Structural insights into the neutralization mechanism of a higher primate antibody against dengue virus

The four serotypes of dengue virus (DENV-1 to -4) cause the most important emerging viral disease. Protein E, the principal viral envelope glycoprotein, mediates fusion of the viral and endosomal membranes during virus entry and is the target of neutralizing antibodies. However, the epitopes of strongly neutralizing human antibodies have not been described despite their importance to vaccine development. The chimpanzee Mab 5H2 potently neutralizes DENV-4 by binding to domain I of E. The crystal structure of Fab 5H2 bound to E from DENV-4 shows that antibody binding prevents formation of the fusogenic hairpin conformation of E, which together with in-vitro assays, demonstrates that 5H2 neutralizes by blocking membrane fusion in the endosome. Furthermore, we show that human sera from patients recovering from DENV-4 infection contain antibodies that bind to the 5H2 epitope region on domain I. This study, thus, provides new information and tools for effective vaccine design to prevent dengue disease.     

Use of national and international growth charts for studying height in European children: development of up-to-date European height-for-age charts

Background

Growth charts based on data collected in different populations and time periods are key tools to assess children’s linear growth. We analyzed the impact of geographic factors and the secular trend on height-for-age charts currently used in European populations, developed up-to-date European growth charts, and studied the effect of using different charts in a sample of growth retarded children.

Methods and Findings

In an international survey we obtained 18 unique national height-for-age charts from 28 European countries and compared them with charts from the World Health Organization (WHO), Euro-Growth reference, and Centers of Disease Control and Prevention (CDC). As an example, we obtained height data from 3,534 children with end-stage renal disease (ESRD) from 13 countries via the ESPN/ERA-EDTA registry, a patient group generally suffering from growth retardation. National growth charts showed a clear secular trend in height (mean height increased on average 0.6 cm/decade) and a North-South height gradient in Europe. For countries without a recent (>1990) national growth chart novel European growth charts were constructed from Northern and Southern European reference populations, reflecting geographic height differences in mean final height of 3.9 cm in boys and 3.8 cm in girls. Mean height SDS of 2- to 17-year-old ESRD patients calculated from recent national or derived European growth charts (−1.91, 95% CI: −1.97 to −1.85) was significantly lower than when using CDC or WHO growth charts (−1.55, 95% CI: −1.61 to −1.49) (P<0.0001).

Conclusion

Differences between height-for-age charts may reflect true population differences, but are also strongly affected by the secular trend in height. The choice of reference charts substantially affects the clinical decision whether a child is considered short-for-age. Therefore, we advocate using recent national or European height-for-age charts derived from recent national data when monitoring growth of healthy and diseased European children.     

Altered glycosylated PrP proteins can have different neuronal trafficking in brain but do not acquire scrapie-like properties

N-Linked glycans have been shown to have an important role in the cell biology of a variety of cell surface glycoproteins, including PrP protein. It has been suggested that glycosylation of PrP can influence the susceptibility to transmissible spongiform encephalopathy and determine the characteristics of the many different strains observed in this particular type of disease. To understand the role of carbohydrates in influencing the PrP maturation, stability, and cell biology, we have produced and analyzed gene-targeted murine models expressing differentially glycosylated PrP. Transgenic mice carrying the PrP substitution threonine for asparagine 180 (G1) or threonine for asparagine 196 (G2) or both mutations combined (G3), which eliminate the first, second, and both glycosylation sites, respectively, have been generated by double replacement gene targeting. An in vivo analysis of altered PrP has been carried out in transgenic mouse brains, and our data show that the lack of glycans does not influence PrP maturation and stability. The presence of one chain of sugar is sufficient for the trafficking to the cell membrane, whereas the unglycosylated PrP localization is mainly intracellular. However, this altered cellular localization of PrP does not lead to any overt phenotype in the G3 transgenic mice. Most importantly, we found that, in vivo, unglycosylated PrP does not acquire the characteristics of the aberrant pathogenic form (PrPSc), as was previously reported using in vitro models.     

