Epitope-blocking enzyme-linked immunosorbent assays for the detection of serum antibodies to West Nile virus in multiple avian species

We report the development of epitope-blocking enzyme-linked immunosorbent assays (ELISAs) for the rapid detection of serum antibodies to West Nile virus (WNV) in taxonomically diverse North American avian species. A panel of flavivirus-specific monoclonal antibodies (MAbs) was tested in blocking assays with serum samples from WNV-infected chickens and crows. Selected MAbs were further tested against serum samples from birds that represented 16 species and 10 families. Serum samples were collected from birds infected with WW or Saint Louis encephalitis virus (SLEV) and from noninfected control birds. Serum samples from SLEV-infected birds were included in these experiments because WNV and SLEV are closely related antigenically, are maintained in similar transmission cycles, and have overlapping geographic distributions. The ELISA that utilized MAb 3.11126 potentially discriminated between WW and SLEV infections, as all serum samples from WNV-infected birds and none from SLEV-infected birds were positive in this assay. Assays with MAbs 2132 and 6B6C-1 readily detected serum antibodies in all birds infected with WNV and SLEV, respectively, and in most birds infected with the other virus. Two other MAbs partially discriminated between infections with these two viruses. Serum samples from most WNV-infected birds but no SLEV-infected birds were positive with MAb 3.676, while almost all serum samples from SLEV-infected birds but few from WNV-infected birds were positive with MAb 6B5A-5. The blocking assays reported here provide a rapid, reliable, and inexpensive diagnostic and surveillance technique to monitor WNV activity in multiple avian species.

Complete genomic sequence of the Australian south-west genotype of Sindbis virus: Comparisons with other Sindbis strains and identification of a unique deletion in the 3 '-untranslated region

Our previous studies have shown that two distinct genotypes of Sindbis (SIN) virus occur in Australia. One of these, the Oriental/Australian type, circulates throughout most of the Australian continent, whereas the recently identified south-west (SW) genetic type appears to be restricted to a distinct geographic region located in the temperate south-west of Australia. We have now determined the complete nucleotide and translated amino acid sequences of a SW isolate of SIN virus (SW6562) and performed comparative analyses with other SIN viruses at the genomic level. The genome of SW6562 is 11,569 nucleotides in length, excluding the cap nucleotide and poly (A) tail. Overall this virus differs from the prototype SIN virus (strain AR339) by 23% in nucleotide sequence and 12.5% in amino acid sequence. Partial sequences of four regions of the genome of four SW isolates were determined and compared with the corresponding sequences from a number of SIN isolates from different regions of the World. These regions are the non-structural protein (nsP3), the E2 gene, the capsid gene, and the repeated sequence elements (RSE) of the 3'UTR. These comparisons revealed that the SW SIN viruses were more closely related to South African and European strains than to other Australian isolates of SIN virus. Thus the SW genotype of SIN virus may have been introduced into this region of Australia by viremic humans or migratory birds and subsequently evolved independently in the region. The sequence data also revealed that the SW genotype contains a unique deletion in the RSE of the 3'UTR region of the genome. Previous studies have shown that deletions in this region of the SIN genome can have significant effects on virus replication in mosquito and avian cells, which may explain the restricted distribution of this genotype of SIN virus.

14-3-3 acts as an intramolecular bridge to regulate cdc25B localization and activity

One of the major regulators of mitosis in somatic cells is cdc25B. cdc25B is tightly regulated at multiple levels. The final activation step involves the regulated binding of 14-3-3 proteins. Previous studies have demonstrated that Ser-323 is a primary 14-3-3 binding site in cdc25B, which influences its activity and cellular localization. 14-3-3 binding to this site appeared to interact with the N-terminal domain of cdc25B to regulate its activity. The presence of consensus 14-3-3 binding sites in the N-terminal domain suggested that the interaction is through direct binding of the 14-3-3 dimer to sites in the N-terminal domain. We have identified Ser-151 and Ser-230 in the N-terminal domain as functional 14-3-3 binding sites utilized by cdc25B in vivo. These low affinity sites cooperate to bind the 14-3-3 dimer bound to the high affinity Ser-323 site, thus forming an intramolecular bridge that constrains cdc25B structure to prevent access of the catalytic site. Loss of 14-3-3 binding to either N-terminal site relaxes cdc25B structure sufficiently to permit access to the catalytic site, and the nuclear export sequence located in the N-terminal domain. Mutation of the Ser-323 site was functionally equivalent to the mutation of all three sites, resulting in the complete loss of 14-3-3 binding, increased access of the catalytic site, and access to nuclear localization sequence.

