Wide spread invasion without sexual reproduction? A case study on European willows in Patagonia,Argentina

Willows of the Salix alba–Salix fragilis complex, native to western Eurasia, represent typical invaders of floodplain ecosystems worldwide. Introduced to South America by European settlers probably at the end of the nineteenth century, their distribution has increased significantly along the rivers in Northern Patagonia. This case study carried out mainly in the area around Lake Nahuel Huapi aims to analyze clonal structures and their spatial distribution using molecular markers as well as to relate the observed patterns to settlement history and life history traits of this species complex. Leaf material from 171 trees was collected along selected river floodplains in Northern Patagonia and genotypes were determined at six microsatellite loci. Including 62 reference samples of the S. alba–S. fragilis complex from German rivers, Probability of Identity (P _ID) was calculated and a Principal Coordinate Analysis (PCoA) conducted. From the altogether thirteen different genotypes detected, one dominant genotype (female) formed monoclonal stands along most of the studied river stretches. The maximum linear distance between the most remote ramets of this clone was 790 km. Evidence arose that the colonizing process so far is exclusively based on vegetative propagation in the focal study area and is obviously attributable to the pronounced brittleness of the hybrid parent S. fragilis. However, outside this area the occurrence of male trees and a diversity of genotypes indicate that evolutionary processes by recombination are involved within the willow complex. Therefore, an increase in genotypes can be assumed when male individuals and therefore sexual reproduction would appear in the area around Lake Nuhuel Huapi. This could be a crucial point for the long-term invasion success of the taxa when climatic and other environmental conditions will change in Southern Argentina.     

High-throughput phenotyping of multicellular organisms: finding the link between genotype and phenotype

High-throughput phenotyping approaches (phenomics) are being combined with genome-wide genetic screens to identify alterations in phenotype that result from gene inactivation. Here we highlight promising technologies for 'phenome-scale' analyses in multicellular organisms.     

A PNA microarray for tomato genotyping

The design and development of a PNA microarray designed for the simultaneous identification of several SNPs characteristic of seven different tomato varieties is described. Highly selective arginine-based monomer containing PNAs (Arg-PNAs) have been used in order to obtain very selective probes. Seven modified PNA probes were synthesised and their binding properties in solution were studied. PNA-microarrays based on these probes were prepared and applied to SNP discrimination in model experiments using oligonucleotide mixtures simulating the different sequences of the seven tomato varieties. The strength and the limitations of such a system for SNP recognition are thoroughly discussed.     

Phospholipase gene expression during Paracoccidioides brasiliensis morphological transition and infection

Phospholipase is an important virulence factor for pathogenic fungi. In this study, we demonstrate the following: (i) the Paracoccidioides brasiliensis pld gene is preferentially expressed in mycelium cells, (ii) the plb1 gene is mostly up-regulated by infection after 6 h of co-infection of MH-S cells or during BALB/c mice lung infection, (iii) during lung infection, plb1, plc and pld gene expression are significantly increased 6-48 h post-infection compared to 56 days after infection, strongly suggesting that phospholipases play a role in the early events of infection, but not during the chronic stages of pulmonary infection by P. brasiliensis.     

Polycystic echinococcosis in the state of Acre,Brazil: contribution to patient diagnosis,treatment and prognosis

The lack of knowledge regarding polycystic hydatid disease results in delayed or even incorrect diagnosis. The lack of systematic information regarding treatment also makes it difficult to assess the results and prognosis in patients with peritoneal and hepatic lesions caused by Echinococcus vogeli. Here we describe the clinical features of patients, propose a radiological classification protocol and describe a therapeutic option for the treatment of hydatid disease that previously had only been used for cases of cystic echinococcosis (Echinococcus granulosus). A prospective cohort study was initiated in 1999 and by 2009 the study included 60 patients. These patients were classified according to the PNM classification (parasite lesion, neighbouring organ invasion and metastases) and placed in one of three therapeutic modalities: (i) chemotherapy with albendazole at a dose of 10 mg/kg/day, (ii) surgical removal of cysts or (iii) percutaneous puncture of the cysts via puncture, aspiration, injection and re-aspiration (PAIR). The results were stratified according to therapeutic outcome: "cure", "clinical improvement", "no improvement", "death" or "no information". The PNM classification was useful in indicating the appropriate therapy in cases of polycystic hydatid disease. In conclusion, surgical therapy produced the best clinical results of all the therapies studied based on "cure" and "clinical improvement" outcomes. The use of PAIR for treatment requires additional study.     

