Glycosylation of Influenza A Virus Hemagglutinin

The genome of a wide range of viruses, including the influenza A virus, are enclosed in a lipid envelope. These envelopes are generally acquired in the final step of virus assembly. During this step viruses bud from regions of the host cell membranes where virally encoded membrane proteins have accumulated. The influenza A envelope is spiked with two glycoproteins: hemagglutinin (HA) and neuraminidase (NA). HA is the most abundant protein on the virus surface. It plays an important role during virus attachment and membrane fusion. The mature HA forms homotrimers of a monomer which is initially present as a single polypeptide precursor (HA0). HA0 is subsequently cleaved into the subunits: HA1 and HA2. Both subunits are glycosylated and each monomer contains 3 to 9 N-linked glycosylation sequins, depending on the virus strain. The structure of the individual glycans depends mainly on virus subtype and on the host cell. The biological function of HA-glycans is still not fully clear. However, it has been demonstrated that some of the glycans shield HA from proteases and neutralize antibodies. In addition, they play an important role in the intracellular transport of HA and the viral replication regulation. Structural modifications of the HA-glycans can influence the virus attachment to the host cell and change viral replication dynamics. HA-glycosylation is effected by the glycosylation machinery of the host cell system and can be influenced during vaccine production by choise of the host cell, fermentation-, inactivation- and downstream processing conditions. Therefore, it is important to monitor and control the glycosylation pattern over the period of the production process. In the present study we determine the HA N-glycosylation sites and glycan structure of the influenza A/PR/8/34 (H1N1) virus produced in Madin Darby canine kidney cells (MDCK) cells. Furthermore, the effect of different fermentation conditions on the HA-glycosylation is described. In this model the glycosylation sites of HA1 and HA2 are predicted ( NetNGlyc 1.0 Server ) to be 11, 23, 125 and 283, and 154 and 312, respectively. These sites are confirmed via mass spectrometry. The glycan structure of the individual sites are characterized via the following procedure. The virus proteins are first separated by SDS-PAGE. N-glycans are removed in gel with PNGase F, an enzyme that cleaves Asn-linked oligosaccharides of oligomannosyl, hybrid and complex type. For further analysis by capillary gel electrophoresis (CGE) the N-glycans are labelled with 8-Aminopyrene-1,3,6-trisulfonic acid trisodium salt (APTS) by reductive amination with NaBH3CN. CGE is performed on a ABI PRISM 3100-Avant genetic analyser (Applied Biosystems) by laser induced fluorescence. With this method, we are able to fingerprint HA N-glycan mixtures from cell culture produced virus with a detection limit of approximately 20 fmol. Additionally HA N-glycans are analyzed by mass spectrometry, carried out on a Quadrupol-quadrupol Time-of-flight hybrid mass spectrometer (QStarXL/Applied Biosystems). For further structural analysis of the HA N-glycans is obtained by sequential sequencing implementing an reagent array analysis method (RAAM). This involves the progressive digestion of the oligosaccharides with exoglycosidases monitored by CGE and mass spectrometry. Together, the methods demonstrated here, represent a promising tool to monitor HA-glycosylation during the most important steps in upstream and downstream processing of influenza virus for vaccine production

The relationship between global distress, mentalizing and well-being in a German teacher sample

Many studies have linked global distress including higher psychological symptom severity and high levels of stress with low levels of well-being among teachers, indicating a need to identify and empirically evaluate protective factors. Mentalizing—the capacity to understand behavior in terms of intentional mental states—may be a candidate protective factor to mediate this association, enhancing well-being in the face of high levels of global distress. The present study examines whether the capacity to mentalize can buffer subjectively experienced stress and psychological symptom severity in a sample of teachers. 215 teachers completed questionnaires measuring self-rated experiences of stress, psychological symptoms, mentalizing capacities and well-being in a cross-sectional design. Structural equation modeling was used to test mediation effects. Our findings show that mentalizing was positively associated with well-being. In addition, mentalizing counteracted the negative influence of stress and psychological symptom severity. However, a structural equation model assessing the mediating effect of global distress on well-being via mentalizing was not significant. Therefore, the data indicate that teachers’ capacity to mentalize, regardless of psychological symptom load and subjective experience of stress, has a positive impact on their well-being. The study highlights the protective function of mentalizing and forms a framework for psychological interventions to increase teachers’ well-being

