Sand flies (Diptera: Psychodidae) in a Cerrado area of the Maranhão state, Brazil

The present paper aims to increase the knowledge on the sand fly fauna in the cerrado areas of Maranh?o state in urban, rural and forest environments. The research was carried out from October 2007 to September 2008, between 18:00h and 06:00h, in the municipality of Chapadinha, northeast Maranh?o. For insect sampling, CDC light traps were set up in peridomicile and domicile areas of urban and rural zones as well as in Cerrado and Gallery forests. The total of 1,401 specimens belonging to 17 species were sampled, all within the genus Lutzomyia. Lutzomyia longipalpis (Lutz & Neiva) (52.5%), Lu. evandroi (Costa Lima & Antunes) (18.3%), Lu. whitmani (Antunes & Coutinho) (12.1%), Lu. lenti (Mangabeira) (4.7%) and Lu. termitophila (Martins, Falc?o & Silva) (4.0%) were the most frequently collected. From an epidemiological viewpoint, five from all of the collected species are vectors of leishmaniasis: Lu. longipalpis, Lu. whitmani, Lu. flaviscutellata (Mangabeira), Lu. gomezi (Nitzulescu) and Lu. chagasi (Costa Lima). Lutzomyia chagasi was registered for the first time in Maranh?o state and Lu. saulensis (Floch & Abonnenc), Lu. monstruosa (Floch & Abonnenc) and Lu. gomezi were found for the first time in the eastern part of the state, since they had been reported only in the Amazonian region of Maranh?o. Regarding to the studied environments, the urban chicken house had the highest number of specimens collected (801), while the Gallery Forest was the most diverse (15 species). This study demonstrates that the northeast cerrado exhibits a mixed sand fly fauna characterized by an extremely important species vectors assortment involved in the epidemiological cycle of leishmaniasis in Maranh?o state.     

Cell-permeable peptides induce dose- and length-dependent cytotoxic effects

We have explored the threshold of tolerance of three unrelated cell types to treatments with potential cytoprotective peptides bound to Tat_48-57 and Antp_43-58 cell-permeable peptide carriers. Both Tat_48-57 and Antp_43-58 are well known for their good efficacy at crossing membranes of different cell types, their overall low toxicity, and their absence of leakage once internalised. Here, we show that concentrations of up to 100 μM of Tat_48-57 were essentially harmless in all cells tested, whereas Antp_43-58 was significantly more toxic. Moreover, all peptides bound to Tat_48-57 and Antp_43-58 triggered significant and length-dependent cytotoxicity when used at concentrations above 10 μM in all but one cell types (208F rat fibroblasts), irrespective of the sequence of the cargo. Absence of cytotoxicity in 208F fibroblasts correlated with poor intracellular peptide uptake, as monitored by confocal laser scanning fluorescence microscopy. Our data further suggest that the onset of cytotoxicity correlates with the activation of two intracellular stress signalling pathways, namely those involving JNK, and to a lesser extent p38 mitogen-activated protein kinases. These responses are of particular concern for cells that are especially sensitive to the activation of stress kinases. Collectively, these results indicate that in order to avoid unwanted and unspecific cytotoxicity, effector molecules bound to Tat_48-57 should be designed with the shortest possible sequence and the highest possible affinity for their binding partners or targets, so that concentrations below 10 μM can be successfully applied to cells without harm. Considering that cytotoxicity associated to Tat_48-57- and Antp_43-58 bound peptide conjugates was not restricted to a particular type of cells, our data provide a general framework for the design of cell-penetrating peptides that may apply to broader uses of intracellular peptide and drug delivery.     

Modeling the interaction between aldolase and the thrombospondin-related anonymous protein, a key connection of the malaria parasite invasion machinery

A complex molecular motor empowers substrate-dependent motility and host cell invasion in malaria parasites. The interaction between aldolase and the transmembrane adhesin thrombospondin-related anonymous protein (TRAP) transduces the motor force across the parasite surface. Here, we analyzed this interaction by using state-of-the-art flexible docking. Besides algorithms to account for induced fit in the side-chains of the Plasmodium falciparum aldolase (PfAldo) structure, we used additional in silico receptors modeled upon crystallographic structures of evolutionarily related aldolases to incorporate enzyme backbone flexibility, and to overcome structure inaccuracies due to the relatively low resolution (3.0 A) of the genuine PfAldo structure. Our results indicate that, in spite of multiple intermolecular contacts, only the six C-terminal residues of the TRAP cytoplasmic tail bind in an ordered manner to PfAldo. This portion of TRAP targets the PfAldo active site, with its n-1 Trp residue, which is essential for this interaction, buried within the PfAldo catalytic pocket. Docking of a TRAP peptide bearing a Trp to Ala mutation rendered the lower energy configurations either bound weakly outside the active site or not bound to PfAldo at all. The position of the bound TRAP peptide, and particularly the close proximity between the carbonyl of its n-2 Asp residue and the experimentally determined position of the phosphate-6 group of fructose 1,6-phosphate bound to mammalian aldolases, predicts an inhibitory effect of TRAP on catalysis. Enzymatic and TRAP-binding assays using mutant PfAldo molecules strongly support the overall structural model. These results might provide the initial framework for the identification of novel antiparasitic compounds.     

