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MO474: Expectation and acceptance of a clinical decision support software by nephrologist end-users: the Ckdnapp survey
No full textComputergestützte Untersuchung der Struktur, Dynamik und Energetik von Proteininteraktionen
Full text linkProtein interactions play an essential function in virtually all biological processes. To understand the underlying molecular principles it is important to characterize the structure and dynamics of proteins on an atomic level. In this work, different protein interactions were analyzed with various in silico and in vitro methods. The coordination of activity in biological systems requires the existence of different signal transduction pathways that interact with one another and must be precisely regulated. The Src-family members of tyrosine kinase, which are found at the beginning of a many signal pathways, differ in their physiological function despite their large structural similarity. In previous work, it was shown that the flexibility of the SH3-SH2 domain linker is critical for the relative orientation of the SH3 and SH2 domains. In this thesis, the global effect of the flexibility of the SH3-SH2 linker on the structure and dynamics of Src kinases was analyzed using of molecular dynamics (MD) simulations. For this purpose, the rigid SH3-SH2 linker sequence of inactive Hck kinase structure was replaced with the more flexible linker sequence from Lck. In the MD simulations, this lead to a higher flexibility in comparison to the wild-type Hck, which, in turn, reduced the regulatory domain pair's ability to fixate the kinase domain in the inactive conformation. Furthermore, the exchange of the SH3-SH2 domain linker lead to conformational changes in the activation segment of the kinase domain. The resulting alternative conformation showed similarities to the conformation of the tyrosine kinase Abl when bound to the inhibitor Glivec. Thus, the sequence of the SH3-SH2 linker can not only modulate the kinase activity, but can also influence the ligand binding properties of the active site of Src kinases. This could explain why Lck is, in contrast to other Src kinases, inhibited by Glivec. One possibility to activate the Src kinases is the binding of ligands to the regulatory domains. To characterize the affinity and specificity determinants of the molecular recognition, MD simulations and, subsequently, energetic analyses were performed. The interaction between the Tip protein of Herpesvirus saimiri and the SH3 domain of tyrosine kinase Lyn was chosen as a model system. Tip binds to the SH3 domain by using the sequence motif (175PTPPLPPRPANLG187), which contains a PPII-helix. The role of all non-prolines was analyzed with the help of an in silico alanine scan. The resulting order of importance with respect to the binding, could be experimentally verified using fluorescence spectroscopy. In addition, by taking entropic contributions into account, a modified approach which allows reliable statements about the effect of terminal ligand truncations was developed. By using this strategy, it was shown that the truncation of the Tip peptide by the L186/G187 residues leads to a lower binding af_nity for LynSH3. In contrast, when binding the truncated Tip peptide to homologous SH3 domains, no changes in the binding affinities were detected. It could furthermore be shown that the interaction between L186/G187 in Tip and H41 in LynSH3 significantly increases the binding affinity. The coordinated interplay between kinases and phosphatases represents a fundamental regulatory mechanism in biological systems. Protein phosphatase 1 (PP1), a major serine/threonine phosphatase, regulates its enzymatic activity and substrate specificity by acquisition of different binding partners. These interactions are primarily mediated by a five residue sequence motif, the properties of which are thus far not well characterized. Detailed knowledge of its sequence properties would greatly facilitate the identification of new interaction partners. In this work, the PP1 docking motif was