Real-time reverse transcription polymerase chain reaction development for rapid detection of Tomato brown rugose fruit virus and comparison with other techniques

Background: Tomato brown rugose fruit virus (ToBRFV) is a highly infectious tobamovirus that causes severe disease in tomato (Solanum lycopersicum L.) crops. In Italy, the first ToBRFV outbreak occurred in 2018 in several provinces of the Sicily region. ToBRFV outbreak represents a serious threat for tomato crops in Italy and the Mediterranean Basin. Methods: Molecular and biological characterisation of the Sicilian ToBRFV ToB-SIC01/19 isolate was performed, and a sensitive and specific Real-time RT-PCR TaqMan minor groove binder probe method was developed to detect ToBRFV in infected plants and seeds. Moreover, four different sample preparation procedures (immunocapture, total RNA extraction, direct crude extract and leaf-disk crude extract) were evaluated. Results: The Sicilian isolate ToB-SIC01/19 (6,391 nt) showed a strong sequence identity with the isolates TBRFV-P12-3H and TBRFV-P12-3G from Germany, Tom1-Jo from Jordan and TBRFV-IL from Israel. The ToB-SIC01/19 isolate was successfully transmitted by mechanical inoculations in S. lycopersicum L. and Capsicum annuum L., but no transmission occurred in S. melongena L. The developed real-time RT-PCR, based on the use of a primer set designed on conserved sequences in the open reading frames3, enabled a reliable quantitative detection. This method allowed clear discrimination of ToBRFV from other viruses belonging to the genus Tobamovirus, minimising false-negative results. Using immunocapture and total RNA extraction procedures, the real-time RT-PCR and end-point RT-PCR gave the same comparable results. Using direct crude extracts and leaf-disk crude extracts, the end-point RT-PCR was unable to provide a reliable result. This developed highly specific and sensitive real-time RT-PCR assay will be a particularly valuable tool for early ToBRFV diagnosis, optimising procedures in terms of costs and time

Mitochondria and the NLRP3 Inflammasome in Alcoholic and Nonalcoholic Steatohepatitis

Alcoholic (ASH) and nonalcoholic steatohepatitis (NASH) are advanced stages of fatty liver disease and two of the most prevalent forms of chronic liver disease. ASH and NASH are associated with significant risk of further progression to cirrhosis and hepatocellular carcinoma (HCC), the most common type of liver cancer, and a major cause of cancer-related mortality. Despite extensive research and progress in the last decades to elucidate the mechanisms of the development of ASH and NASH, the pathogenesis of both diseases is still poorly understood. Mitochondrial damage and activation of inflammasome complexes have a role in inducing and sustaining liver damage. Mitochondrial dysfunction produces inflammatory factors that activate the inflammasome complexes. NLRP3 inflammasome (nucleotide-binding oligomerization domain-like receptor protein 3) is a multiprotein complex that activates caspase 1 and the release of pro-inflammatory cytokines, including interleukin-1? (IL-1?) and interleukin-18 (IL-18), and contributes to inflammatory pyroptotic cell death. The present review, which is part of the issue "Mitochondria in Liver Pathobiology", provides an overview of the role of mitochondrial dysfunction and NLRP3 activation in ASH and NASH

Myristic acid potentiates palmitic acid-induced lipotoxicity and steatohepatitis associated with lipodystrophy by sustaning de novo ceramide synthesis.

Palmitic acid (PA) induces hepatocyte apoptosis and fuels de novo ceramide synthesis in the endoplasmic reticulum (ER). Myristic acid (MA), a free fatty acid highly abundant in copra/palmist oils, is a predictor of nonalcoholic steatohepatitis (NASH) and stimulates ceramide synthesis. Here we investigated the synergism between MA and PA in ceramide synthesis, ER stress, lipotoxicity and NASH. Unlike PA, MA is not lipotoxic but potentiated PA-mediated lipoapoptosis, ER stress, caspase-3 activation and cytochrome c release in primary mouse hepatocytes (PMH). Moreover, MA kinetically sustained PA-induced total ceramide content by stimulating dehydroceramide desaturase and switched the ceramide profile from decreased to increased ceramide 14:0/ceramide16:0, without changing medium and long-chain ceramide species. PMH were more sensitive to equimolar ceramide14:0/ceramide16:0 exposure, which mimics the outcome of PA plus MA treatment on ceramide homeostasis, than to either ceramide alone. Treatment with myriocin to inhibit ceramide synthesis and tauroursodeoxycholic acid to prevent ER stress ameliorated PA plus MA induced apoptosis, similar to the protection afforded by the antioxidant BHA, the pan-caspase inhibitor z-VAD-Fmk and JNK inhibition. Moreover, ruthenium red protected PMH against PA and MA-induced cell death. Recapitulating in vitro findings, mice fed a diet enriched in PA plus MA exhibited lipodystrophy, hepatosplenomegaly, increased liver ceramide content and cholesterol levels, ER stress, liver damage, inflammation and fibrosis compared to mice fed diets enriched in PA or MA alone. The deleterious effects of PA plus MA-enriched diet were largely prevented by in vivo myriocin treatment. These findings indicate a causal link between ceramide synthesis and ER stress in lipotoxicity, and imply that the consumption of diets enriched in MA and PA can cause NASH associated with lipodystrophy

