Humoral immune response kinetics in Philander opossum and Didelphis marsupialis infected and immunized by Trypanosoma cruzi employing an immunofluorescence antibody test.

Philander opossum and Didelphis marsupialis considered the most ancient mammals and an evolutionary success, maintain parasitism by Trypanosoma cruzi without developing any apparent disease or important tissue lesion. In order to elucidate this well-balanced interaction, we decided to compare the humoral immune response kinetics of the two didelphids naturally and experimentally infected with T. cruzi and immunized by different schedules of parasite antigens, employing an indirect fluorescence antibody test (IFAT). Both didelphids responded with high serological titers to different immunization routes, while the earliest response occurred with the intradermic route. Serological titers of naturally infected P. opossum showed a significant individual variation, while those of D. marsupialis remained stable during the entire follow-up period. The serological titers of the experimentally infected animals varied according to the inoculated strain. Our data suggest that (1) IFAT was sensitive for follow-up of P. opossum in natural and experimental T. cruzi infections; (2) both P. opossum and D. marsupialis are able to mount an efficient humoral immune response as compared to placental mammals; (3) experimentally infected P. opossum and D. marsupialis present distinct patterns of infection, depending on the subpopulation of T. cruzi, (4) the differences observed in the humoral immune responses between P. opossum and D. marsupialis, probably, reflect distinct strategies selected by these animals during their coevolution with T. cruzi.     

Trypanosoma cruzi: Insights into naphthoquinone effects on growth and proteinase activity

In this study we compared the effects of naphthoquinones (α-lapachone, β-lapachone, nor-β-lapachone and Epoxy-α-lap) on growth of Trypanosoma cruzi epimastigotes forms, and on viability of VERO cells. In addition we also experimentally analyzed the most active compounds inhibitory profile against T. cruzi serine- and cysteine-proteinases activity and theoretically evaluated them against cruzain, the major T. cruzi cysteine proteinase by using a molecular docking approach. Our results confirmed β-lapachone and Epoxy-α-lap with a high trypanocidal activity in contrast to α-lapachone and nor-β-lapachone whereas Epoxy-α-lap presented the safest toxicity profile against VERO cells. Interestingly the evaluation of the active compounds effects against T. cruzi cysteine- and serine-proteinases activities revealed different targets for these molecules. β-Lapachone is able to inhibit the cysteine-proteinase activity of T. cruzi proteic whole extract and of cruzain, similar to E-64, a classical cysteine-proteinase inhibitor. Differently, Epoxy-α-lap inhibited the T. cruzi serine-proteinase activity, similar to PMSF, a classical serine-proteinase inhibitor. In agreement to these biological profiles in the enzymatic assays, our theoretical analysis showed that E-64 and β-lapachone interact with the cruzain specific S2 pocket and active site whereas Epoxy-α-lap showed no important interactions. Overall, our results infer that β-lapachone and Epoxy-α-lap compounds may inhibit T. cruzi epimastigotes growth by affecting T. cruzi different proteinases. Thus the present data shows the potential of these compounds as prototype of protease inhibitors on drug design studies for developing new antichagasic compounds.     

Structural and ecophysiological adaptations to forest gaps

To survive new microclimatic conditions of a forest gap environment, plant species must physiologically and structurally adjust. A morpho-anatomical, ultrastructural and ecophysiological study was performed at three different times in a forest gap that was created by illegal selective logging. The study followed the early successional Actinostemon verticillatus and the late-successional Metrodorea brevifolia, to elucidate the adaptive strategies of acclimation to gaps. Additionally, Schinus terebinthifolius was included in the study in order to test the plasticity of a pioneer species that grows on forest edges, where this species had higher values of leaf thickness, leaf mass area and succulence. M. brevifolia had succulent leaves, high leaf area and a thin cuticle. A. verticillatus presented the densest leaves and was the only species to show leaf morpho-anatomical plasticity. Ultrastructural and physiological differences were observed only in A. verticillatus and M. brevifolia leaves from the gap: increase in the stroma volume, oil droplets, plastoglobuli, photochemical and non-photochemical quenching. Photosynthetic efficiency showed that the early stages of gap formation are the most critical. Acclimation strategies of A. verticillatus suggest this species invests in the efficiency of photosynthesis by increasing its leaf thickness, leaf mass area and in water content maintenance by increasing the density of its leaves, at the expense of gas exchange, was compensated by a high density of stomata. M. brevifolia compensates for the higher cost of leaves and lower leaf plasticity with ultrastructural changes that are used to adjust the photosynthetic process, which promotes a shorter leaf payback time.     

