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Real-time PCR was developed to quantify Leishmania infantum kinetoplast DNA and optimized to achieve a sensitivity of 1 parasite/mL. For this purpose, we cloned the conserved kDNA fragment of 120 bp into competent cells and correlated them with serial dilutions of DNA extracted from reference parasite cultures calculating that a parasite cell contains approximately 36 molecules of kDNA. This assay was applied to estimate parasite load in clinical samples from visceral, cutaneous leishmaniasis patients and infected dogs and cats comparing with conventional diagnosis. The study aimed to propose a real-time PCR for the detection of Leishmania DNA from clinical samples trying to solve the diagnostic problems due to the low sensitivity of microscopic examination or the low predictive values of serology and resolve problems related to in vitro culture. The quantitative PCR assay in this study allowed detection of Leishmania DNA and quantification of considerably low parasite loads in samples that had been diagnosed negative by conventional techniques. In conclusion, this quantitative PCR can be used for the diagnosis of both human, canine and feline Leishmaniasis with high sensitivity and specificity, but also for evaluating treatment and the endpoint determination of leishmaniasis.

Background: Chagas disease, also known as American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite Trypanosoma cruzi. No progress in the treatment of this pathology has been made since Nifurtimox was introduced more than fifty years ago, and this drug is considered very aggressive and may cause several adverse effects. This drug currently has severe limitations, including a high frequency of undesirable side effects and limited efficacy and availability, so research to discover new drugs for the treatment of Chagas disease is imperative. Many drugs available on the market are natural products as found in nature or compounds designed based on the structure and activity of these natural products. Methods: This study evaluated the in vitro antiparasitic activity of a series of previously synthesized stilbene and terphenyl compounds in T. cruzi epimastigotes and intracellular amastigotes. The action of the most selective compounds was investigated by flow cytometric analysis to evaluate the mechanism of cell death. The ability to induce apoptosis or caspase-1 inflammasomes was assayed in macrophages infected with T. cruzi after treatment, comparing it with that of Nifurtimox. Results: The stilbene ST18 was the most potent compound of the series. It was slightly less active than Nifurtimox in epimastigotes but most active in intracellular amastigotes. Compared to Nifurtimox, it was markedly less cytotoxic when tested in vitro on normal cells. ST18 was able to induce a marked increase in parasites positive for Annexin V and monodansylcadaverine. Moreover, ST18 induced the activation, in infected macrophages, of caspase-1, a conserved enzyme that plays a major role in controlling parasitemia, host survival and the onset of the adaptive immune response in Trypanosoma infection. Conclusions: The antiparasitic activity of ST18 together with its ability to activate caspase-1 in infected macrophages and its low toxicity toward normal cells makes this compound interesting for further clinical investigation.

