Potential secondary structure at translation-initiation sites.

Since translational start codons also occur internally, more-complex features within mRNA must determine initiation. We compare the potential secondary structure of 123 prokaryotic mRNA start regions to that of regions coding for internal methionines. The latter display an unexpectedly-uniform, almost-periodic pattern of pairing potential. In contrast, sequences 5' to start codons have little self-pairing, and do not pair extensively with the proximal coding region. Pairing potential surrounding start codons was found to be less than half of that found near internal AUGs. In groups of random sequences where the distribution of nucleotides at each position, or of trinucleotides at each in-frame codon position, matched the observed natural distribution, there was no periodicity in the pairing potential of the internal sequences. Randomized internal sequences had less pairing: the ratio of pairing intensity between internals and starts was reduced from 2.0 to 1.6 by randomization. We propose that the transition from the relatively-unstructured start domains to the highly-structured internal sequences may be an important determinant of translational start-site recognition.     

Organic acid profile of isovaleric acidemia: a comprehensive metabolomics approach

Isovaleric acidemia (IVA, MIM 248600) can be a severe and potentially life-threatening disease in affected neonates, but with a positive prognosis on treatment for some phenotypes. This study presents the first application of metabolomics to evaluate the metabolite profiles derived from urine samples of untreated and treated IVA patients as well as of obligate heterozygotes. All IVA patients carried the same homozygous c.367 G > A nucleotide change in exon 4 of the IVD gene but manifested phenotypic diversity. Concurrent class analysis (CONCA) was used to compare all the metabolites from the original complete data set obtained from the three case and two control groups used in this investigation. This application of CONCA has not been reported previously, and is used here to compare four different modes of scaling of all metabolites. The variables important in discrimination from the CONCA thus enabled the recognition of different metabolic patterns encapsulated within the data sets that would not have been revealed by using only one mode of scaling. Application of multivariate and univariate analyses disclosed 11 important metabolites that distinguished untreated IVA from controls. These included well-established diagnostic biomarkers of IVA, endogenous detoxification markers, and 3-hydroxycaproic acid, an indicator of ketosis, but not reported previously for this disease. Nine metabolites were identified that reflected the effect of treatment of IVA. They included detoxification products and indicators related to the high carbohydrate and low protein diet which formed the hallmark of the treatment. This investigation also provides the first comparative metabolite profile for heterozygotes of this inherited metabolic disorder. The detection of informative metabolites in even very low concentrations in all three experimental groups highlights the potential advantage of the holistic mode of analysis of inherited metabolic diseases in a metabolomics investigation.     

A DNA Vaccine against Yellow Fever Virus: Development and Evaluation

Attenuated yellow fever (YF) virus 17D/17DD vaccines are the only available protection from YF infection, which remains a significant source of morbidity and mortality in the tropical areas of the world. The attenuated YF virus vaccine, which is used worldwide, generates both long-lasting neutralizing antibodies and strong T-cell responses. However, on rare occasions, this vaccine has toxic side effects that can be fatal. This study presents the design of two non-viral DNA-based antigen formulations and the characterization of their expression and immunological properties. The two antigen formulations consist of DNA encoding the full-length envelope protein (p/YFE) or the full-length envelope protein fused to the lysosomal-associated membrane protein signal, LAMP-1 (pL/YFE), aimed at diverting antigen processing/presentation through the major histocompatibility complex II precursor compartments. The immune responses triggered by these formulations were evaluated in H2b and H2d backgrounds, corresponding to the C57Bl/6 and BALB/c mice strains, respectively. Both DNA constructs were able to induce very strong T-cell responses of similar magnitude against almost all epitopes that are also generated by the YF 17DD vaccine. The pL/YFE formulation performed best overall. In addition to the T-cell response, it was also able to stimulate high titers of anti-YF neutralizing antibodies comparable to the levels elicited by the 17DD vaccine. More importantly, the pL/YFE vaccine conferred 100% protection against the YF virus in intracerebrally challenged mice. These results indicate that pL/YFE DNA is an excellent vaccine candidate and should be considered for further developmental studies.     

Cost-Effectiveness Analysis of Universal Vaccination of Adults Aged 60 Years with 23-Valent Pneumococcal Polysaccharide Vaccine versus Current Practice in Brazil

Objective

To evaluate the cost-effectiveness of introducing universal vaccination of adults aged 60 years with the 23-valent pneumococcal polysaccharide vaccine (PPV23) into the National Immunization Program (NIP) in Brazil.