MAFCO: A Compression Tool for MAF Files

In the last decade, the cost of genomic sequencing has been decreasing so much that researchers all over the world accumulate huge amounts of data for present and future use. These genomic data need to be efficiently stored, because storage cost is not decreasing as fast as the cost of sequencing. In order to overcome this problem, the most popular general-purpose compression tool, gzip, is usually used. However, these tools were not specifically designed to compress this kind of data, and often fall short when the intention is to reduce the data size as much as possible. There are several compression algorithms available, even for genomic data, but very few have been designed to deal with Whole Genome Alignments, containing alignments between entire genomes of several species. In this paper, we present a lossless compression tool, MAFCO, specifically designed to compress MAF (Multiple Alignment Format) files. Compared to gzip, the proposed tool attains a compression gain from 34% to 57%, depending on the data set. When compared to a recent dedicated method, which is not compatible with some data sets, the compression gain of MAFCO is about 9%. Both source-code and binaries for several operating systems are freely available for non-commercial use at: http://bioinformatics.ua.pt/software/mafco.     

MreC and MreD Proteins Are Not Required for Growth of Staphylococcus aureus

The transmembrane proteins MreC and MreD are present in a wide variety of bacteria and are thought to be involved in cell shape determination. Together with the actin homologue MreB and other morphological elements, they play an essential role in the synthesis of the lateral cell wall in rod-shaped bacteria. In ovococcus, which lack MreB homologues, mreCD are also essential and have been implicated in peripheral cell wall synthesis. In this work we addressed the possible roles of MreC and MreD in the spherical pathogen Staphylococcus aureus. We show that MreC and MreD are not essential for cell viability and do not seem to affect cell morphology, cell volume or cell cycle control. MreC and MreD localize preferentially to the division septa, but do not appear to influence peptidoglycan composition, nor the susceptibility to different antibiotics and to oxidative and osmotic stress agents. Our results suggest that the function of MreCD in S. aureus is not critical for cell division and cell shape determination.     

Pattern of cytokine and chemokine production by THP-1 derived macrophages in response to live or heat-killed Mycobacterium bovis bacillus Calmette-Guérin Moreau strain

Tuberculosis has great public health impact with high rates of mortality and the only prophylactic measure for it is the Mycobacterium bovisbacillus Calmette-Guérin (BCG) vaccine. The present study evaluated the release of cytokines [interleukin (IL)-1, tumour necrosis factor and IL-6] and chemokines [macrophage inflammatory protein (MIP)-1α and MIP-1β] by THP-1 derived macrophages infected with BCG vaccine obtained by growing mycobacteria in Viscondessa de Moraes Institute medium medium (oral) or Sauton medium (intradermic) to compare the effects of live and heat-killed (HK) mycobacteria. Because BCG has been reported to lose viability during the lyophilisation process and during storage, we examined whether exposing BCG to different temperatures also triggers differences in the expression of some important cytokines and chemokines of the immune response. Interestingly, we observed that HK mycobacteria stimulated cytokine and chemokine production in a different pattern from that observed with live mycobacteria.     

Exercise training reduces insulin resistance and upregulates the mTOR/p70S6k pathway in cardiac muscle of diet‐induced obesity rats