thoroughly characterized with a combination of in silico (molecular modeling, MD simulations) and in vitro (GST pull-down) analysis. It was shown that the amino acid at position +1 represents a key anchor point of the motif, as it forms a network of salt bridges and hydrogen bonds to PP1. In addition, it could be shown that positions +1 and +2 of the docking motif prefer positively charged and/or polar residues. In contrast, the position +3 clearly favors the hydrophobic amino acid valine. The largest sequence variability was observed at position +4, whereas position +5 only tolerates the unpolar and aromatic side chains of phenylalanine and tryptophan. By using a combined approach and performing a systematic analysis of each individual position of the PP11 docking motif, the specific interaction motif [HKR][ACHKMNQRSTV][V][CHKNQRST][FW] could be defined. The usefulness of this motif for genome-wide searches was subsequently tested by identifying and experimentally verifying previously unknown PP1 interactors. The HIV-1 protease, an enzyme of the HI virus, is essential for replication and assembly of the virus. This property makes it an important target for the design of antiviral agents for AIDS treatment. A major problem of the current HIV therapies is the rapid development of resistance to antiretroviral drugs by genetic mutation. It is necessary to understand the resistance mechanism in order to develop more effective and longer lasting treatment regimens. In this thesis, the mutation E35D, which has been described in context with the inhibitor amprenavir, was analyzed. To gain insight into the resistance mechanism, molecular dynamics simulations of the wild-type's as well as the mutant's free, and ligand complexed proteases were performed. In the course of the simulations, an increased flexibility of the mutated HIV protease's flaps, which are parts of the binding pocket, could be observed. This affects the conformational equilibrium between the closed and semiopen conformations of the free protease, whereas in the ligand bound protease some intermolecular interactions are disrupted. Energetic analysis showed that the mutation also causes a significant reduction of the calculated ligand binding energies, thus giving a plausible explanation for its ability to develop resistance. Further analysis showed that the amino acid E35 is also located in an epitope which is important for the recognition of the virus by the immune system. Mutations in the epitope allow the virus to hamper the recognition process, thereby suppressing an immune system response. The results of the investigation suggest that the mutation conveys resistance against protease inhibitors and also allows the virus to escape immune system recognition.Proteininteraktionen spielen praktisch bei allen biologischen Prozessen eine wesentliche Rolle. Um die zugrunde liegenden molekularen Vorgänge zu verstehen, ist es notwendig die Struktur und Dynamik von Proteinen auf atomarer Ebene zu charakterisieren. In der vorliegenden Arbeit wurde eine Reihe von Proteininteraktionen eingehend mit verschiedenen in silico und in vitro Methoden untersucht. Die Koordination von Abläufen in biologischen Systemen erfordert das Vorhandensein verschiedener Signalübertragungswege, die untereinander in Wechselwirkung stehen und exakt reguliert werden müssen. Die Tyrosinkinasen der Src-Familie, die am Anfang vieler Signalwege stehen unterscheiden sich trotz ihrer großen strukturellen Ähnlichkeit in ihren physiologischen Funktionen. In früheren Arbeiten wurde bereits gezeigt, dass die Flexibilität des SH3-SH2 Domänenlinkers kritisch für die relative Orientierung von SH3- und SH2-Domäne ist. Im Rahmen dieser Arbeit wurde mit Hilfe von Moleküldynamik(MD)-Simulationen untersucht, ob die Flexibilität des SH3-SH2 Linkers einen globalen Einfluss auf die Struktur und Dynamik von Src-Kinasen hat. Hierzu wurde die starre SH3-SH2 Linkersequenz aus Hck in der Struktur der inaktiven Kinase durch die flexiblere Linkersequenz aus Lck ersetzt. Dies führte in MD-Simulationen zu einer erhöhten Flexibilität im Vergleich