Repair of three pathologic fractures in a dog with multiple myeloma

Three pathological fractures occurred secondary to osteolytic lesions of multiple myeloma. Two long bone fractures were each stabilised using interlocking nail fixation augmented with polymethyl meth acral ate bone cement. One vertebral fracture was stabilised using Steinmann pins and PMMA. Successful stabilisation, rapid return to function and improvement in quality of life occurred in all fractures. The patient survived approximately eight months on concurrent chemotherapy.

Multiple ordered phases in Langmuir-Blodgett films of cadmium arachidate at elevated temperatures

Using synchrotron X-ray grazing incidence diffraction, superlattice structures have been observed to develop in Langmuir-Blodgett films of cadmium arachidate as the temperature is raised. The previously reported superstructure in the stacked lamellae at room temperature changes at about 70 degreesC and there are further changes at about 90 and 103 degreesC before the major phase transition from stacked lamellae to hexagonally packed rods occurs at 107 degreesC (Langmuir 1997, 13, 1602). Between 70 and 103 degreesC there is a 1 x 10 one-dimensional in-plane superstructure, which is commensurate with the local structure and has an interlayer shift along [01] by a distance of b (of the local structure) at lower temperatures, and a further shift at about 90 degreesC. At lower (

Mecanismos de governança corporativa na pequena empresa familiar

Esta dissertação tem como objetivo descrever e analisar como o fenômeno da governança corporativa articula mecanismos de gestão e impacta na dinâmica de uma pequena organização familiar. Para isso, utilizou-se as perspectivas de Fan (2001) e Silva Junior (2006), que tratam da governança corporativa, e de Chandler (1994), para empresas familiares. Através de abordagem qualitativa com ênfase em um estudo de caso, uma triangulação de dados foi realizada, utilizando a entrevista semiestruturada, observação assistemática ou livre e pesquisa de documentos, e a análise dos dados foi realizada por análise de conteúdo. Verificou-se a presença do fenômeno da governança por intermédio da identificação dos fatores de diferenciação, resultando na implantação de mecanismos de governança ao longo do ciclo de vida da organização familiar, o que possibilitou mudanças em seu controle, no seu processo sucessório e na sua profissionalização. Quanto ao controle, foram verificados que os mecanismos de “manutenção do status quo”, “aconselhamento profissional”, “sinergia de interesses”, “regulamentação financeira da sociedade”, “atribuições e responsabilidades”, “alinhamento de interesses do negócio” e “proteção do empreendimento familiar” possibilitaram a modificação e manutenção do controle da empresa com os sócios. Quanto ao eixo da profissionalização, foi verificado que os mecanismos de “regulamentação financeira da sociedade”, “atribuições e responsabilidades”, “alinhamento de interesses do negócio”, “proteção do empreendimento familiar” e “atenção aos interesses dos stakeholders” foram responsáveis por prover as modificações neste quesito e indicar que a empresa caminha no sentido de se profissionalizar. Quanto ao processo sucessório, verificou-se que a terceira geração não tem interesse na empresa, o que podem resultar na contratação de um profissional externo para gerir a mesma, respeitando a questão dos valores familiares e a cultura organizacional ou conduzir à sucessão recursiva. Aparentemente, observou-se que a segunda questão foi mais evidente, visto que a saída do último irmão do negócio culmina com a venda da empresa familiar, gerando duas alternativas: a compra da empresa por um grupo de sócios ou outra empresa não familiar, o que resultaria na “morte” da empresa enquanto empresa familiar; ou a sua compra por outra família ou empresa familiar, preservando sua classificação inicial.

