Studies on survival, biological activities and behavior of Biomphalaria glabrata, the host snail of Schistosomiasis, submitted to increased hydrostatic pressure: a technique

To study changes in survival, in biological activities and behavior of planorbids submitted to increased hydrostatic pressure, we developed a technique using two transparent chambers and a hydraulic piston. The apparatus permitted renewal of the liquid medium without substantial variations in pressure, thus eliminating excretion products and maintaining the desired O2 level and thereby permitting us to evaluate the effects of pressure independently of the occurrence of anoxia. Pressure was maintained without any contact of the liquid medium with compressed air, a situation which reproduced with relative fidelity what occurs in nature and assured the presence of the same amounts of gases in the two observation chambers (Control and Experimental). Biomphalaria glabrata was found to be able to survive at least 48 hours when submitted to 49.02 x 10**4 Pa (equivalent to a water depth of 48.8 m), continuing to day egg masses and showing few behavioral changes when compared with the control group.

Biological aspects of the DM28C clone of Trypanosoma cruzi after metacylogenesis in chemically defined media

The biological characterization of the Trypanosoma cruzi clone Dm 28c in terms of its growth in LIT medium, cell-cycle, infectivity to mice and interaction with professional and non-professional phagocytic cells shows that it behaves as a bona fide T. cruzi representant. The biological properties of this myotropic clone do not change according to the origin of the trypomastigote forms (i. e., from triatomines, infected mice, cell-culture or from the chemically defined TAUP and TAU3AAG media). In addition Dm 28c metacyclic trypomastigotes from TAU3AAG medium display a high infectivity level to fibroblasts and muscle cells. Experiments on binding of cationized ferritin to trypomastigotes surface show the existence of cap-like structures of ferritin in regions near the kinetoplast. However the nature and role of these anionic sites remain to be determined. The results indicate that metacyclic trypomastigotes from Dm 28c clone obtained under chemically defined conditions reproduce the biological behaviour of T. cruzi, rendering this system very suitable for the study of cell-parasite interactions and for the isolation of trypanosome relevant macromolecules.

Plasmodium falciparum merozoite surface protein 2: epitope mapping and biological activity analysis of specific antibodies

Background: Plasmodium falciparum(P. falciparum) merozoite surfaceprotein 2 (MSP-2) is one of bloodstage proteins that are associated withprotection from malaria. MSP-2 consistsof a highly polymorphic centralrepeat region flanked by a dimorphicregion that defines the two allelicfamilies, 3D7 and FC27; N- and Cterminalregions are conserved domains.Long synthetic peptides (LSP)representing the two allelic familiesof MSP-2 and constant regions arerecognized by sera from donors livingin endemic areas; and specific antibodies(Abs) are associated with protectionand active in antibody dependentcellular inhibition (ADCI) in vitro.However, the fine specificity ofAb response to the two allelic familiesof MSP-2 is unknown. Methods: Peptidesrepresenting dimorphic regionof 3D7 and FC27 families and theirC-terminal (common fragment to thetwo families) termed 3D7-D (88 aa),FC27-D (48 aa) and C (40 aa) respectivelywere synthesized. Overlapping20 mer peptides covering dimorphicand constant regions of two familieswere also synthesized for epitopemapping. Human sera were obtainedfrom donors living in malaria endemicareas. SpecificDand CregionsAbs were purified from single or poolhuman sera. Sera from mice were obtainedafter immunization with thetwo families LSP mixture in three differentadjuvants: alhydrogel (Alum),Glucopyranosyl Lipid Adjuvant-Stableoil-in-water Emulsion (GLA-SE)and Virosome. For ADCI, P. falciparum(strain 3D7) parasite wasmaintained in culture at 0.5% parasitemiaand 4% hematocrit in air tightbox at love oxygen (2%) and 37 ºC.Results: We identified several epitopesfrom the dimorphic and constantregions of both families of MSP-2, inmice and humans (adults and children).In human, most recognizedepitopes were the same in differentendemic regions for each domain ofthe two families of MSP-2. In mice,the differential recognition of epitopewas depending on the strain of mouseand interestingly on the adjuvantused. GLA-SE and alum as adjuvantswere more often associated with therecognition of multiple epitopes thanvirosomes. Epitope-specific Abs recognizednative merozoites of P.falciparum and were active in ADCIto block development of parasite.Conclusion: The delineation of a limitednumber of epitopes could be exploitedto develop MSP-2 vaccinesactive on both allelic families ofMSP-2.

