A single residue substitution in the receptor-binding domain of H5N1 hemagglutinin is critical for packaging into pseudotyped lentiviral particles

© 2012 Tang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background: Serological studies for influenza infection and vaccine response often involve microneutralization and hemagglutination inhibition assays to evaluate neutralizing antibodies against human and avian influenza viruses, including H5N1. We have previously characterized lentiviral particles pseudotyped with H5-HA (H5pp) and validated an H5pp-based assay as a safe alternative for high-throughput serological studies in BSL-2 facilities. Here we show that H5-HAs from different clades do not always give rise to efficient production of H5pp and the underlying mechanisms are addressed. Methodology/Findings: We have carried out mutational analysis to delineate the molecular determinants responsible for efficient packaging of HA from A/Cambodia/40808/2005 (H5Cam) and A/Anhui/1/2005 (H5Anh) into H5pp. Our results demonstrate that a single A134V mutation in the 130-loop of the receptor binding domain is sufficient to render H5Anh the ability to generate H5Anh-pp efficiently, whereas the reverse V134A mutation greatly hampers production of H5Cam-pp. Although protein expression in total cell lysates is similar for H5Anh and H5Cam, cell surface expression of H5Cam is detected at a significantly higher level than that of H5Anh. We further demonstrate by several independent lines of evidence that the behaviour of H5Anh can be explained by a stronger binding to sialic acid receptors implicating residue 134. Conclusions: We have identified a single A134V mutation as the molecular determinant in H5-HA for efficient incorporation into H5pp envelope and delineated the underlying mechanism. The reduced binding to sialic acid receptors as a result of the A134V mutation not only exerts a critical influence in pseudotyping efficiency of H5-HA, but has also an impact at the whole virus level. Because A134V substitution has been reported as a naturally occurring mutation in human host, our results may have implications for the understanding of human host adaptation of avian influenza H5N1 virusesThis work was supported by grants from the Research Fund for the Control of Infectious Diseases of Hong Kong (RFCID#08070972), the Area of Excellence Scheme of the University Grants Committee (grant AoE/M-12/-06 of the Hong Kong Special Administrative Region, China), the French Ministry of Health, and the RESPARI project of the Institut Pasteur International Network

The disease management program for type 2 diabetes in Germany enhances process quality of diabetes care - a follow-up survey of patient's experiences

<p>Abstract</p> <p>Background</p> <p>In summer 2003 a disease management program (DMP) for type 2 diabetes was introduced on a nationwide basis in Germany. Patient participation and continuity of care within the DMP are important factors to achieve long-term improvements in clinical endpoints. Therefore it is of interest, if patients experience any positive or negative effects of the DMP on their treatment that would support or hamper further participation. The main objective of the study was to find out if the German Disease Management Program (DMP) for type 2 diabetes improves process and outcome quality of medical care for patients in the light of their subjective experiences over a period of one year.</p> <p>Methods</p> <p>Cohort study with a baseline interview and a follow-up after 10.4 ± 0.64 months. Data on process and outcome measures were collected by telephone interviews with 444 patients enrolled and 494 patients not enrolled in the German DMP for type 2 diabetes. Data were analyzed by multivariate logistic regression analyses.</p> <p>Results</p> <p>DMP enrolment was significantly associated with a higher process quality of care. At baseline enrolled patients more often reported that they had attended a diabetes education course (OR = 3.4), have ≥ 4 contacts/year with the attending physician (OR = 3.3), have at least one annual foot examination (OR = 3.1) and one referral to an ophthalmologist (OR = 3.4) and possess a diabetes passport (OR = 2.4). Except for the annual referral to an ophthalmologist these parameters were also statistically significant at follow-up. In contrast, no differences between enrolled and not enrolled patients were found concerning outcome quality indicators, e.g. self-rated health, Glycated hemoglobin (GHb) and blood pressure. However, 16-36% of the DMP participants reported improvements of body weight and/or GHb and/or blood pressure values due to enrolment - unchanged within one year of follow-up.</p> <p>Conclusions</p> <p>In the light of patient's experiences the DMP enhances the process quality of medical care for type 2 diabetes in Germany. The lack of significant differences in outcome quality between enrolled and not enrolled patients might be due to the short program duration. Our data suggest that the DMP for type 2 diabetes should not be withdrawn unless an evidently more promising approach is found.</p