Unknown Radial Distortion Centers in Multiple View Geometry Problems

The radial undistortion model proposed by Fitzgibbon and the radial fundamental matrix were early steps to extend classical epipolar geometry to distorted cameras. Later minimal solvers have been proposed to find relative pose and radial distortion, given point correspondences between images. However, a big drawback of all these approaches is that they require the distortion center to be exactly known. In this paper we show how the distortion center can be absorbed into a new radial fundamental matrix. This new formulation is much more practical in reality as it allows also digital zoom, cropped images and camera-lens systems where the distortion center does not exactly coincide with the image center. In particular we start from the setting where only one of the two images contains radial distortion, analyze the structure of the particular radial fundamental matrix and show that the technique also generalizes to other linear multi-view relationships like trifocal tensor and homography. For the new radial fundamental matrix we propose different estimation algorithms from 9,10 and 11 points. We show how to extract the epipoles and prove the practical applicability on several epipolar geometry image pairs with strong distortion that - to the best of our knowledge - no other existing algorithm can handle properly.

Effect of fiber alignment in gelled electrospun membranes based on poly(vinylidene fluoride) for battery applications

Battery separators based on electrospun membranes of poly(vinylidene fluoride) (PVDF) have been prepared in order to study the effect of fiber alignment on the performance and characteristics of the membrane. The prepared membranes show an average fiber diameter of 272 nm and a degree of porosity of 87 %. The gel polymer electrolytes are prepared by soaking the membranes in the electrolyte solution. The alignment of the fibers improves the mechanical properties for the electrospun membranes. Further, the microstructure of the membrane also plays an important role in the ionic conductivity, being higher for the random electrospun membrane due to the lower tortuosity value. Independently of the microstructure, both membranes show good electrochemical stability up to 5.0 V versus Li/Li+. These results show that electrospun membranes based on PVDF are appropriate for battery separators in lithium-ion battery applications, the random membranes showing a better overall performance.

Multiple centroid method to evaluate the adaptability of alfalfa genotypes

This study aimed to evaluate the efficiency of multiple centroids to study the adaptability of alfalfa genotypes (Medicago sativa L.). In this method, the genotypes are compared with ideotypes defined by the bissegmented regression model, according to the researcher's interest. Thus, genotype classification is carried out as determined by the objective of the researcher and the proposed recommendation strategy. Despite the great potential of the method, it needs to be evaluated under the biological context (with real data). In this context, we used data on the evaluation of dry matter production of 92 alfalfa cultivars, with 20 cuttings, from an experiment in randomized blocks with two repetitions carried out from November 2004 to June 2006. The multiple centroid method proved efficient for classifying alfalfa genotypes. Moreover, it showed no unambiguous indications and provided that ideotypes were defined according to the researcher's interest, facilitating data interpretation.

Surface plasmon resonance as a tool in the functional analisys of an immunodominant site in foot-and-mouth disease virus