Landscape context on generalist and specialist aphid natural enemies

Report for the scientific sojourn at the University of Lund, Sweden, between May and September 2007. A landscape-scale research approach has been highlighted by a growing body of literature as essential for understanding important ecosystem services as biological control. Aphids are victims of a diversity of enemies making the aphid-enemy interaction a nice example for the role of enemy diversity for the functioning of biological control. Here it is examined the effects of landscape complexity on cereal aphids and associated natural enemies that varied in the degree of specialization. Parasitoids wasps abundance did not differ between landscape types but was strongly negatively related to the percentage of arable land. In contrast, abundances of generalist predators like Coccinellidae were significantly higher in simple landscapes since can benefit from the high availability of a variety of alternative resources within cropping systems. Consequently coccinellidae-to-aphid ratio was significantly higher in fields in homogenous landscapes as compared to fields included in an heterogeneous landscape, suggesting that enemy pressure on cereal aphids increases with landscape simplification. The landscape effect will depend mainly on the degree of specialization of functionally dominant natural enemies, so that the results imply that conservation actions aiming to optimise abundance for one taxonomic group in the agricultural landscape will not automatically increase abundance of other groups. Given that the strength of natural enemy impact on biocontrol depends on landscape features and the role of functionally dominant natural enemies. So, therefore it is essential to focus the future empirical work in examining the schedule of agricultural landscapes that maintain a diversity of generalist and specialist natural enemies.

Spatial modelling of biodiversity at the community level

1. Statistical modelling is often used to relate sparse biological survey data to remotely derived environmental predictors, thereby providing a basis for predictively mapping biodiversity across an entire region of interest. The most popular strategy for such modelling has been to model distributions of individual species one at a time. Spatial modelling of biodiversity at the community level may, however, confer significant benefits for applications involving very large numbers of species, particularly if many of these species are recorded infrequently. 2. Community-level modelling combines data from multiple species and produces information on spatial pattern in the distribution of biodiversity at a collective community level instead of, or in addition to, the level of individual species. Spatial outputs from community-level modelling include predictive mapping of community types (groups of locations with similar species composition), species groups (groups of species with similar distributions), axes or gradients of compositional variation, levels of compositional dissimilarity between pairs of locations, and various macro-ecological properties (e.g. species richness). 3. Three broad modelling strategies can be used to generate these outputs: (i) 'assemble first, predict later', in which biological survey data are first classified, ordinated or aggregated to produce community-level entities or attributes that are then modelled in relation to environmental predictors; (ii) 'predict first, assemble later', in which individual species are modelled one at a time as a function of environmental variables, to produce a stack of species distribution maps that is then subjected to classification, ordination or aggregation; and (iii) 'assemble and predict together', in which all species are modelled simultaneously, within a single integrated modelling process. These strategies each have particular strengths and weaknesses, depending on the intended purpose of modelling and the type, quality and quantity of data involved. 4. Synthesis and applications. The potential benefits of modelling large multispecies data sets using community-level, as opposed to species-level, approaches include faster processing, increased power to detect shared patterns of environmental response across rarely recorded species, and enhanced capacity to synthesize complex data into a form more readily interpretable by scientists and decision-makers. Community-level modelling therefore deserves to be considered more often, and more widely, as a potential alternative or supplement to modelling individual species.

Tumor necrosis factor (TNF) signaling, but not TWEAK (TNF-like weak inducer of apoptosis)-triggered cIAP1 (cellular inhibitor of apoptosis protein 1) degradation, requires cIAP1 RING dimerization and E2 binding.