Supplementation of vitamin E and C prevent granulomatosis in meagre larvae

Systemic granulomatosis has already been reported in meagre larvae with an adequate feeding protocol and enrichment media preventing its appearance in the first weeks of life. Afterwards, the control of this disease could be prevented through nutritional components of the inert food, being the antioxidants the key to success. For this reason, in the present study, meagre larvae were reared from 30 days post hatching (dph) with five isonitrogenous and isolipidic experimental microdiets with different levels of vitamin E and C: C- (40 mg kg-1 E, 100 mg kg-1 C), C+ (400 mg kg-1 E, 1,000 mg kg-1 C), Krill (400 mg kg-1 E, 1,000 mg kg-1 C and substitution of fish oil by krill oil), EC (200 mg kg-1 E, 500 mg kg-1 C) and EECC (800 mg kg-1 E, 2,000 mg kg-1 C). Prior to this, larvae were co-fed with rotifers and Artemia following a protocol which prevented the appearance of granulomas, as previously demonstrated. The substitution of fish oil by krill oil significantly increased levels of eicosapentaenoic acid (EPA, 16.6 %) and docosahexaenoic acid (DHA, 17.6 %) in meagre, consequently increasing the peroxidation index, which in turn translated into a higher incidence of granulomas. Although even low levels of vitamin E and C (40 mg kg-1 E, 100 mg kg-1 C; C-) allowed the adequate growth of larvae, these levels were not enough to prevent the appearance of granulomas, requiring superior levels of both antioxidant vitamins (800 mg kg-1 E and 2,000 mg kg-1 C) to mitigate systemic granulomatosis. This mitigation was simultaneous with the reduction of thiobarbituric acid reactive substances TBARs content in larvae, which were highly correlated with the appearance of granulomas (R2=0.892, y=0.0446x+0.0756). A strong negative correlation was observed between the dietary levels of vitamin E (y = -0.0098x + 11.174, R2 = 0.8766, p value = 0.019, r = -0.93) and vitamin C (y = -0.0022x + 6.4777, R2 = 0.9278, p value = 0.003, r = -0.96) and the percentage of larvae with granulomas. The results showed that the occurrence of systemic granulomatosis seems to be associated to the larvae peroxidation status, so that high dietary levels of vitamin E and C (800 and 2,000 mg kg-1, respectively; Diet EECC), reduced lipid peroxidation and completely prevented the appearance of granulomas in meagre larvae at 44 dph

Ianus: an Adpative FPGA Computer

Dedicated machines designed for specific computational algorithms can outperform conventional computers by several orders of magnitude. In this note we describe {\it Ianus}, a new generation FPGA based machine and its basic features: hardware integration and wide reprogrammability. Our goal is to build a machine that can fully exploit the performance potential of new generation FPGA devices. We also plan a software platform which simplifies its programming, in order to extend its intended range of application to a wide class of interesting and computationally demanding problems. The decision to develop a dedicated processor is a complex one, involving careful assessment of its performance lead, during its expected lifetime, over traditional computers, taking into account their performance increase, as predicted by Moore's law. We discuss this point in detail

An in-depth view of the microscopic dynamics of Ising spin glasses at fixed temperature

