Survey of Sand Flies (Diptera: Psychodidae) in an Environmentally Protected Area in Brazil

Brazil is one of the most important endemic areas for leishmaniasis worldwide. Protected areas that are tourist attractions likely present an important risk of transmission of cutaneous leishmaniasis (CL). Furthermore, with the geographical expansion of visceral leishmaniasis (VL), several studies have recorded the occurrence of its vector, Lutzomyia longipalpis, and cases of human and canine VL in such tourist areas. The Parque Estadual do Sumidouro is an environmentally protected area located in the Brazilian Cerrado biome and in an important area endemic for leishmaniasis in the state of Minas Gerais. The purpose of this study was to monitor the sand fly fauna in areas of tourist activity in the park. Sampling was performed every month, from September 2011 to August 2013, using CDC light traps at six sites of differing environmental characteristics. Sampled specimens were identified following Galati (2003), and females were submitted to molecular techniques for the detection and identification of Leishmania DNA. A total of 4,675 sand fly specimens of 25 species belonging to nine genera were collected. The most abundant species were Micropygomyia quinquefer, Lutzomyia renei and Pintomyia pessoai, although only Pi. pessoai is implicated in the transmission of Leishmania braziliensis. The species accumulation curve reached saturation on the 16th sampling event. Species richness, diversity and evenness differed among the sampled areas. The seasonal curve was not determined by a single unique species, and no single species was the most abundant in all environments sampled. The main vector of Leishmania (Leishmania) infantum, Lutzomyia longipalpis, accounted for only 5.35% of the specimens collected. Proven or suspected vectors of Leishmania (Viannia) braziliensis were recorded, and one female of the cortellezzii complex tested positive for Le. braziliensis DNA. Even with a low infection rate (0.62%), these data indicate the circulation of the parasite and reinforce the need for entomological and epidemiological surveillance in the park and its surroundings.     

A Multiplex Assay for Detection of Staphylococcal and Streptococcal Exotoxins

Staphylococcal and streptococcal exotoxins, also known as superantigens, mediate a range of diseases including toxic shock syndrome, and they exacerbate skin, pulmonary and systemic infections caused by these organisms. When present in food sources they can cause enteric effects commonly known as food poisoning. A rapid, sensitive assay for the toxins would enable testing of clinical samples and improve surveillance of food sources. Here we developed a bead-based, two-color flow cytometry assay using single protein domains of the beta chain of T cell receptors engineered for high-affinity for staphylococcal (SEA, SEB and TSST-1) and streptococcal (SpeA and SpeC) toxins. Site-directed biotinylated forms of these high-affinity agents were used together with commercial, polyclonal, anti-toxin reagents to enable specific and sensitive detection with SD₅₀ values of 400 pg/ml (SEA), 3 pg/ml (SEB), 25 pg/ml (TSST-1), 6 ng/ml (SpeA), and 100 pg/ml (SpeC). These sensitivities were in the range of 4- to 80-fold higher than achieved with standard ELISAs using the same reagents. A multiplex format of the assay showed reduced sensitivity due to higher noise associated with the use of multiple polyclonal agents, but the sensitivities were still well within the range necessary for detection in food sources or for rapid detection of toxins in culture supernatants. For example, the assay specifically detected toxins in supernatants derived from cultures of Staphylococcus aureus. Thus, these reagents can be used for simultaneous detection of the toxins in food sources or culture supernatants of potential pathogenic strains of Staphylococcus aureus and Streptococcus pyogenes.     

“Vision for Action” in Young Children Aligning Multi-Featured Objects: Development and Comparison with Nonhuman Primates

Effective vision for action and effective management of concurrent spatial relations underlie skillful manipulation of objects, including hand tools, in humans. Children’s performance in object insertion tasks (fitting tasks) provides one index of the striking changes in the development of vision for action in early life. Fitting tasks also tap children’s ability to work with more than one feature of an object concurrently. We examine young children’s performance on fitting tasks in two and three dimensions and compare their performance with the previously reported performance of adult individuals of two species of nonhuman primates on similar tasks. Two, three, and four year-old children routinely aligned a bar-shaped stick and a cross-shaped stick but had difficulty aligning a tomahawk-shaped stick to a matching cut-out. Two year-olds were especially challenged by the tomahawk. Three and four year-olds occasionally held the stick several inches above the surface, comparing the stick to the surface visually, while trying to align it. The findings suggest asynchronous development in the ability to use vision to achieve alignment and to work with two and three spatial features concurrently. Using vision to align objects precisely to other objects and managing more than one spatial relation between an object and a surface are already more elaborated in two year-old humans than in other primates. The human advantage in using hand tools derives in part from this fundamental difference in the relation between vision and action between humans and other primates.     

Extending glacial refugia for a European tree: genetic markers show that Iberian populations of white elm are native relicts and not introductions

Conservation policies usually focus on in situ protection of native populations, a priority that requires accurate assessment of population status. Distinction between native and introduced status can be particularly difficult (and at the same time, is most important) for species whose natural habitat has become both rare and highly fragmented. Here, we address the status of the white elm (Ulmus laevis Pallas), a European riparian tree species whose populations have been fragmented by human activity and is protected wherever it is considered native. Small populations of this species are located in Iberia, where they are unprotected because they are considered introductions due to their rarity. However, Iberia and neighbouring regions in southwestern France have been shown to support discrete glacial refuge populations of many European trees, and the possibility remains that Iberian white elms are native relicts. We used chloroplast RFLPs and nuclear microsatellites to establish the relationship between populations in Iberia and the Central European core distribution. Bayesian approaches revealed significant spatial structure across populations. Those in Iberia and southwestern France shared alleles absent from Central Europe, and showed spatial population structure within Iberia common in recognized native taxa. Iberian populations show a demographic signature of ancient population bottlenecks, while those in Central European show a signature of recent population bottlenecks. These patterns are not consistent with historical introduction of white elm to Iberia, and instead strongly support native status, arguing for immediate implementation of conservation measures for white elm populations in Spain and contiguous areas of southern France.     