Direct versus indirect facilitation (herbivore mediated) among woody plants in a semiarid Chaco forest: A spatial association approach

In arid environments, direct facilitation (microhabitat amelioration) and indirect facilitation (‘associational resistance’ via protection from herbivory) among plants of different species may act simultaneously. Little is known about their relative effects. One way to disentangle the effects is by evaluating spatial associations. We examined the relative importance of these two mechanisms of facilitation in the semiarid Chaco vegetation of north‐central Argentina, through an eight‐way observational study in which we quantified the degree of spatial association between saplings of each of two key tree species, Schinopsis lorentzii (Anacardiaceae) and Aspidosperma quebracho‐blanco (Apocynaceae), with shrub neighbours either possessing spines or without spines and in both an ungrazed site and a site with a long history of cattle grazing. We analysed data across 400 subparcels at each site with spatial analysis by distance indices. Saplings of both tree species showed positive spatial associations with spiny shrubs in the grazed site but not in the ungrazed site, and never with non‐spiny shrubs. This result suggests that spiny shrubs may indeed provide associational resistance for saplings of key tree species in grazed habitats in these dry subtropical forests, that is, that indirect facilitation may predominate over direct facilitation. If confirmed by experimental studies, this result can have implications for the silvopastoral management of rapidly expanding ranches in the semiarid Chaco, where current practice includes the near elimination of native shrubs.     

A novel series of IKKβ inhibitors part II: description of a potent and pharmacologically active series of analogs

A novel series of (E)-1-((2-(1-methyl-1H-imidazol-5-yl) quinolin-4-yl) methylene) thiosemicarbazides was discovered as potent inhibitors of IKKβ. In this Letter we document our efforts at further optimization of this series, culminating in 2 with submicromolar potency in a HWB assay and efficacy in a CIA mouse model.     

Immobilized pH gradient-driven paper-based IEF: a new method for fractionating complex peptide mixtures before MS analysis

Introduction  

The vast difference in the abundance of different proteins in biological samples limits the determination of the complete proteome of a cell type, requiring fractionation of proteins and peptides before MS analysis.     

Mcl-1 downregulation by pro-inflammatory cytokines and palmitate is an early event contributing to β-cell apoptosis

Pancreatic β-cell apoptosis is a key feature of diabetes mellitus and the mitochondrial pathway of apoptosis is a major mediator of β-cell death. We presently evaluated the role of the myeloid cell leukemia sequence 1 (Mcl-1), an antiapoptotic protein of the Bcl-2 family, in β-cells following exposure to well-defined β-cell death effectors, for example, pro-inflammatory cytokines, palmitate and chemical endoplasmic reticulum (ER) stressors. All cytotoxic stresses rapidly and preferentially decreased Mcl-1 protein expression as compared with the late effect observed on the other antiapoptotic proteins, Bcl-2 and Bcl-xL. This was due to ER stress-mediated inhibition of translation through eIF2α phosphorylation for palmitate and ER stressors and through the combined action of translation inhibition and JNK activation for cytokines. Knocking down Mcl-1 using small interference RNAs increased apoptosis and caspase-3 cleavage induced by cytokines, palmitate or thapsigargin, whereas Mcl-1 overexpression partly prevented Bax translocation to the mitochondria, cytochrome c release, caspase-3 cleavage and apoptosis induced by the β-cell death effectors. Altogether, our data suggest that Mcl-1 downregulation is a crucial event leading to β-cell apoptosis and provide new insights into the mechanisms linking ER stress and the mitochondrial intrinsic pathway of apoptosis. Mcl-1 is therefore an attractive target for the design of new strategies in the treatment of diabetes.     

Human anti-V3 HIV-1 monoclonal antibodies encoded by the VH5-51/VL lambda genes define a conserved antigenic structure

Preferential usage of immunoglobulin (Ig) genes that encode antibodies (Abs) against various pathogens is rarely observed and the nature of their dominance is unclear in the context of stochastic recombination of Ig genes. The hypothesis that restricted usage of Ig genes predetermines the antibody specificity was tested in this study of 18 human anti-V3 monoclonal Abs (mAbs) generated from unrelated individuals infected with various subtypes of HIV-1, all of which preferentially used pairing of the VH5-51 and VL lambda genes. Crystallographic analysis of five VH5-51/VL lambda-encoded Fabs complexed with various V3 peptides revealed a common three dimensional (3D) shape of the antigen-binding sites primarily determined by the four complementarity determining regions (CDR) for the heavy (H) and light (L) chains: specifically, the H1, H2, L1 and L2 domains. The CDR H3 domain did not contribute to the shape of the binding pocket, as it had different lengths, sequences and conformations for each mAb. The same shape of the binding site was further confirmed by the identical backbone conformation exhibited by V3 peptides in complex with Fabs which fully adapted to the binding pocket and the same key contact residues, mainly germline-encoded in the heavy and light chains of five Fabs. Finally, the VH5-51 anti-V3 mAbs recognized an epitope with an identical 3D structure which is mimicked by a single mimotope recognized by the majority of VH5-51-derived mAbs but not by other V3 mAbs. These data suggest that the identification of preferentially used Ig genes by neutralizing mAbs may define conserved epitopes in the diverse virus envelopes. This will be useful information for designing vaccine immunogen inducing cross-neutralizing Abs.