Carboxy-fluorescein diacetate, succinimidyl ester labeled papillomavirus virus-like particles fluoresce after internalization and interact with heparan sulfate for binding and entry

Human papillomaviruses (HPVs) infect epithelial cells and are associated with genital carcinoma. Most epithelial cell lines express cell-surface glycosaminoglycans (GAGs) usually found attached to the protein core of proteoglycans. Our aim was to study how GAGs influenced HPV entry. Using a human keratinocyte cell line (HaCaT), preincubation of HPV virus-like particles (VLPs) with GAGs showed a dose-dependent inhibition of binding. The IC50 (50% inhibition) was only 0.5 mug/ml for heparin, 1 mug/ml for dextran sulfate, and 5-10 mug/ml for heparan sulfate from mucosal origin. Mutated chinese hamster ovary (CHO) cell lines lacking heparan sulfate or all GAGs were unable to bind HPV VLPs. Here we also report a method to study internalization by using VLPs labeled with carboxy-fluorescein diacetate, succinimidyl ester, a fluorochrome that is only activated after cell entry. Pretreatment of labeled HPV VLPs with heparin inhibited uptake, suggesting a primary interaction between HPV and cell-surface heparan sulfate. (C) 2003 Elsevier Science (USA). All rights reserved.

Epitope map for a growth hormone receptor agonist monoclonal antibody, MAb 263

Monoclonal antibody (MAb) 263 is a widely used monoclonal antibody that recognizes the extracellular domain (ECD) of the GH receptor. It has been shown to act as a GH agonist both in vitro and in vivo, and we report here that it must be divalent to exert its effect on the full-length receptor. To understand the mechanism of its agonist action, we have determined the precise epitope for this antibody using a novel random PCR mutagenesis approach together with expression screening in yeast. A library of 5200 clones of rabbit GH receptor ECD mutants were screened both with MAb 263 and with an anticarboxy-tag antibody to verify complete ECD expression. Sequencing for clones that expressed complete ECD but were not MAb 263 positive identified 20 epitope residues distributed in a discontinuous manner throughout the ECD. The major part of the epitope, as revealed after mapping onto the crystal structure model of the ECD molecule, was located on the side and upper portion of domain 1, particularly within the D - E strand disulfide loop 79 - 96. Molecular dynamics docking of an antibody of the same isotype as MAb 263 was used to dock the bivalent antibody to the 1528-Angstrom(2) epitope and to visualize the likely consequences of MAb binding. The minimized model enables the antibody to grasp two receptors in a pincer-like movement from opposite sides, facilitating alignment of the receptor dimerization domains in a manner similar to, but not identical with, GH.

Characterization of Treponema phagedenis-like spirochetes isolated from papillomatous digital dermatitis lesions in dairy cattle

Four spirochete strains were isolated from papillomatous digital dermatitis (PDD) lesions in Iowa dairy cattle and compared with two previously described spirochete strains isolated from dairy cattle in California. These six strains shared an identical 16S ribosomal DNA sequence that was 98% similar to Treponema phagedenis and 99% similar to the uncultivated PDD spirochete sequence DDLK-4. The whole-cell protein profiles resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these six strains were similar. However, these strains showed differences in the antigenic diversity of lipopolysaccharide (LPS). Genetic diversity was also detected by pulsed-field gel electrophoresis of genomic DNA digests, revealing differences among five of the six strains. Serum immunoglobulin G antibodies from dairy cattle with active PDD lesions reacted with the LPS of all but one PDD spirochete strain. Likewise, peripheral blood mononuclear cells from cattle with active PDD lesions produced blastogenic responses to one of the two California isolates. Both antibody and lymphocyte blastogenic responses were reduced in convalescent dairy cattle, suggesting the immune response to these spirochetes has short duration. These results demonstrate genetic and antigenic diversity among T. phagedenis-like treponemes and provide further evidence for the involvement of these spirochetes in the pathogenesis of PDD.

What does the field of International Relations look like in South America?

Abstract This article provides a comprehensive picture of IR in South America by applying content analysis to 7,857 articles published in 35 journals from six South American countries from 2006 to 2014 in order to discover what the predominant theories, methods and research areas in this field are, how scholars tend to combine them in their research designs, and what the profiles of regional journals are, regarding their epistemological, methodological and subject preferences. The findings reveal a predominantly Positivist and largely Qualitative discipline, resembling North American and European IR.

