Solution structure of the equine infectious anemia virus p9 protein: a rationalization of its different ALIX binding requirements compared to the analogous HIV-p6 protein

<p>Abstract</p> <p>Background</p> <p>The equine infection anemia virus (EIAV) p9 Gag protein contains the late (L-) domain required for efficient virus release of nascent virions from the cell membrane of infected cell.</p> <p>Results</p> <p>In the present study the p9 protein and N- and C-terminal fragments (residues 1-21 and 22-51, respectively) were chemically synthesized and used for structural analyses. Circular dichroism and ¹H-NMR spectroscopy provide the first molecular insight into the secondary structure and folding of this 51-amino acid protein under different solution conditions. Qualitative ¹H-chemical shift and NOE data indicate that in a pure aqueous environment p9 favors an unstructured state. In its most structured state under hydrophobic conditions, p9 adopts a stable helical structure within the C-terminus. Quantitative NOE data further revealed that this α-helix extends from Ser-27 to Ser-48, while the N-terminal residues remain unstructured. The structural elements identified for p9 differ substantially from that of the functional homologous HIV-1 p6 protein.</p> <p>Conclusions</p> <p>These structural differences are discussed in the context of the different types of L-domains regulating distinct cellular pathways in virus budding. EIAV p9 mediates virus release by recruiting the ALG2-interacting protein X (ALIX) via the YPDL-motif to the site of virus budding, the counterpart of the YPX_nL-motif found in p6. However, p6 contains an additional PTAP L-domain that promotes HIV-1 release by binding to the tumor susceptibility gene 101 (Tsg101). The notion that structures found in p9 differ form that of p6 further support the idea that different mechanisms regulate binding of ALIX to primary versus secondary L-domains types.</p

GERBIL: General Entity Annotator Benchmarking Framework

We present GERBIL, an evaluation framework for semantic entity annotation. The rationale behind our framework is to provide developers, end users and researchers with easy-to-use interfaces that allow for the agile, fine-grained and uniform evaluation of annotation tools on multiple datasets. By these means, we aim to ensure that both tool developers and end users can derive meaningful insights pertaining to the extension, integration and use of annotation applications. In particular, GERBIL provides comparable results to tool developers so as to allow them to easily discover the strengths and weaknesses of their implementations with respect to the state of the art. With the permanent experiment URIs provided by our framework, we ensure the reproducibility and archiving of evaluation results. Moreover, the framework generates data in machine-processable format, allowing for the efficient querying and post-processing of evaluation results. Finally, the tool diagnostics provided by GERBIL allows deriving insights pertaining to the areas in which tools should be further refined, thus allowing developers to create an informed agenda for extensions and end users to detect the right tools for their purposes. GERBIL aims to become a focal point for the state of the art, driving the research agenda of the community by presenting comparable objective evaluation results

Development and evaluation of robust molecular markers linked to disease resistance in tomato for distinctness, uniformity and stability testing

Molecular markers linked to phenotypically important traits are of great interest especially when traits are difficult and/or costly to be observed. In tomato where a strong focus on resistance breeding has led to the introgression of several resistance genes, resistance traits have become important characteristics in distinctness, uniformity and stability (DUS) testing for Plant Breeders Rights (PBR) applications. Evaluation of disease traits in biological assays is not always straightforward because assays are often influenced by environmental factors, and difficulties in scoring exist. In this study, we describe the development and/or evaluation of molecular marker assays for the Verticillium genes Ve1 and Ve2, the tomato mosaic virusTm1 (linked marker), the tomato mosaic virus Tm2 and Tm22 genes, the Meloidogyne incognita Mi1-2 gene, the Fusarium I (linked marker) and I2 loci, which are obligatory traits in PBR testing. The marker assays were evaluated for their robustness in a ring test and then evaluated in a set of varieties. Although in general, results between biological assays and marker assays gave highly correlated results, marker assays showed an advantage over biological tests in that the results were clearer, i.e., homozygote/heterozygote presence of the resistance gene can be detected and heterogeneity in seed lots can be identified readily. Within the UPOV framework for granting of PBR, the markers have the potential to fulfil the requirements needed for implementation in DUS testing of candidate varieties and could complement or may be an alternative to the pathogenesis tests that are carried out at present

