Infection of human airway epithelium by human and avian strains of influenza a virus

We describe the characterization of influenza A virus infection of an established in vitro model of human pseudostratified mucociliary airway epithelium (HAE). Sialic acid receptors for both human and avian viruses, alpha-2,6- and alpha-2,3-linked sialic acids, respectively, were detected on the HAE cell surface, and their distribution accurately reflected that in human tracheobronchial tissue. Nonciliated cells present a higher proportion of alpha-2,6-linked sialic acid, while ciliated cells possess both sialic acid linkages. Although we found that human influenza viruses infected both ciliated and nonciliated cell types in the first round of infection, recent human H3N2 viruses infected a higher proportion of nonciliated cells in HAE than a 1968 pandemic-era human virus, which infected proportionally more ciliated cells. In contrast, avian influenza viruses exclusively infected ciliated cells. Although a broad-range neuraminidase abolished infection of HAE by human parainfluenza virus type 3, this treatment did not significantly affect infection by influenza viruses. All human viruses replicated efficiently in HAE, leading to accumulation of nascent virus released from the apical surface between 6 and 24 h postinfection with a low multiplicity of infection. Avian influenza A viruses also infected HAE, but spread was limited compared to that of human viruses. The nonciliated cell tropism of recent human H3N2 viruses reflects a preference for the sialic acid linkages displayed on these cell types and suggests a drift in the receptor binding phenotype of the H3 hemagglutinin protein as it evolves in humans away from its avian virus precursor.

Adjuvant-free immunization with hemagglutinin-Fc fusion proteins as an approach to influenza vaccines

The hemagglutinins (HAs) of human H1 and H3 influenza viruses and avian H5 influenza virus were produced as recombinant fusion proteins with the human immunoglobulin Fc domain. Recombinant HA-human immunoglobulin Fc domain (HA-HuFc) proteins were secreted from baculovirus-infected insect cells as glycosylated oligomer HAs of the anticipated molecular mass, agglutinated red blood cells, were purified on protein A, and were used to immunize mice in the absence of adjuvant. Immunogenicity was demonstrated for all subtypes, with the serum samples demonstrating subtype-specific hemagglutination inhibition, epitope specificity similar to that seen with virus infection, and neutralization. HuFc-tagged HAs are potential candidates for gene-to-vaccine approaches to influenza vaccination.

Haplotype analysis of vernalization loci in European barley germplasm reveals novel VRN-H1 alleles and a predominant winter VRN-H1/VRN-H2 multi-locus haplotype

In barley, variation in the requirement for vernalization (an extended period of low temperature before flowering can occur) is determined by the VRN-H1, -H2 and -H3 loci. In European cultivated germplasm, most variation in vernalization requirement is accounted for by alleles at VRN-H1 and VRN-H2 only, but the range of allelic variation is largely unexplored. Here we characterise VRN-H1 and VRN-H2 haplotypes in 429 varieties representing a large portion of the acreage sown to barley in Western Europe over the last 60 years. Analysis of genotype, intron I sequencing data and growth habit tests identified three novel VRN-H1 alleles and determined the most frequent VRN-H1 intron I rearrangements. Combined analysis of VRN-H1 and VRN-H2 alleles resulted in the classification of seventeen VRN-H1/VRN-H2 multi-locus haplotypes, three of which account for 79% of varieties. The molecular markers employed here represent powerful diagnostic tools for prediction of growth habit and assessment of varietal purity. These markers will also allow development of germplasm to test the behaviour of individual alleles with the aim of understanding the relationship between allelic variation and adaptation to specific agri-environments.

