In Vitro HIV-1 Evolution in Response to Triple Reverse Transcriptase Inhibitors & In Silico Phenotypic Analysis

Background Effectiveness of ART regimens strongly depends upon complex interactions between the selective pressure of drugs and the evolution of mutations that allow or restrict drug resistance. Methods Four clinical isolates from NRTI-exposed, NNRTI-naive subjects were passaged in increasing concentrations of NVP in combination with 1 µM 3 TC and 2 µM ADV to assess selective pressures of multi-drug treatment. A novel parameter inference procedure, based on a stochastic viral growth model, was used to estimate phenotypic resistance and fitness from in vitro combination passage experiments. Results Newly developed mathematical methods estimated key phenotypic parameters of mutations arising through selective pressure exerted by 3 TC and NVP. Concentrations of 1 µM 3 TC maintained the M184V mutation, which was associated with intrinsic fitness deficits. Increasing NVP concentrations selected major NNRTI resistance mutations. The evolutionary pathway of NVP resistance was highly dependent on the viral genetic background, epistasis as well as stochasticity. Parameter estimation indicated that the previously unrecognized mutation L228Q was associated with NVP resistance in some isolates. Conclusion Serial passage of viruses in the presence of multiple drugs may resemble the selection of mutations observed among treated individuals and populations in vivo and indicate evolutionary preferences and restrictions. Phenotypic resistance estimated here “in silico” from in vitro passage experiments agreed well with previous knowledge, suggesting that the unique combination of “wet-” and “dry-lab” experimentation may improve our understanding of HIV-1 resistance evolution in the future

Ein auf stochastischer Dynamik basierender Modellierungsansatz

A key feature and a central driving force behind biological evolution is the capability of adaption to changing environmental conditions. Noise-induced transitions play a central role in these decision making processes allowing for a natural stochastic sampling between various evolutionary strategies. Mathematical analysis of such mechanisms requires experimental data, which represent the multimodal stochastic probabilities assigned to these strategies, being sampled at a sufficiently high resolution. However, in most applications the available experimental measurements are temporally and spatially too sparse for this objective. In this thesis different mathematical methods are derived for dissecting the mechanisms underlying such decision making processes despite the sparsity of data. The key idea is based on a compensation of lacking direct experimental observations using indirect inference from other, coupled system variables measured with a higher accuracy. One of the multistable systems studied here is the mutational dynamics conferring drug resistance to HIV. Since the likelihood of constitutive mutations is strongly associated with their phenotypic impact, time-discrete measurements of intrinsically stochastic viral population growth are used for inferring the principles underlying the mutational dynamical system. Furthermore, a similar idea is applied for analysing the phenotypic bistability of a stress-induced signaling network in E. Coli, giving rise to biofilm synthesis. Although direct single-cell measurements of E. Coli within the two modes of the probability distribution are not yet available, qualitative measurements of gene and protein interactions of the underlying signaling system are used for analysing dynamical properties of the bistable biofilm regulation. As a unifying framework, the theory of biochemical reactions based on Markov jump processes is adapted to the described problems and the resulting practical implications are discussed.Ein entscheidener Aspekt und eine zentrale Antriebskraft hinter der biologischen Evolution ist die Fähigkeit der Adaptation an sich verändernde äussere Bedingungen. Zufallsbedingte Zustandsübergänge spielen eine Schlüsselrolle in diesen Entscheidungsprozessen und ermöglichen eine natürliche stochastische Suche unter verschiedenen evolutionären Strategien. Die mathematische Analyse von solchen Mechanismen benötigt experimentelle Daten, die multimodale stochastische Wahrscheinlichkeitsverteilungen repräsentieren, gemessen mit einer hinreichend hohen Auflösung. In den meisten Anwendungen ist jedoch die zeitliche und räumliche Auflösung von experimentellen Messungen hierfür zu gering. In dieser Arbeit werden unterschiedliche mathematische Methoden hergeleitet, um Mechanismen hinter solchen Entscheidungsprozessen zu analysieren, trotz der unzureichenden Menge an Daten. Die zentrale Idee basiert auf einer Kompensation von fehlenden direkten experimentellen Messungen durch eine indirekte Schätzung, mit Hilfe von anderen gekoppelten Systemvariablen mit höherer Messhäufigkeit. Eins der multistabilen Systeme, die hier betrachtet werden, ist die Mutationsdynamik von HIV, welche Wirkstoffresistenzen verursachen kann. Da die Wahrscheinlichkeit von sich festsetzenden Mutationsereignissen eng an deren phenotypische Auswirkungen gekoppelt ist, werden zeit-diskrete Messungen von intrinsisch stochastischem viralen Populationswachstum verwendet, um Rückschlüsse auf Prinzipien der Dynamik von Mutationsereignissen zu ziehen. Weiterhin wird eine ähnliche Idee angewandt, um die phenotypische Bistabilität der durch Stress aktivierten Signalkaskade zu analysieren, die in E. Coli zur Biofilmbildung führt. Trotz des Fehlens von Einzelzellmessungen von E. Coli innherhalb der beiden Modi der Wahrscheinlichkeitverteilung, werden qualitative Messungen von Gen- und Proteininteraktionen der zugrundeliegenden Signalkaskade verwendet, um die Eigenschaften der bistabilen Biofilmregulation zu untersuchen. Als ein vereinigendes methodisches Gerüst, wird die auf Markov Sprungprozessen basierende Theorie von biochemischen Reaktionssystemen auf die beschriebenen Problemstellungen angewandt und resultierende praktische Aspekte werden diskutiert

