DC-SIGN as an attachment factor mediates Japanese encephalitis virus infection of human dendritic cells via interaction with a single high-mannose residue of viral E glycoprotein

AbstractThe skin-resident dendritic cells (DCs) are thought to be the first defender to encounter incoming viruses and likely play a role in Japanese encephalitis virus (JEV) early infection. In the current study, following the demonstration of JEV productive infection in DCs, we revealed that the interaction between JEV envelope glycoprotein (E glycoprotein) and DC-SIGN was important for such infection as evidenced by antibody neutralization and siRNA knockdown experiments. Moreover, the high-mannose N-linked glycan at N154 of E glycoprotein was shown to be crucial for JEV binding to DC-SIGN and subsequent internalization, while mutation of DC-SIGN internalization motif did not affect JEV uptake and internalization. These data together suggest that DC-SIGN functions as an attachment factor rather than an entry receptor for JEV. Our findings highlight the potential significance of DC-SIGN in JEV early infection, providing a basis for further understanding how JEV exploits DC-SIGN to gain access to dendritic cells

Management Aspects of Software Clone Detection and Analysis

Copying a code fragment and reusing it by pasting with or without minor modifications is a common practice in software development for improved productivity. As a result, software systems often have similar segments of code, called software clones or code clones. Due to many reasons, unintentional clones may also appear in the source code without awareness of the developer. Studies report that significant fractions (5% to 50%) of the code in typical software systems are cloned. Although code cloning may increase initial productivity, it may cause fault propagation, inflate the code base and increase maintenance overhead. Thus, it is believed that code clones should be identified and carefully managed. This Ph.D. thesis contributes in clone management with techniques realized into tools and large-scale in-depth analyses of clones to inform clone management in devising effective techniques and strategies. To support proactive clone management, we have developed a clone detector as a plug-in to the Eclipse IDE. For clone detection, we used a hybrid approach that combines the strength of both parser-based and text-based techniques. To capture clones that are similar but not exact duplicates, we adopted a novel approach that applies a suffix-tree-based k-difference hybrid algorithm, borrowed from the area of computational biology. Instead of targeting all clones from the entire code base, our tool aids clone-aware development by allowing focused search for clones of any code fragment of the developer's interest. A good understanding on the code cloning phenomenon is a prerequisite to devise efficient clone management strategies. The second phase of the thesis includes large-scale empirical studies on the characteristics (e.g., proportion, types of similarity, change patterns) of code clones in evolving software systems. Applying statistical techniques, we also made fairly accurate forecast on the proportion of code clones in the future versions of software projects. The outcome of these studies expose useful insights into the characteristics of evolving clones and their management implications. Upon identification of the code clones, their management often necessitates careful refactoring, which is dealt with at the third phase of the thesis. Given a large number of clones, it is difficult to optimally decide what to refactor and what not, especially when there are dependencies among clones and the objective remains the minimization of refactoring efforts and risks while maximizing benefits. In this regard, we developed a novel clone refactoring scheduler that applies a constraint programming approach. We also introduced a novel effort model for the estimation of efforts needed to refactor clones in source code. We evaluated our clone detector, scheduler and effort model through comparative empirical studies and user studies. Finally, based on our experience and in-depth analysis of the present state of the art, we expose avenues for further research and development towards a versatile clone management system that we envision

How Accurate Is Coarse-grained Clone Detection?: Comparision with Fine-grained Detectors

Research on clone detection has been quite successful over the past two decades, which produced a number of state-of-the-art clone detectors.However, it has been still challenging to detect clones, even with such successful detectors, across multiple projects or on thousands of revisions of code in limited time.A simple and coarse-grained detector will be an alternative of detectors using fine-grained analysis.It will drastically reduce time required for detection although it may miss some of clones that fine-grained detectors can detect.Hence, it should be adequate for a tentative analysis of clones if it has an acceptable accuracy.However, it is not clear how accurate such a coarse-grained approach is.This paper evaluates the accuracy of a coarse-grained clone detector compared with some fine-grained clone detectors.Our experiment provides an empirical evidence about acceptable accuracy of such a coarse-grained approach.Thus, we conclude that coarse-grained detection is adequate to make a summary of clone analysis and to be a starter of detailed analysis including manual inspections and bug detection

Dancing with dendritic cells:Targeting human skin dendritic cells for anti-tumor immunity