Circulating insulin-like factor 3 (INSL3) in healthy and infertile women

STUDY QUESTION: How does insulin-like factor 3 (INSL3) concentration in blood vary across the menstrual cycle in women? SUMMARY ANSWER: INSL3 is secreted by the theca interna cells of growing antral follicles and is phasic in its expression. WHAT IS KNOWN ALREADY: The relaxin-like hormone INSL3 is known to be expressed in follicles of several mammal species, and was recently shown in cows to be specifically secreted into the bloodstream by growing antral follicles, corresponding to follicular waves. In males INSL3 is known to be acutely independent of the hormones of the hypothalamic-pituitary-gonadal axis, suggesting that in women INSL3 might be a novel biomarker for antral follicle recruitment and development. STUDY DESIGN, SIZE, DURATION: Two cohorts of women were studied. First, 18 healthy women of reproductive age were followed longitudinally for one and a half cycles, with blood sampling and hormone measurement every 2-3 days. A second cohort comprised a cross-sectional study of 909 women attending an infertility clinic, with a single blood sample taken at entry, together with other clinical and hormonal parameters. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blood samples from both retrospective cohorts were analyzed for INSL3 using a highly sensitive time-resolved fluorescent immunoassay, and data were analyzed in comparison with other clinical and hormonal parameters. MAIN RESULT AND THE ROLE OF CHANCE: For young healthy women of reproductive age, we showed a phasic expression of INSL3 corresponding to antral follicle growth in both the follicular and luteal phases of the cycle, which was significantly (P < 0.05) elevated compared with that during menses. For women attending an infertility clinic, those with diagnosed polycystic ovarian syndrome indicated significantly (P < 0.0005) greater circulating INSL3 levels and those with low ovarian reserve showed significantly (P < 0.002) decreased INSL3 values. LIMITATIONS, REASONS FOR CAUTION: These were retrospective studies and the results were obtained from natural cycles only, with their inherent variability. WIDER IMPLICATIONS OF THE FINDINGS: We show for the first time that INSL3 in women does vary across the menstrual cycle, and appears to reflect the number of growing antral follicles recruited within both follicular and luteal phases. STUDY FUNDING/COMPETING INTEREST(S): The present retrospective study was largely supported by departmental funds. There were no competing interests.

An epigenetic signature of developmental potential in neural stem cells and early neurons.

A cardinal property of neural stem cells (NSCs) is their ability to adopt multiple fates upon differentiation. The epigenome is widely seen as a read-out of cellular potential and a manifestation of this can be seen in embryonic stem cells (ESCs), where promoters of many lineage-specific regulators are marked by a bivalent epigenetic signature comprising trimethylation of both lysine 4 and lysine 27 of histone H3 (H3K4me3 and H3K27me3, respectively). Bivalency has subsequently emerged as a powerful epigenetic indicator of stem cell potential. Here, we have interrogated the epigenome during differentiation of ESC-derived NSCs to immature GABAergic interneurons. We show that developmental transitions are accompanied by loss of bivalency at many promoters in line with their increasing developmental restriction from pluripotent ESC through multipotent NSC to committed GABAergic interneuron. At the NSC stage, the promoters of genes encoding many transcriptional regulators required for differentiation of multiple neuronal subtypes and neural crest appear to be bivalent, consistent with the broad developmental potential of NSCs. Upon differentiation to GABAergic neurons, all non-GABAergic promoters resolve to H3K27me3 monovalency, whereas GABAergic promoters resolve to H3K4me3 monovalency or retain bivalency. Importantly, many of these epigenetic changes occur before any corresponding changes in gene expression. Intriguingly, another group of gene promoters gain bivalency as NSCs differentiate toward neurons, the majority of which are associated with functions connected with maturation and establishment and maintenance of connectivity. These data show that bivalency provides a dynamic epigenetic signature of developmental potential in both NSCs and in early neurons. Stem Cells 2013;31:1868-1880.

Optimal steady motions for oriented vehicles

Motivated by the motion planning problem for oriented vehicles travelling in a 3-Dimensional space; Euclidean space E3, the sphere S3 and Hyperboloid H3. For such problems the orientation of the vehicle is naturally represented by an orthonormal frame over a point in the underlying manifold. The orthonormal frame bundles of the space forms R3,S3 and H3 correspond with their isometry groups and are the Euclidean group of motion SE(3), the rotation group SO(4) and the Lorentzian group SO(1; 3) respectively. Orthonormal frame bundles of space forms coincide with their isometry groups and therefore the focus shifts to left-invariant control systems defined on Lie groups. In this paper a method for integrating these systems is given where the controls are time-independent. For constant twist motions or helical motions, the corresponding curves g(t) 2 SE(3) are given in closed form by using the well known Rodrigues’ formula. However, this formula is only applicable to the Euclidean case. This paper gives a method for computing the non-Euclidean screw/helical motions in closed form. This involves decoupling the system into two lower dimensional systems using the double cover properties of Lie groups, then the lower dimensional systems are solved explicitly in closed form.

