erillisinä tapauksina ja näistä tyyppikuormituksista johdettiin rakenteen normaalia käyttöä vastaava kuormitushistoria käyttäen painokertoimia eri tyyppikuormien kesken. Murskaimen runkorakennetta tutkittiin FE-analyysillä käyttäen kuormituksena kenttämittauksilla saatua todellista kuormitusta. FE-menetelmällä tutkittiin väsymisen kannalta kriittisiä kohtia rakenteesta. Tulosten perusteella kriittisiin yksityiskohtiin laadittiin parannusehdotuksia, joiden perusteella yhteistyössä työn teettäjän kanssa laadittiin uudet rakenneratkaisut. Rakenteen kestoikä määritettiin väsymisvaurion kannalta kriittisimmän yksityiskohdan mukaan. Kestoiän määrittämiseen käytettiin Palmgren-Miner menetelmää ja Palmgren-Miner menetelmästä johdettua ekvivalentin jännitysvaihtelun menetelmää. Muutosten jälkeen rakenne täyttää sille asetetun suunnittelukestoiän käytettyjen menetelmien perusteella.

Structural and functional Studies of angucycline tailoring enzymes

Polyketides are a diverse group of natural products produced in many bacteria, fungi and plants. These metabolites have diverse biological activities and several members of this group are in clinical use as antibiotics, anticancer agents, antifungals and immunosuppressants. The different polyketides are produced by polyketide synthases, which catalyze the condensation of extender units into various polyketide scaffolds. After the biosynthesis of the polyketide backbone, more versatility is created to the molecule by tailoring enzymes catalyzing for instance hydroxylations, methylations and glycosylations. Flavoprotein monooxygenases (FPMO) and short-chain alcohol dehydrogenases/reductases (SDR) are two enzyme families that catalyze unusual tailoring reactions in the biosynthesis of natural products. In the experimental section, functions of homologous FPMO and SDR tailoring enzymes from five different angucycline pathways were studied in vitro. The results revealed how different angucyclinones are produced from a common intermediate and that FPMO JadH and SDR LanV are responsible for the divergence of jadomycins and landomycins, respectively, from other angucyclines. Structural studies of these tailoring enzymes revealed differences between homologous enzymes and enabled the use of structure-based protein engineering. Mutagenesis experiments gave important information about the enzymes behind the evolution of distinct angucycline metabolites. These experiments revealed a correlation between the substrate inhibition and bi-functionality in JadH homologue PgaE. In the case of LanV, analysis of mutagenesis results revealed that the difference between the stereospecificities of LanV and its homologues CabV and UrdMred is unexpectedly related to the conformation of the substrate rather than to the structure of the enzyme. Altogether, the results presented here have improved our knowledge about different steps of angucycline biosynthesis and the reaction mechanisms used by the tailoring enzymes behind these steps. This information can hopefully be used to modify these enzymes to produce novel metabolites, which have new biological targets or possess novel modes-of-action. The understanding of these unusual enzyme mechanisms is also interesting to enzymologists outside the field of natural product research.

Phylogenetic Analysis of Avidins and Expression of Novel Avidins from Lactrodectus hesperus and Hoeflea phototrophica

Avidins (Avds) are homotetrameric or homodimeric glycoproteins with typically less than 130 amino acid residues per monomer. They form a highly stable, non-covalent complex with biotin (vitamin H) with Kd = 10-15 M (for chicken Avd). The best-studied Avds are the chicken Avd from Gallus gallus and streptavidin from Streptomyces avidinii, although other Avd studies have also included Avds from various origins, e.g., from frogs, fishes, mushrooms and from many different bacteria. Several engineered Avds have been reported as well, e.g., dual-chain Avds (dcAvds) and single-chain Avds (scAvds), circular permutants with up to four simultaneously modifiable ligand-binding sites. These engineered Avds along with the many native Avds have potential to be used in various nanobiotechnological applications. In this study, we made a structure-based alignment representing all currently available sequences of Avds and studied the evolutionary relationship of Avds using phylogenetic analysis. First, we created an initial multiple sequence alignment of Avds using 42 closely related sequences, guided by the known Avd crystal structures. Next, we searched for non-redundant Avd sequences from various online databases, including National Centre for Biotechnology Information and the Universal Protein Resource; the identified sequences were added to the initial alignment to expand it to a final alignment of 242 Avd sequences. The MEGA software package was used to create distance matrices and a phylogenetic tree. Bootstrap reproducibility of the tree was poor at multiple nodes and may reflect on several possible issues with the data: the sequence length compared is relatively short and, whereas some positions are highly conserved and functional, others can vary without impinging on the structure or the function, so there are few informative sites; it may be that periods of rapid duplication have led to paralogs and that the differences among them are within the error limit of the data; and there may be other yet unknown reasons. Principle component analysis applied to alternative distance data did segregate the major groups, and success is likely due to the multivariate consideration of all the information. Furthermore, based on our extensive alignment and phylogenetic analysis, we expressed two novel Avds, lacavidin from Lactrodectus Hesperus, a western black widow spider, and hoefavidin from Hoeflea phototrophica, an aerobic marine bacterium, the ultimate aim being to determine their X-ray structures. These Avds were selected because of their unique sequences: lacavidin has an N-terminal Avd-like domain but a long C-terminal overhang, whereas hoefavidin was thought to be a dimeric Avd. Both these Avds could be used as novel scaffolds in biotechnological applications.