The membranotropic activity of N-terminal peptides from the pore-forming proteins sticholysin I and II is modulated by hydrophobic and electrostatic interactions as well as lipid composition

The sea anemone Stichodactyla helianthus produces two pore-forming proteins, sticholysins I and II (St I and St II). Despite their high identity (93%), these toxins exhibit differences in hemolytic activity that can be related to those found in their N-terminal. To clarify the contribution of the N-terminal amino acid residues to the activity of the toxins, we synthesized peptides spanning residues 1-31 of St I (StI1-31) or 1-30 of St II (StII1-30) and demonstrated that StII1-30 promotes erythrocyte lysis to a higher extent than StI1-31. For a better understanding of the molecular mechanism underlying the peptide activity, here we studied their binding to lipid monolayers and pemeabilizing activity in liposomes. For this, we examined the effect on peptide membranotropic activity of including phospatidic acid and cholesterol in a lipid mixture of phosphatidylcholine and sphingomyelin. The results suggest the importance of continuity of the 1-10 hydrophobic sequence in StII1-30 for displaying higher binding and activity, in spite of both peptides' abilities to form pores in giant unilamellar vesicles. Thus, the different peptide membranotropic action is explained in terms of the differences in hydrophobic and electrostatic peptide properties as well as the enhancing role of membrane inhomogeneities.     

Application of the modular approach to an in-house validation study of real-time PCR methods for the detection and serogroup determination of verocytotoxigenic Escherichia coli

European Commission regulation 2073/2005 on the microbiological criteria for food requires that Escherichia coli is monitored as an indicator of hygienic conditions. Since verocytotoxigenic E. coli (VTEC) strains often cause food-borne infections by the consumption of raw food, the Biological Hazards (BIOHAZ) panel of the European Food Safety Authority (EFSA) recommended their monitoring in food as well. In particular, VTEC strains belonging to serogroups such as O26, O103, O111, O145, and O157 are known causative agents of several human outbreaks. Eight real-time PCR methods for the detection of E. coli toxin genes and their variants (stx(1), stx(2)), the intimin gene (eae), and five serogroup-specific genes have been proposed by the European Reference Laboratory for VTEC (EURL-VTEC) as a technical specification to the European Normalization Committee (CEN TC275/WG6). Here we applied a "modular approach" to the in-house validation of these PCR methods. The modular approach subdivides an analytical process into separate parts called "modules," which are independently validated based on method performance criteria for a limited set of critical parameters. For the VTEC real-time PCR module, the following parameters are being assessed: specificity, dynamic range, PCR efficiency, and limit of detection (LOD). This study describes the modular approach for the validation of PCR methods to be used in food microbiology, using single-target plasmids as positive controls and showing their applicability with food matrices.     

Fast and easy colorimetric tests for single mismatch recognition by PNA-DNA duplexes with the diethylthiadicarbocyanine dye and succinyl-beta-cyclodextrin

The 3,3'-diethylthiacarbocyanine (DiSC(2)(5)) dye is able to aggregate on full matched PNA-DNA duplexes by changing its absorption properties, which are manifested by an instantaneous colour shift from blue to purple. However the spontaneous aggregation of the dye also on mismatched duplexes and even on free PNA strands makes the test quite aspecific. Here it is demonstrated that the addition of succinyl-beta-cyclodextrin (Succ-beta-CyD) to the solutions containing PNA-DNA duplexes and the dye strongly enhances the specificity of the colour shift, allowing for a fast, very specific and extremely sensitive visual recognition of mismatches in DNA strands by using PNA probes in combination with the DiSC(2)(5) dye. The phenomenon has been studied by CD and NMR spectroscopies. The method has been optimized and preliminarily applied for the recognition of an apoE gene mutation in human DNA samples.