Electrospun silk-elastin-like fiber mats for tissue engineering applications

Protein-based polymers are present in a wide variety of organisms fulfilling structural and mechanical roles. Advances in protein engineering and recombinant DNA technology allow the design and production of recombinant protein-based polymers (rPBPs) with an absolute control of its composition. Although the application of recombinant proteins as biomaterials is still an emerging technology, the possibilities are limitless and far superior to natural or synthetic materials, as the complexity of the structural design can be fully customized. In this work, we report the electrospinning of two new genetically engineered silk-elastin-like proteins (SELPs) consisting of alternate silk- and elastin-like blocks. Electrospinning was performed with formic acid and aqueous solutions at different concentrations without addition of further agents. The size and morphology of the electrospun structures was characterized by scanning electron microscopy showing to be dependent of concentration and solvent used. Treatment with air saturated with methanol was employed to stabilize the structure and promote water insolubility through a time-dependent conversion of random coils into β-sheets (FTIR). The resultant methanol-treated electrospun mats were characterized for swelling degree (570-720%), water vapour transmission rate (1083 g/m2/day) and mechanical properties (modulus of elasticity of ~126 MPa). Furthermore, the methanol-treated SELP fiber mats showed no cytotoxicity and were able to support adhesion and proliferation of normal human skin fibroblasts. Adhesion was characterized by a filopodia-mediated mechanism. These results demonstrate that SELP fiber mats can provide promising solutions for the development of novel biomaterials suitable for tissue engineering applications.

Impact of a 1755-like tsunami in Huelva, Spain

Coastal areas are highly exposed to natural hazards associated with the sea. In all cases where there is historical evidence for devastating tsunamis, as is the case of the southern coasts of the Iberian Peninsula, there is a need for quantitative hazard tsunami assessment to support spatial planning. Also, local authorities must be able to act towards the population protection in a preemptive way, to inform 'what to do' and 'where to go' and in an alarm, to make people aware of the incoming danger. With this in mind, we investigated the inundation extent, run-up and water depths, of a 1755-like event on the region of Huelva, located on the Spanish southwestern coast, one of the regions that was affected in the past by several high energy events, as proved by historical documents and sedimentological data. Modelling was made with a slightly modified version of the COMCOT (Cornell Multi-grid Coupled Tsunami Model) code. Sensitivity tests were performed for a single source in order to understand the relevance and influence of the source parameters in the inundation extent and the fundamental impact parameters. We show that a 1755-like event will have a dramatic impact in a large area close to Huelva inundating an area between 82 and 92 km(2) and reaching maximum run-up around 5 m. In this sense our results show that small variations on the characteristics of the tsunami source are not too significant for the impact assessment. We show that the maximum flow depth and the maximum run-up increase with the average slip on the source, while the strike of the fault is not a critical factor as Huelva is significantly far away from the potential sources identified up to now. We also show that the maximum flow depth within the inundated area is very dependent on the tidal level, while maximum run-up is less affected, as a consequence of the complex morphology of the area.

Potential inundation of Lisbon downtown by a 1755-like tsunami

In this study, we present 10 m resolution tsunami flooding maps for Lisbon downtown and the Tagus estuary. To compute these maps we use the present bathymetry and topographic maps and a reasonable estimate for the maximum credible tsunami scenario. Tsunami modeling was made with a non-linear shallow water model using four levels of nested grids. The tsunami flood is discussed in terms of flow depth, run-up height and maximum inundation area. The results show that, even today, in spite of the significant morphologic changes in the city river front after the 1755 earthquake, a similar event would cause tsunami flow depths larger than one meter in a large area along the Tagus estuary and Lisbon downtown. Other areas along the estuary with a high population density would also be strongly affected. The impact of the tide on the extent of tsunami inundation is discussed, due to the large amplitude range of the tide in Lisbon, and compared with the historical descriptions of the 1755 event. The results presented here can be used to identify the potential tsunami inundation areas in Lisbon; this identification comprises a key element of the Portuguese tsunami emergency management system.

Can power laws help us understand gene and proteome information?

Proteins are biochemical entities consisting of one or more blocks typically folded in a 3D pattern. Each block (a polypeptide) is a single linear sequence of amino acids that are biochemically bonded together. The amino acid sequence in a protein is defined by the sequence of a gene or several genes encoded in the DNA-based genetic code. This genetic code typically uses twenty amino acids, but in certain organisms the genetic code can also include two other amino acids. After linking the amino acids during protein synthesis, each amino acid becomes a residue in a protein, which is then chemically modified, ultimately changing and defining the protein function. In this study, the authors analyze the amino acid sequence using alignment-free methods, aiming to identify structural patterns in sets of proteins and in the proteome, without any other previous assumptions. The paper starts by analyzing amino acid sequence data by means of histograms using fixed length amino acid words (tuples). After creating the initial relative frequency histograms, they are transformed and processed in order to generate quantitative results for information extraction and graphical visualization. Selected samples from two reference datasets are used, and results reveal that the proposed method is able to generate relevant outputs in accordance with current scientific knowledge in domains like protein sequence/proteome analysis.