A fast and direct surface plasmon resonance (SPR) method for the kinetic analysis of the interactions between peptide antigens and immobilised monoclonal antibodies (mAb) has been established. Protocols have been developed to overcome the problems posed by the small size of the analytes (< 1600 Da). The interactions were well described by a simple 1:1 bimolecular interaction and the rate constants were self-consistent and reproducible. The key features for the accuracy of the kinetic constants measured were high buffer flow rates, medium antibody surface densities and high peptide concentrations. The method was applied to an extensive analysis of over 40 peptide analogues towards two distinct anti-FMDV antibodies, providing data in total agreement with previous competition ELISA experiments. Eleven linear 15-residue synthetic peptides, reproducing all possible combinations of the four replacements found in foot-and-mouth disease virus (FMDV) field isolate C-S30, were evaluated. The direct kinetic SPR analysis of the interactions between these peptides and three anti-site A mAbs suggested additivity in all combinations of the four relevant mutations, which was confirmed by parallel ELISA analysis. The four-point mutant peptide (A15S30) reproducing site A from the C-S30 strain was the least antigenic of the set, in disagreement with previously reported studies with the virus isolate. Increasing peptide size from 15 to 21 residues did not significantly improve antigenicity. Overnight incubation of A15S30 with mAb 4C4 in solution showed a marked increase in peptide antigenicity not observed for other peptide analogues, suggesting that conformational rearrangement could lead to a stable peptide-antibody complex. In fact, peptide cyclization clearly improved antigenicity, confirming an antigenic reversion in a multiply substituted peptide. Solution NMR studies of both linear and cyclic versions of the antigenic loop of FMDV C-S30 showed that structural features previously correlated with antigenicity were more pronounced in the cyclic peptide. Twenty-six synthetic peptides, corresponding to all possible combinations of five single-point antigenicity-enhancing replacements in the GH loop of FMDV C-S8c1, were also studied. SPR kinetic screening of these peptides was not possible due to problems mainly related to the high mAb affinities displayed by these synthetic antigens. Solution affinity SPR analysis was employed and affinities displayed were generally comparable to or even higher than those corresponding to the C-S8c1 reference peptide A15. The NMR characterisation of one of these multiple mutants in solution showed that it had a conformational behaviour quite similar to that of the native sequence A15 and the X-ray diffraction crystallographic analysis of the peptide ? mAb 4C4 complex showed paratope ? epitope interactions identical to all FMDV peptide ? mAb complexes studied so far. Key residues for these interactions are those directly involved in epitope ? paratope contacts (141Arg, 143Asp, 146His) as well as residues able to stabilise a particular peptide global folding. A quasi-cyclic conformation is held up by a hydrophobic cavity defined by residues 138, 144 and 147 and by other key intrapeptide hydrogen bonds, delineating an open turn at positions 141, 142 and 143 (corresponding to the Arg-Gly-Asp motif).

Renormalization-group constraints on Yukawa alignment in multi-Higgs-doublet models

We write down the renormalization-group equations for the Yukawa-coupling matrices in a general multi-Higgs-doublet model. We then assume that the matrices of the Yukawa couplings of the various Higgs doublets to right-handed fermions of fixed quantum numbers are all proportional to each other. We demonstrate that, in the case of the two-Higgs-doublet model, this proportionality is preserved by the renormalization-group running only in the cases of the standard type-I, II, X, and Y models. We furthermore show that a similar result holds even when there are more than two Higgs doublets: the Yukawa-coupling matrices to fermions of a given electric charge remain proportional under the renormalization-group running if and only if there is a basis for the Higgs doublets in which all the fermions of a given electric charge couple to only one Higgs doublet.

The importance of quality of life in patients with multiple sclerosis: do they need auxiliary walking devices?

Objective: To describe the importance of training multiple sclerosis (MS) patients with auxiliary walking devices (walking stick, crutch, or wheelchair) in a way that achieves a better quality of life (QOL). Design: Exploratory and descriptive. Setting: General hospital in Portugal.

Risk assessment of exposure to multiple mycotoxins in food

Moulds may produce a diversity of toxins such as aflatoxins, ochratoxins, trichothecenes, zearalenone, fumonisins and others. Although toxicological, environmental and epidemiological studies have addressed the problem of these toxins one by one, more than one mycotoxin are found usually in the same contaminated food. Risk assessment for humans potentially exposed to multimycotoxins suffers very much from the lack of adequate food consumption data. Furthermore, for a given mycotoxin, synergism and antagonism with other mycotoxins, found in the same food commodities, are not taken into account. Aflatoxin B1 and ochratoxin A belong to the most frequently occurring mycotoxins. This has repeatedly been demonstrated, however, normally, the risk resulting from their simultaneous occurrence is not considered. A descriptive study was developed to monitor air fungal contamination in one hospital food unit.