Towards sustainable peace : peace-building and human security

Failed and fragile states that result from intrastate war pose severe threats to the security of both the international system and individual states alike. In the post-Cold War era, the international community has come to recognize the reality of these threats and the difficulty involved in ending violence and building sustainable peace in failed and fragile states. This work focuses upon the development of a comprehensive strategy for sustainable peace-building by incorporating the tenets of the human security doctrine into the peace-building process. Through the use of case studies of The Former Yugoslav Republic of Macedonia and East Timor, the development and refinement of the doctrine of human security will occur, as well as, an understanding of how and where human security fits into the sustainable peace-building equation. The end result of the analysis is the development of a hierarchical pyramid formation that brings together human security and peace-building into one framework that ultimately creates the foundation and structure of sustainable peace-building. With the development of a sustainable peace-building structure based upon the human security doctrine, the role of Canada in the support of sustainable peace-building is analyzed in relation to the form and level of involvement that Canada undertakes and contributes to in the implementation and support of sustainable peace-building initiatives. Following from this, recommendations are provided regarding what role(s) Canada should undertake in the sustainable peace-building process that take into consideration the present and likely future capabilities of Canada to be involved in various aspects of the peace-building process. ii This paper outlines the need for a peace-building strategy that is designed to be sustainable in order that failed and fragile states resulting from intrastate conflict do not regress or collapse back into a condition of civil war, and subsequently designs such a strategy. The linking of peace-building and human security creates the required framework from which sustainable peace-building is derived. Creating sustainable peace is necessary in order to increase the likelihood that both present and future generations existing in failed and fragile states will be spared from the scourge of intrastate war.

To see and to touch : God, honour, and irony in Shakespeare's English histories /

Failed and fragile states that result from intrastate war pose severe threats to the security of both the international system and individual states alike. In the post-Cold War era, the international community has come to recognize the reality of these threats and the difficulty involved in ending violence and building sustainable peace in failed and fragile states. This work focuses upon the development of a comprehensive strategy for sustainable peace-building by incorporating the tenets of the human security doctrine into the peace-building process. Through the use of case studies of The Former Yugoslav Republic of Macedonia and East Timor, the development and refinement of the doctrine of human security will occur, as well as, an understanding of how and where human security fits into the sustainable peace-building equation. The end result of the analysis is the development of a hierarchical pyramid formation that brings together human security and peace-building into one framework that ultimately creates the foundation and structure of sustainable peace-building. With the development of a sustainable peace-building structure based upon the human security doctrine, the role of Canada in the support of sustainable peace-building is analyzed in relation to the form and level of involvement that Canada undertakes and contributes to in the implementation and support of sustainable peace-building initiatives. Following from this, recommendations are provided regarding what role(s) Canada should undertake in the sustainable peace-building process that take into consideration the present and likely future capabilities of Canada to be involved in various aspects of the peace-building process. ii This paper outlines the need for a peace-building strategy that is designed to be sustainable in order that failed and fragile states resulting from intrastate conflict do not regress or collapse back into a condition of civil war, and subsequently designs such a strategy. The linking of peace-building and human security creates the required framework from which sustainable peace-building is derived. Creating sustainable peace is necessary in order to increase the likelihood that both present and future generations existing in failed and fragile states will be spared from the scourge of intrastate war.