Synthesis and structure of viral regulatory proteins

Ein Schwerpunkt dieser Arbeit war die Synthese von langen viralen regulatorischen Peptiden sowie von Fragmenten dieser Peptide. Neben der Synthese standen dabei die Optimierung von bekannten Synthesestrategien und die Anwendung von neuen Konzepten, wie beispielsweise die „Native Chemical Ligation“ (NCL), bei den im Rahmen dieser Arbeit erstmalig synthetisch hergestellten Peptiden im Vordergrund. So wurden vom Influenza A Virus (IAV) Protein PB1-F2 zwei humane H1N1 Varianten synthetisiert. Ein PB1-F2 Peptid entstammt einem Isolat vom Mount Sinai [1], im Weiteren bezeichnet als PR8. Das zweite humane PB1-F2 Peptid entstammt der „Spanischen Grippe“ [2], im Weiteren bezeichnet als SF2. Eine weitere aviäre H5N1 Variante [3] des PB1-F2 Peptids, bezeichnet als BF2, wurde als Vertreter der „Vogelgrippe“ synthetisiert. Vom Humanen Immundefizienzvirus Typ-1 wurden zum einen das Gag Protein p6 [4] synthetisiert. Von diesem Peptid wurden zur Herstellung von verschiedenen p6-Mutanten (Ser14,25,40&#61664;Asn) die Peptidkupplungen bei höheren Temperaturen unter Verwendung eines Mikrowellensyntheseautomaten (Liberty, Firma CEM) durchgeführt. Eine von Khurana et al. [5] modifizierte p6 Sequenz (in dieser Arbeit als p6_neu bezeichnet) wurde für Vergleichsstudien synthetisiert, die von Patricia Klingler in der Gruppe von Prof. Dr. U. Schubert (Universität Erlangen-Nürnberg) durchgeführt wurden. Ein weiteres Gag Peptid p9 mit einer Länge von 51 Aminosäuren vom Equine Infectious Anemia Virus wurde mittels step-by-step Methode erstmalig synthetisch hergestellt [6,7]. Im Rahmen der vorliegenden Arbeit wurde ebenfalls das HIV-1 Protein Vpr mit einer Kettenlänge von 96 Aminosäuren synthetisch hergestellt, wobei hier die bekannte Synthese [8] durch die Verwendung von Pseudoprolinen optimiert wurde. Dieses Peptid wurde im Rahmen dieser Arbeit erstmalig durch die Methode der „Native Chemical Ligation“ erfolgreich synthetisiert. Neben der Synthese dieser Peptide wurde im Rahmen der Arbeit von einem ausgewählten Peptid, dem PB1-F2 Peptid der „Spanischen Grippe“, genannt SF2, die Struktur in Lösung unter hydrophoben Bedingungen (TFE:Wasser, 1:1) berechnet. Dazu wurden von den überlappenden Fragmenten SF2(1-40), SF2(30-70) und SF2(50-90) 1H NMR spektroskopische Untersuchungen durchgeführt und die erhaltenen quantitativen und qualitativen NOE-Signale für die weitere Berechnung verwendet. Diese für das Peptid SF2 erhaltenen Strukturinformationen wurden anschließend mit der von Bruns et al. für das PB1-F2 Peptid PR8 publizierten Struktur verglichen [9]. Zur Vermeidung von Schwierigkeiten bei der Synthese von langen Peptiden, hervorgerufen durch die Aggregation der wachsenden Peptidkette infolge von Wasserstoffbrückenbindungen, werden im Allgemeinen „Backbone“ geschützte Aminosäurebausteine eingesetzt. Die am häufigsten in der Literatur eingesetzten Bausteine enthalten dabei die 2-Hydroxy-4-methoxybenzyl (Hmb) [10,11,12] bzw. deren Weiterentwicklung die 2,4-Dimethoxybenzyl (Dmb) Schutzgruppe [12,13]. Auf Grund der relativ stark sauren Bedingungen zur Abspaltung der Hmb und Dmb-Gruppe und der Erkenntnis, dass bei der Verwendung der Hmb-Gruppe an deren freien Hydroxylfunktion ungewünschte Nebenreaktionen auftreten können, wurde nach einer neuen Schutzgruppe für die Peptidbindung des Peptidrückgrates gesucht. Als besonders geeignet erwies sich die von