Comparison of PCR protocols for detecting Histoplasma capsulatum DNA through a multicenter study

BackgroundA multicenter study was conducted. A panel containing DNA from Histoplasma capsulatum, as well as negative and cross-reaction controls, was sent to five different laboratories, members of the MICOMOL network from CYTED Program.AimsThe objective was to assess the accuracy of different PCR protocols to detect H. capsulatum DNA.MethodsSeven different PCR protocols were tested. They were based on PCR techniques and used unicopy and multicopy targets.ResultsMost of these protocols (4/7) were able to detect the smallest amounts of fungal DNA (10² fg/μl). Overall sensitivity was 86% and specificity was 100%. The protocol based on a unicopy target (SCAR₂₂₀) presented lower sensitivity (43%) but 100% specificity. The real-time protocols tested were highly reproducible, sensitive, and specific. Neither false positives nor cross-reactions were detected in any protocol.ConclusionsAll laboratories were able to amplify H. capsulatum DNA, and real-time PCR seems to be a promising tool to efficiently detect this pathogen in clinical samples.     

Molecular cytogenetic characterisation of Salix viminalis L. using repetitive DNA sequences

Salix viminalis L. (2n?=?38) is a diploid dicot species belonging to the Salix genus of the Salicaceae family. This short-rotation woody crop is one of the most important renewable bioenergy resources worldwide. In breeding for high biomass productivity, limited knowledge is available on the molecular cytogenetics of willow, which could be combined with genetic linkage mapping. The present paper describes the adaptation of a fluorescence in situ hybridisation (FISH) protocol as a new approach to analyse the genomic constitution of Salix viminalis using the heterologous DNA clones pSc119.2, pTa71, pTa794, pAs1, Afa-family, pAl1, HT100.3, ZCF1 and the GAA microsatellite marker. Three of the nine probes showed unambiguous signals on the metaphase chromosomes. FISH analysis with the pTa71 probe detected one major 18S-5.8S-26S rDNA locus on the short arm of one chromosome pair; however, the pTa794 rDNA site was not visible. One chromosome pair showed a distinct signal around the centromeric region after FISH with the telomere-specific DNA clone HT100.3. Two chromosome pairs were found to have pAs1 FISH signals, which represent a D-genome-specific insert from Aegilops tauschii. Based on the FISH study, a set of chromosomes with characteristic patterns is presented, which could be used to establish the karyotype of willow species.     

Effect of lead stress on mineral content and growth of wheat (Triticum aestivum) and spinach (Spinacia oleracea) seedlings

Lead (Pb) is the most common heavy metal contaminant in the environment. Pb is not an essential element for plants, but they absorb it when it is present in their environment, especially in rural areas when the soil is polluted by automotive exhaust and in fields contaminated with fertilizers containing heavy metal impurities. To investigate lead effects on nutrient uptake and metabolism, two plant species, spinach (Spinacia oleracea) and wheat (Triticum aestivum), were grown under hydroponic conditions and stressed with lead nitrate, Pb(NO₃)₂, at three concentrations (1.5, 3, and 15 mM).Lead is accumulated in a dose-dependent manner in both plant species, which results in reduced growth and lower uptake of all mineral ions tested. Total amounts and concentrations of most mineral ions (Na, K, Ca, P, Mg, Fe, Cu and Zn) are reduced, although Mn concentrations are increased, as its uptake is reduced less relative to the whole plant’s growth. The deficiency of mineral nutrients correlates in a strong decrease in the contents of chlorophylls a and b and proline in both species, but these effects are less pronounced in spinach than in wheat. By contrast, the effects of lead on soluble proteins differ between species; they are reduced in wheat at all lead concentrations, whereas they are increased in spinach, where their value peaks at 3 mM Pb.The relative lead uptake by spinach and wheat, and the different susceptibility of these two species to lead treatment are discussed.     

De Novo Assembly and Functional Annotation of the Olive (Olea europaea) Transcriptome

Olive breeding programmes are focused on selecting for traits as short juvenile period, plant architecture suited for mechanical harvest, or oil characteristics, including fatty acid composition, phenolic, and volatile compounds to suit new markets. Understanding the molecular basis of these characteristics and improving the efficiency of such breeding programmes require the development of genomic information and tools. However, despite its economic relevance, genomic information on olive or closely related species is still scarce. We have applied Sanger and 454 pyrosequencing technologies to generate close to 2 million reads from 12 cDNA libraries obtained from the Picual, Arbequina, and Lechin de Sevilla cultivars and seedlings from a segregating progeny of a Picual × Arbequina cross. The libraries include fruit mesocarp and seeds at three relevant developmental stages, young stems and leaves, active juvenile and adult buds as well as dormant buds, and juvenile and adult roots. The reads were assembled by library or tissue and then assembled together into 81 020 unigenes with an average size of 496 bases. Here, we report their assembly and their functional annotation.     

Ddi1-like protein from Leishmania major is an active aspartyl proteinase

Eukaryotic cells respond to DNA damage by activating damage checkpoint pathways, which arrest cell cycle progression and induce gene expression. We isolated a full-length cDNA encoding a 49-kDa protein from Leishmania major, which exhibited significant deduced amino acid sequence homology with the annotated Leishmania sp. DNA damage-inducible (Ddi1-like) protein, as well as with the Ddi1 protein from Saccharomyces cerevisiae. In contrast to the previously described Ddi1 protein, the protein from L. major displays three domains: (1) an NH2-terminal ubiquitin like; (2) a COOH terminal ubiquitin-associated; (3) a retroviral aspartyl proteinase, containing the typical D[S/T]G signature. The function of the L. major Ddi1-like recombinant protein was investigated after expression in baculovirus/insect cells and biochemical analysis, revealing preferential substrate selectivity for aspartyl proteinase A₂ family substrates, with optimal activity in acidic conditions. The proteolytic activity was inhibited by aspartyl proteinase inhibitors. Molecular modeling of the retroviral domain of the Ddi1-like Leishmania protein revealed a dimer structure that contained a double Asp-Ser-Gly-Ala amino acid sequence motif, in an almost identical geometry to the exhibited by the homologous retroviral aspartyl protease domain of yeast Ddi1 protein. Our results indicate that the isolated Ddi1-like protein is a functional aspartyl proteinase in L. major, opening possibility to be considered as a potential target for novel antiparasitic drugs.     

Concentration by ultrafiltration and stabilization of phytase produced by solid-state fermentation

Ultrafiltration is an attractive downstream processing technique for concentrating enzymes and could be considered the primary step of purification. However, the efficiency of this process is often limited by protein fouling and shear-induced enzyme inactivation, which decreases permeate flux and results in the loss of enzyme activity. Although the rejection of phytase was higher than 99%, the loss of the enzyme activity was 14% during operation, indicating that the shear forces generated in the filter have significant influences on the enzyme activity. Two preparations using glycerol (25% and 35%, v/v) as a cryo-protecting agent at different temperatures were studied. The preparation containing 35% glycerol retained 70% of the initial enzyme activity at 70 °C after 1 h and had more than 3 and 6 months storage half-life at 29 °C and 4 °C, respectively.