zur Wildtyp-Hck und somit zu einer verringerten Fähigkeit des regulatorischen Domänenpaars die Kinasedomäne in der inaktiven Konformation zu fixieren. Des Weiteren führte der Austausch des SH3-SH2 Domänenlinkers zu konformationellen Änderungen im Aktivierungssegment der Kinasedomäne. Die hierbei eingenommenen alternativen Konformationen zeigten Ähnlichkeiten zur Konformation der Tyrosinkinase Abl, die nach Bindung des Inhibitors Glivec eingenommen wird. Die Sequenz des SH3-SH2 Linkers könnte demzufolge nicht nur die Modulation der Kinaseaktivität, sondern auch die Ligandenbindungseigenschaften im aktiven Zentrum von Src-Kinasen beeinflussen. Dies könnte erklären, warum Lck im Gegensatz zu den anderen Src-Kinasen durch Glivec gehemmt werden kann. Die Aktivierung der Src-Kinasen kann unter anderem durch die Bindung von Liganden an die regulatorischen Domänen vermittelt werden. Zur Charakterisierung der Determinanten der Affinität und Spezifität der molekularen Erkennung wurden MD-Simulationen und darauf aufbauende energetische Analysen durchgeführt. Als Modellsystem wurde hier die Wechselwirkung zwischen dem Tip-Protein aus Herpesvirus saimiri und der SH3-Domäne der Tyrosinkinase Lyn gewählt. Tip bindet mit dem Sequenzmotiv (175PTPPLPPRPANLG187), welches eine PPII-Helix beinhaltet, an die SH3-Domäne. Mit Hilfe eines in silico Alanin-Scans wurde die Rolle aller Nicht-Proline bei der LynSH3-Bindung analysiert. Die dabei erhaltene Reihenfolge der Wichtigkeit für die Bindung konnte anschließend experimentell mittels Fluoreszenzspektroskopischer Messungen bestätigt werden. Außerdem konnte ein modifizierter Ansatz entwickelt werden, der durch die Berücksichtigung entropischer Beiträge auch für die Deletion von terminalen Aminosäuren zuverlässige Aussagen über Bindungsaffinitäten liefert. Mit dieser Strategie konnte gezeigt werden, dass die Verkürzung des Tip-Peptids um die Reste L186/G187 bei der LynSH3-Bindung zu einer schlechteren Bindung führt, wohingegen bei homologen SH3-Domänen kein Effekt beobachtet wurde. Es konnte weiter gezeigt werden, dass die Wechselwirkungen zwischen L186/G187 in Tip und H41 in LynSH3 maßgeblich zu einer Steigerung der Bindungsaffinität beitragen. Das koordinierte Zusammenspiel zwischen Kinasen und Phosphatasen stellt einen wichtigen regulatorischen Mechanismus in biologischen Systemen dar. Die Proteinphosphatase 1 (PP1), eine der bedeutendsten Serin-/Threonin-Phosphatasen, erreicht ihre enzymatische Aktivität und Substratspezifität durch die Bindung verschiedener Interaktionspartner. Diese Interaktion wird primär über ein fünf Aminosäuren langes Sequenzmotiv vermittelt, dessen Eigenschaften bislang nur unzureichend charakterisiert waren, was die Identifizierung neuer Wechselwirkungspartner erschwerte. In der vorliegenden Arbeit wurde das PP1-Bindemotiv mit Hilfe von in silico (Modelling und MD) und in vitro (GST-Pulldown) Analysen eingehend charakterisiert. Es konnte gezeigt werden, dass die Aminosäure an Position +1 einen Ankerpunkt repräsentiert, da sie ein Netzwerk von Salz- und Wasserstoffbrücken zur PP11 bildet. Des Weiteren konnte gezeigt werden, dass die Positionen +1 und +2 im Bindemotiv positiv geladene bzw. polare Aminosäuren tolerieren. Im Gegensatz dazu ließ die Position +3 eine deutliche Präferenz für Valin erkennen. Die größte Variabilität innerhalb des Bindemotivs konnte an der Position +4 beobachtet werden, wohingegen an Position +5 nur die Seitenketten der unpolaren und aromatischen Aminosäuren Phenylalanin und Tryptophan toleriert wurden. Durch den kombinierten Ansatz und die systematische Analyse jeder einzelnen Position des PP1-Bindemotivs war es somit möglich ein spezifisches Interaktionsmotiv [HKR][ACHKMNQRSTV][V][CHKNQRST][FW] aufzustellen. Die Brauchbarkeit dieses Motivs für genomweite Suchen konnte anschließend durch die Identifizierung und experimentelle Verifizierung neuer PP1-Interaktionspartner bestätigt werden. Die HIV-1 Protease, ein Enzym des HI-Virus, ist essentiell für die Bildung reifer Viruspartikel. Somit stellt die Protease ein äußerst wichtiges Zielmolekül für die Entwicklung