Carpino für den Schutz der Amidfunktion von Glutamin und Asparagin sowie als Carboxylschutzgruppe entwickelte Dicyclopropylmethyl (Dcpm) Gruppe [14]. Im Rahmen dieser Arbeit wurden zu diesem Zweck die drei Dcpm geschützten Aminosäuren Glycin, Alanin und Leucin synthetisiert und für die Peptidsynthese eingesetzt. Die Einführung der Fmoc-Gruppe erwies sich auf Grund der Säurelabilität der eingeführten Dcpm-Gruppe als schwierig. Zur Verbesserung der Ausbeute wurde nach einem alternativen Syntheseweg für die Synthese der Fmoc geschützten (Dcpm)-Aminosäuren in Lösung [15] unter Verwendung anderer Methoden zur Einführung der Fmoc-Gruppe an der festen Phase gesucht. Als günstigste Methode stellte sich die Umsetzung unter neutralen Bedingungen mit einem in unserer Arbeitsgruppe (Henklein et al.) entwickelten Einführungsreagenz Fmoc-OAt heraus. Von Vorteil erwies sich, dass kein HCl während der Reaktion generiert wird, wie es im Gegensatz zur Umsetzung mit Fmoc-Cl der Fall ist. Neben der Vermeidung von Strukturproblemen durch die Hmb- und Dmb-Gruppe wird die Verwendung dieser Gruppen als Dipeptidbaustein Fmoc-Asp(OtBu)-(Hmb)Gly-OH bzw. Fmoc-Asp(OtBu)-(Dmb)Gly-OH zur Verhinderung der Aspartimid-Bildung während der Synthese von Peptiden mit dem Sequenzmotiv Asp-Gly beschrieben [16,17,18]. Wegen den bereits erwähnten Nachteilen der Hmb-Gruppe schien es daher interessant, die zuvor bereits als „Backbone“ Schutzgruppe mit Erfolg eingesetzte Dcpm-Gruppe in einen derartigen Dipeptidbaustein zu integrieren. Zu diesem Zweck wurde der Synthesebaustein Fmoc-Asp(OtBu)-(Dcpm)Gly-OH entwickelt und bei der Synthese von Peptiden erfolgreich getestet [19]. Ein Vorteil der Dcpm-Gruppe besteht darin, dass während der Abspaltung mit TFA keine Nebenreaktionen mit dem zu synthetisierenden Peptid auftreten. Es konnte auch keine Bildung von eventuell störenden Kationen beobachtet werden. Außerdem sind die entstehenden Produkte aus der Abspaltung der Dcpm-Gruppe entweder flüchtig oder leicht abtrennbar.A main topic was the synthesis of long viral regulation Peptids and fragments thereof. Besides these syntheses the optimisation of known synthetic strategies and the usage of new concepts like the “Native Chemical Ligation” (NCL) was our focus of research for the Peptids made within the framework of this dissertation for the first time. From the Influenza A Virus (IAV) protein PB1-F2 two different human H1N1 strains were synthesized; one from the Mount Sinai [1], named as PR8, and the second from the 1918/19 “Spanish flu” [2], named as SF2. Additionally one avian H5N1 strain [3], named as BF2, was synthesized. From the human immunodeficiency virus typ 1 the Gag protein p6 was synthesized [4]. For the synthesis of various mutants (Ser&#61664;Asn) of the p6 protein the coupling reactions were also carried out at higher temperatures by using a microwave assisted automated Peptids synthesizer (Liberty, company CEM). A recently by Khurana et al. described modified sequence (named as p6_neu) [5], was synthsized for comparable experiments done by Patricia Klinger in the group of Prof. Dr. U. Schubert (university of Erlangen-Nürnberg). One additional 51 amino acids long Gag protein p9 from the Equine Infectious Anemia Virus was synthesized for the first time via the “step-by-step” method [6,7]. In the content of this dissertation the HIV-1 regulatory protein Vpr with a length of 96 amino acids was synthesized by