In the studies described in this thesis we explored the potential of human skin DC to induce anti-tumor immunity. By the design of various vaccines we explored how the multiple human skin DC subsets responded for the induction of robust adaptive anti-tumor immunity. For intradermally injected particulate-vaccines, we need to ensure that the tumor-antigen containing particle will reach the targeted DC subset to facilitate the induction of cytotoxic CD8+ T-cells with tumor cell killing capacity. For this DC need to shuttle exogenously derived antigens into the endogenous presentation pathway, better known as cross-presentation. The expression of DC specific CLR gives us the opportunity to both specifically target skin DC whilst inducing cross-presentatio in combinaiton with TLR activation. In chapter 2 we show Langerin to be superior in the activation of specific CD8+ T-cells. In order to explore whether targeting dermal DC is beneficial in facilitating cross-presentation, we used DC-SIGN as target candidate. In chapter 3 we elucidated the exact intracellular routingof DC-SIGN and its cargo. Furthermore, we show that simultaneous triggering of DC-SIGN using an antibody conjugated to the melanoma specific gp100 SLP and TLR4 using LPS, efficiently enhances cross-presentation. This dual stimulation ensured SLP degradation by the proteasome and antigen processing for MHC I loading. In chapter 4, we used the overlapping glycan binding profiles for the Lewis Y type antigens of Langerin and DC-SIGN to target both receptors. Using Lewis Y (LeY ) as targeting glycan we aimed to generate a single glycovaccine targeting multiple human skin DC subsets simultaneously. As carrier system we used PAMAM-dendrimers, to covalently link the gp100 SLP and create two differentially sized vaccines. We show glycodendrimers of approximately 50nm efficiently target both Langerin and DC-SIGN,thereby reaching multiple human skin DC subsets when injected in situ. Furthermore,this enhanced activation of tumor specific CD4+ and CD8+ T-cells compared to nontargeting dendrimers. In order to investigate whether we could further optimize our cancer vaccine, we investigated whether we could improve DC maturation for cytokine skewing and co-stimulation and antigen processing, by combining various PRR agonists. TLR and NOD-like receptors (NLR) that are expressed by different human skin DC and can synergize for DC maturation and antigen handling. In chapter 5 we used the PAMAMdendrimer, to develop a multivalent antigenic vaccine containing both the gp100 SLP and NOD2 agonist MDP. A synthetic NOD2-agonist, was covalently linked to the multivalent antigenic dendrimer. We show that the combination of the NLR agonist-antigen complex with the soluble TLR4 agonist MPLA enhances cytokine secretion within the skin micromilieu. Furthermore, it enhances cross-presentation by human skin DC for CD8+ T-cell activation. Intradermal vaccine delivery is usually achieved through injection, though efforts are made to design systems that simplify intradermal vaccine delivery. In chapter 6 we made use of an ablative fractional laser to verify whether it might benefit vaccination with our anti-tumor vaccine particles. We show that in our human skin explant model intradermal injection was more efficient for vaccine delivery to and uptake by skin DC, resulting in higher level CD8+ T-cell activation. This thesis aimed to develop a human skin DC targeting cancer vaccine, exploiting the expression by DC of the CLR Langerin and DC-SIGN. By the design of a multivalent glyco-vaccine incorporating melanoma specific gp100 epitopes and the targeting moiety LeY we could efficiently target multiple human skin DC for enhanced (cross)- presentation using a single vaccine formulation. This dual targeting, multivalent vaccine can be used for inclusion of a multitude of epitopes and PRR agonists. Thereby we developed a flexible intradermal vaccine platform which has merit for clinical studies aiming to cure different types of cancer