Species concepts in Cercospora: spotting the weeds among the roses

The genus Cercospora contains numerous important plant pathogenic fungi from a diverse range of hosts. Most species of Cercospora are known only from their morphological characters in vivo. Although the genus contains more than 5 000 names, very few cultures and associated DNA sequence data are available. In this study, 360 Cercospora isolates, obtained from 161 host species, 49 host families and 39 countries, were used to compile a molecular phylogeny. Partial sequences were derived from the internal transcribed spacer regions and intervening 5.8S nrRNA, actin, calmodulin, histone H3 and translation elongation factor 1-alpha genes. The resulting phylogenetic clades were evaluated for application of existing species names and five novel species are introduced. Eleven species are epi-, lecto- or neotypified in this study. Although existing species names were available for several clades, it was not always possible to apply North American or European names to African or Asian strains and vice versa. Some species were found to be limited to a specific host genus, whereas others were isolated from a wide host range. No single locus was found to be the ideal DNA barcode gene for the genus, and species identification needs to be based on a combination of gene loci and morphological characters. Additional primers were developed to supplement those previously published for amplification of the loci used in this study. TAXONOMIC NOVELTIES: New species - Cercospora coniogrammes Crous & R.G. Shivas, Cercospora delaireae C. Nakash., Crous, U. Braun & H.D. Shin, Cercospora euphorbiae-sieboldianae C. Nakash., Crous, U. Braun & H.D. Shin, Cercospora pileicola C. Nakash., Crous, U. Braun & H.D. Shin, Cercospora vignigena C. Nakash., Crous, U. Braun & H.D. Shin. Typifications: epitypifications - Cercospora alchemillicola U. Braun & C.F. Hill, Cercospora althaeina Sacc., Cercospora armoraciae Sacc., Cercospora corchori Sawada, Cercospora mercurialis Pass., Cercospora olivascens Sacc., Cercospora violae Sacc.; neotypifications - Cercospora fagopyri N. Nakata & S. Takim., Cercospora sojina Hara.

Changes in histone acetylation and methylation that are important for persistent but not transient expression of CCR4 in human CD4(+) T cells

Although regulation of CXCR3 and CCR4 is related to Th1 and Th2 differentiation, respectively, many CXCR3(+) and CCR4(+) cells do not express IFN-gamma and/or IL-4, suggesting that the chemokine receptor genes might be inducible by mechanisms that are lineage-independent. We investigated the regulation of CXCR3 versus IFNG, and CCR4 versus IL4 in human CD4(+) T cells by analyzing modifications of histone H3. In naive cord-blood cells, under nonpolarizing conditions not inducing IL4, CCR4 was induced to high levels without many of the activation-associated changes in promoter histone H3 found for both IL4 and CCR4 in Th2 cells. Importantly, CCR4 expression was stable in Th2 cells, but fell in nonpolarized cells after the cells were rested; this decline could be reversed by increasing histone acetylation using sodium butyrate. Patterns of histone H3 modifications in CXCR3(+) CCR4(-) and CXCR3(-) CCR4(+) CD4(+) T-cell subsets from adult blood matched those in cells cultured under polarizing conditions in vitro. Our data show that high-level lineage-independent induction of CCR4 can occur following T-cell activation without accessibility-associated changes in histone H3, but that without such changes expression is transient rather than persistent.

