Development of a minor groove binding probe based real-time PCR for the diagnosis and quantification of Leishmania infantum in dog specimens.

A new quantitative real-time PCR (qPCR) assay based on Taqman® technology and minor groove binding (MGB) probe was developed for the diagnosis of leishmaniosis and quantification of Leishmania infantum DNA in infected dogs. This method was based on the amplification of a 122bp fragment of the highly conserved kDNA minicircles of L. infantum. The reaction was performed using the StepOnePlus™ system with StepOne software™. This assay was able to detect the presence of protozoan parasite DNA in amounts as low as 0.03 parasites per reaction. The standard curve designed for the quantification of parasites showed linearity over seven log DNA concentration range with a correlation coefficient >0.999 and both intra- and inter-assay variability demonstrated the high efficiency and reproducibility of the assay. The qPCR also proved to be successfully applicable to different clinical samples including blood, bone marrow, lymph node aspirates and conjunctival swabs.

Genetic typing of bovine viral diarrhoea virus: evidence of an increasing number of variants in Italy.

Bovine Viral Diarrhoea Virus (BVDV) is responsible worldwide for severe economic losses on cattle farms. BVDV is an RNA virus with a high genome variability having practical consequences on epidemiology, diagnosis and disease control. Genetic monitoring was suggested as the first step in BVDV control. Thirty-seven Bovine Viral Diarrhoea Viruses were identified in persistently infected cattle, mucosal disease-affected animals and in bulk milk, and were characterised genetically. The 5'UTR region was amplified and sequenced, and a phylogenetic analysis was carried out comparing all the Italian sequences of BVDV available from the Genbank database. An unusual number of persistent infected animals was evidenced on more than one farm. Phylogenetic analysis attributed all our viruses to BVDV type I and distinguished four different subgroups inside this genotype. Analysis of old and new viruses revealed the circulation of viruses classified in subgroups BVDV Ia and Ij never reported in Italy.

Paclitaxel and docetaxel resistance: molecular mechanisms and development of new generation taxanes.

Taxanes represent one of the most promising classes of anticancer agents. Unfortunately, their clinical success has been limited by the insurgence of cellular resistance, mainly mediated by the expression of the MDR phenotype or by microtubule alterations. However, the remarkable relevance of paclitaxel and docetaxel in clinical oncology stimulated intensive efforts in the last decade to identify new derivatives endowed with improved activities towards resistant tumor cells, resulting in a huge number of novel natural and synthetic taxanes. Among them, several structurally different derivatives were found to exhibit a promising behavior against the MDR phenotype in terms of either MDR inhibiting properties, or enhanced cytotoxicity compared to parental drugs, or both. On the other hand, only in more recent years have the first taxanes retaining activity against resistant cancer cells bearing alterations of the tubulin/microtubule system emerged. This review describes the main molecular mechanisms of resistance to paclitaxel and docetaxel identified so far, focusing on the advances achieved in the development of new taxanes potentially useful for the treatment of resistant tumors.