Effects of galectins and antibodies in HIV infection; Novel Assays

The high variability of the HIV envelope glycoproteins (Env), and their heavy glycan coating, contributes to the limited host immune control. Still, broadly neutralizing antibodies (bnAbs) are found in some chronically infected HIV-infected individuals, which has spurred the research on antibody-based vaccines. An important tool in detecting and studying bnAbs, are neutralization assays. Here we developed an image-based, high-content automated version of a plaque reduction (PR) assay, which uses green fluorescent protein expression as a reporter of HIV infection. This permitted simultaneous detection of antibodies mediating neutralization and inhibition of virus induced cell-cell fusion. In a multicenter study, Neutnet II, the assay compared well with other neutralization assays and was suggested to be an alternative to the traditional peripheral blood monocyte (PBMC)-based assay and the TZMbl assay. The glycans of Env can also take part in HIV host cell adhesion and infection, via host glycan-binding protein, such as galectins. To explore this, we examined the interaction of gp120 (the surface Env protein) with a panel of galectins, by adapting the fluorescent anisotropy (FA) assay to microscale. Galectin-8, a galectin with two carbohydrate recognition domains (CRDs), had high affinity for gp120 as well as the HIV receptor CD4. The N-terminal CRD mediated the strongest interaction with gp120. The results of the FAassay correlated well with binding of whole virions screened in another assay against the same panel of galectins, now immobilized on beads. In the PR assay described above, here used as an infectivity assay, added intact galectin-8 enhanced infectivity of some HIV-1- strains, while this was not seen with the N-CRD, demonstrating that both CRDs of galectin-8 are required for the effect on infectivity. The enhancement effect mediated by galectin-8 was most pronounced with HIV-1 isolates obtained during the relative immune competent chronic phase, as compared to viruses isolated after AIDS onset. Hence, galectin-8 binding carbohydrate motifs on Env appear to be altered at severe immunodeficiency, adding to the knowledge on the evolution of Env glycosylation patterns related to HIV pathogenesis. These results add to the basic knowledge of virus-host interactions, which hopefully could be used for identification of antibodies and galectin-inhibitors effective in HIV prophylactic interventions

Identifying developers’ habits and expectations in copy and paste programming practice

Máster Universitario en Investigación e Innovación en Inteligencia Computacional y Sistemas InteractivosBoth novice and experienced developers rely more and more in external sources of code to include into their programs by copy and paste code snippets. This behavior differs from the traditional software design approach where cohesion was achieved via a conscious design effort. Due to this fact, it is essential to know how copy and paste programming practices are actually carried out, so that IDEs (Integrated Development Environments) and code recommenders can be designed to fit with developer expectations and habit

Broad Antiviral Activity of Carbohydrate-Binding Agents Against Dengue Virus Infection

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Membrane protein nanoclustering as a functional unit of immune cells : from nanoscopy to single molecule dynamics

