Improvement of Legionnaires' disease diagnosis using real-time PCR assay: a retrospective analysis, Italy, 2010 to 2015

AimTo evaluate real-time PCR as a diagnostic method for Legionnaires' disease (LD). Detection of Legionella DNA is among the laboratory criteria of a probable LD case, according to the European Centre for Disease Prevention and Control, although the utility and advantages, as compared to culture, are widely recognised.MethodsTwo independent laboratories, one using an in-house and the other a commercial real-time PCR assay, analysed 354 respiratory samples from 311 patients hospitalised with pneumonia between 2010-15. The real-time PCR reliability was compared with that of culture and urinary antigen tests (UAT). Concordance, specificity, sensitivity and positive and negative predictive values (PPV and NPV, respectively) were calculated.ResultsOverall PCR detected eight additional LD cases, six of which were due to Legionella pneumophila (Lp) non-serogroup 1. The two real-time PCR assays were concordant in 99.4% of the samples. Considering in-house real-time PCR as the reference method, specificity of culture and UAT was 100% and 97.9% (95% CI: 96.2-99.6), while the sensitivity was 63.6% (95%CI: 58.6-68.6) and 77.8% (95% CI: 72.9-82.7). PPV and NPV for culture were 100% and 93.7% (95% CI: 91.2-96.3). PPV and NPV for UAT were 87.5% (95% CI: 83.6-91.4) and 95.8% (95% CI: 93.5-98.2).ConclusionRegardless of the real-time PCR assay used, it was possible to diagnose LD cases with higher sensitivity than using culture or UAT. These data encourage the adoption of PCR as routine laboratory testing to diagnose LD and such methods should be eligible to define a confirmed LD case

Elongation Factor 1 alpha interacts with phospho-Akt in breast cancer cells and regulates their proliferation, survival and motility

BACKGROUND: Akt/PKB is a serine/threonine kinase that has attracted much attention because of its central role in regulating cell proliferation, survival, motility and angiogenesis. Activation of Akt in breast cancer portends aggressive tumour behaviour, resistance to hormone-, chemo-, and radiotherapy-induced apoptosis and it is correlated with decreased overall survival. Recent studies have identified novel tumor-specific substrates of Akt that may provide new diagnostic and prognostic markers and serve as therapeutic targets. This study was undertaken to identify pAkt-interacting proteins and to assess their biological roles in breast cancer cells. RESULTS: We confirmed that one of the pAkt interacting proteins is the Elongation Factor EF1alpha. EF1alpha contains a putative Akt phosphorylation site, but is not phosphorylated by pAkt1 or pAkt2, suggesting that it may function as a modulator of pAkt activity. Indeed, downregulation of EF1alpha expression by siRNAs led to markedly decreased expression of pAkt1 and to less extent of pAkt2 and was associated with reduced proliferation, survival and invasion of HCC1937 cells. Proliferation and survival was further reduced by combining EF1alpha siRNAs with specific pAkt inhibitors whereas EF1alpha downregulation slightly attenuated the decreased invasion induced by Akt inhibitors. CONCLUSION: We show here that EF1alpha is a pAkt-interacting protein which regulates pAkt levels. Since EF1alpha is often overexpressed in breast cancer, the consequences of EF1alpha increased levels for proliferation, survival and invasion will likely depend on the relative concentration of Akt1 and Akt2

Current Understanding of the Role of Cytoskeletal Cross-Linkers in the Onset and Development of Cardiomyopathies

Cardiomyopathies affect individuals worldwide, without regard to age, sex and ethnicity and are associated with significant morbidity and mortality. Inherited cardiomyopathies account for a relevant part of these conditions. Although progresses have been made over the years, early diagnosis and curative therapies are still challenging. Understanding the events occurring in normal and diseased cardiac cells is crucial, as they are important determinants of overall heart function. Besides chemical and molecular events, there are also structural and mechanical phenomena that require to be investigated. Cell structure and mechanics largely depend from the cytoskeleton, which is composed by filamentous proteins that can be cross-linked via accessory proteins. Alpha-actinin 2 (ACTN2), filamin C (FLNC) and dystrophin are three major actin cross-linkers that extensively contribute to the regulation of cell structure and mechanics. Hereby, we review the current understanding of the roles played by ACTN2, FLNC and dystrophin in the onset and progress of inherited cardiomyopathies. With our work, we aim to set the stage for new approaches to study the cardiomyopathies, which might reveal new therapeutic targets and broaden the panel of genes to be screened

STRUCTURAL STUDIES OF PYRIMIDO[2,1-B][1,3]THIAZINE AND THIAZOLO[3,2-A]PYRIMIDINE RINGS - AN X-RAY AND MULTINUCLEAR (H-1, C-13, N-15) APPROACH

1-H, 13-C, and 15-N NMR spectroscopy and X-ray diffraction were carried out on some derivatives of 8 amino-3,4-dihydro[2,1-b][thiazin-6-one and 7-amino-2,3-dihydrothiazolo[3,2-a]pyrimidin-5-one. The ring conformations of thiazine and thiazolo are in good accordance with the X-ray structures

BCR/ABL activates mdm2 rnRNA translation via the La antigen

In a BCR/ABL-expressing myeloid precursor cell line, p53 levels were markedly downmodulated. Expression of MDM2, the negative regulator of p53, was upregulated in a tyrosine kinase-dependent manner in growth factor-independent BCR/ABL-expressing cells, and in accelerated phase and blast crisis CML samples. Increased MDM2 expression was associated with enhanced mdm2 mRNA translation, which required the interaction of the La antigen with mdm2 5' UTR. Expression of MDM2 correlated with that of La and was suppressed by La siRNAs and by a dominant negative La mutant, which also enhanced the susceptibility to drug-induced apoptosis of BCR/ABL-transformed cells. By contrast, La overexpression led to increased MDM2 levels and enhanced resistance to apoptosis. Thus, La-dependent activation of mdm2 translation might represent an important molecular mechanism involved in BCR/ABL leukemogenesis