Differential Antitumor Effects of IgG and IgM Monoclonal Antibodies and Their Synthetic Complementarity-Determining Regions Directed to New Targets of B16F10-Nex2 Melanoma Cells

Malignant melanoma has increased incidence worldwide and causes most skin cancer-related deaths. A few cell surface antigens that can be targets of antitumor immunotherapy have been characterized in melanoma. This is an expanding field because of the ineffectiveness of conventional cancer therapy for the metastatic form of melanoma. In the present work, antimelanoma monoclonal antibodies (mAbs) were raised against B16F10 cells (subclone Nex4, grown in murine serum), with novel specificities and antitumor effects in vitro and in vivo. MAb A4 (IgG2ak) recognizes a surface antigen on B16F10-Nex2 cells identified as protocadherin beta(13). It is cytotoxic in vitro and in vivo to B16F10-Nex2 cells as well as in vitro to human melanoma cell lines. MAb A4M (IgM) strongly reacted with nuclei of permeabilized murine tumor cells, recognizing histone 1. Although it is not cytotoxic in vitro, similarly with mAb A4, mAb A4M significantly reduced the number of lung nodules in mice challenged intravenously with B16F10-Nex2 cells. The V(H) CDR3 peptide from mAb A4 and V(L) CDR1 and CDR2 from mAb A4M showed significant cytotoxic activities in vitro, leading tumor cells to apoptosis. A cyclic peptide representing A4 CDR H3 competed with mAb A4 for binding to melanoma cells. MAb A4M CDRs L1 and L2 in addition to the antitumor effect also inhibited angiogenesis of human umbilical vein endothelial cells in vitro. As shown in the present work, mAbs A4 and A4M and selected CDR peptides are strong candidates to be developed as drugs for antitumor therapy for invasive melanoma.

Analysis of Agonist and Antagonist Effects on Thyroid Hormone Receptor Conformation by Hydrogen/Deuterium Exchange

Thyroid hormone receptors (TRs) are ligand-gated transcription factors with critical roles in development and metabolism. Although x-ray structures of TR ligand-binding domains (LBDs) with agonists are available, comparable structures without ligand (apo-TR) or with antagonists are not. It remains important to understand apo-LBD conformation and the way that it rearranges with ligands to develop better TR pharmaceuticals. In this study, we conducted hydrogen/deuterium exchange on TR LBDs with or without agonist (T(3)) or antagonist (NH(3)). Both ligands reduce deuterium incorporation into LBD amide hydrogens, implying tighter overall folding of the domain. As predicted, mass spectroscopic analysis of individual proteolytic peptides after hydrogen/deuterium exchange reveals that ligand increases the degree of solvent protection of regions close to the buried ligand-binding pocket. However, there is also extensive ligand protection of other regions, including the dimer surface at H10-H11, providing evidence for allosteric communication between the ligand-binding pocket and distant interaction surfaces. Surprisingly, C-terminal activation helix H12, which is known to alter position with ligand, remains relatively protected from solvent in all conditions suggesting that it is packed against the LBD irrespective of the presence or type of ligand. T(3), but not NH(3), increases accessibility of the upper part of H3-H5 to solvent, and we propose that TR H12 interacts with this region in apo-TR and that this interaction is blocked by T(3) but not NH(3.) We present data from site-directed mutagenesis experiments and molecular dynamics simulations that lend support to this structural model of apo-TR and its ligand-dependent conformational changes. (Molecular Endocrinology 25: 15-31, 2011)

A quantum chemical study on a set of non-imidazole H(3) antihistamine molecules

Molecular orbital calculations were carried out on a set of 28 non-imidazole H(3) antihistamine compounds using the Hartree-Fock method in order to investigate the possible relationships between electronic structural properties and binding affinity for H3 receptors (pK(i)). It was observed that the frontier effective-for-reaction molecular orbital (FERMO) energies were better correlated with pK(i) values than highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy values. Exploratory data analysis through hierarchical cluster (HCA) and principal component analysis (PCA) showed a separation of the compounds in two sets, one grouping the molecules with high pK(i) values, the other gathering low pK(i) value compounds. This separation was obtained with the use of the following descriptors: FERMO energies (epsilon(FERMO)), charges derived from the electrostatic potential on the nitrogen atom (N(1)), electronic density indexes for FERMO on the N(1) atom (Sigma((FERMO))c(i)(2)). and electrophilicity (omega`). These electronic descriptors were used to construct a quantitative structure-activity relationship (QSAR) model through the partial least-squares (PLS) method with three principal components. This model generated Q(2) = 0.88 and R(2) = 0.927 values obtained from a training set and external validation of 23 and 5 molecules, respectively. After the analysis of the PLS regression equation and the values for the selected electronic descriptors, it is suggested that high values of FERMO energies and of Sigma((FERMO))c(i)(2), together with low values of electrophilicity and pronounced negative charges on N(1) appear as desirable properties for the conception of new molecules which might have high binding affinity. 2010 Elsevier Inc. All rights reserved.