Exploiting the pathway structure of metabolism to reveal high-order epistasis

<p>Abstract</p> <p>Background</p> <p>Biological robustness results from redundant pathways that achieve an essential objective, e.g. the production of biomass. As a consequence, the biological roles of many genes can only be revealed through multiple knockouts that identify a <it>set </it>of genes as essential for a given function. The identification of such "epistatic" essential relationships between network components is critical for the understanding and eventual manipulation of robust systems-level phenotypes.</p> <p>Results</p> <p>We introduce and apply a network-based approach for genome-scale metabolic knockout design. We apply this method to uncover over 11,000 minimal knockouts for biomass production in an <it>in silico </it>genome-scale model of <it>E. coli</it>. A large majority of these "essential sets" contain 5 or more reactions, and thus represent complex epistatic relationships between components of the <it>E. coli </it>metabolic network.</p> <p>Conclusion</p> <p>The complex minimal biomass knockouts discovered with our approach illuminate robust essential systems-level roles for reactions in the <it>E. coli </it>metabolic network. Unlike previous approaches, our method yields results regarding high-order epistatic relationships and is applicable at the genome-scale.</p

Optimized two-dimensional thin layer chromatography to monitor the intracellular concentration of acetyl phosphate and other small phosphorylated molecules

Acetyl phosphate (acetyl-P) serves critical roles in coenzyme A recycling and ATP synthesis. It is the intermediate of the Pta-AckA pathway that inter-converts acetyl-coenzyme A and acetate. Acetyl-P also can act as a global signal by donating its phosphoryl group to specific two-component response regulators. This ability derives from its capacity to store energy in the form of a high-energy phosphate bond. This bond, while critical to its function, also destabilizes acetyl-P in cell extracts. This lability has greatly complicated biochemical analysis, leading in part to widely varying acetyl-P measurements. We therefore developed an optimized protocol based on two-dimensional thin layer chromatography that includes metabolic labeling under aerated conditions and careful examination of the integrity of acetyl-P within extracts. This protocol results in greatly improved reproducibility, and thus permits precise measurements of the intracellular concentration of acetyl-P, as well as that of other small phosphorylated molecules

Comparative analysis of involvement of UGT1 and UGT2 splice variants of UDP-galactose transporter in glycosylation of macromolecules in MDCK and CHO cell lines

Nucleotide sugar transporters deliver nucleotide sugars into the Golgi apparatus and endoplasmic reticulum. This study aimed to further characterize mammalian UDP-galactose transporter (UGT) in MDCK and CHO cell lines. MDCK-RCAr and CHO-Lec8 mutant cell lines are defective in UGT transporter, although they exhibit some level of galactosylation. Previously, only single forms of UGT were identified in both cell lines, UGT1 in MDCK cells and UGT2 in CHO cells. We have identified the second UGT splice variants in CHO (UGT1) and MDCK (UGT2) cells. Compared to UGT1, UGT2 is more abundant in nearly all examined mammalian tissues and cell lines, but MDCK cells exhibit different relative distribution of both splice variants. Complementation analysis demonstrated that both UGT splice variants are necessary for N- and O-glycosylation of proteins. Both mutant cell lines produce chondroitin-4-sulfate at only a slightly lower level compared to wild-type cells. This defect is corrected by overexpression of both UGT splice variants. MDCK-RCAr mutant cells do not produce keratan sulfate and this effect is not corrected by either UGT splice variant, overexpressed either singly or in combination. Here we demonstrate that both UGT splice variants are important for glycosylation of proteins. In contrast to MDCK cells, MDCK-RCAr mutant cells may possess an additional defect within the keratan sulfate biosynthesis pathway

The PARAChute project: remote monitoring of posture and gait for fall prevention

Falls in the elderly are a major public health problem due to both their frequency and their medical and social consequences. In France alone, more than two million people aged over 65 years old fall each year, leading to more than 9 000 deaths, in particular in those over 75 years old (more than 8 000 deaths). This paper describes the PARAChute project, which aims to develop a methodology that will enable the detection of an increased risk of falling in community-dwelling elderly. The methods used for a remote noninvasive assessment for static and dynamic balance assessments and gait analysis are described. The final result of the project has been the development of an algorithm for movement detection during gait and a balance signature extracted from a force plate. A multicentre longitudinal evaluation of balance has commenced in order to validate the methodologies and technologies developed in the project

Public appraisal of government efforts and participation intent in medico-ethical policymaking in Japan: a large scale national survey concerning brain death and organ transplant