Cellular inhibitor of apoptosis (cIAP) proteins, cIAP1 and cIAP2, are important regulators of tumor necrosis factor (TNF) superfamily (SF) signaling and are amplified in a number of tumor types. They are targeted by IAP antagonist compounds that are undergoing clinical trials. IAP antagonist compounds trigger cIAP autoubiquitylation and degradation. The TNFSF member TWEAK induces lysosomal degradation of TRAF2 and cIAPs, leading to elevated NIK levels and activation of non-canonical NF-kappaB. To investigate the role of the ubiquitin ligase RING domain of cIAP1 in these pathways, we used cIAP-deleted cells reconstituted with cIAP1 point mutants designed to interfere with the ability of the RING to dimerize or to interact with E2 enzymes. We show that RING dimerization and E2 binding are required for IAP antagonists to induce cIAP1 degradation and protect cells from TNF-induced cell death. The RING functions of cIAP1 are required for full TNF-induced activation of NF-kappaB, however, delayed activation of NF-kappaB still occurs in cIAP1 and -2 double knock-out cells. The RING functions of cIAP1 are also required to prevent constitutive activation of non-canonical NF-kappaB by targeting NIK for proteasomal degradation. However, in cIAP double knock-out cells TWEAK was still able to increase NIK levels demonstrating that NIK can be regulated by cIAP-independent pathways. Finally we show that, unlike IAP antagonists, TWEAK was able to induce degradation of cIAP1 RING mutants. These results emphasize the critical importance of the RING of cIAP1 in many signaling scenarios, but also demonstrate that in some pathways RING functions are not required.

On the fate of sexual traits under asexuality.

Environmental shifts and life-history changes may result in formerly adaptive traits becoming non-functional or maladaptive. In the absence of pleiotropy and other constraints, such traits may decay as a consequence of neutral mutation accumulation or selective processes, highlighting the importance of natural selection for adaptations. A suite of traits are expected to lose their adaptive function in asexual organisms derived from sexual ancestors, and the many independent transitions to asexuality allow for comparative studies of parallel trait maintenance versus decay. In addition, because certain traits, notably male-specific traits, are usually not exposed to selection under asexuality, their decay would have to occur as a consequence of drift. Selective processes could drive the decay of traits associated with costs, which may be the case for the majority of sexual traits expressed in females. We review the fate of male and female sexual traits in 93 animal lineages characterized by asexual reproduction, covering a broad taxon range including molluscs, arachnids, diplopods, crustaceans and eleven different hexapod orders. Many asexual lineages are still able occasionally to produce males. These asexually produced males are often largely or even fully functional, revealing that major developmental pathways can remain quiescent and functional over extended time periods. By contrast, for asexual females, there is a parallel and rapid decay of sexual traits, especially of traits related to mate attraction and location, as expected given the considerable costs often associated with the expression of these traits. The level of decay of female sexual traits, in addition to asexual females being unable to fertilize their eggs, would severely impede reversals to sexual reproduction, even in recently derived asexual lineages. More generally, the parallel maintenance versus decay of different trait types across diverse asexual lineages suggests that neutral traits display little or no decay even after extended periods under relaxed selection, while extensive decay for selected traits occurs extremely quickly. These patterns also highlight that adaptations can fix rapidly in natural populations of asexual organisms, in spite of their mode of reproduction.

MARCKS-related protein (MRP) is a substrate for the Leishmania major surface protease leishmanolysin (gp63).

Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP; MacMARCKS) are protein kinase C substrates in diverse cell types. Activation of murine macrophages by cytokines increases MRP expression, but infection with Leishmania promastigotes during activation results in MRP depletion. We therefore examined the effect of Leishmania major LV39 on recombinant MRP. Both live promastigotes and a soluble fraction of LV39 lysates degraded MRP to yield lower molecular weight fragments. Degradation was independent of MRP myristoylation and was inhibited by protein kinase C-dependent phosphorylation of MRP. MRP was similarly degraded by purified leishmanolysin (gp63), a Leishmania surface metalloprotease. Degradation was evident at low enzyme/substrate ratios, over a broad pH range, and was inhibited by 1,10-phenanthroline and by a hydroxamate dipeptide inhibitor of leishmanolysin. Using mass spectrometric analysis, cleavage was shown to occur within the effector domain of MRP between Ser(92) and Phe(93), in accordance with the substrate specificity of leishmanolysin. Moreover, an MRP construct in which the effector domain had been deleted was resistant to cleavage. Thus, Leishmania infection may result in leishmanolysin-dependent hydrolysis of MRP, a major protein kinase C substrate in macrophages.