Structural, physical, and chemical modifications induced by microwave heating on native agar-like galactans

Native agars from Gracilaria vermiculophylla produced in sustainable aquaculture systems (IMTA) were extracted under conventional (TWE) and microwave (MAE) heating. The optimal extracts from both processes were compared in terms of their properties. The agars’ structure was further investigated through Fourier transform infrared and NMR spectroscopy. Both samples showed a regular structure with an identical backbone, β-D-galactose (G) and 3,6-anhydro-α-L-galactose (LA) units; a considerable degree of methylation was found at C6 of the G units and, to a lesser extent, at C2 of the LA residues. The methylation degree in the G units was lower for MAEopt agar; the sulfate content was also reduced. MAE led to higher agar recoveries with drastic extraction time and solvent volume reductions. Two times lower values of [η] and Mv obtained for the MAEopt sample indicate substantial depolymerization of the polysaccharide backbone; this was reflected in its gelling properties; yet it was clearly appropriate for commercial application in soft-texture food products.

Identification of neuronal alterations induced by Shank3 mutations using iPS cells from Phelan-McDermid Patients' Fibroblasts

A família de proteínas Shank é o principal conjunto de proteinas de suporte e está localizada na densidade pós-sináptica das sinapses excitatórias. Existem 3 genes na família Shank, Shank1, Shank2 e Shank3 e são caracterizados por múltiplos domínios repetidos de anquirina próximo ao N-terminal seguido pelos domínios Src homologo 3 e PDZ, uma região longa rica em prolina e um domínio de motivo α estéril próximo ao C-terminal. Shank proteínas conectam duas subunidades de receptors glutamatérgicos, recetores NMDA e recetores metabotrópicos de glutamato do tipo-I (mGluRs). O domínio PDZ da Shank conecta-se ao C-terminal do GKAP e este, liga-se, ao complexo recetor PSD-95-NMDA. Por outro lado, a proteína Homer interage com o domínio rico em prolina para confirmar a associação entre a proteína Shank com o mGluR tipo-I. A proteína específica em estudo, Shank3, é haploinsuficiente em pacientes com sindrome Phelan-McDermid devido à deleções no braço comprido do cromossoma 22 levando à danos intelectuais, ausência ou atraso no discurso, comportamentos semelhantes ao autismo, hipotonia e características dismórficas. Neste trabalho, investigamos o papel da Shank3 na função sináptica para compreender a relação entre alterações nesta proteína e as características neurológicas presente em Pacientes com síndrome Phelan-McDermid. Foram utilizados dois modelos diferentes, ratinhos knockout Shank3 e hiPSC de pacientes com PMS. Ratinhos geneticamente modificados são ferramentas uteis no estudo de genes e na compreensão dos mecanismos que experiências in vitro não são capazes de reproduzir, mas de maneira a compreender melhor as patologias humanas, decidimos trabalhar também com células humanas. Os fibroblastos dos pacientes com síndrome Phelan-McDermid fora reprogramados em hiPS cells, diferenciados em neurónios e comparados com os neurónios obtidos a partir de doadores saudavéis e da mesma idade. A reprogramação em iPSC foi realizada por infecção de lentivirus com quatro genes de reprogramação OCT4, c-MYC, SOX2 e KFL4 para posteriormente serem diferenciados em neurónios, com cada passo sendo positivamente confirmado através de marcadores neuronais. Através dos neurónios diferenciados, analisamos a expressão de proteínas sinápticas. Pacientes com haploinsuficiencia na proteína Shank3 apresentam níveis elevados de proteína mGluR5 e decrescidos de proteína Homer sugerindo que a haploinsuficiencia leva a desregulação do complexo mGluR5-Homer-Shank3 conduzindo também, a defeitos na maturação sináptica. Assim, a expressão da proteína mGluR5 está alterada nos pacientes com PMS podendo estar relacionada com defeitos encontrados na diferenciação neuronal e maturação sináptica observados nos neurónios de pacientes. Conclusivamente, iPS cells representam um modelo fundamental no estudo da proteína Shank3 e a sua influência no sindrome de Phelan-McDermid.