Biomechanics, behaviour dynamics and archaeology : Integrative attempts to study animal domestication and husbandry

Aquesta tesi està centrada en l'estudi dels inicis de la ramaderia. En concret, s'avalua la gestió de la mobilitat dels primers ramats de bovins domèstics, especialment en si l'aprofitament de la força animal fou un aspecte essencial en aquestes economies. Per fer-ho, s'han estudiat tres jaciments neolítics que permeten abordar el tema diacrònicament i sincrònica. Tell Halula (Síria) és un jaciment amb una ocupació continuada durant més de 2000 anys on s'assisteix a l'adopció de la ramaderia d'aquesta espècie i la seva consolidació. La Draga (Banyoles) és un jaciment lacustre amb una conservació idònia de la matèria orgànica. Cova de les Pixarelles (Tavertet) se situa en un entorn abrupte i escarpat, que contrasta amb l'ambient lacustre de Banyoles, òptima per avaluar com les variables antròpiques, ecològiques i topogràfiques van influir en les característiques biomecàniques dels bòvids neolítics. El treball amb jaciments dels dos extrems del Mediterrani també ha permès analitzar un cas de domesticació autòctona (Tell Halula) i un segon cas en què l'espècie arriba de forma domèstica (la Draga). D'acord amb la problemàtica històrica definida, s'ha treballat amb dues de les espècies animals amb un paper rellevant a les pràctiques ramaderes prehistòriques, i que són claus per a la comprensió del Neolític: els bovins i els suins. La gestió d'aquestes espècies és un element important del procés de neolitització i va contribuir significativament al canvi econòmic, social i ideològic durant la Revolució Neolítica. A partir de l'anàlisi de les restes de Bos taurus, s'ha abordat la qüestió dels orígens de l'explotació de l'energia animal. L'ús de la força animal va suposar una innovació important pel que fa al moviment i a les tasques que impliquen una quantitat significativa de força. Des del punt de vista econòmic, va permetre augmentar la productivitat, aplicar tècniques agrícoles més intensives i recórrer distàncies més llargues transportant càrregues. Aquestes innovacions van suposar potents accions i pressions selectives sobre els animals, fins a aconseguir animals plenament especialitzats en aquestes activitats. En el cas del porc, s'ha creat un referencial de porcs senglars per a la investigació sobre la domesticació i la ramaderia d'aquesta espècie. La col·lecció de referència inclou 50 senglars d'edat, sexe i origen coneguts, amb la intenció d'examinar la influència del sexe i l'edat en la variabilitat morfològica. Fins ara, aquesta col·lecció és l'única d'aquestes característiques a la Península Ibèrica. La seva caracterització exhaustiva basada en la morfometria geomètrica constitueix una aportació original i un instrument de gran valor per a la investigació arqueozoològica i la domesticació d'aquesta espècie. S'han dissenyat i desenvolupat noves eines analítiques que faciliten l'anàlisi dels materials arqueològics en totes les seves dimensions, contribuint als avenços metodològics i tècnics en arqueologia. Així, s'ha desenvolupat un programari de codi obert, ArchaeoToolbox, que permet fer morfometria geomètrica en 3D. ArchaeoToolbox permet digitalitzar punts de referència, semipunts de referència corbs i superficials, anàlisis de Procrustes completes i parcials, anàlisis de components principals dels residus de Procrustes, destacar els punts de referència més importants de cada morfoespai i crear mapes tèrmics exportables per mostrar disparitats morfològiques. Aquest treball ha posat a prova l'abast de la morfometria geomètrica i l'anàlisi d'elements finits per investigar la proporció desconeguda de variància en les dades zooarqueològiques. La flexibilitat de la morfometria geomètrica per detectar canvis en els patrons morfològics, com la disminució o exageració d'una asimetria estructural, s'ha utilitzat amb èxit per destacar els canvis induïts per les comunitats neolítiques al mecanoambient dels bovins neolítics i separar-los dels efectes dels factors anatòmics. L'aplicació de l'anàlisi d'elements finits ha permés verificar si les disparitats observades als residus de Procrustes tenen l'origen en activitats relacionades amb la ramaderia inicial.Esta tesis se centra en el estudio de los inicios de la ganadería. En concreto, se evalúa la gestión de la movilidad de los primeros rebaños de bovinos domésticos, especialmente en si el aprovechamiento de la fuerza animal fue un aspecto esencial en estas economías. Para ello, se han estudiado tres yacimientos neolíticos que permiten abordar el tema diacrónica y sincrónica. Tell Halula (Siria) tiene una ocupación continuada durante más de 2000 años donde se asiste a la adopción de la ganadería de esta especie y su consolidación. La Draga (Banyoles) es un yacimiento lacustre con una conservación idónea de la materia orgánica. Cova de las Pixarelles (Tavertet) está en un entorno abrupto y escarpado, que contrasta con el ambiente lacustre de Banyoles, óptimo para evaluar cómo las variables antrópicas, ecológicas y topográficas influyeron en las características biomecánicas de los bóvidos neolíticos. El trabajo con yacimientos de ambos extremos del Mediterráneo también ha permitido analizar un caso de domesticación autóctona (Tell Halula) y uno de adopción de la forma doméstica (Draga). De acuerdo con la problemática histórica definida, se ha trabajado con bovinos y suinos, dos de las especies animales involucradas en las prácticas ganaderas prehistóricas, y claves para entender el Neolítico. Su gestión contribuyó significativamente al cambio económico, social e ideológico en la Revolución Neolítica. Con el análisis de Bos taurus, se ha abordado los orígenes de la explotación de la energía animal. El uso de la fuerza animal supuso una innovación importante en lo que se refiere al movimiento ya las tareas que implican una cantidad significativa de fuerza. Desde el punto de vista económico, permitió aumentar la productividad, aplicar técnicas agrícolas más intensivas y recorrer distancias más largas