Yellow Fever 17DD Vaccine Virus Infection Causes Detectable Changes in Chicken Embryos

The yellow fever (YF) 17D vaccine is one of the most effective human vaccines ever created. The YF vaccine has been produced since 1937 in embryonated chicken eggs inoculated with the YF 17D virus. Yet, little information is available about the infection mechanism of YF 17DD virus in this biological model. To better understand this mechanism, we infected embryos of Gallus gallus domesticus and analyzed their histopathology after 72 hours of YF infection. Some embryos showed few apoptotic bodies in infected tissues, suggesting mild focal infection processes. Confocal and super-resolution microscopic analysis allowed us to identify as targets of viral infection: skeletal muscle cells, cardiomyocytes, nervous system cells, renal tubular epithelium, lung parenchyma, and fibroblasts associated with connective tissue in the perichondrium and dermis. The virus replication was heaviest in muscle tissues. In all of these specimens, RT-PCR methods confirmed the presence of replicative intermediate and genomic YF RNA. This clearer characterization of cell targets in chicken embryos paves the way for future development of a new YF vaccine based on a new cell culture system.     

Ethanol from sugarcane in Brazil: a ‘midway’ strategy for increasing ethanol production while maximizing environmental benefits

This article reviews the history and current state of ethanol production from sugarcane in Brazil and presents a strategy for improving ecosystem services and production. We propose that it is possible to produce ethanol from sugarcane while maintaining or even recovering some of Brazil's unique neotropical biodiversity and ecosystem climate services. This approach to the future of sustainable and responsible ethanol production is termed the ‘midway’ strategy. The ‘midway’ strategy involves producing the necessary biotechnology to increase productivity while synergistically protecting and regenerating rainforest. Three main areas of scientific and technological advance that are key to realizing the ‘midway’ strategy are: (i) improving the quality of scientific data on sugarcane biology as pertains to its use as a bioenergy crop; (ii) developing technologies for the use of bagasse for cellulosic ethanol; and (iii) developing policies to improve the ecosystem services associated with sugarcane landscapes. This article discusses these three issues in the general context of biofuels production and highlights examples of scientific achievements that are already leading towards the ‘midway’ strategy.     

In silico and in vitro characterization of phospholipase A2 isoforms from soybean (Glycine max)

At the present, no secreted phospholipase A₂ (sPLA₂) from soybean (Glycine max) was investigated in detail. In this work we identified five sequences of putative secreted sPLA₂ from soybean after a BLAST search in G. max database. Sequence analysis showed a conserved PA2c domain bearing the Ca²⁺ binding loop and the active site motif. All the five mature proteins contain 12 cysteine residues, which are commonly conserved in plant sPLA₂s. We propose a phylogenetic tree based on sequence alignment of reported plant sPLA₂s including the novel enzymes from G. max. According to PLA₂ superfamily, two of G. max sPLA₂s are grouped as XIA and the rest of sequences as XIB, on the basis of differences found in their molecular weights and deviating sequences especially in the N- and C-terminal regions of the isoenzymes. Furthermore, we report the cloning, expression and purification of one of the putative isoenzyme denoted as GmsPLA₂-XIA-1. We demonstrate that this mature sPLA₂ of 114 residues had PLA₂ activity on Triton:phospholipid mixed micelles and determine the kinetic parameters for this system. We generate a model based on the known crystal structure of sPLA₂ from rice (isoform II), giving first insights into the three-dimensional structure of folded GmsPLA₂-XIA-1. Besides describing the spatial arrangement of highly conserved pair HIS-49/ASP-50 and the Ca⁺² loop domains, we propose the putative amino acids involved in the interfacial recognition surface. Additionally, molecular dynamics simulations indicate that calcium ion, besides its key function in the catalytic cycle, plays an important role in the overall stability of GmsPLA₂-XIA-1 structure.     

Synthesis and antifungal activity of diverse C-2 pyridinyl and pyridinylvinyl substituted quinolines

Diverse 2-pyridinyl quinolines 6–12 and 2-pyridinilvinyl quinolines 13–17 were prepared using a straightforward synthesis based on the BiCl₃-catalyzed multicomponent imino Diels–Alder (imino DA) reaction or a novel tandem imino DA/catalytic tetrahydroquinoline ring oxidation/Perkin condensation sequential process. All members of the series showed activities against dermatophytes and some of them possessed a broad spectrum of action. 2-(Pyridin-4-yl)quinoline 9 and 2-(2-pyridin-4-yl)vinyl)quinoline 16 showed the best MIC₈₀ and MIC₅₀ against the clinically important fungi Candida albicans and non-albicans Candida species. In turn, 6-ethyl-2-(pyridin-2-yl)quinoline 6 showed the best properties against standardized as well as clinical strains of Cryptococcus neoformans.     

