Traffic of Leukocytes and Cytokine Up-regulation in the Central Nervous System in a Murine Model of Neuroparacoccidioidomycosis

Background

Paracoccidioidomycosis is the most important systemic mycosis in South America. In the last decades, it was observed that central nervous system involvement is frequent, occurring in 12.5 % of the cases. The aim of this study was to report the early inflammatory changes associated with an experimental model of neuroparacoccidioidomycosis (NPCM).

Methods

C57BL/6 mice were infected by intracranial route with 10⁶ yeast cells of PB18 strain of Paracoccidioides brasiliensis. Leukocyte–endothelium interactions were assessed by intravital microscopy 1, 2, 4, and 8 weeks post-infection (p.i.). Chemokine/cytokine levels in the brain and histopathological changes were assessed 4 and 8 weeks p.i..

Results

Intravital microscopy analysis revealed a progressive increase in leukocyte recruitment in the vessels of pia mater with a peak 4 weeks p.i. The chemokine CXCL9 was increased at 4 and 8 weeks p.i., while CCL2, CCL3, and CCL5 were increased at 8 weeks p.i. Histopathological analysis revealed the infiltration of inflammatory cells and the development of progressive granulomatous meningoencephalitis. CCL3 levels correlated with clinical manifestations of disease, as measured by the SHIRPA battery.

Conclusions

The experimental model of NPCM showed increased leukocyte recruitment associated with increased expression of chemokines and nervous tissue inflammation which correlated with clinical manifestations of disease.     

Optimal control strategies and cost-effectiveness analysis of a malaria model

The aim of this paper is to investigate the effectiveness and cost-effectiveness of three malaria preventive measures (use of treated bednets, spray of insecticides and a possible treatment of infective humans that blocks transmission to mosquitoes). For this, we consider a mathematical model for the transmission dynamics of the disease that includes these measures. We first consider the constant control parameters’ case, we calculate the basic reproduction number and investigate the existence and stability of equilibria; the model is found to exhibit backward bifurcation. We then assess the relative impact of each of the constant control parameters measures by calculating the sensitivity index of the basic reproductive number to the model's parameters. In the time-dependent constant control case, we use Pontryagin's Maximum Principle to derive necessary conditions for the optimal control of the disease. We also calculate the Infection Averted Ratio (IAR) and the Incremental Cost-Effectiveness Ratio (ICER) to investigate the cost-effectiveness of all possible combinations of the three control measures. One of our findings is that the most cost-effective strategy for malaria control, is the combination of the spray of insecticides and treatment of infective individuals. This strategy requires a 100% effort in both treatment (for 20 days) and spray of insecticides (for 57 days). In practice, this will be extremely difficult, if not impossible to achieve. The second most cost-effective strategy which consists of a 100% use of treated bednets and 87% treatment of infective individuals for 42 and 100 days, respectively, is sustainable and therefore preferable.     

Genotypic variation of nodules’ enzymatic activities in symbiotic nitrogen fixation among common bean (Phaseolus vulgaris L.) genotypes grown under salinity constraint

The effect of salt stress, under glasshouse conditions, was studied on plant biomass, nodulation, and activities of acid phosphatases (APase, EC 3.1.3.2) and trehalose 6-phosphate phosphatase (TPP, EC 3.1.3.12) in the symbiosis common bean (Phaseolus vulgaris L.)-rhizobia nodules. Four common bean recombinant inbred lines (147, 115, 104 and 83) were separately inoculated, with CIAT 899 or RhM11 strains and grown in hydroaeroponic culture. Two NaCl levels (0 and 25 mM NaCl plant^?1 week^?1 corresponding, respectively, to the control and the salt treatment) were applied and the culture was assessed during 42 days after their transplantation. The results showed that the nodulation of these lines was not affected by salinity except for the line 83 inoculated with CIAT 899, whose nodule dry weight decreased by 48.24 % compared with the corresponding controls. For the other symbiotic combinations, shoot and root biomasses were not significantly affected by salt constraint. Salinity stress generally reduced acid phosphatise and trehalose phosphate phosphatase activities in nodules that were less affected in plants inoculated with RhM11. Based on our data, it appears that nodule phosphatase activity may be involved in salinity tolerance in common beans and the levels of salt tolerance depend principally on specific combination of the rhizobial strain and the host cultivar.     

O-Heterocyclic Embeurekols from Embellisia eureka,an Endophyte of Cladanthus arabicus

Three new polyketides (-)-1 , (+)-1 , and 2 ) were isolated from the EtOAc extract of the fungus Embellisia eureka, an endophyte of the Moroccan plant Cladanthus arabicus (Asteraceae). The structures of these new compounds were determined on the basis of one- and two-dimensional NMR spectroscopy as well as by high-resolution mass spectrometry. The absolute configurations of (-)-1 , (+)-1 , and 2 were determined by TDDFT ECD calculations of solution conformers, online HPLC-ECD analysis, and the modified Mosher method. Chirality 25:250–256, 2013. © 2013 Wiley Periodicals, Inc.     