Abstract Background Chagas disease, also known as American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite Trypanosoma cruzi. No progress in the treatment of this pathology has been made since Nifurtimox was introduced more than fifty years ago and is considered very aggressive and may cause several adverse effects. Currently, this drug has severe limitations, including high frequency of undesirable side effects and limited efficacy and availability and the research to discover new drugs for the treatment of Chagas disease is imperative. Many drugs available in the market are natural products as found in nature or compounds designed based on the structure and activity of these natural products. Methodology/Principal Findings This study evaluated the in vitro antiparasitic activity in T. cruzi epimastigotes and intracellular amastigotes of a series of stilbene and terphenyl compounds previously synthesized. The action of the most selective compounds has been investigated by flow cytometry analysis to evaluate the mechanism of cell death. The ability to induce apoptosis or caspase-1 inflammasome were assayed in macrophages infected with T. cruzi after treatment comparing with Nifurtimox. Conclusions/Significance The stilbene ST18 was the most potent compound of the series. It was slightly less active than Nifurtimox in epimastigotes but most active in intracellular amastigotes. Compared to Nifurtimox, it was markedly less cytotoxic when tested in vitro on normal cells. ST18 was able to induce a marked increase of parasites positive to Annexin V and monodansylcadaverine. Moreover, ST18 induced the activation in infected macrophages of caspase-1, a conserved enzyme which plays a main role in controlling parasitemia, host survival, and the onset of adaptive immune response in Trypanosoma infection. The antiparasitic activity of ST18 together to its ability to activate caspase-1 in infected macrophages and its low toxicity on normal cells makes this compound interesting for further clinical investigations. Author Summary Chagas disease is a pathology caused by the protozoan parasite Trypanosoma cruzi. No progress in the treatment of this pathology has been made since benznidazole and Nifurtimox were introduced more than fifty years ago. However, these drugs have severe limitations and the research to discover new drugs for the treatment of Chagas disease is imperative. We evaluated the in vitro antiparasitic activity in T. cruzi epimastigotes of a series of stilbene and terphenyl compounds previously synthesized. The stilbene ST18 was the most potent compound of the series. It was slightly less active than nifurtimox in epimastigotes but most active in intracellular amastigotes. Compared to Nifurtimox, it was markedly less cytotoxic when tested in vitro on normal cells. ST18 was able to induce a marked increase of parasites positive to Annexin V and monodansylcadaverine. Moreover, this compound induced the activation in infected macrophages of caspase-1, an evolutionarily conserved enzyme which plays a main role in controlling parasitemia, host survival, and the onset of adaptive immune response in T. cruzi infection. The antiparasitic activity of ST18 together to its ability to activate caspase-1 in infected macrophages and its low toxicity on normal cells makes this compound interesting for further clinical investigations.

The Water-Cooled Lead–Lithium Breeding Blanket (WCLL BB) is one of the two blanket concept candidates to become the driver blanket of the EU-DEMO reactor. The design was enacted with a holistic approach. The influence that neutronics, thermal-hydraulics (TH), thermo-mechanics (TM) and magneto-hydro-dynamics (MHD) may have on the design were considered at the same time. This new approach allowed for the design team to create a WCLL BB layout that is able to comply with different foreseen requirements in terms of integration, tritium self-sufficiency, and TH and TM needs. In this paper, the rationale behind the design choices and the main characteristics of the WCLL BB needed for the EU-DEMO are reported and discussed. Finally, the main achievements reached during the pre-conceptual design phase and some remaining open issues to be further investigated in the upcoming conceptual design phase are reported as well.

The water-cooled lead lithium breeding blanket (WCLL BB) is one of two BB candidate concepts to be chosen as the driver blanket of the EU-DEMO fusion reactor. Research activities carried out in the past decade, under the umbrella of the EUROfusion consortium, have allowed a quite advanced reactor architecture to be achieved. Moreover, significant efforts have been made in order to develop the WCLL BB pre-conceptual design following a holistic approach, identifying interfaces between components and systems while respecting a system engineering approach. This paper reports a description of the current WCLL BB architecture, focusing on the latest modifications in the BB reference layout aimed at evolving the design from its pre-conceptual version into a robust conceptual layout. In particular, the main rationale behind design choices and the BB’s overall performances are highlighted. The present paper also gives an overview of the integration between the BB and the different in-vessel systems interacting with it. In particular, interfaces with the tritium extraction and removal (TER) system and the primary heat transfer system (PHTS) are described. Attention is also paid to auxiliary systems devoted to heat the plasma, such as electron cyclotron heating (ECH). Indeed, the integration of this system in the BB will strongly impact the segment design since it envisages the introduction of significant cut-outs in the BB layout. A preliminary CAD model of the central outboard blanket (COB) segment housing the ECH cut-out has been set up and is reported in this paper. The chosen modeling strategy, adopted loads and boundary conditions, as well as obtained results, are reported in the paper and critically discussed.

