<Emphasis Type="Italic">In vitro</Emphasis> and cryogenic preservation of plant biodiversity in Brazil

The Brazilian biomes (Amazon, Atlantic Forest, Cerrado, Caatinga, Pantanal, and Pampa) comprise one of the highest levels of plant diversity in the world; however, non-sustainable practices, deforestation, and land use have resulted in significant losses and fragmentation of the native forests. These ecosystems are now threatened and protection of their native plants through ex situ conservation is an urgent necessity. Cryopreservation and in vitro conservation are complementary options for securing and protecting Brazil’s native plant species because their potential economic value is critically important to develop strategies that will (1) support their sustainable utilization, (2) protect against the over-exploitation of species growing in natural habitats, and (3) conserve the genetic diversity of germplasm from species of different provenances. Biotechnological approaches will help to address future economic and environmental demands placed upon already at-risk species. Conserving seed germplasm ex situ provides an additional safeguard against the risks (e.g., loss due to disease, climate change) of field conservation. Moreover, seed banks and cryobanks permit the long-term conservation of a wider genetic base; this offsets the labor and space intensive costs of conserving in the active growing state. This paper is a compilation of the current status of strategies applied for conserving Brazilian native plant species.     

Mucopolysaccharidoses in northern Brazil: Targeted mutation screening and urinary glycosaminoglycan excretion in patients undergoing enzyme replacement therapy

Mucopolysaccharidoses (MPS) are rare lysosomal disorders caused by the deficiency of specific lysosomal enzymes responsible for glycosaminoglycan (GAG) degradation. Enzyme Replacement Therapy (ERT) has been shown to reduce accumulation and urinary excretion of GAG, and to improve some of the patients' clinical signs. We studied biochemical and molecular characteristics of nine MPS patients (two MPS I, four MPS II and three MPS VI) undergoing ERT in northern Brazil. The responsiveness of ERT was evaluated through urinary GAG excretion measurements. Patients were screened for eight common MPS mutations, using PCR, restriction enzyme tests and direct sequencing. Two MPS I patients had the previously reported mutation p.P533R. In the MPS II patients, mutation analysis identified the mutation p.R468W, and in the MPS VI patients, polymorphisms p.V358M and p.V376M were also found. After 48 weeks of ERT, biochemical analysis showed a significantly decreased total urinary GAG excretion in patients with MPS I (p < 0.01) and MPS VI (p < 0.01). Our findings demonstrate the effect of ERT on urinary GAG excretion and suggest the adoption of a screening strategy for genotyping MPS patients living far from the main reference centers.     

Guanidine Alkaloids from Monanchora arbuscula: Chemistry and Antitumor Potential

Five guanidine alkaloids, mirabilin B ( 1 ), 8bβ‐hydroxyptilocaulin ( 2 ), ptilocaulin ( 3 ), and a mixture of the 8β‐ and 8α‐epimers, 4 and 5 , of 8‐hydroxymirabilin (1,8a;8b,3a‐didehydro‐8‐hydroxyptilocaulin), were isolated from Monanchora arbuscula colonies collected off the northeastern Brazilian coast. All structures were elucidated by spectroscopic analysis, including 1D (¹H‐, ¹³C‐ (BB), and ¹³C‐DEPT) and 2D (COSY, HSQC, and HMBC) NMR experiments, and comparison with the literature data. The cytotoxicity of the isolated compounds were evaluated against four tumor cell lines, showing that mirabilin B ( 1 ) and the two epimers were inactive, while 8bβ‐hydroxyptilocaulin ( 2 ) and ptilocaulin ( 3 ) presented IC₅₀ values in the range of 7.9 to 61.5 μM , and 5.8 to 40.0 μM , respectively. Further studies on the mechanism of action of ptilocaulin, using HL‐60 leukemia cells, demonstrated that this guanidine compound induced apoptosis of the treated cells.     