CYP21 mutations in Brazilian patients with 21-hydroxylase deficiency

Congenital adrenal hyperplasia caused by 21-hydroxylase deficiency is a common autosomal recessive disorder resulting from mutations in the 21-hydroxylase (CYP21) gene. To develop a strategy to screen for the most commonly occurring CYP21 mutations in Brazil, we performed molecular genotype analysis on 73 children with CAH representing 71 unrelated families. The techniques used for CYP21 molecular genotype analysis were: restriction fragment length polymorphism, single-strand conformational polymorphism, allele-specific oligonucleotide hybridization, allele-specific polymerase chain reaction amplification, and heteroduplex analyses. Mutations were identified on all but eight affected alleles. The intron 2 splicing mutation was the most frequently identified mutation. Screening for the most common mutations detected at least one mutation on 132/142 (93%) alleles. Multiple CYP21 mutations were detected on 16.2% of alleles. The high frequency of multiple mutations on a single allele emphasizes the importance of thorough and accurate molecular genotype analysis of the complex CYP21 locus.     

Lipolytic enzymes with improved activity and selectivity upon adsorption on polymeric nanoparticles

Nanostructured polystyrene (PS) and polymethylmethacrylate (PMMA) were used as carriers for the preparation of bioconjugates with lipolytic enzymes, such as Candida rugosa lipase (CRL) and Pseudomonas cepacia lipase (PCL). Simple addition of the lipase solution to the polymeric nanoparticles under protein-friendly conditions (pH 7.6) led to the formation of polymer-enzyme bioconjugates. Energy filtered-transmission electron microscopy (EF-TEM) performed on immuno-gold labeled samples revealed that the enzyme preferentially binds to the polymer nanoparticles and that the binding does not affect the nanostructured features of the carriers. The studies performed on the activity of the bioconjugates pointed out that the lipases adsorbed onto polymeric nanoparticles show an improved performance in terms of activity and selectivity with respect to those shown by lipases adsorbed on the same non-nanostructured carriers. The residual activities of CRL and PCL immobilized on nanostructured PMMA and PS reached 60% and 74%, respectively. Moreover, we found that enantioselectivity and pH and thermal stability increase upon immobilization. These results highlight the fact that new protein conformers with improved enantioselectivity stabilized after adsorption on nanoparticles are obtained. On the basis of the chemical structures of the selected polymers and the slopes of the adsorption isotherms, a hydrophobic binding model for lipase/nanostructured polymers is suggested.     

Selenite-reducing capacity of the copper-containing nitrite reductase of Rhizobium sullae

Rhizobium sullae strain HCNT1 contains a nitric oxide-producing nitrite reductase of unknown function due to the absence of a complementary nitric oxide reductase. HCNT1 had the ability to grow on selenite concentrations as high as 50 mM, and during growth, selenite was reduced to the less toxic elemental selenium. An HCNT1 mutant lacking nitrite reductase grew poorly in the presence of 5 mM selenite, was unable to grow in the presence of 25 or 50 mM selenite and also showed no evidence of selenite reduction. A naturally occurring nitrite reductase-deficient R. sullae strain, CC1335, also showed little growth on the higher concentrations of selenite. Mobilization of a plasmid containing the HCNT1 gene encoding nitrite reductase into CC1335 increased its resistance to selenite. To confirm that this ability to grow in the presence of high concentrations of selenite correlated with nitrite reductase activity, a new nitrite reductase-containing strain was isolated from the same location where HCNT1 was isolated. This strain was also resistant to high concentrations of selenite. Inactivation of the gene encoding nitrite reductase in this strain increased selenite sensitivity. These data suggest that the nitrite reductase of R. sullae provides resistance to selenite and offers an explanation for the radically truncated denitrification found uniquely in this bacterium.     