using pseudoprolines to optimise the known strategy [8]. Besides for the synthesis of the Vpr protein we used successfully the strategy of the „Native Chemical Ligation“for the first time. Besides the syntheses of all these Peptids, the structure of the PB1-F2 IAV peptide from the “Spanish flu”, named SF2, was calculated under hydrophobic conditions (TFE:water 1:1). For this 1H NMR analysis was done of the three overlapping fragments SF2(1-40), SF2(30-70) und SF2(50-90) and the quantitative and qualitative NOE signals received from the spectra were taken for the further calculation. The obtained results for the structure of the SF2 peptide were then compared with known structure of the PB1-F2 IAV peptide PR8 published recently by Bruns et al. [9]. The protection of the “Backbone” amide position of presynthesized amino acid building blocks is normally done to overcome or to prevent problems during the synthesis of long Peptids like the aggregation of the growing peptide chain caused by hydrogen bonds between them. The mostly cited building blocks in the literature contain the 2-Hydroxy-4-methoxybenzyl (Hmb) [10,11,12] or their improvement the 2,4-Dimethoxybenzyl (Dmb) protecting group [12,13]. Because of the strong acidic conditions for deblocking the Hmb- and Dmb-group and the fact that by using the Hmb group side reactions could occur with their free hydroxyl group we searched for a new protecting group for the backbone amide position in Peptids. As the most promising group we found the Dicyclopropylmethyl (Dcpm) protecting group, which was originally developed by Carpino for the protection of the amide function of glutamine and asparagine and as a general protection for the carboxyl function [14]. In the content of this work the three Dcpm protected amino acids glycine, alanine and leucine were synthesized and used for the peptide synthesis. The introduction of the Fmoc group via Fmoc-Cl analogue to the work of Carpino was difficult because of the acid sensitivity of the introduced Dcpm group. To increase the yield an alternative strategy was the aim of research for the synthesis of the Fmoc protected Dcpm-amino acids in solution [15] by using other methods for the introduction of the Fmoc group on the solid support. The best method was the reaction under neutral conditions with the new reagent Fmoc-OAt developed in our group (Henklein et al.). It was advantageous that no HCl gas was generated during the reaction compared the the conditions by using Fmoc-Cl. Besides the prevention of structure problems by using the Hmb and Dmb group both groups were also used as the dipeptide building block Fmoc-Asp(OtBu)-(Hmb)Gly-OH or Fmoc-Asp(OtBu)-(Dmb)Gly-OH to prevent the aspartimide side reaction in the synthesis of Peptids containing the Asp-Gly motiv [16,17,18]. Because of the mentioned disadvantages of the Hmb group it was interesting to incorporate the Dcpm group, which was used successfully as backbone protecting group, in such a dipeptide building block. For that purpose the new dipeptide building block Fmoc-Asp(OtBu)-(Dcpm)Gly-OH was developed and tested successful in peptide synthesis [19]. One advantage of the Dcpm group is that during the cleavage with TFA no side reactions with the desired peptide occur and also no creation of maybe violating cations were observed. Besides the products of the deblocking process of the Dcpm group are either volatile or easy to remove