transportando cargas. Estas innovaciones supusieron potentes acciones y presiones selectivas sobre los animales, hasta conseguir animales plenamente especializados en estas actividades. En el caso del cerdo, se ha creado un referencial de jabalíes para la investigación sobre la domesticación y la ganadería de esta especie. La colección de referencia incluye 50 jabalíes de edad, sexo y origen conocidos, con la intención de examinar la influencia del sexo y la edad en la variabilidad morfológica. Hasta ahora, esta colección es la única de estas características en la Península Ibérica. Su exhaustiva caracterización basada en la morfometría geométrica constituye un aporte original y un instrumento de gran valor para la investigación arqueozoológica y la domesticación de esta especie. Se han diseñado y desarrollado nuevas herramientas analíticas que facilitan el análisis de los materiales arqueológicos en todas sus dimensiones, contribuyendo a los avances metodológicos y técnicos en arqueología. Así, se ha desarrollado un software de código abierto, ArchaeoToolbox, que permite realizar morfometría geométrica en 3D. ArchaeoToolbox permite digitalizar puntos de referencia, semipuntos de referencia curvos y superficiales, análisis de Procrustas completas y parciales, análisis de componentes principales de los residuos de Procrustes, destacar los puntos de referencia más importantes de cada morfoespacio y crear mapas térmicos exportables para mostrar disparidades morfológicas. Este trabajo ha puesto a prueba el alcance de la morfometría geométrica y el análisis de elementos finitos para investigar la proporción desconocida de varianza en los datos zooarqueológicos. La flexibilidad de la morfometría geométrica para detectar cambios en los patrones morfológicos, como la disminución o exageración de una asimetría estructural, se ha utilizado con éxito para destacar los cambios inducidos por las comunidades neolíticas en el mecanoambiente de los bovinos neolíticos y separarlos de los efectos de los factores anatómicos. La aplicación del análisis de elementos finitos ha permitido verificar si las disparidades observadas en los residuos de Procrustes tienen su origen en actividades relacionadas con la ganadería inicial.This thesis focuses on studying early husbandry practices. Specifically, the management of the mobility of the first herds of domestic bovines is evaluated, especially if the use of animal power was an essential aspect in these economies. To do this, three Neolithic sites have been studied that allow addressing the diachronic and synchronous issue. Tell Halula (Syria) has a continuous occupation for more than 2000 years where the adoption of the livestock of this species and its consolidation is witnessed. La Draga (Banyoles) is a lake site with ideal conservation of organic matter. Cova de las Pixarelles (Tavertet) is in an abrupt and steep environment, which contrasts with the lake environment of Banyoles, optimal for evaluating how anthropogenic, ecological and topographic variables influenced the biomechanical characteristics of Neolithic bovids. Working with samples from both ends of the Mediterranean allowed analysing a case of autochthonous domestication (Tell Halula) and one of adoption of the domestic form (Draga). In accordance with the defined historical problems, work has been done with bovines and pigs, two of the animal species involved in prehistoric livestock practices, and keys to understanding the Neolithic. His management contributed significantly to the economic, social and ideological change in the Neolithic Revolution. With the analysis of Bos taurus, the origins of the exploitation of animal energy have been addressed. The use of animal power was a major innovation when it comes to movement and tasks that involve a significant amount of force. From an economic point of view, it made it possible to increase productivity, apply more intensive agricultural techniques and travel longer distances transporting loads. These innovations implied powerful actions and selective pressures on the animals until fully specialized animals were obtained in these activities. In the case of the pig, a wild boar reference has been created for research on the domestication and livestock of this species. The reference collection includes 50 wild boars of known age, sex and origin, with the intention of examining the influence of sex and age on morphological variability. Nowadays, this collection is the only one of these characteristics in the Iberian Peninsula. Its exhaustive characterization based on geometric morphometry constitutes an original contribution and an instrument of great value for archaeozoological research and the domestication of this species. New analytical tools have been designed and developed that facilitate the analysis of archaeological materials in all their dimensions, contributing to methodological and technical advances in archaeology. Thus, this work has designed "ArchaeoToolbox", an open-source software that allows performing geometric morphometrics in 3D. ArchaeoToolbox allows digitizing landmarks, curved and surface semi-landmarks, full and partial Procruste analysis, principal component analysis of Procrustes residues, highlighting the most important landmarks in each morphospace, and creating exportable heatmaps to show morphological disparities. This work has tested the power of geometric morphometry and finite element analysis to investigate the unknown proportion of variance in zooarchaeological data. The flexibility of geometric morphometry to detect changes in morphological patterns, such as the decrease or exaggeration of a structural asymmetry, has been used successfully to highlight changes induced by Neolithic communities in the Neolithic bovine mechanoenvironment and to separate them from the effects of anatomical factors. The application of finite element analysis has made it possible to verify if the disparities observed in the Procrustes residues originate in activities related to the initial livestock