Timing analysis of an inter-cell mobility procedure for a wired/wireless PROFIBUS network

Recently, there have been a few research efforts towards extending the capabilities of fieldbus networks to encompass wireless support. In previous works we have proposed a hybrid wired/wireless PROFIBUS network solution where the interconnection between the heterogeneous communication media was accomplished through bridge-like interconnecting devices. The resulting networking architecture embraced a Multiple Logical Ring (MLR) approach, thus with multiple independent tokens, where the communication between different domains was supported by the Inter-Domain Protocol (IDP). The proposed architecture also supports mobility of stations between different wireless cells. To that hybrid wired/wireless networking architecture we have proposed a worst-case response timing analysis of the IDP, without considering inter-cell mobility (or handoff) of stations. In this paper, we advance that previous work by proposing a worst-case timing analysis of the mobility procedure.

Pattern operators for grid

The definition and programming of distributed applications has become a major research issue due to the increasing availability of (large scale) distributed platforms and the requirements posed by the economical globalization. However, such a task requires a huge effort due to the complexity of the distributed environments: large amount of users may communicate and share information across different authority domains; moreover, the “execution environment” or “computations” are dynamic since the number of users and the computational infrastructure change in time. Grid environments, in particular, promise to be an answer to deal with such complexity, by providing high performance execution support to large amount of users, and resource sharing across different organizations. Nevertheless, programming in Grid environments is still a difficult task. There is a lack of high level programming paradigms and support tools that may guide the application developer and allow reusability of state-of-the-art solutions. Specifically, the main goal of the work presented in this thesis is to contribute to the simplification of the development cycle of applications for Grid environments by bringing structure and flexibility to three stages of that cycle through a commonmodel. The stages are: the design phase, the execution phase, and the reconfiguration phase. The common model is based on the manipulation of patterns through pattern operators, and the division of both patterns and operators into two categories, namely structural and behavioural. Moreover, both structural and behavioural patterns are first class entities at each of the aforesaid stages. At the design phase, patterns can be manipulated like other first class entities such as components. This allows a more structured way to build applications by reusing and composing state-of-the-art patterns. At the execution phase, patterns are units of execution control: it is possible, for example, to start or stop and to resume the execution of a pattern as a single entity. At the reconfiguration phase, patterns can also be manipulated as single entities with the additional advantage that it is possible to perform a structural reconfiguration while keeping some of the behavioural constraints, and vice-versa. For example, it is possible to replace a behavioural pattern, which was applied to some structural pattern, with another behavioural pattern. In this thesis, besides the proposal of the methodology for distributed application development, as sketched above, a definition of a relevant set of pattern operators was made. The methodology and the expressivity of the pattern operators were assessed through the development of several representative distributed applications. To support this validation, a prototype was designed and implemented, encompassing some relevant patterns and a significant part of the patterns operators defined. This prototype was based in the Triana environment; Triana supports the development and deployment of distributed applications in the Grid through a dataflow-based programming model. Additionally, this thesis also presents the analysis of a mapping of some operators for execution control onto the Distributed Resource Management Application API (DRMAA). This assessment confirmed the suitability of the proposed model, as well as the generality and flexibility of the defined pattern operators

sMapReduce: a programming pattern for wireless sensor networks

Wireless Sensor Networks (WSNs) are increasingly used in various application domains like home-automation, agriculture, industries and infrastructure monitoring. As applications tend to leverage larger geographical deployments of sensor networks, the availability of an intuitive and user friendly programming abstraction becomes a crucial factor in enabling faster and more efficient development, and reprogramming of applications. We propose a programming pattern named sMapReduce, inspired by the Google MapReduce framework, for mapping application behaviors on to a sensor network and enabling complex data aggregation. The proposed pattern requires a user to create a network-level application in two functions: sMap and Reduce, in order to abstract away from the low-level details without sacrificing the control to develop complex logic. Such a two-fold division of programming logic is a natural-fit to typical sensor networking operation which makes sensing and topological modalities accessible to the user.

Static-priority scheduling over wireless networks with multiple broadcast domains

We propose a wireless medium access control (MAC) protocol that provides static-priority scheduling of messages in a guaranteed collision-free manner. Our protocol supports multiple broadcast domains, resolves the wireless hidden terminal problem and allows for parallel transmissions across a mesh network. Arbitration of messages is achieved without the notion of a master coordinating node, global clock synchronization or out-of-band signaling. The protocol relies on bit-dominance similar to what is used in the CAN bus except that in order to operate on a wireless physical layer, nodes are not required to receive incoming bits while transmitting. The use of bit-dominance efficiently allows for a much larger number of priorities than would be possible using existing wireless solutions. A MAC protocol with these properties enables schedulability analysis of sporadic message streams in wireless multihop networks.