Microvascular oxygen delivery and consumption following treatment with verapamil

The microvascular distribution of oxygen was studied in the arterioles and venules of the awake hamster window chamber preparation to determine the contribution of vascular smooth muscle relaxation to oxygen consumption of the microvascular wall during verapamil-induced vasodilatation. Verapamil HCl delivered in a 0.1 mg/kg bolus injection followed by a continuous infusion of 0.01 mg.kg(-1).min(-1) caused significant arteriolar dilatation, increased microvascular flow and functional capillary density, and decreased arteriolar vessel wall transmural Po(2) difference. Verapamil caused tissue Po(2) to increase from 25.5 +/- 4.1 mmHg under control condition to 32.0 +/- 3.7 mmHg during verapamil treatment. Total oxygen released by the microcirculation to the tissue remained the same as at baseline. Maintenance of the same level of oxygen release to the tissue, increased tissue Po(2), and decreased wall oxygen concentration gradient are compatible if vasodilatation significantly lowers vessel wall oxygen consumption, which in this model appears to constitute an important oxygen-consuming compartment. These findings show that treatment with verapamil, which increases oxygen supply through vasodilatation, may further improve tissue oxygenation by lowering oxygen consumption of the microcirculation.     

Milbemectin resistance in <Emphasis Type="Italic">Tetranychus urticae</Emphasis> (Acari: Tetranychidae): selection,stability and cross-resistance to abamectin

Studies on artificial laboratory selections with milbemectin, stability of milbemectin resistance and possible cross-resistance with abamectin were carried out with Tetranychus urticae Koch to provide basic information for a milbemectin resistance management program. Selections for resistance and susceptibility to milbemectin were performed in a population of T. urticae, collected from a commercial chrysanthemum field in the State of São Paulo, Brazil. After six selections for resistance and five selections for susceptibility, susceptible (S) and resistant (R) strains of T. urticae to milbemectin were obtained. The resistance ratio (R/S) at the LC₅₀ reached 409-fold value. The stability of milbemectin resistance was also studied under laboratory conditions, using a population with initial frequency of 75% of resistant mites. The frequencies of milbemectin resistance were evaluated monthly for a period of 7 months. In order to observe possible correlation between milbemectin and abamectin resistance, the frequencies of abamectin resistance were also evaluated for that population, during the same period. The frequency of milbemectin resistance decreased from 75 to 14.5%, while the percentage of abamectin resistant mites decreased from 57 to 9.1%, in 7 months. The frequencies of milbemectin and abamectin resistance were also evaluated in 25 field populations of T. urticae, collected from several crops in the State of São Paulo. The frequencies of milbemectin resistance varied from 4.1 to 89.5%, and of abamectin, from 7.0 to 90.5%. A positive and significant correlation was observed between the frequencies of milbemectin and abamectin resistance, indicating positive cross-resistance between these acaricides. The results indicate that abamectin should be avoided for managing milbemectin resistance in T. urticae. This is the first report on milbemectin resistance in T. urticae in Brazil.     

Bromide does not bind to the Mn4Ca complex in its S1 state in Cl(-)-depleted and Br(-)-reconstituted oxygen-evolving photosystem II: evidence from X-ray absorption spectroscopy at the Br K-edge

Chloride is an important cofactor in photosynthetic water oxidation. It can be replaced by bromide with retention of the oxygen-evolving activity of photosystem II (PSII). Binding of bromide to the Mn(4)Ca complex of PSII in its dark-stable S(1) state was studied by X-ray absorption spectroscopy (XAS) at the Br K-edge in Cl(-)-depleted and Br(-)-substituted PSII membrane particles from spinach. The XAS spectra exclude the presence of metal ions in the first and second coordination spheres of Br(-). EXAFS analysis provided tentative evidence of at least one metal ion, which may be manganese or calcium, at a distance of approximately 5 A to Br(-). The native Cl(-) ion may bind at a similar distance. Accordingly, water oxidation may not require binding of a halide directly to the metal ions of the Mn complex in its S(1) state.     

Purification of betulinic acid from Eugenia florida (Myrtaceae) by high-speed counter-current chromatography

A high yield of betulinic acid (up to 17% from the ethanolic extract) was found in the leaves of Eugenia florida collected in south-eastern Brazil, making this species a potential commercial source of the title compound. Extracts of E. florida were subjected to solvent partition, and rapid high-speed counter-current chromatography (HSCCC) was applied to the semi-crude extracts to afford betulinic acid in high purity. The mobile and stationary phases were derived from the two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (10:5:2.5:1). The developing solvent system (stationary and mobile phases) for optimum HSCCC separation was chosen by dissolving the fraction to be chromatographed in the proposed solvent mixture and determining the amount of betulinic acid in each phase by densitometric TLC. Purified betulinic acid was characterized by 13C-NMR, GC-MS and co-injection of its methyl ester with standards in GC-FID. The HSCCC technique is commonly employed to isolate triterpene glycosides, but is applied in this study to an aglycone.