Increasing genomic information in bivalves through new EST collections in four species: development of new genetic markers for environmental studies and genome evolution

The generation of EST information is an essential step in the genomic characterisation of species. In the context of the European Network Marine Genomics, a common goal was to significantly increase the amount of ESTs in commercial marine mollusk species and more specifically in the less studied but ecologically and commercially important groups, such as mussel and clam genera. Normalized cDNA libraries were constructed for four different relevant bivalves species (Crassostrea gigas, Mytilus edulis, Ruditapes decussatus and Bathymodiolus azoricus), using numerous tissues and physiological conditions. In this paper, we present the analysis of the 13,013 expressed sequence tags (ESTs) generated. Each EST library was independently assembled and 1300-3000 unique sequences were identified in each species. For the different species, functional categories could be assigned to only about 16 to 27% of ESTs using the GO annotation tool. All sequences have been incorporated into a publicly available database and form the basis for subsequent microarray design, SNP detection and polymorphism analysis, and the placement of novel markers on genetic linkage maps.     

Following Darwin’s footsteps: Evaluating the impact of an activity designed for elementary school students to link historically important evolution key concepts on their understanding of natural selection

While several researchers have suggested that evolution should be explored from the initial years of schooling, little information is available on effective resources to enhance elementary school students’ level of understanding of evolution by natural selection (LUENS). For the present study, we designed, implemented, and evaluated an educational activity planned for fourth graders (9 to 10 years old) to explore concepts and conceptual fields that were historically important for the discovery of natural selection. Observation field notes and students’ productions were used to analyze how the students explored the proposed activity. Additionally, an evaluation framework consisting of a test, the evaluation criteria, and the scoring process was applied in two fourth‐grade classes (N = 44) to estimate elementary school students’ LUENS before and after engaging in the activity. Our results show that our activity allowed students to link the key concepts, resulting in a significant increase of their understanding of natural selection. They also reveal that additional activities and minor fine‐tuning of the present activity are required to further support students’ learning about the concept of differential reproduction.     

Detection of Mineralized Structures in Early Stages of Development of Marine Teleostei Using a Modified Alcian Blue-Alizarin Red Double Staining Technique for Bone and Cartilage

We have developed a procedure for staining cartilage and bone in fish larvae as small as 2 mm (notochord length), for which standard alcian blue/alizarin red procedures did not give positive and/or consistent results. Small calcified structures only 100-200 ixm in length can be clearly visualized. The method is suitable for both onto-genic studies during early stages of skeletal development in most marine fishes (e.g., Sporus aurata L., Solea senegalensis Kaup), whose larvae at hatching are often only a few millimeters long and for detecting skeletal abnormalities in small larvae. This procedure can also be used for specimens that have been preserved in 100% ethanol for up to two years.     

Enhanced DNA transfer into fish bone cells using polyethylenimine

The use of in vitro cell culture systems to assess gene function largely depends on the successful transfer of DNA into target cells. Well developed in mammals, transfection methods are still to be optimized for non-mammalian cell culture systems, like fish. Here we describe a rapid, cost-efficient, and successful method to transfer DNA into a fish bone-derived cell line using polyethylenimine (PEI) as the DNA carrier. Using this method, DNA transfer was remarkably enhanced in comparison with commercially available reagents, as demonstrated by the increased activity of both luciferase and green fluorescent protein observed in the transfected cells. Its efficiency in transferring DNA intoa wide range of cell types, including non-mammalian and hard-to-transfect cells, in addition to a low cost, show that PEI is a reagent of choice for nonviral vector transfection.     

Nitric oxide biosynthesis by Leishmania amazonensis promastigotes containing a high percentage of metacyclic forms

Due to the diversity of its physiological and pathophysiological functions and general ubiquity, the study of nitric oxide (NO) has become of great interest. In this work, it was demonstrated that Leishmania amazonensis promastigotes produces NO, a free radical synthesized from l-arginine by nitric oxide synthase (NOS). A soluble NOS was purified from L. amazonensis promastigotes by affinity chromatography (2′, 5′-ADP-agarose) and on SDS-PAGE the enzyme migrates as a single protein band of 116.2 (±6) kDa. Furthermore, the presence of a constitutive NOS was detected through indirect immunofluorescence using anti-cNOS and in NADPH consumption assays. The present work show that NO production, detected as nitrite in culture supernatant, is prominent in promastigotes preparations with high number of metacyclic forms, suggesting an association with the differentiation and the infectivity of the parasite.     