Characterization of natural polymorphic sites of the HIV-1 integrase before the introduction of HIV-1 integrase inhibitors in Germany

Introduction: The aim of our study was to analyze the occurrence and evolution of HIV-1 integrase polymorphisms during the HIV-1 epidemic in Germany prior to the introduction of the first integrase inhibitor raltegravir in 2007. Materials and Methods: Plasma samples from drug-naïve HIV-1 infected individuals newly diagnosed between 1986 and 2006 were used to determine PCR-based population sequences of the HIV-1 integrase (amino acids 1–278). The HIV-1 subtype was determined using the REGA HIV-1 subtyping tool. We calculated the frequency of amino acids at each position of the HIV-1 integrase in 337 subtype B strains for the time periods 1986–1989, 1991–1994, 1995–1998, 1999–2002, and 2003–2006. Positions were defined as polymorphic if amino acid variation was >1% in any period. Logistic regression was used to identify trends in amino acid variation over time. Resistance-associated mutations were identified according to the IAS 2013 list and the HIVdb, ANRS and GRADE algorithms. Results: Overall, 56.8% (158/278) amino acid positions were polymorphic and 15.8% (25/158) of these positions exhibited a significant trend in amino acid variation over time. Proportionately, most polymorphic positions (63.3%, 31/49) were detected in the N-terminal zinc finger domain of the HIV-1 integrase. Motifs and residues essential for HIV-1 integrase activity were little polymorphic, but within the minimal non-specific DNA binding region I220-D270 up to 18.1% amino acid variation was noticed, including four positions with significant amino acid variation over time (S230, D232, D256, A265). No major resistance mutations were identified, and minor resistance mutations were rarely observed without trend over time. E157Q considered by HIVdb, ANRS, and GRADE algorithms was the most frequent resistance-associated polymorphism with an overall prevalence of 2.4%. Conclusions: Detailed knowledge of the evolutionary variation of HIV-1 integrase polymorphisms is important to understand the development of resistance in the presence of the drug. Our results will contribute to define the relevance of integrase polymorphisms in HIV-strains resistant to integrase inhibitors and to improve resistance interpretation algorithms