Obesity and insulin resistance are rapidly expanding public health problems. These disturbances are related to many diseases, including heart pathology. Acting through the Akt/mTOR pathway, insulin has numerous and important physiological functions, such as the induction of growth and survival of many cell types and cardiac hypertrophy. However, obesity and insulin resistance can alter mTOR/p70S6k. Exercise training is known to induce this pathway, but never in the heart of diet‐induced obesity subjects. To evaluate the effect of exercise training on mTOR/p70S6k in the heart of obese Wistar rats, we analyzed the effects of 12 weeks of swimming on obese rats, induced by a high‐fat diet. Exercise training reduced epididymal fat, fasting serum insulin and plasma glucose disappearance. Western blot analyses showed that exercise training increased the ability of insulin to phosphorylate intracellular molecules such as Akt (2.3‐fold) and Foxo1 (1.7‐fold). Moreover, reduced activities and expressions of proteins, induced by the high‐fat diet in rats, such as phospho‐JNK (1.9‐fold), NF‐kB (1.6‐fold) and PTP‐1B (1.5‐fold), were observed. Finally, exercise training increased the activities of the transduction pathways of insulin‐dependent protein synthesis, as shown by increases in Raptor phosphorylation (1.7‐fold), p70S6k phosphorylation (1.9‐fold), and 4E‐BP1 phosphorylation (1.4‐fold) and a reduction in atrogin‐1 expression (2.1‐fold). Results demonstrate a pivotal regulatory role of exercise training on the Akt/mTOR pathway, in turn, promoting protein synthesis and antagonizing protein degradation. J. Cell. Physiol. 226: 666–674, 2011. © 2010 Wiley‐Liss, Inc.     

The removal of a disulfide bridge in CotA-laccase changes the slower motion dynamics involved in copper binding but has no effect on the thermodynamic stability

The contribution of the disulfide bridge in CotA-laccase from Bacillus subtilis is assessed with respect to the enzyme’s functional and structural properties. The removal of the disulfide bond by site-directed mutagenesis, creating the C322A mutant, does not affect the spectroscopic or catalytic properties and, surprisingly, neither the long-term nor the thermodynamic stability parameters of the enzyme. Furthermore, the crystal structure of the C322A mutant indicates that the overall structure is essentially the same as that of the wild type, with only slight alterations evident in the immediate proximity of the mutation. In the mutant enzyme, the loop containing the C322 residue becomes less ordered, suggesting perturbations to the substrate binding pocket. Despite the wild type and the C322A mutant showing similar thermodynamic stability in equilibrium, the holo or apo forms of the mutant unfold at faster rates than the wild-type enzyme. The picosecond to nanosecond time range dynamics of the mutant enzyme was not affected as shown by acrylamide collisional fluorescence quenching analysis. Interestingly, copper uptake or copper release as measured by the stopped-flow technique also occurs more rapidly in the C322A mutant than in the wild-type enzyme. Overall the structural and kinetic data presented here suggest that the disulfide bridge in CotA-laccase contributes to the conformational dynamics of the protein on the microsecond to millisecond timescale, with implications for the rates of copper incorporation into and release from the catalytic centres.     

Host Specificity and Genetic Diversity of Didymella bryoniae from Cucurbitaceae in Brazil

Thirty‐seven isolates of Didymella bryoniae from three Cucurbitaceae species were collected in Brazil and tested for pathogenicity to watermelon. All isolates were pathogenic but differed in aggressiveness levels. Seven representative isolates were used in cross‐pathogenicity tests against 10 cucurbitaceous hosts. Most isolates were pathogenic to most host species tested, except to Sechium edule. Among the susceptible species, Citrullus and Cucumis species were the most susceptible hosts, while pumpkin and Luffa purgans were the most resistant. Host of origin affected the pattern of aggressiveness on each host. Isolates from watermelon were very aggressive to their original host, but much less aggressive or not pathogenic at all to some Cucurbita. Two previously described random‐amplified polymorphic DNA (RAPD)‐specific primers indicated that 81% of the isolates could be classified into the so‐called RG I group, while the remaining isolates could not be classified into any of the described RG groups. All 37 isolates were further characterized by RAPD fingerprinting and compared with three US isolates representative of RG I and RG II groups. The Brazilian D. bryoniae isolates could be separated into genetically similar clusters. The majority of the isolates were grouped in cluster DB Ia, which contained only isolates of Citrullus lanatus and Cucumis melo. Two of the American isolates used as controls clustered with this group at 68% similarity level. The DB Ib cluster included three Brazilian isolates obtained from melon and watermelon and the American representative for RG II, at a lower similarity level (43%). Two isolates from watermelon clustered with one isolate from melon in a separate group (DB II), while one single isolate from pumpkin (DB III) showed the lowest genetic similarity to all other isolates. Didymella bryoniae isolates from Brazil showed, therefore, a level of genetic diversity higher than previously reported for the species. RAPD fingerprinting allowed for geographical distinction of D. bryoniae isolates but no correlation between genetic distance, aggressiveness or origin of the isolate was found.