Development of a lectin-affinity chromatography step for the downstream processing of influenza virus vaccines

Influenza remains due to its annual death rate and potential to cause pandemics a major public health concern. Efforts to control the annual spread of influenza have centered on prophylactic vaccinations. Human influenza vaccines are traditionally produced in embryonated hen s eggs. However, major constraints with this method, e.g. allergic reactions induced by egg proteins and lack of scalability have lead to the development of cell culture based production processes. In recent years, several continuous cell lines such as the Madin Darby canine kidney (MDCK) or the African green monkey kidney Vero cells have been successfully established for the production of influenza vaccines in cell culture. These processes require the modification of existing but also the development of new downstream strategies to account for the changed upstream technology. Downstream processing of biological products is conventionally subdivided into three steps: capture or concentration, separation or fractionation and polishing. The capture step is commonly the most expensive unit operation. Hence, the efficiency of this step has a large impact on the total process economics. The presented study focuses on the development of a proficient capture step based on lectin-affinity chromatography. Lectins are a class of carbohydrate specific proteins of non-immune origin that have a selective affinity for a carbohydrate or a group of carbohydrates. Immobilized lectins have been used successfully for many years to separate and isolate glycoconjugates, polysaccharides, soluble cell components, and cells containing glycoproteins with specific carbohydrate structures on its surface. The influenza A virus contains two spike glycoproteins on its surface: hemagglutinin (HA) and neuraminidase (NA). HA is the most abundant surface protein. It is a trimeric glycoprotein containing per subunit 3 to 9 N-linked glycosylation sites depending on the viral strain. Here the influenza A/PR/8/34 virus has been selected as a model. The HA molecule of this particular virus contains according to the NetNGlyc 1.0 Server prediction six glycosylation sites. Detailed analysis of these sites and their individual glycan structures are presently performed. Based on preliminary structural glycan analysis studies and literature data several HA-binding lectins are selected for a pre-screening via lectin-blots. The most promising lectinblot results are obtained from lectins specific for terminal galactose e.g. Erythrina cristagalli (ECL), Arachis hypogaea (PNA). Lectins, by which lectin-blot analysis suggests an interaction with viral membrane proteins, are currently screened for their suitability as an affinity matrix ligand. Therefore, centrifuged cultivation broths of influenza A/PR/8/34 virus infected MDCK cells are applied to various agaroseimmobilized lectins. Components interfering with the immobilized lectins are selectively adsorbed. Non or weak binding components are washed from the column. Subsequently, bound components are dissociated from the lectin by competitive elution with suitable hapten carbohydrates. This fraction contains the influenza virus particles and virally encoded membrane proteins, which have to be further processed for vaccine manufacturing. The extend of the subsequent purification depends on the specificity of the lectin binding to virally encoded surface proteins. Lectins with weak or no interaction with host cell proteins or medium components and strong interaction with viral membrane glycoproteins represent a powerful tool to concentrate and purify viral surface proteins from contaminating nucleic acids, medium components, and non-virally encoded host cell proteins

Longitudinal associations of importance of religion and frequency of service attendance with depression risk among adolescents in Nova Scotia