Multivariate analysis of morphological characteristics of two species of passion flower with ornamental potential and of hybrids between them

We estimated genetic parameters through multivariate analysis of two species of Passiflora and their hybrids, considered of ornamental potential, based on the morphological characteristics: flower diameter, corona diameter, corona filament size, flower peduncle length, petal length and width, sepal length and width, internode length, stem diameter, leaf length, leaf width (mm), and leaf area (cm(2)). Five specimens of Passiflora sublanceolata [ex P. palmeri var. sublanceolata], five of P. foetida var. foetida and 20 F(1) hybrids between the two were evaluated. A randomized block design with four replications was used. The data were submitted to variance analysis and multivariate procedures, principal components analysis and unweighted pair group method with arithmetic mean grouping. We found significant differences between genotypes for all these morphological parameters. The hybrid plants had the highest variability, making them the most indicated for future improvement programs. The various multivariate techniques gave similar results, allowing separation of the plants into three distinct groups, these being the two paternal species and the hybrids. The hybrids were closer to the male genitors, revealing a paternal effect on the inheritance of vegetative and floral characters. Based on estimates of genetic parameters, the floral characteristics are the most indicated for the selection of plants for ornamental purposes, since these characteristics displayed greatest variability, a variation index of more than one, and high genotypic determination coefficients.     

International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients

Background

A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation.

Methodology/Findings

An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05–0.5 parasites/mL whereas specific kDNA tests detected 5.10⁻³ par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3–94.4%, specificity of 85–95%, accuracy of 86.8–89.5% and kappa index of 0.7–0.8 compared to consensus PCR reports of the 16 good performing tests and 63–69%, 100%, 71.4–76.2% and 0.4–0.5, respectively compared to serodiagnosis. Method LbD2 used solvent extraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extraction followed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extraction followed by TaqMan Real Time PCR targeted to Sat-DNA (cruzi 1/cruzi 2 and cruzi 3 TaqMan probe) and the fourth method (LbQ) used solvent DNA extraction followed by conventional hot-start PCR targeted to kDNA (primer pairs 121/122). These four methods were further evaluated at the coordinating laboratory in a subset of human blood samples, confirming the performance obtained by the participating laboratories.

Conclusion/Significance

This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.     

Tenascin-C in the extracellular matrix promotes the selection of highly proliferative and tubulogenesis-defective endothelial cells

The extracellular matrix (ECM) contains important cues for tissue homeostasis and morphogenesis. The matricellular protein tenascin-C (TN-C) is overexpressed in remodeling tissues and cancer. In the present work, we studied the effect of different ECM—which exhibited a significant diversity in their TN-C content—in endothelial survival, proliferation and tubulogenic differentiation: autologous (endothelial) ECM devoid of TN-C, but bearing large amounts of FN; fibroblast ECM, bearing both high TN-C and FN contents; and finally, glioma-derived matrices, usually poor in FN, but very rich in TN-C. HUVECs initially adhered to the immobilized matrix produced by U373 MG glioma cells, but significantly detached and died by anoikis (50 to 80%) after 24 h, as compared with cells incubated with endothelial and fibroblast matrices. Surviving endothelial cells (20 to 50%) became up to 6-fold more proliferative and formed 74–97% less tube-like structures in vitro than cells grown on non-tumoral matrices. An antibody against the EGF-like repeats of tenascin-C (TN-C) partially rescued cells from the tubulogenic defect, indicating that this molecule is responsible for the selection of highly proliferative and tubulogenic defective endothelial cells. Interestingly, by using defined substrata, in conditions that mimic glioma and normal cell ECM composition, we observed that fibronectin (FN) modulates the TN-C-induced selection of endothelial cells. Our data show that TN-C is able to modulate endothelial branching morphogenesis in vitro and, since it is prevalent in matrices of injured and tumor tissues, also suggest a role for this protein in vascular morphogenesis, in these physiological contexts.