Methods

Economic evaluation using a Markov model to compare two strategies: (1) universal vaccination of adults aged 60 years with one dose of PPV23 and 2) current practice (vaccination of institutionalized elderly and elderly with underlying diseases). The perspective was from the health system and society. Temporal horizon was 10 years. Discount rate of 5% was applied to costs and benefits. Clinical syndromes of interest were invasive pneumococcal disease (IPD) including meningitis, sepsis and others and pneumonia. Vaccine efficacy against IPD was obtained from a meta-analysis of randomized control trials and randomized studies, whereas vaccine effectiveness against pneumonia was obtained from cohort studies. Resource utilization and costs were obtained from the Brazilian Health Information Systems. The primary outcome was cost per life year saved (LYS). Univariate and multivariate sensitivity analysis were performed.

Results

The universal vaccination strategy avoided 7,810 hospitalizations and 514 deaths, saving 3,787 years of life and costing a total of USD$31,507,012 and USD$44,548,180, respectively, from the health system and societal perspective. The universal immunization would result in ICERs of USD$1,297 per LYS, from the perspective of the health system, and USD$904 per LYS, from the societal perspective.

Conclusion

The results suggest that universal vaccination of adults aged 60 years with the 23-valent pneumococcal polysaccharide vaccine (PPV23) is a very cost-effective intervention for preventing hospitalization and deaths for IPD and pneumonia is this age group in Brazil.     

Artemisinin-quinoline hybrid-dimers: synthesis and in vitro antiplasmodial activity

Novel artemisinin-quinoline hybrid-dimers were synthesized from dihydroartemisinin and different aminoquinolines at elevated temperatures (90-110°C). All compounds were obtained as the β-isomers and were tested against both chloroquine sensitive and resistant strains of Plasmodium falciparum. Hybrid-dimer 8 showed the highest antiplasmodial activity, inheriting the optimum chain length of three carbon atoms.     

Developmental and reproductive patterns of Triatoma brasiliensis infected with Trypanosoma cruzi under laboratory conditions

The aim of this work was to study the interaction between Trypanosoma cruzi-1 and Triatoma brasiliensis. A group of 1st instar nymphs was initially fed on T. cruzi-infected mice and a control group was fed on uninfected mice. From the second feeding onwards, both groups were otherwise fed on non-infected mice. The resulting adults were grouped in pairs: infected male/uninfected female, uninfected male/infected female, infected male and female and uninfected male/uninfected female. The infection affected only the 1st instar nymphs, which took significantly more time to reach the 2nd instar than uninfected nymphs. The differences in the molting time between the infected and uninfected nymphs from the 2nd to the 5th instars were not statistically significant. Both groups presented similar rates of nymphal mortality and reproductive performance was not significantly affected by infection in any of the treatments.     

Expression of the sucrose binding protein from soybean: renaturation and stability of the recombinant protein

The sucrose binding protein (SBP) belongs to the cupin family of proteins and is structurally related to vicilin-like storage proteins. In this investigation, a SBP isoform (GmSBP2/S64) was expressed in E. coli and large amounts of the protein accumulated in the insoluble fraction as inclusion bodies. The renatured protein was studied by circular dichroism (CD), intrinsic fluorescence, and binding of the hydrophobic probes ANS and Bis-ANS. The estimated content of secondary structure of the renatured protein was consistent with that obtained by theoretical modeling with a large predominance of beta-strand structure (42%) over the alpha-helix (9.9%). The fluorescence emission maximum of 303 nm for SBP2 indicated that the fluorescent tryptophan was completely buried within a highly hydrophobic environment. We also measured the equilibrium dissociation constant (K(d)) of sucrose binding by fluorescence titration using the refolded protein. The low sucrose binding affinity (K(d)=2.79+/-0.22 mM) of the renatured protein was similar to that of the native protein purified from soybean seeds. Collectively, these results indicate that the folded structure of the renatured protein was similar to the native SBP protein. As a member of the bicupin family of proteins, which includes highly stable seed storage proteins, SBP2 was fairly stable at high temperatures. Likewise, it remained folded to a similar extent in the presence or absence of 7.6M urea or 6.7 M GdmHCl. The high stability of the renatured protein may be a reminiscent property of SBP from its evolutionary relatedness to the seed storage proteins.     

Full-length infectious clone of a low passage dengue virus serotype 2 from Brazil

Full-length dengue virus (DENV) cDNA clones are an invaluable tool for many studies, including those on the development of attenuated or chimeric vaccines and on host-virus interactions. Furthermore, the importance of low passage DENV infectious clones should be highlighted, as these may harbour critical and unique strain-specific viral components from field-circulating isolates. The successful construction of a functional Brazilian low passage DENV serotype 2 full-length clone through homologous recombination reported here supports the use of a strategy that has been shown to be highly useful by our group for the development of flavivirus infectious clones and replicons.