Synthesis and biological evaluation of tricarbonyl Re(I) and Tc(I) complexes anchored by poly(azolyl)borates: application on the design of radiopharmaceuticals for the targeting of 5-HT<Subscript>1A</Subscript> receptors

The building blocks fac-[^99mTc{κ³-HB(tim^Me)₃}(CO)₃] and fac-[^99mTc{κ³-R(μ-H)B(tim^Me)₂}(CO)₃] [R is H (4a), Ph (5a); tim^Me is 2-mercapto-1-methylimidazolyl] were obtained almost quantitatively by reacting fac-[^99mTc(CO)₃(H₂O)₃]⁺ with the corresponding scorpionate. These compounds cross the intact blood–brain barrier in mice, with significant retention in the case of 4a and 5a. Using 4a as the lead structure, we have synthesized the functionalized complexes fac-[M{κ³-H(μ-H)B(tim^Bu-pip)₂}(CO)₃] [M is Re (8), ^99mTc (8a); tim^Bu-pip is methyl[4-((2-methoxyphenyl)-1-piperazinyl)butyl](2-mercapto-1-methylimidazol-5-yl)methanamide] and fac-[M{κ ³-H(μ-H)B(tim^Me)(tim^Bu-pip)}(CO)₃] [M is Re (9), ^99mTc (9a)] and evaluated their potential as radioactive probes for the targeting of brain 5-HT_1A serotonergic receptors. The Re complexes exhibit excellent affinity [IC₅₀=0.172 ± 0.003 nM (8); IC₅₀=0.65 ± 0.01 nM (9)] for the 5-HT_1A receptor. The radioactive congeners (^99mTc) have shown an initial brain uptake of 1.38 ± 0.46%ID g⁻¹ (8a) and 0.43 ± 0.12%ID g⁻¹ (9a), but suffer from a relatively fast washout.     

Infection-associated FUT2 (Fucosyltransferase 2) genetic variation and impact on functionality assessed by in vivo studies

The secretor (Se)/nonsecretor (se) histo-blood group variation depends on the action of the FUT2 enzyme and has major implications for human susceptibility to infections. To characterize the functionality of FUT2 variants, we assessed the correlation between saliva phenotypes and sequence variation at the FUT2 gene in sixty seven individuals from northern Portugal. While most non-secretor haplotypes were found to carry the 428G > A nonsense mutation in association with a 739G > A missense substitution, we have also identified a recombinant haplotype carrying the 739*A allele together with the efficient 428*G variant in individuals with the Se phenotype. This finding suggested, in contrast to previous results, that the 739*A allele encodes an efficient Se allele. To test this hypothesis we evaluated the in vivo enzyme activity of full coding expression constructs in transient transfection of CHO-K1 cells using FACS (fluorescence-activated cell sorting) analysis and expression of type 2 and type 3 chain H structures as read out. We detected FUT2 activity for the 739*A expression construct, demonstrating that the 739G > A substitution is indeed not inactivating. In accordance with the hypothesis that FUT2 is under long standing balancing selection, we estimated that the time depth of FUT2 global genetic variation is as old as 3 million years. Age estimates of specific variants suggest that the 428G > A mutation occurred at least 1.87 million years ago while the 739G > A substitution is about 816,000 years old. The 385A > T missense mutation underlying the non-secretor phenotype in East Asians appears to be more recent and is likely to have occurred about 256,000 years ago.     

Effect of Mexican propolis extracts from Apis mellifera on Candida albicans in vitro growth

Propolis is a resinous substance collected by bees (Apis mellifera) from different trees and bushes. Due to its antifungal, antibacterial, antiviral and antiparasitic properties, it has continued to be very popular throughout the time showing variable activity depending on its geographical origin. In Mexico, information about this product is very limited. The aim of this work was to evaluate the antifungal activity of four propolis ethanolic extracts from three different Mexican states, and four commercial extracts on Candida albicans growth. A reference strain (ATCC 10231) and 36 clinical isolates of C. albicans were used. The Minimal Inhibitory Concentration (MIC) was determined by the dilution on agar method. Growth curves on Sabouraud Dextrose broth with and without different propolis ethanolic extracts concentrations were performed. In addition, whether the effect was fungistatic or fungicide was determined. The propolis ethanolic extract obtained from Cuautitlán Izcalli, State of Mexico, showed the best biological activity, inhibiting 94.4% from the clinical isolates at 0.8 mg/ml; the reference strain was inhibited at 0.6 mg/ml. The propolis effect was fungistatic in low concentrations and fungicide in concentrations higher to MIC. The Mexican propolis ethanolic extract could be further investigated for its alternative use for the treatment of some C. albicans infections.     

Antifungal and cytotoxic activities of some N-substituted aniline derivatives bearing a hetaryl fragment

Diverse N-substituted anilines bearing hetaryl fragments were easily prepared from corresponding aldimines derived from commercially available aromatic aldehydes and anilines. 2-Furyl substituted anilines showed very good antifungal activities against dermatophytes, particularly against Trichophyton rubrum (MIC=3.12-6.25microg/mL). In addition, all active compounds, 45-47, 73, and 74, were tested for cytotoxic activities against breast (MCF-7), lung (H-460), and central nervous system (SF-268) human cancer cell lines with the NCI-anticancer-drug screen. The activity of amines described in this paper, along with the low toxicity of most of them, shows promise for the future development of non-toxic new antimycotic agents.