High-yield production,refolding and a molecular modelling of the catalytic module of (1,3)-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucan (curdlan) synthase from <Emphasis Type="Italic">Agrobacterium</Emphasis> sp.

Biosynthesis of the (1,3)-β-d-glucan (curdlan) in Agrobacterium sp., is believed to proceed by the repetitive addition of glucosyl residues from UDP-glucose by a membrane-embedded curdlan synthase (CrdS) [UDP-glucose: (1,3)-β-d-glucan 3-β-d-glucosyltransferase; EC 2.4.1.34]. The catalytic module of CrdS (cm-CrdS) was expressed in good yield from a cDNA encoding cm-CrdS cloned into the pET-32a(+) vector, containing a coding region for thioredoxin, and from the Champion™ pET SUMO system that possesses a coding region of a small ubiquitin-related modifier (SUMO) partner protein. The two DNA fusions, designated pET-32a_cm-CrdS and SUMO_cm-CrdS were expressed as chimeric proteins. High yields of inclusion bodies were produced in E. coli and these could be refolded to form soluble proteins, using a range of buffers and non-detergent sulfobetaines. A purification protocol was developed, which afforded a one-step on-column refolding and simultaneous purification of the recombinant 6xHis-tagged SUMO_cm-CrdS protein. The latter protein was digested by a specific protease to yield intact cm-CrdS in high yields. The refolded SUMO_cm-CrdS protein did not exhibit curdlan synthase activity, but showed a circular dischroism spectrum, which had an α/β-type-like conformation. Amino acid sequences of tryptic fragments of the SUMO_cm-CrdS fusion and free cm-CrdS proteins, determined by MALDI/TOF confirmed that the full-length proteins were synthesized by E. coli, and that no alterations in amino acid sequences occurred. A three-dimensional model of cm-CrdS predicted the juxtaposition of highly conserved aspartates D156, D208, D210 and D304, and the QRTRW motif, which are likely to play roles in donor and acceptor substrate binding and catalysis.     

Would SYNTAX have been a positive trial if XIENCE V had been used instead of TAXUS?: A meta-analysis of a first-generation vs. a second-generation drug-eluting stent system

Treatment options for coronary revascularisation include percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG). In the ‘synergy between PCI with TAXUS and cardiac surgery (SYNTAX)’ trial, PCI and CABG using state-of-the-art techniques (using paclitaxel-eluting stents and arterial grafts, respectively) were compared in the treatment of complex coronary artery disease. In Syntax, PCI was inferior to CABG at one year, entirely due to an increased repeat intervention rate. We hypothesised that the use of a superior drug-eluting stent system could reduce the need for repeat intervention. (Neth Heart J 2010;18:451-3.)     

The second-shell metal ligands of human arginase affect coordination of the nucleophile and substrate

The active sites of eukaryotic arginase enzymes are strictly conserved, especially the first- and second-shell ligands that coordinate the two divalent metal cations that generate a hydroxide molecule for nucleophilic attack on the guanidinium carbon of l-arginine and the subsequent production of urea and l-ornithine. Here by using comprehensive pairwise saturation mutagenesis of the first- and second-shell metal ligands in human arginase I, we demonstrate that several metal binding ligands are actually quite tolerant to amino acid substitutions. Of >2800 double mutants of first- and second-shell residues analyzed, we found more than 80 unique amino acid substitutions, of which four were in first-shell residues. Remarkably, certain second-shell mutations could modulate the binding of both the nucleophilic water/hydroxide molecule and substrate or product ligands, resulting in activity greater than that of the wild-type enzyme. The data presented here constitute the first comprehensive saturation mutagenesis analysis of a metallohydrolase active site and reveal that the strict conservation of the second-shell metal binding residues in eukaryotic arginases does not reflect kinetic optimization of the enzyme during the course of evolution.     

Embryonic inbreeding depression varies among populations and by mating system in Witheringia solanacea (Solanaceae)

? Premise of the study: Embryonic inbreeding depression is a key influence on mating system evolution and can be difficult to estimate in self-incompatible species. A pollen chase experiment was used to estimate the magnitude of embryonic inbreeding depression in Costa Rican Witheringia solanacea, a species polymorphic for self-incompatibility (SI). In a pollen chase experiment, bud self-pollinations are followed after anthesis by outcross pollinations, with a comparable pair of outcross pollinations used as a control. Lowered seed set for the self-precedence treatment indicates embryonic inbreeding depression. ? Methods: Embryonic inbreeding depression was assayed for self-compatible (SC) individuals and for SI plants from two populations that differ quantitatively in the onset and enzymatic activity of their SI response. Microsatellite markers were used to assay the selfing rate of a sample of surviving progeny from the prior self-pollination treatment. ? Key results: SC individuals showed no evidence of embryonic inbreeding depression. In SI plants, prior self-pollination reduced seed number by 28-70%, depending on population. Microsatellite genotyping revealed that embryonic inbreeding depression was even more severe than estimated by the phenotypic data: for mature fruits resulting from self-pollination precedence, the majority of the progeny were the result of outcross fertilization. ? Conclusions: Lineage-specific purging of recessive lethals has accompanied the evolution of SC in this species. SI populations show contrasting levels of embryonic inbreeding depression, with nearly complete embryonic lethality upon selfing in the Monteverde population. In the face of high embryonic inbreeding depression, an increase in selfing rate can evidently occur only under severe pollen limitation.