Phenoscope: an automated large‐scale phenotyping platform offering high spatial homogeneity

Increased phenotyping accuracy and throughput are necessary to improve our understanding of quantitative variation and to be able to deconstruct complex traits such as those involved in growth responses to the environment. Still, only a few facilities are known to handle individual plants of small stature for non‐destructive, real‐time phenotype acquisition from plants grown in precisely adjusted and variable experimental conditions. Here, we describe Phenoscope, a high‐throughput phenotyping platform that has the unique feature of continuously rotating 735 individual pots over a table. It automatically adjusts watering and is equipped with a zenithal imaging system to monitor rosette size and expansion rate during the vegetative stage, with automatic image analysis allowing manual correction. When applied to Arabidopsis thaliana, we show that rotating the pots strongly reduced micro‐environmental disparity: heterogeneity in evaporation was cut by a factor of 2.5 and the number of replicates needed to detect a specific mild genotypic effect was reduced by a factor of 3. In addition, by controlling a large proportion of the micro‐environmental variance, other tangible sources of variance become noticeable. Overall, Phenoscope makes it possible to perform large‐scale experiments that would not be possible or reproducible by hand. When applied to a typical quantitative trait loci (QTL) mapping experiment, we show that mapping power is more limited by genetic complexity than phenotyping accuracy. This will help to draw a more general picture as to how genetic diversity shapes phenotypic variation.     

Historical Shifts in Brazilian P. falciparum Population Structure and Drug Resistance Alleles

Previous work suggests that Brazilian Plasmodium falciparum has limited genetic diversity and a history of bottlenecks, multiple reintroductions due to human migration, and clonal expansions. We hypothesized that Brazilian P. falciparum would exhibit clonal structure. We examined isolates collected across two decades from Amapá, Rondônia, and Pará state (n = 190). By examining more microsatellites markers on more chromosomes than previous studies, we hoped to define the extent of low diversity, linkage disequilibrium, bottlenecks, population structure, and parasite migration within Brazil. We used retrospective genotyping of samples from the 1980s and 1990s to explore the population genetics of SP resistant dhfr and dhps alleles. We tested an existing hypothesis that the triple mutant dhfr mutations 50R/51I/108N and 51I/108N/164L developed in southern Amazon from a single origin of common or similar parasites. We found that Brazilian P. falciparum had limited genetic diversity and isolation by distance was rejected, which suggests it underwent bottlenecks followed by migration between sites. Unlike Peru, there appeared to be gene flow across the Brazilian Amazon basin. We were unable to divide parasite populations by clonal lineages and pairwise FST were common. Most parasite diversity was found within sites in the Brazilian Amazon, according to AMOVA. Our results challenge the hypothesis that triple mutant alleles arose from a single lineage in the Southern Amazon. SP resistance, at both the double and triple mutant stages, developed twice and potentially in different regions of the Brazilian Amazon. We would have required samples from before the 1980s to describe how SP resistance spread across the basin or describe the complex internal migration of Brazilian parasites after the colonization efforts of past decades. The Brazilian Amazon basin may have sufficient internal migration for drug resistance reported in any particular region to rapidly spread to other parts of basin under similar drug pressure.     

Characterization of a thermoactive endoglucanase isolated from a biogas plant metagenome

A metagenomic library from DNA isolated from a biogas plant was constructed and screened for thermoactive endoglucanases to gain insight into the enzymatic diversity involved in plant biomass breakdown at elevated temperatures. Two cellulase-encoding genes were identified and the corresponding proteins showed sequence similarities of 59% for Cel5A to a putative cellulase from Anaerolinea thermolimosa and 99% for Cel5B to a characterized endoglucanase isolated from a biogas plant reactor. The cellulase Cel5A consists of one catalytical domain showing sequence similarities to glycoside hydrolase family 5 and comprises 358 amino acids with a predicted molecular mass of 41.2 kDa. The gene coding for cel5A was successfully cloned and expressed in Escherichia coli C43(DE3). The recombinant protein was purified to homogeneity using affinity chromatography with a specific activity of 182 U/mg, and a yield of 74%. Enzymatic activity was detectable towards cellulose and mannan containing substrates and over a broad temperature range from 40 °C to 70 °C and a pH range from 4.0 to 7.0 with maximal activity at 55 °C and pH 5.0. Cel5A showed high thermostability at 60 °C without loss of activity after 24 h. Due to the enzymatic characteristics, Cel5A is an attractive candidate for the degradation of lignocellulosic material.

     

Restoration of human dystrophin following transplantation of exon-skipping-engineered DMD patient stem cells into dystrophic mice

Duchenne muscular dystrophy (DMD) is a hereditary disease caused by mutations that disrupt the dystrophin mRNA reading frame. In some cases, forced exclusion (skipping) of a single exon can restore the reading frame, giving rise to a shorter, but still functional, protein. In this study, we constructed lentiviral vectors expressing antisense oligonucleotides in order to induce an efficient exon skipping and to correct the initial frameshift caused by the DMD deletion of CD133+ stem cells. The intramuscular and intra-arterial delivery of genetically corrected CD133 expressing myogenic progenitors isolated from the blood and muscle of DMD patients results in a significant recovery of muscle morphology, function, and dystrophin expression in scid/mdx mice. These data demonstrate that autologous engrafting of blood or muscle-derived CD133+ cells, previously genetically modified to reexpress a functional dystrophin, represents a promising approach for DMD.