Using the dedicated computer Janus, we follow the nonequilibrium dynamics of the Ising spin glass in three dimensions for eleven orders of magnitude. The use of integral estimators for the coherence and correlation lengths allows us to study dynamic heterogeneities and the presence of a replicon mode and to obtain safe bounds on the Edwards-Anderson order parameter below the critical temperature. We obtain good agreement with experimental determinations of the temperature-dependent decay exponents for the thermoremanent magnetization. This magnitude is observed to scale with the much harder to measure coherence length, a potentially useful result for experimentalists. The exponents for energy relaxation display a linear dependence on temperature and reasonable extrapolations to the critical point. We conclude examining the time growth of the coherence length, with a comparison of critical and activated dynamics.Comment: 38 pages, 26 figure

Elevation and cholera: an epidemiological spatial analysis of the cholera epidemic in Harare, Zimbabwe, 2008-2009

BACKGROUND: In highly populated African urban areas where access to clean water is a challenge, water source contamination is one of the most cited risk factors in a cholera epidemic. During the rainy season, where there is either no sewage disposal or working sewer system, runoff of rains follows the slopes and gets into the lower parts of towns where shallow wells could easily become contaminated by excretes. In cholera endemic areas, spatial information about topographical elevation could help to guide preventive interventions. This study aims to analyze the association between topographic elevation and the distribution of cholera cases in Harare during the cholera epidemic in 2008 and 2009. METHODS: We developed an ecological study using secondary data. First, we described attack rates by suburb and then calculated rate ratios using whole Harare as reference. We illustrated the average elevation and cholera cases by suburbs using geographical information. Finally, we estimated a generalized linear mixed model (under the assumption of a Poisson distribution) with an Empirical Bayesian approach to model the relation between the risk of cholera and the elevation in meters in Harare. We used a random intercept to allow for spatial correlation of neighboring suburbs. RESULTS: This study identifies a spatial pattern of the distribution of cholera cases in the Harare epidemic, characterized by a lower cholera risk in the highest elevation suburbs of Harare. The generalized linear mixed model showed that for each 100 meters of increase in the topographical elevation, the cholera risk was 30% lower with a rate ratio of 0.70 (95% confidence interval=0.66-0.76). Sensitivity analysis confirmed the risk reduction with an overall estimate of the rate ratio between 20% and 40%. CONCLUSION: This study highlights the importance of considering topographical elevation as a geographical and environmental risk factor in order to plan cholera preventive activities linked with water and sanitation in endemic areas. Furthermore, elevation information, among other risk factors, could help to spatially orientate cholera control interventions during an epidemic

Evaluation of native microalgae from Tunisia using the pulse-amplitude-modulation measurement of chlorophyll fluorescence and a performance study in semi-continuous mode for biofuel production

Background: Microalgae are attracting much attention as a promising feedstock for renewable energy production, while simultaneously providing environmental benefits. So far, comparison studies for microalgae selection for this purpose were mainly based on data obtained from batch cultures, where the lipid content and the growth rate were the main selection parameters. The present study evaluates the performance of native microalgae strains in semi-continuous mode, considering the suitability of the algal-derived fatty acid composition and the saponifiable lipid productivity as selection criteria for microalgal fuel production. Evaluation of the photosynthetic performance and the robustness of the selected strain under outdoor conditions was conducted to assess its capability to grow and tolerate harsh environmental growth conditions. Results: In this study, five native microalgae strains from Tunisia (one freshwater and four marine strains) were isolated and evaluated as potential raw material to produce biofuel. Firstly, molecular identification of the strains was performed. Then, experiments in semi-continuous mode at different dilution rates were carried out. The local microalgae strains were characterized in terms of biomass and lipid productivity, in addition to protein content, and fatty acid profile, content and productivity. The marine strain Chlorella sp. showed, at 0.20 1/day dilution rate, lipid and biomass productivities of 35.10 mg/L day and 0.2 g/L day, respectively. Moreover, data from chlorophyll fluorescence measurements demonstrated the robustness of this strain as it tolerated extreme outdoor conditions including high (38 ° C) and low (10 ° C) temperature, and high irradiance (1600 µmol/m2 s). Conclusions: Selection of native microalgae allows identifying potential strains suitable for use in the production of biofuels. The selected strain Chlorella sp. demonstrated adequate performance to be scaled up to outdoor conditions. Although experiments were performed at laboratory conditions, the methodology used in this paper allows a robust evaluation of microalgae strains for potential market applications.This study was supported by the Marine Microalgae Biotechnology Group at the University of Almer'a (BIO 173) and the Campus de Excelencia Internacional Agroalimentario (ceiA3) within the joint framework of supervised theses between the University of Almeria, Spain and the University of Sfax, Tunisia.Scopu