Objective: To examine the directionality of associations between self-reported religious importance or worship attendance and depression among adolescents, and to determine whether social supports or general self-efficacy are mechanisms of observed associations. Method: A cohort (n = 976) of Canadian high school students were surveyed in Grade 10 (2000 to 2001) and 2 years later (2002 to 2003). Logistic regression was conducted separately among adolescents with and without elevated depressive symptoms to examine associations between baseline religious attendance and religious importance with later depression, adjusting for confounding factors. Effects of reverse causation were also assessed, determining associations between baseline depression and follow-up religious attendance and importance. Results: Girls who were not depressed at baseline and who attended religious services had lower odds of later depression (adjusted odds ratio [AOR] 0.46; 95% CI 0.22 to 0.95, P < 0.05), which was accounted for by general self-efficacy. Boys who were depressed at baseline who attended religious services had lower odds of still being depressed at followup (AOR 0.23; 95% CI 0.06 to 0.80, P < 0.01). Depression at baseline predicted lower attendance at follow-up among boys (AOR 0.26; 95% CI 0.09 to 0.75, P < 0.01). Conclusions: Religious attendance independently predicts lower depression at followup among girls, and may do so by increasing self-efficacy. Among boys with depression, religious attendance predicts a lower likelihood of still being depressed at follow-up. The relation between religious attendance and depression in boys is bidirectional

Sterile inflammation as a factor in human male infertility: Involvement of Toll like receptor 2, biglycan and peritubular cells

Changes in the wall of seminiferous tubules in men with impaired spermatogenesis imply sterile inflammation of the testis. We tested the hypothesis that the cells forming the wall of seminiferous tubules, human testicular peritubular cells (HTPCs), orchestrate inflammatory events and that Toll like receptors (TLRs) and danger signals from the extracellular matrix (ECM) of this wall are involved. In cultured HTPCs we detected TLRs, including TLR2. A TLR-2 ligand (PAM) augmented interleukin 6 (IL-6), monocyte chemo-attractant protein-1 (MCP-1) and pentraxin 3 (PTX3) in HTPCs. The ECM-derived proteoglycan biglycan (BGN) is secreted by HTPCs and may be a TLR2-ligand at HTPCs. In support, recombinant human BGN increased PTX3, MCP-1 and IL-6 in HTPCs. Variable endogenous BGN levels in HTPCs derived from different men and differences in BGN levels in the tubular wall in infertile men were observed. In testes of a systemic mouse model for male infertility, testicular sterile inflammation and elevated estradiol (E2) levels, BGN was also elevated. Hence we studied the role of E2 in HTPCs and observed that E2 elevated the levels of BGN. The anti-estrogen ICI 182,780 blocked this action. We conclude that TLR2 and BGN contribute to sterile inflammation and infertility in man

Sterile inflammation as a factor in human male infertility: Involvement of Toll like receptor 2, biglycan and peritubular cells

Changes in the wall of seminiferous tubules in men with impaired spermatogenesis imply sterile inflammation of the testis. We tested the hypothesis that the cells forming the wall of seminiferous tubules, human testicular peritubular cells (HTPCs), orchestrate inflammatory events and that Toll like receptors (TLRs) and danger signals from the extracellular matrix (ECM) of this wall are involved. In cultured HTPCs we detected TLRs, including TLR2. A TLR-2 ligand (PAM) augmented interleukin 6 (IL-6), monocyte chemo-attractant protein-1 (MCP-1) and pentraxin 3 (PTX3) in HTPCs. The ECM-derived proteoglycan biglycan (BGN) is secreted by HTPCs and may be a TLR2-ligand at HTPCs. In support, recombinant human BGN increased PTX3, MCP-1 and IL-6 in HTPCs. Variable endogenous BGN levels in HTPCs derived from different men and differences in BGN levels in the tubular wall in infertile men were observed. In testes of a systemic mouse model for male infertility, testicular sterile inflammation and elevated estradiol (E2) levels, BGN was also elevated. Hence we studied the role of E2 in HTPCs and observed that E2 elevated the levels of BGN. The anti-estrogen ICI 182,780 blocked this action. We conclude that TLR2 and BGN contribute to sterile inflammation and infertility in man