State-of-the-art biophysical techniques featuring high temporal and spatial resolution have allowed for the first time the direct visualization of individual transmembrane proteins on the cell membrane. These techniques have revealed that a large amount of molecular components of the cell membrane do not organize in a random manner but they rather grouped together forming so-called clusters at the nanoscale. Moreover, the lateral behavior of these clusters shows a great dependence on the compartmentalization of the cell membrane by, e.g., the actin cytoskeleton at multiple temporal and spatial scales. Since these lateral and temporal organizations have been shown to be crucial for the regulation of the biological activity by these transmembrane proteins, the understanding of the spatiotemporal behavior of membrane receptors, and of proteins in general, is a necessary step towards understanding the biology of the cell. Protein nanoclustering and membrane compartmentalization have been shown to play a crucial role on leukocytes, particularly on the surface of antigen presenting cells. Hence, the direct visualization of membrane proteins on the cell membrane of antigen presenting proteins represents a crucial step in understanding how an immune response can be controlled by leukocytes at the molecular level. In Chapter 1, the immune system, the membrane receptor DC-SIGN and the antigen presenting protein CD1d are briefly introduced. Moreover, recent advances in superresolution microscopy and single particle tracking techniques which allow the study of membrane proteins at the nanoscale are discussed. Finally, an updated review of protein nanoclustering on the cell membrane shows examples of the importance of protein nanoclustering in regulating biological function in the immune system. Chapter 2 presents the quantitative methodology for analyzing STED nanoscopy images and multi-color single particle tracking data used throughout this thesis. Chapter 2 also describes the single-molecule fluorescence sensitive microscopes implemented in this thesis for multi-color single particle tracking experiments and the corresponding data analysis. At the end of Chapter 2, cartography maps combining high temporal with micron-scale spatial information on the basis of single-molecule detection are presented. The following chapters in this thesis describe the major results obtained on two important receptors of the immune system. In Chapter 3, we address the role of the neck region of DC-SIGN in fine-tuning the nanoclustering degree of DC-SIGN on the cell membrane. Moreover, Chapter 3 also links the nanoclustering capability of DC-SIGN with its virus binding capability. The meso-scale organization of DC-SIGN and its dependence on a glycan-based connectivity is addressed on Chapter 4. This glycosylation network enhances the interaction between DC-SIGN and clathrin beyond stochastic random encountering. In Chapter 5, we showed that DC-SIGN shows subdiffusive behavior and weak ergodicity breaking (wEB) that cannot be described using the continuous time random walk (CTRW) model. Instead, our data are more consistent with a model in which the plasma membrane is composed of "patches" that change in space in time. In Chapter 6, we demonstrate that the antigen presenting protein CD1d organizes in nanoclusters on the cell membrane of antigen presenting cells whose size and density are tightly controlled by the actin cytoskeleton. Moreover, we also showed that this cytoskeletal control of the CD1d nanoclustering predominantly occurs on the pool of CD1d that has undergone lysosomal recycling, including under inflammatory conditions. Finally, in Chapter 7 we summarize the main results of this thesis and highlight future experiments that will expand the knowledge obtained so far regarding the role of plasma membrane organization and biological regulation.Gracias a su alta resolución temporal y espacial, las técnicas biofísicas de última generación han permitido la observación directa de proteínas de transmembrana de forma individual en la membrana celular. Estas técnicas han mostrado que la organización de una gran parte de las proteínas de transmembrana no es aleatoria sino que éstas están agrupadas en la membrana celular formando nano-agregados, o "clusters". En el caso concreto del sistema inmune, se ha demostrado que el agrupamiento de proteínas y los compartimentos de la membrana celular juegan un papel determinante en las células presentadoras de antígenos a la hora de controlar la iniciación de una respuesta inmune. Por tanto, la visualización directa de proteínas de membrana en células presentadoras de antígenos a la escala nanométrica representa un paso crucial en el entendimiento del sistema inmune y en un futuro desarrollo de terapias basadas en el sistema inmune humano. En el primer capítulo de esta tesis, se presentará al lector una breve introducción del sistema inmune y una descripción general de las dos proteínas que se han estudiado extensivamente en esta tesis: el receptor reconocedor de patógenos DC-SIGN y la proteína presentadora de antígenos glicolipídicos CD1d. Se discutirán además los últimos avances en técnicas de microscopía de fluorescencia con alta resolución temporal y espacial que permiten el estudio de proteínas a la escala nanométrica. Finalmente, el primer capítulo concluye con una revisión de los últimos avances en la caracterización de la organización lateral de proteínas de membrana mostrando cómo dicha organización determina la función biológica de estas proteínas. En el capítulo 2, se presentan los distintos tipos de metodología utilizados en esta tesis para cuantificar imágenes de microscopía de super-resolución STED así como para analizar datos provenientes del seguimiento de partículas individuales usando varios colores. Al final del capítulo 2 se presenta una nueva metodología desarrollada en esta tesis que permite el estudio lateral de proteínas de membrana con una alta resolución temporal y una escala espacial de orden de micras y a la que hemos denominado mapas cartográficos. Los siguientes capítulos de esta tesis se enfocan en el estudio de dos importantes proteínas involucradas en el sistema inmune. En el capítulo 3 se describe como la parte central de la estructura del receptor captador de patógenos DC-SIGN determina su grado de nano-agrupamiento sobre la membrana celular. A su vez, este agrupamiento tiene una incidencia clave en la capacidad de DC-SIGN en unirse a partículas virales. La organización de DC-SIGN a la escala mesoscópica y la dependencia de dicha organización de una conectividad en la membrana celular basada en la glicosilación de proteínas es descrita en el capítulo 4. En el capítulo 5 descubrimos que DC-SIGN tiene un comportamiento que no solo es sub-difusivo en la membrana celular sino que también conlleva a la ruptura de ergodicidad por parte de este receptor. Esta rotura de ergodicidad no puede ser descrita por el modelo "continous time random walk" (CTRW) sino por un modelo nuevo donde la difusión de la partícula cambia constantemente en el espacio y en el tiempo. En el capítulo 6 de esta tesis describimos como la molécula CD1d forma nano-agrupamientos en la membrana celular cuyo tamaño y densidad son controlados por el citoesqueleto de actina. Además, observamos que dicho control mayoritariamente sucede cuando CD1d ha sido reciclado a través de compartimentos lisosomales, incluyendo procesos inflamatorios. Finalmente, en el capítulo 7 se discuten las conclusiones generales de esta tesis y se sugieren experimentos a futuro de manera de incrementar, en base a los resultados obtenidos en esta tesis, nuestro conocimiento de la membrana celular y el papel que la organización espacial y temporal juega en el control del sistema inmune