Background/Objectives: Astroblastoma is a rare glial neoplasm more frequent in young female patients, with unclear clinical behaviors and outcomes. The diagnostic molecular alteration is a rearrangement of the Meningioma 1 (MN1) gene. MicroRNAs (miRNAs) are important gene expression regulators with strong implications in biological processes. Here, we investigated microRNA expression, regulation, and biological processes correlated to target genes of deregulated miRNAs in MN1-altered astroblastoma. Methods: A cohort of 14 tumor samples, histologically classified as astroblastoma, was retrospectively collected and analyzed through their DNA methylation profiles. MiRNA expression profiles were then detected on MN1-altered astroblastomas (n = 8) and normal brain controls (n = 2) by Nanostring technology and validated by RT-qPCR; then, the expression of deregulated miRNAs was correlated with clinical-pathological characteristics. Subsequently, the methylation status of promoters of deregulated miRNAs was investigated through a methylation profiling microarray. Finally, bioinformatics analysis was conducted to explore the biological processes (BPs) and target genes of differentially expressed miRNAs. Results: Eight MN-altered astroblastoma were identified. Thirty-nine miRNAs were deregulated in tumor samples compared to normal brain tissue. Downregulated microRNAs exhibited an association with an increased risk of recurrence. The promoter methylation status was investigated in 32/39 miRNAs: 14/32 were epigenetically deregulated. None of them were genetically regulated. Conclusions: MN1-altered astroblastomas have an miRNA expression signature that identifies specific BPs and pathways. Our findings suggested that the involved pathways could be associated with clinical and pathological characteristics of MN1-altered astroblastomas. Also, the biology of this rare tumor could have potential implications on prognostic markers and therapy.

ObjectivesTo describe the long-term prognosis of epilepsy and prognostic patterns in a large cohort of newly diagnosed patients and identify prognostic factors.MethodsStudy participants were 13 Italian epilepsy centres with accessible records dating back to 2005 or earlier, complete data on seizure outcome and treatments, precise epilepsy diagnosis, and follow-up of at least 10 years. Records were examined by trained neurology residents for demographics, seizure characteristics, neurological signs, psychiatric comorbidity, first electroencephalogram (EEG) and MRI/CT, epilepsy type and aetiology, antiepileptic drugs (AEDs), and 1-year, 2-year, 5-year and 10-year seizure remissions. Five predefined prognostic patterns were identified: early remission, late remission, relapsing-remitting course, worsening course and no remission. Prognostic factors were assessed using multinomial logistic regression models.Results1006 children and adults were followed for 17 892 person-years (median 16 years; range 10–57). During follow-up, 923 patients (91.7%) experienced 1-year remission. 2-year, 5-year and 10-year remissions were present in 89.5%, 77.1% and 44.4% of cases. 5-year remission was associated with one to two seizures at diagnosis, generalised epilepsy, no psychiatric comorbidity, and treatment with one or two AEDs during follow-up. 10-year remission was associated with one or two AEDs. The most common prognostic pattern was relapsing-remitting (52.2%), followed by early remission (24.5%). 8.3% of cases experienced no remission. Predictors of a relapsing-remitting course were <6 seizures at diagnosis, (presumed) genetic aetiology and no psychiatric comorbidity.ConclusionsFew seizures at diagnosis, generalised epilepsy and no psychiatric comorbidity predict early or late seizure freedom in epilepsy. Achieving remission at any time after the diagnosis does not exclude further relapses.

Antinuclear antibodies (ANAs) are significant biomarkers in the diagnosis of autoimmune diseases in humans, done by mean of Indirect ImmunoFluorescence (IIF) method, and performed by analyzing patterns and fluorescence intensity. This paper introduces the AIDA Project (autoimmunity: diagnosis assisted by computer) developed in the framework of an Italy-Tunisia cross-border cooperation and its preliminary results. A database of interpreted IIF images is being collected through the exchange of images and double reporting and a Gold Standard database, containing around 1000 double reported images, has been settled. The Gold Standard database is used for optimization of a CAD (Computer Aided Detection) solution and for the assessment of its added value, in order to be applied along with an Immunologist as a second Reader in detection of autoantibodies. This CAD system is able to identify on IIF images the fluorescence intensity and the fluorescence pattern. Preliminary results show that CAD, used as second Reader, appeared to perform better than Junior Immunologists and hence may significantly improve their efficacy; compared with two Junior Immunologists, the CAD system showed higher Intensity Accuracy (85,5% versus 66,0% and 66,0%), higher Patterns Accuracy (79,3% versus 48,0% and 66,2%), and higher Mean Class Accuracy (79,4% versus 56,7% and 64.2%).