Locus of Controls inverkan på strategier för konflikthantering i kärleksrelationer

Syftet var att undersöka om personers Locus of Control påverkar deras konflikthanteringsstrategier i kärleksrelationer. Hypoteserna var: H1: Det finns skillnader mellan personer med extern och intern Locus of Control när det gäller vilka typer av strategier de använder för att hantera kärleksrelationsproblem. H2: Personer med intern Locus of Control har större benägenhet att använda sig av relationskonflikthanteringsstrategin Kompromiss i sina kärleksrelationer. H3: Kvinnor påverkas mer än män av Locus of Control när det gäller vilka typer av strategier de använder för att hantera kärleksrelationsproblem. Deltagarna var 172 studenter. Data insamlades med en webbenkät innehållande svenska versioner av Rotters I-E skala och RPCS. Resultatet visade en signifikant skillnad mellan personer med intern och extern Locus of Control i tre konflikthanteringsstrategier: Kompromiss, Underkastelse och Undvikande. Vidare använde personer med intern Locus of Control Kompromiss mer. Personer med extern Locus of Control använde Underkastelse och Undvikande mer. Skillnaderna fanns bara bland kvinnorna. Samtliga hypoteser kunde därmed godtas. Undersökningen visade att Locus of Control är en möjlig anledning till varför personer använder sig av vissa typer av konflikthanteringsstrategier i kärleksrelationer.

Den psykosociala arbetsmiljön utifrån modellen; krav, kontroll och socialt stöd : En jämförande studie mellan personal på offentligt och privat äldreboende

Studier har visat att äldre personer i behov av vård ökar vilket kan leda till att vårdpersonalen upplever högre krav. Detta kan i sin tur resultera i arbetsrelaterad stress och ohälsa hos personalen. Utifrån Robert Karasek och Töres Theorells modell om krav, kontroll och socialt stöd kan man undersöka hur den psykosociala arbetsmiljön upplevs på en arbetsplats. Enkäter delades ut i samarbete med enhetschefer på de båda äldreboendena. 40 undersökningsdeltagare ingick i studien. Syftet med föreliggande studie är att försöka urskilja om det finns skillnader mellan personalen på offentligt och privat äldreboende gällande den psykosociala arbetsmiljön utifrån modellen krav, kontroll och socialt stöd samt upplevelsen av arbetsrelaterad stress. H1, Det finns en relation mellan upplevelse av krav och upplevd arbetsrelaterad stress. H2: Det finns en relation mellan upplevelse av kontroll och upplevd arbetsrelaterad stress. H3: Det finns en relation mellan upplevelse av socialt stöd och upplevd arbetsrelaterad stress. H4: Det finns en signifikant skillnad i upplevelsen av krav mellan offentlig och privat personal. H5: Det finns en signifikant skillnad i upplevelsen av kontroll mellan offentlig och privat personal. H6: Det finns en signifikant skillnad i upplevelsen av socialt stöd mellan offentlig och privat personal. H7: Det finns en signifikant skillnad i upplevelsen av stress mellan offentlig och privat personal. H1-4 kan godtas. H5-7 kan förkastas. Resultaten diskuteras utifrån Robert Karasek och Töres Theorells modell.