Molecular evolution of HIV-1 integrase during the 20 years prior to the first approval of integrase inhibitors

Abstract Background Detailed knowledge of the evolutionary potential of polymorphic sites in a viral protein is important for understanding the development of drug resistance in the presence of an inhibitor. We therefore set out to analyse the molecular evolution of the HIV-1 subtype B integrase at the inter-patient level in Germany during a 20-year period prior to the first introduction of integrase strand inhibitors (INSTIs). Methods We determined 337 HIV-1 integrase subtype B sequences (amino acids 1–278) from stored plasma samples of antiretroviral treatment-naïve individuals newly diagnosed with HIV-1 between 1986 and 2006. Shannon entropy was calculated to determine the variability at each amino acid position. Time trends in the frequency of amino acid variants were identified by linear regression. Direct coupling analysis was applied to detect covarying sites. Results Twenty-two time trends in the frequency of amino acid variants demonstrated either single amino acid exchanges or variation in the degree of polymorphy. Covariation was observed for 17 amino acid variants with a temporal trend. Some minor INSTI resistance mutations (T124A, V151I, K156 N, T206S, S230 N) and some INSTI-selected mutations (M50I, L101I, T122I, T124 N, T125A, M154I, G193E, V201I) were identified at overall frequencies >5%. Among these, the frequencies of L101I, T122I, and V201I increased over time, whereas the frequency of M154I decreased. Moreover, L101I, T122I, T124A, T125A, M154I, and V201I covaried with non-resistance-associated variants. Conclusions Time-trending, covarying polymorphisms indicate that long-term evolutionary changes of the HIV-1 integrase involve defined clusters of possibly structurally or functionally associated sites independent of selective pressure through INSTIs at the inter-patient level. Linkage between polymorphic resistance- and non-resistance-associated sites can impact the selection of INSTI resistance mutations in complex ways. Identification of these sites can help in improving genotypic resistance assays, resistance prediction algorithms, and the development of new integrase inhibitors

Selection dynamics.

<p>A: Average number of mutations per passage in experiments with NVP (experimental set-ups C, D, E & F). Asterisks indicate whether there were significantly more mutations (Wilcoxon rank sum test) than in the NVP-free experiments (experimental set-ups A & B). *p<0.1, **p<0.05, ***p<0.01. B: Cumulative probability of detecting no mutation. The blue and red lines show the cumulative probability of not detecting a mutation after the indicated numbers of passages (x-axis) in experiments where NVP was added with increasing concentrations (blue line; experimental set-ups C, D, E & F) vs. experiments where no NVP was added (red line; experimental set-ups A & B).</p

Baseline amino acid substitutions in relation to reference sequence (Hxb2) from the Stanford HIVDB [28].

<p>Baseline amino acid substitutions in relation to reference sequence (Hxb2) from the Stanford HIVDB <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061102#pone.0061102-Rhee1" target="_blank">[28]</a>.</p

Estimated relative fitness deficit <i>f(q)</i> of mutations present in the genetic background of baseline isolates #1,#2/3,#4,#5.

<p>A small value (close to 0) denotes a large fitness loss, whereas a value close to 1 denotes no fitness deficit. Values indicated are medians of all parameter estimates. The 5^th and 95^th percentile of estimates are indicated in brackets. 'n.ds’ means ‘not deselected’ and n.i. means parameter ‘not identifiable’. Parameters <i>f</i>(67S), <i>f</i>(208H), <i>f</i>(35I) and <i>f</i>(210W/211K) could not be reliably estimated from the data or were not significantly different from the value 1.</p