Open Knowledge Extraction Challenge 2017

Comunicació presentada al 4th SemWebEval Challenge at ESWC 2017, celebrat entre els dies 28 de maig i 1 de juny de 2017 a Portoroz, Eslovènia.The Open Knowledge Extraction Challenge invites researchers and practitioners from academia as well as industry to compete to the aim of pushing further the state of the art of knowledge extraction from text for the Semantic Web. The challenge has the ambition to provide a reference framework for research in this field by redefining a number of tasks typically from information and knowledge extraction by taking into account Semantic Web requirements and has the goal to test the performance of knowledge extraction systems. This year, the challenge goes in the third round and consists of three tasks which include named entity identification, typing and disambiguation by linking to a knowledge base depending on the task. The challenge makes use of small gold standard datasets that consist of manually curated documents and large silver standard datasets that consist of automatically generated synthetic documents. The performance measure of a participating system is twofold base on (1) Precision, Recall, F1-measure and on (2) Precision, Recall, F1-measure with respect to the runtime of the system.This work has been supported by the H2020 project HOBBIT (GA no. 688227) as well as the EuroStars projects DIESEL (project no. 01QE1512C) and QAMEL (project no. 01QE1549C). Also this work was partially funded by the Spanish Ministry of Economy and Competitiveness under the Maria de Maeztu Units of Excellence Programme (MDM-2015–0502)

Open Knowledge Extraction Challenge 2017

Comunicació presentada al 4th SemWebEval Challenge at ESWC 2017, celebrat entre els dies 28 de maig i 1 de juny de 2017 a Portoroz, Eslovènia.The Open Knowledge Extraction Challenge invites researchers and practitioners from academia as well as industry to compete to the aim of pushing further the state of the art of knowledge extraction from text for the Semantic Web. The challenge has the ambition to provide a reference framework for research in this field by redefining a number of tasks typically from information and knowledge extraction by taking into account Semantic Web requirements and has the goal to test the performance of knowledge extraction systems. This year, the challenge goes in the third round and consists of three tasks which include named entity identification, typing and disambiguation by linking to a knowledge base depending on the task. The challenge makes use of small gold standard datasets that consist of manually curated documents and large silver standard datasets that consist of automatically generated synthetic documents. The performance measure of a participating system is twofold base on (1) Precision, Recall, F1-measure and on (2) Precision, Recall, F1-measure with respect to the runtime of the system.This work has been supported by the H2020 project HOBBIT (GA no. 688227) as well as the EuroStars projects DIESEL (project no. 01QE1512C) and QAMEL (project no. 01QE1549C). Also this work was partially funded by the Spanish Ministry of Economy and Competitiveness under the Maria de Maeztu Units of Excellence Programme (MDM-2015–0502)

NeuLAND: The high-resolution neutron time-of-flight spectrometer for R3B at FAIR

NeuLAND (New Large-Area Neutron Detector) is the next-generation neutron detector for the R3B (Reactions with Relativistic Radioactive Beams) experiment at FAIR (Facility for Antiproton and Ion Research). NeuLAND detects neutrons with energies from 100 to 1000 MeV, featuring a high detection efficiency, a high spatial and time resolution, and a large multi-neutron reconstruction efficiency. This is achieved by a highly granular design of organic scintillators: 3000 individual submodules with a size of 5 × 5 × 250 cm3 are arranged in 30 double planes with 100 submodules each, providing an active area of 250 × 250 cm2 and a total depth of 3 m. The spatial resolution due to the granularity together with a time resolution of 150 ps ensures high-resolution capabilities. In conjunction with calorimetric properties, a multi-neutron reconstruction efficiency of 50% to 70% for four-neutron events will be achieved, depending on both the emission scenario and the boundary conditions allowed for the reconstruction method. We present in this paper the final design of the detector as well as results from test measurements and simulations on which this design is based