A interatividade como medida de eficiência : um estudo sobre efeitos cognitivos resultantes da interação com web sites e suas implicações para o marketing da web

A pesquisa realizada avalia a relação entre o projeto de um Web Site1 comercial e o Flow, e a influência desse estado cognitivo sobre a comunicação de marketing do Site. É desenvolvida a partir de três perspectivas: perspectiva tecnológica do meio, mercadológica da organização e psicológica do usuário. O enfoque da pesquisa está voltado para a perspectiva humana, onde sugere que o Flow, um estado cognitivo decorrente de um processo de interação, traz reflexos positivos às comunicações de marketing de Web Sites comerciais. As três hipóteses levantadas para responder a questão de pesquisa são: H1: As características do design gráfico e arquitetura do Web Site influenciam no nível da experimentação do Flow. H2: O usuário que experimenta o Flow interage com mais profundidade com o ambiente mediado, visitando mais páginas e permanecendo mais tempo no Web Site e, por conseqüência, ficando mais exposto as comunicações emanadas pelo Site. H3: A experimentação do Flow aumenta a probabilidade do usuário avaliar positivamente o Site, aumenta a sua predisposição em retornar ao Site ou recomendá-lo a terceiros. A pesquisa empregou um método quase experimental, utilizando como instrumento um site experimental e para os testes e análises um grupo de teste e um grupo de controle. Os resultados obtidos concluem que, primeiro, as características do design e da 1 Os termos em língua inglesa apresentados ao longo deste trabalho encontram-se no glossário. VIII arquitetura do Web Site estimulam a experimentação do Flow. Segundo, o indivíduo que experimenta o fenômeno detém-se por mais tempo no Site. E terceiro, o usuário que experimenta o Flow tende a uma atitude positiva em relação ao Site.

Qualidade das projeções dos analistas Sell Side: evidência empírica do mercado brasileiro

A presente dissertação analisa o erro de projeção dos analistas de investimentos do sell side, definido como a diferença entre o consenso das projeções dos analistas e o resultado reportado pela empresa. O tamanho do erro de projeção é uma medida da qualidade das projeções dos analistas de um determinado mercado de capitais. Uma vasta literatura acadêmica mostra que uma melhora na qualidade das projeções dos analistas, medida através de uma diminuição do tamanho do erro de projeção, está relacionada com a redução da assimetria de informação e com um aumento do valor de mercado das empresas. São testadas duas regressões, nas quais características das empresas, como setor, tamanho, endividamento e variabilidade do lucro, e características do ambiente de informação da empresa, como listagem de ADR, número de analistas que acompanham a empresa e convergência das projeções, são testadas contra duas métricas do erro de projeção, acurácia e viés. Nossas hipóteses são que existem fatores que influenciam de maneira significativa o tamanho do erro de projeção (acurácia) e o viés das projeções (viés). Estas hipóteses foram confirmadas, isto é, nossas regressões apresentaram pelo menos um fator que se mostrou significativo estatisticamente para influenciar o tamanho do erro de projeção (hipóteses H1 e H2) ou o seu viés (hipótese H3). Entretanto, os resultados mostram que vários fatores que se mostram significativos em testes conduzidos em mercados desenvolvidos – tais como tamanho, endividamento e variabilidade do lucro – não se mostraram significativos no mercado brasileiro. Por outro lado, os fatores relacionados com o resultado do ano projetado ou do ano anterior se mostraram fortemente significativos. Acreditamos que os resultados podem ser explicados de três maneiras: 1) ou a capacidade de adicionar valor dos analistas em relação a modelos estatísticos de projeção é muito pequena, devido à sua falta de habilidade; ou 2) a instabilidade macroeconômica é tão grande domina todos os outros fatores que poderiam influenciar o tamanho do erro de projeção; ou 3) os resultados das empresas nos mercados desenvolvidos são tão administrados, isto é, tão estáveis, que permitem que fatores mais sutis como o tamanho, o nível de endividamento e a variabilidade do lucro se tornem significativos. Esta dissertação não permite distinguir qual das explicações é a correta. Uma de suas limitações é não incluir variáveis referentes à habilidade e experiência dos analistas e, também, variáveis relacionadas a fatores como governança corporativa e disclosure de informações. Em uma linha de pesquisa muito extensa nos países desenvolvidos, mas praticamente inexistente no Brasil, esperamos que estudos futuros supram estas lacunas e nos permitam entender melhor a questão da qualidade das projeções de resultados no contexto brasileiro.