BACKGROUND: Public satisfaction with policy process influences the legitimacy and acceptance of policies, and conditions the future political process, especially when contending ethical value judgments are involved. On the other hand, public involvement is required if effective policy is to be developed and accepted. METHODS: Using the data from a large-scale national opinion survey, this study evaluates public appraisal of past government efforts to legalize organ transplant from brain-dead bodies in Japan, and examines the public's intent to participate in future policy. RESULTS: A relatively large percentage of people became aware of the issue when government actions were initiated, and many increasingly formed their own opinions on the policy in question. However, a significant number (43.3%) remained unaware of any legislative efforts, and only 26.3% of those who were aware provided positive appraisals of the policymaking process. Furthermore, a majority of respondents (61.8%) indicated unwillingness to participate in future policy discussions of bioethical issues. Multivariate analysis revealed the following factors are associated with positive appraisals of policy development: greater age; earlier opinion formation; and familiarity with donor cards. Factors associated with likelihood of future participation in policy discussion include younger age, earlier attention to the issue, and knowledge of past government efforts. Those unwilling to participate cited as their reasons that experts are more knowledgeable and that the issues are too complex. CONCLUSIONS: Results of an opinion survey in Japan were presented, and a set of factors statistically associated with them were discussed. Further efforts to improve policy making process on bioethical issues are desirable

A Functional Genomics Approach to Establish the Complement of Carbohydrate Transporters in Streptococcus pneumoniae

The aerotolerant anaerobe Streptococcus pneumoniae is part of the normal nasopharyngeal microbiota of humans and one of the most important invasive pathogens. A genomic survey allowed establishing the occurrence of twenty-one phosphotransferase systems, seven carbohydrate uptake ABC transporters, one sodium∶solute symporter and a permease, underlining an exceptionally high capacity for uptake of carbohydrate substrates. Despite high genomic variability, combined phenotypic and genomic analysis of twenty sequenced strains did assign the substrate specificity only to two uptake systems. Systematic analysis of mutants for most carbohydrate transporters enabled us to assign a phenotype and substrate specificity to twenty-three transport systems. For five putative transporters for galactose, pentoses, ribonucleosides and sulphated glycans activity was inferred, but not experimentally confirmed and only one transport system remains with an unknown substrate and lack of any functional annotation. Using a metabolic approach, 80% of the thirty-two fermentable carbon substrates were assigned to the corresponding transporter. The complexity and robustness of sugar uptake is underlined by the finding that many transporters have multiple substrates, and many sugars are transported by more than one system. The present work permits to draw a functional map of the complete arsenal of carbohydrate utilisation proteins of pneumococci, allows re-annotation of genomic data and might serve as a reference for related species. These data provide tools for specific investigation of the roles of the different carbon substrates on pneumococcal physiology in the host during carriage and invasive infection

Improving the iMM904 S. cerevisiae metabolic model using essentiality and synthetic lethality data

<p>Abstract</p> <p>Background</p> <p><it>Saccharomyces cerevisiae </it>is the first eukaryotic organism for which a multi-compartment genome-scale metabolic model was constructed. Since then a sequence of improved metabolic reconstructions for yeast has been introduced. These metabolic models have been extensively used to elucidate the organizational principles of yeast metabolism and drive yeast strain engineering strategies for targeted overproductions. They have also served as a starting point and a benchmark for the reconstruction of genome-scale metabolic models for other eukaryotic organisms. In spite of the successive improvements in the details of the described metabolic processes, even the recent yeast model (i.e., <it>i</it>MM904) remains significantly less predictive than the latest <it>E. coli </it>model (i.e., <it>i</it>AF1260). This is manifested by its significantly lower specificity in predicting the outcome of grow/no grow experiments in comparison to the <it>E. coli </it>model.</p> <p>Results</p> <p>In this paper we make use of the automated GrowMatch procedure for restoring consistency with single gene deletion experiments in yeast and extend the procedure to make use of synthetic lethality data using the genome-scale model <it>i</it>MM904 as a basis. We identified and vetted using literature sources 120 distinct model modifications including various regulatory constraints for minimal and YP media. The incorporation of the suggested modifications led to a substantial increase in the fraction of correctly predicted lethal knockouts (i.e., specificity) from 38.84% (87 out of 224) to 53.57% (120 out of 224) for the minimal medium and from 24.73% (45 out of 182) to 40.11% (73 out of 182) for the YP medium. Synthetic lethality predictions improved from 12.03% (16 out of 133) to 23.31% (31 out of 133) for the minimal medium and from 6.96% (8 out of 115) to 13.04% (15 out of 115) for the YP medium.</p> <p>Conclusions</p> <p>Overall, this study provides a roadmap for the computationally driven correction of multi-compartment genome-scale metabolic models and demonstrates the value of synthetic lethals as curation agents.</p