A character‐based approach in the Mexican cycads supports diverse multigene combinations for DNA barcoding

A DNA barcoding study was conducted to determine the optimal combination of loci needed for successful species‐level molecular identification in three extant cycad genera—Ceratozamia, Dioon, and Zamia—that occur in Mexico. Based on conclusions of a previous multigene study in representative species of all genera in the Cycadales, we tested the DNA barcoding performance of seven chloroplast coding (matK, rpoB, rpoC1, and rbcL) and non‐coding (atpF/H, psbK/I, and trnH‐psbA) regions, plus sequences of the nuclear internal transcribed spacer. We analysed data under the assumptions of the “character attributes organization system” (CAOS), a character‐based approach in which species are identified through the presence of ‘DNA diagnostics’. In Ceratozamia, four chloroplast regions and one nuclear region were needed to achieve > 70% unique species identification. In contrast, the two‐gene combination atpF/H + psbK/I and the four‐gene combination atpF/H + psbK/I + rpoC1 + ITS2 were needed to reach 79% and 75% unique species identification in Dioon and Zamia, respectively. The combinations atpF/H + psbK/I and atpF/H + psbK/I + rpoC1 + ITS2 include loci previously considered by the international DNA barcoding community. However, none of the three combinations of potential DNA barcoding loci found to be optimal with a character‐based approach in the Mexican cycads coincides with the ‘core barcode’ of chloroplast markers (matK + rbcL) recently proposed for universal use in the plant kingdom.     

β-Gal gene expression MRI reporter in melanoma tumor cells. Design, synthesis, and in vitro and in vivo testing of a Gd(III) containing probe forming a high relaxivity, melanin-like structure upon β-Gal enzymatic activation

The aim of this work is to design and test an MRI probe (Gd-DOTAtyr-gal) able to report on the gene expression of β-galactosidase (β-Gal) in melanoma cells. The probe consists of a Gd-DOTA reporter bearing on its surface a tyrosine-galactose-pyranose functionality that, upon the release of the sugar moiety, readily transforms, in the presence of tyrosinase, into melanin oligomeric/polymeric mixture. The formation of Gd-DOTA-containing melanin oligomers and polymers is accompanied by a marked increase of the water proton relaxation rate. The steps involving the release of the galactose-pyranose group and the formation of the melanin-like structure have been carefully investigated in vitro by relaxometric and UV-vis measurements. Cellular uptake studies of Gd-DOTAtyr-gal by melanoma cells have shown that the probe enters the cells, and it appears not to be confined in endosomal vesicles. Using B16-F10LacZ transfected cells, the fast formation of paramagnetic melanin-Gd(III)-containing species has been assessed by the measurement of increased longitudinal relaxation rates of the cellular pellets suspensions. The in vitro results have been confirmed in in vivo MRI investigations on murine melanoma tumor bearing mice. Upon direct injection of Gd-DOTAtyr-gal, a good contrast is observed after 5 h post injection in B16-F10LacZ tumors, but not in B16-F10 tumors lacking the β-Gal enzyme. Gd-DOTAtyr-gal in combination with tyrosinase introduces a novel approach for the detection of β-Gal expression by MRI in vivo.     

Genetic diversity of the genus Malus and implications for linkage mapping with SNPs

Knowledge about the sequence-based genetic diversity of a crop species is important in order to develop highly informative genotyping assays, which will eventually positively impact breeding practice. Diversity data were obtained from two pools of 185 and 75 accessions each, representing most of the species belonging to the genus Malus, by re-sequencing 27 gene-specific amplicons and by screening 237 Malus × domestica SNPs using the multiplex genotyping technology SNPlex™. Nucleotide diversity and insertion/deletion rates in M. × domestica were estimated as π = 0.0037 and 1/333 bp, respectively. The SNP frequency was estimated as 0.0194 (1 SNP/52 bp) while within a single apple cultivar an average of one SNP in every 455 bp was found. We also investigated transferability (T _SNP) of the heterozygous state of SNPs across the species M. × domestica and the genus Malus. Raw re-sequencing showed that 12–15% of M. × domestica SNPs are transferable to a second M. × domestica cultivar, however T _SNP rose to ∼41% with SNPs selected for high minor allele frequency. T _SNP of chosen SNPs averaged ∼27% in the two M. × domestica-related species, Malus sieversii and Malus sylvestris, but was much lower in more distantly related species. On the basis of T _SNP, simulations, and empirical results, we calculated that a close-design, multiplexed genotyping array with at least 2,000 SNPs is required for building a highly saturated linkage maps within any M. × domestica cross. The same array would gradually lose informativeness in increasingly phylogenetically distant Malus species.     