Biological control of Biomphalaria tenagophila (Mollusca, Planorbidae), a schistosomiasis vector, using the fish Geophagus brasiliensis (Pisces, Cichlidae) in the laboratory or in a seminatural environment

In order to investigate a possible method of biological control of schistosomiasis, we used the fish Geophagus brasiliensis (Quoy & Gaimard, 1824) which is widely distributed throughout Brazil, to interrupt the life cycle of the snail Biomphalaria tenagophila (Orbigny, 1835), an intermediate host of Schistosoma mansoni. In the laboratory, predation eliminated 97.6% of the smaller snails (3-8 mm shell diameter) and 9.2% of the larger ones (12-14 mm shell diameter). Very promising results were also obtained in a seminatural environment. Studies of this fish in natural snail habitats should be further encouraged.

Combinatorial Synthesis and Biological Evaluation of Inhibitors of D-Ala-D-Ala-Ligase

Report for the scientific sojourn carried out at the Max Planck Institut of Molecular Phisiology, Germany, from 2006 to 2008.The work carried out during this postdoctoral stage was focused on two different projects. Firstly, identification of D-Ala D-Ala Inhibitors and the development of new synthethic approaches to obtain lipidated peptides and proteins and the use of these lipidated proteins in biological and biophysical studies. In the first project, new D-Ala D-Ala inhibitors were identified by using structural alignments of the ATP binding sites of the bacterial ligase DDl and protein and lipid kinases in complex with ATP analogs. We tested a series of commercially available kinase inhibitors and found LFM-A13 and Tyrphostine derivatives to inhibit DDl enzyme activity. Based on the initial screening results we synthesized a series of malononitrilamide and salicylamide derivatives and were able to confirm the validity of these scaffolds as inhibitors of DDl. From this investigation we gained a better understanding of the structural requirements and limitations necessary for the preparation of ATP competitive DDl inhibitors. The compounds in this study may serve as starting points for the development of bi-substrate inhibitors that incorporate both, an ATP competitive and a substrate competitive moiety. Bisubstrate inhibitors that block the ATP and D-Ala binding sites should exhibit enhanced selectivity and potency profiles by preferentially inhibiting DDl over kinases. In the second project, an optimized synthesis for tha alkylation of cysteins using the thiol ene reaction was establisehd. This new protocol allowed us to obtain large amounts of hexadecylated cysteine that was required for the synthesis of differently lipidated peptides. Afterwards the synthesis of various N-ras peptides bearing different lipid anchors was performed and the peptides were ligated to a truncated N-ras protein. The influence of this differently lipidated N-ras proteins on the partioning and association of N-Ras in model membrane subdomains was studied using Atomic Force Microscopy.

Estudos bionomicos de Cavernicola lenti Barrett & Arias, 1985 (Hemiptera, Reduviidae, Triatominae)

In the biological study, the development of the biological cycle of the insect under different feeding conditions was evaluated. The insects were fed on either mouse blood (c) or pigeon blood (p) using two types of rearing techniques (individual or group) at 28(graus) C and 90% relative humidity (which is equivalent to their natural environment). A fifth cycle studied, was fed on mouse blood reared on group, and mantained at laboratory environmental temperature. In the five cycles analyzed, it was found that groups on mouse blood at near natural conditions developed more rapidly (between 60 and 73 days) and had a lower rate of mortality (16.66%). The daily handling and changes in environment, of the individually reared insects, for observation of biological characteristics (no. and duration of bloodmeal, defecation and first fed of each stage) had a negative influence. None of the individuals fed on mouse or pigeon blood reached adult hood. It was found that the longevity and the fertility rate were significantly superior on couples maintained individually on mouse blood. These males had an average lifespan of 110,26 days and the females had an average lifespan of 104,46 days. The average number of eggs laid by each female was 21,26. Four couples kept in groups (five couples in each group) under the same condition, the longevity for males was 51,86 days and for females was 81,06 days. An average of 10,5 eggs were laid by each female. However, the percentage of fertile eggs was higher in couples kept in groups (72.15%) than in the individual couples (57.68%).