von Wirkstoffen gegen die Immunschwächekrankheit AIDS dar. Eines der Hauptprobleme der antiretroviralen Therapie ist die schnelle Entwicklung von Resistenzen gegen die eingesetzten Wirkstoffe, welche häufig auf Mutationen der HIV-1 Protease zurückzuführen sind. Zur Entwicklung effektiver und lang anhaltender Wirkstoffe ist es wichtig die Resistenzmechanismen zu verstehen. In dieser Arbeit wurde die Mutation E35D, welche unter anderem im Zusammenhang mit dem Inhibitor Amprenavir beschrieben wird, untersucht. Um einen Einblick in den Resistenzmechanismus zu erhalten, wurden sowohl für den Wildtyp als auch für die Mutante MD-Simulationen der freien und ligandengebundenen Protease durchgeführt. Im Verlauf der Simulationen wurde für die mutierte HIV-1 Protease eine höhere Flexibilität von Schleifenbereichen sichtbar, die Teil der Ligandenbindungstasche sind. Dies wirkte sich bei der freien Protease auf das Gleichgewicht zwischen geschlossener und halboffener Konformation aus, wohingegen bei der ligandengebundenen Protease intermolekulare Wechselwirkungen gestört wurden. Energetische Analysen zeigten, dass die Mutation zu einer signifikanten Erniedrigung der Bindungsenergien für Liganden führt und somit die beobachtete Resistenzentstehung erklären kann. Weiterführende Analysen zeigten, dass die Aminosäure E35 auch in einem Epitop liegt, das für die Erkennung des Virus durch das Immunsystem wichtig ist. Das Virus kann durch Mutationen im Epitop den Erkennungsprozess erschweren und so zu einer Unterdrückung der Immunantwort führen. Die Ergebnisse der Untersuchungen deuten darauf hin, dass die Mutation sowohl Resistenz gegen Proteaseinhibitoren vermittelt als auch eine Flucht vor der Erkennung durch das Immunsystem ermöglicht
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Interactive exploration of adverse events and multimorbidity in CKD
No full textAbstract
Background and hypothesis
Persons with chronic kidney disease (CKD) are at increased risk of adverse events, early mortality, and multimorbidity. A detailed overview of adverse event types and rates from a large CKD cohort under regular nephrological care is missing. We generated an interactive tool to enable exploration of adverse events and their combinations in the prospective, observational German CKD (GCKD) study.
Methods
The GCKD study enrolled 5217 participants under regular nephrological care with an estimated glomerular filtration rate of 30–60 or >60 mL/min/1.73m2 and an overt proteinuria. Cardio-, cerebro- and peripheral vascular, kidney, infection, and cancer events, as well as deaths were adjudicated following a standard operation procedure. We summarized these time-to-event data points for exploration in interactive graphs within an R shiny app. Multivariable adjusted Cox models for time to first event were fitted. Cumulative incidence functions, Kaplan-Meier curves and intersection plots were used to display main adverse events and their combinations by sex and CKD etiology.
Results
Over a median of 6.5 years, 10 271 events occurred in total and 680 participants (13.0%) died while 2947 participants (56.5%) experienced any event. The new publicly available interactive platform enables readers to scrutinize adverse events and their combinations as well as mortality trends as a gateway to better understand multimorbidity in CKD: incident rates per 1000 patient-years varied by event type, CKD etiology, and baseline characteristics. Incidence rates for the most frequent events and their recurrence were 113.6 (cardiovascular), 75.0 (kidney), and 66.0 (infection). Participants with diabetic kidney disease and men were more prone to experiencing events.
Conclusion
This comprehensive explorative tool to visualize adverse events (https://gckd.diz.uk-erlangen.de/), their combination, mortality, and multimorbidity among persons with CKD may manifest as a valuable resource for patient care, identification of high-risk groups, health services, and public health policy planning
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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