The Brazilian legal framework on the scientific use of animals

Brazil has an exceptionally dynamic research sector in Latin America in health, biotechnology, and pharmacology, backed by defined government policies on science and technology and a health research agenda focusing on important neglected diseases: malaria, leishmaniasis, Chagas disease, turberculosis, leprosy, and dengue. The Brazilian health research policy promotes partnerships and networks among scientists in academic institutions in both wealthy industrialized and disease-endemic countries, and in these efforts the government's guidelines for animal use in biomedical research are considered fundamental to guarantee both animal welfare and the quality of research. Given international discussions of animal experimentation regulations and guidelines, in this article we describe current Brazilian legislation governing the use of animals in scientific investigations. We conclude that, despite advances in the implementation of the 3Rs (reduction, refinement, replacement), the new regulatory framework does not sufficiently incorporate ethical considerations, lacking explicit reference to the 3Rs as well as measures for their full application. The more humane use of animals in research will depend on the approach adopted by Brazil's National Council for the Control of Animal Experimentation to promote the 3Rs and to improve internal regulations as well as data collection and analysis in research institutions. In Brazil as elsewhere, one of the greatest challenges to policymakers is to harmonize the myriad and intertwined legal provisions without hindering biomedical research.     

Drosophila katanin is a microtubule depolymerase that regulates cortical-microtubule plus-end interactions and cell migration

Regulation of microtubule dynamics at the cell cortex is important for cell motility, morphogenesis and division. Here we show that the Drosophila katanin Dm-Kat60 functions to generate a dynamic cortical-microtubule interface in interphase cells. Dm-Kat60 concentrates at the cell cortex of S2 Drosophila cells during interphase, where it suppresses the polymerization of microtubule plus-ends, thereby preventing the formation of aberrantly dense cortical arrays. Dm-Kat60 also localizes at the leading edge of migratory D17 Drosophila cells and negatively regulates multiple parameters of their motility. Finally, in vitro, Dm-Kat60 severs and depolymerizes microtubules from their ends. On the basis of these data, we propose that Dm-Kat60 removes tubulin from microtubule lattice or microtubule ends that contact specific cortical sites to prevent stable and/or lateral attachments. The asymmetric distribution of such an activity could help generate regional variations in microtubule behaviours involved in cell migration.     

A study on the pretreatment of a sugarcane bagasse sample with dilute sulfuric acid

Experiments based on a 2³ central composite full factorial design were carried out in 200-ml stainless-steel containers to study the pretreatment, with dilute sulfuric acid, of a sugarcane bagasse sample obtained from a local sugar–alcohol mill. The independent variables selected for study were temperature, varied from 112.5°C to 157.5°C, residence time, varied from 5.0 to 35.0 min, and sulfuric acid concentration, varied from 0.0% to 3.0% (w/v). Bagasse loading of 15% (w/w) was used in all experiments. Statistical analysis of the experimental results showed that all three independent variables significantly influenced the response variables, namely the bagasse solubilization, efficiency of xylose recovery in the hemicellulosic hydrolysate, efficiency of cellulose enzymatic saccharification, and percentages of cellulose, hemicellulose, and lignin in the pretreated solids. Temperature was the factor that influenced the response variables the most, followed by acid concentration and residence time, in that order. Although harsher pretreatment conditions promoted almost complete removal of the hemicellulosic fraction, the amount of xylose recovered in the hemicellulosic hydrolysate did not exceed 61.8% of the maximum theoretical value. Cellulose enzymatic saccharification was favored by more efficient removal of hemicellulose during the pretreatment. However, detoxification of the hemicellulosic hydrolysate was necessary for better bioconversion of the sugars to ethanol.     

Fix your eyes in the space you could reach: neurons in the macaque medial parietal cortex prefer gaze positions in peripersonal space

Interacting in the peripersonal space requires coordinated arm and eye movements to visual targets in depth. In primates, the medial posterior parietal cortex (PPC) represents a crucial node in the process of visual-to-motor signal transformations. The medial PPC area V6A is a key region engaged in the control of these processes because it jointly processes visual information, eye position and arm movement related signals. However, to date, there is no evidence in the medial PPC of spatial encoding in three dimensions. Here, using single neuron recordings in behaving macaques, we studied the neural signals related to binocular eye position in a task that required the monkeys to perform saccades and fixate targets at different locations in peripersonal and extrapersonal space. A significant proportion of neurons were modulated by both gaze direction and depth, i.e., by the location of the foveated target in 3D space. The population activity of these neurons displayed a strong preference for peripersonal space in a time interval around the saccade that preceded fixation and during fixation as well. This preference for targets within reaching distance during both target capturing and fixation suggests that binocular eye position signals are implemented functionally in V6A to support its role in reaching and grasping.