Macroscopic Equations of Motion for Two Phase Flow in Porous Media

The established macroscopic equations of motion for two phase immiscible displacement in porous media are known to be physically incomplete because they do not contain the surface tension and surface areas governing capillary phenomena. Therefore a more general system of macroscopic equations is derived here which incorporates the spatiotemporal variation of interfacial energies. These equations are based on the theory of mixtures in macroscopic continuum mechanics. They include wetting phenomena through surface tensions instead of the traditional use of capillary pressure functions. Relative permeabilities can be identified in this approach which exhibit a complex dependence on the state variables. A capillary pressure function can be identified in equilibrium which shows the qualitative saturation dependence known from experiment. In addition the new equations allow to describe the spatiotemporal changes of residual saturations during immiscible displacement.Comment: 15 pages, Phys. Rev. E (1998), in prin

Associations between general self-efficacy and health-related quality of life among 12-13-year-old school children: a cross-sectional survey

<p>Abstract</p> <p>Background</p> <p>While research on school children's health has mainly focused on risk factors and illness, few studies have examined aspects of health promotion. Thus, this study focuses on health promotional factors including general self-efficacy (GSE) and health-related quality of life (HRQOL). GSE refers to a global confidence in coping ability across a wide range of demanding situations, and is related to health. The purpose of this study was to examine associations between GSE and HRQOL, and associations between HRQOL and socio-demographic characteristics. Knowledge of these associations in healthy school children is currently lacking.</p> <p>Methods</p> <p>During 2006 and 2007, 279 school children in the seventh grade across eastern Norway completed a survey assessing their GSE and HRQOL. The children were from schools that had been randomly selected using cluster sampling. T-tests were computed to compare mean subscale values between HRQOL and socio-demographic variables. Single and multiple regression analyses were performed to explore associations among GSE, HRQOL and socio-demographic variables.</p> <p>Results</p> <p>Regression analyses showed a significant relationship between increasing degrees of GSE and increasing degrees of HRQOL. In analyses adjusted for socio-demographic variables, boys scored higher than girls on self-esteem. School children from single-parent families had lower scores on HRQOL than those from two-parent families, and children who had relocated within the last five years had lower scores on HRQOL than those who had not relocated.</p> <p>Conclusion</p> <p>The strong relationship between GSE and HRQOL indicates that GSE might be a resource for increasing the HRQOL for school children.</p

Male factors determining the outcome of intracytoplasmic sperm injection with epididymal and testicular spermatozoa

Summary. During a period of 8 years, 1079 intracytoplasmic sperm injection (ICSI) procedures with aspirated epididymal or testicular spermatozoa were performed. Epididymal spermatozoa were used in 172 cycles and testicular spermatozoa or spermatids in 907 cycles. Multiple biopsies were obtained from at least two different locations in the testes. Retrieved spermatozoa were used after cryopreservation (frozen) or immediately after aspiration (fresh). Three hundred patients had obstructive azoospermia (OA) or ejaculation failure. In 414 cases, azoospermia was caused by impaired spermatogenesis resulting from maldescended testes, chemotherapy/radiotherapy, or by Sertoli-cellonly syndrome, genetic disorders or unknown aetiology. Transfer rates, pregnancy rates and birth rates per ICSI cycle showed no statistically significant differences between testicular and epididymal spermatozoa in men with OA (28% average birth rates in both cases). However, birth rates differed significantly with regard to the status of spermatogenesis. Treatment of men with nonobstructive azoospermia (NOA) resulted in a birth rate of 19% per cycle. In all patient groups, there was no difference in the birth rates achieved with fresh and cryopreserved spermatozoa. While testicular volume, follicle-stimulating hormone level and age of the male patient are no statistically significant prognostic factors, the underlying cause of azoospermia is the most important factor determining the outcome of ICSI with epididymal and testicular spermatozoa. The pregnancy rate is lower in NOA patients than in those with OA