IEEE 802.11b Ad Hoc Networks: Performance Measurements

In this paper we investigate the performance of IEEE 802.11b ad hoc networks by means of an experimental study. An extensive literature, based on simulation studies, there exists on the performance of IEEE 802.11 ad hoc networks. Our analysis reveals several aspects that are usually neglected in previous simulation studies. Firstly, since different transmission rates are used for control and data frames, different transmission ranges and carrier-sensing ranges may exist at the same time in the network. In addition, the transmission ranges are in practice much shorter than usually assumed in simulation analysis, not constant but highly variable (even in the same session) and depends on several factors. Finally, the results presented in this paper indicate that for correctly understanding the behavior of an 802.11b network operating in ad hoc mode, several different ranges must be considered. In addition to the transmission range, the physical carrier sensing range is very important. The transmission range is highly dependent on the data rate and is up to 100 m, while the physical carrier sensing range is almost independent from the data rate and is approximately 200 m. Furthermore, even though stations are outside from their respective physical carrier sensing range, they may still interfere if their distance is lower than 350 m.Giuseppe Anastasi received the Laurea degree in Electronics Engineering and the Ph.D. degree in Computer Engineering both from the University of Pisa, Italy, in 1990 and 1995, respectively. He is currently an associate professor of Computer Engineering at the Department of Information Engineering of the University of Pisa. His research interests include architectures and protocols for mobile computing, energy management, QoS in mobile networks, and ad hoc networks. He was a co-editor of the book Advanced Lectures in Networking (LNCS 2497, Springer, 2002), and published more than 50 papers, both in international journals and conference proceedings, in the area of computer networking. He served in the TPC of several international conferences including IFIP Networking 2002 and IEEE PerCom 2003. He is a member of the IEEE Computer Society.Eleonora Borgia received the Laurea degree in Computer Engineering from the University of Pisa, Italy, in 2002. She is currently working toward her Ph.D. degree at the IIT Institute of the Italian National Research Council (CNR). Her research interests are in the area of the wireless and mobile networks with particular attention to MAC protocols and routing algorithms for ad hoc networks.Marco Conti received the Laurea degree in Computer Science from the University of Pisa, Italy, in 1987. In 1987 he joined the Italian National Research Council (CNR). He is currently a senior researcher at CNR-IIT. His research interests include Internet architecture and protocols, wireless networks and ad hoc networking, mobile computing, and QoS in packet switching networks. He co-authored the book Metropolitan Area Networks (Springer, London, 1997), and published in journal and conference proceedings more than 100 research papers related to design, modeling, and performance evaluation of computer-network architectures and protocols. He served as the technical program committee chair of the IFIP-TC6 conferences Networking 2002 and PWC 2003, and technical program committee co-chair of ACM WoWMoM 2002. He is serving as technical program committee co-chair of the IEEE Symposium on a World of Wireless, Mobile and Multimedia Networks (WoWMoM 2005). He served as guest editor for the Cluster Computing Journal (special issue on Mobile Ad Hoc Networking), IEEE Transactions on Computers (special issue on Quality of Service issues in Internet Web Services), and ACM/Kluwer Mobile Networks & Applications Journal (special issue on Mobile Ad hoc Networks). He is member of IFIP WGs 6.2, 6.3 and 6.8.Enrico Gregori received the Laurea in electronic engineering from the University of Pisa in 1980. He joined CNUCE, an institute of the Italian National Research Council (CNR) in 1981. He is currently a CNR research director. In 1986 he held a visiting position in the IBM research center in Zurich working on network software engineering and on heterogeneous networking. He has contributed to several national and international projects on computer networking. He has authored more than 100 papers in the area of computer networks and has published in international journals and conference proceedings and is co-author of the book Metropolitan Area Networks (Springer, London, 1997). He was the General Chair of the IFIP TC6 conferences: Networking2002 and PWC2003 (Personal Wireless Communications). He served as guest editor for the Networking2002 journal special issues on: Performance Evaluation, Cluster Computing and ACM/Kluwer Wireless Networks Journals. He is a member of the board of directors of the Create-Net association, an association with several Universities and research centres that is fostering research on networking at European level. He is on the editorial board of the Cluster Computing, of the Computer Networks and of the Wireless Networks Journals. His current research interests include: Wireless access to Internet, Wireless LANs, Quality of service in packet-switching networks, Energy saving protocols, Evolution of TCP/IP protocols.