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The expansion of Integrated Pest Management (IPM), including biological control, has had several positive consequences for the agricultural environment and participants in the production chain. To enable successful operation and applications of biological control, production of insects used for rearing natural enemies (parasitoids and predators) must be optimized to reduce time and costs and improve production both qualitatively and quantitatively. The present study evaluated the effect of wheat varieties, the main component of artificial diets for Ephestia kuehniella , on the reproductive performance and biological parameters of this flour moth, which is used for mass production of Trichogramma spp. (Hymenoptera: Trichogrammatidae) and other parasitoids and predators. Four varieties of wheat were compared: BRS 327, BRS Marcante, BRS Parrudo, and KBR, with and without the addition of corn E. kuehniella reared on 97% BRS 327 wheat flour + 3% nutritional yeast had the best biological parameters and substitution of corn for about half of the wheat increased the number of eggs per female. [ABSTRACT FROM AUTHOR]

Chinese Justice, the Fiction: Law and Literature in Modern China, by Jeffrey C. Kinkley. Stanford, California, Stanford University Press, 2000. xi, 497 pp. $69.50 U.S. (cloth), $24.95 U.S. (paper). The [...]

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Introduction: The aim of our study is to evaluate the feasibility, safety and efficacy of the percutaneous closure of PFO (abnormal communication between the right and left atrium). Methods: Between July 2009 and October 2012 percutaneous closure was performed in 37 patients. The presence of PFO was diagnosed through the use of ultrasound techniques: transcranial doppler with contrast (cTCD), transthoracic echocardiography(TTE) and transesophageal echocardiography (TEE). Follow-up was composed consisted of a Holter ECG 7 days after the closure with a 24 hour heart rhythm monitoring, to evaluate eventual arrhythmia cases and programmed controls which included a TTE at 1-3 months, TTE+ cTCD at 6-12 months, to evaluate the right positioning of the device and the complete closure of the defect. Results: We obtained 100% of procedural success (correct and stable implantation of the device in a perfect position on the interatrial septum).No complications were recorded during the procedure and no new onset atrial fibrillation was detected in any patients after the PFO closure. The follow up with cTCD and TEE reported a closing rate of 86.7%. No new clinical cerebrovascular events occurred in treated patients until now. Conclusion: Our experience describes the percutaneous PFO procedure as feasible, safe and effective with a high rate of procedural success, with an absence of significant adverse events and a high rate of complete closure. [ABSTRACT FROM AUTHOR]

Background: Intramammary infection is the result of invasion and multiplication of microorganisms in the mammary gland and commonly leads to mastitis in dairy animals. Although much has been done to improve cows' udder health, mastitis remains a significant and costly health issue for dairy farmers, especially if subclinical. In this study, quarter milk samples from clinically healthy cows were harvested to detect pathogens via quantitative PCR (qPCR) and evaluate changes in individual milk traits according to the number of quarters infected and the type of microorganism(s). A commercial qPCR kit was used for detection of Mycoplasma bovis, Mycoplasma spp., Staphylococcus aureus, coagulase-negative staphylococci (CNS), Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Prototheca spp., Escherichia coli, Klebsiella spp., Enterococcus spp. and Lactococcus lactis ssp. lactis. Quarter and pooled milk information of 383 Holstein, 132 Simmental, 129 Rendena, and 112 Jersey cows in 9 Italian single-breed herds was available., Results: Among the cows with pathogen(s) present in at least 1 quarter, CNS was the most commonly detected DNA, followed by Streptococcus uberis, Mycoplasma bovis, and Streptococcus agalactiae. Cows negative to qPCR were 206 and had the lowest milk somatic cell count. Viceversa, cows with DNA isolated in ≥ 3 quarters were those with the highest somatic cell count. Moreover, when major pathogens were isolated in ≥ 3 quarters, milk had the lowest casein index and lactose content. In animals with pathogen(s) DNA isolated, the extent with whom milk yield and major solids were impaired did not significantly differ between major and minor pathogens., Conclusions: The effect of the number of affected quarters on the pool milk quality traits was investigated in clinically healthy cows using a commercial kit. Results remark the important negative effect of subclinical udder inflammations on milk yield and quality, but more efforts should be made to investigate the presence of untargeted microorganisms, as they may be potentially dangerous for cows. For a smarter use of antimicrobials, analysis of milk via qPCR is advisable - especially in cows at dry off - to identify quarters at high risk of inflammation and thus apply a targeted/tailored treatment., (© 2024. The Author(s).)

In this study we evaluated the prevalence of pathogens detected via quantitative PCR (qPCR) in milk from apparently healthy cows to identify the most common etiological agents present in Italian dairy farms. Milk samples were collected using a sterile protocol at quarter-level (3239 samples, 822 cows) and a conventional protocol at udder level as composite milk from the functional quarters of each cow (5464 samples, 5464 cows). The qPCR commercial kit detected Mycoplasma bovis , Mycoplasma spp., Staphylococcus aureus , coagulase-negative staphylococci (CNS), Streptococcus agalactiae , Streptococcus dysgalactiae , Streptococcus uberis , Prototheca spp., Escherichia coli , Klebsiella spp., Enterococcus spp. and Lactococcus lactis ssp. lactis as well as DNA from the penicillin resistance β-lactamase gene from staphylococci. The prevalence of specific DNA was calculated based on its presence or absence in the samples, factoring in both the sampling protocols and herds. Regardless of the sampling protocol used, the most frequently detected pathogens were CNS (26.6% in sterile and 13.9% in conventional protocol) and Streptococcus uberis (9.6% and 16.5%, respectively). These results underscore the necessity for pathogen-specific interventions at the farm level to enhance the udder health of dairy cows via management recommendations.

Milk differential somatic cell count (DSCC) represents the percentage of polymorphonuclear neutrophils and lymphocytes out of the total somatic cell count (SCC) and has been proposed in recent years as a proxy for udder health in dairy cows. We investigated phenotypic factors affecting SCC and DSCC using 3978 records of 212 Alpine Grey and 426 Burlina cows farmed in Northern Italy. The linear mixed model accounted for the fixed effects of breed, parity, lactation stage, sampling season, and first-order interactions of breed with the other effects. Cow, herd-test-date nested within breed were random. Subsequently, four udder health status groups (UHS) were created by combining SCC and DSCC to assess the UHS impact on milk yield and quality. DSCC was greater in Alpine Grey (66.2 ± 0.8%) than Burlina cows (63.2 ± 0.6%) and, similarly to SCC, it increased with days in milk and parity regardless of breed. Milk yield and composition were affected by UHS in both breeds. These results suggest that also udder health of local breeds can be monitored on a large scale through SCC and DSCC for reduction in biodiversity loss and increased farm profitability. However, in addition to milk data, the introduction of mastitis recording and monitoring plans is advisable.

Somatic gene translocations are key to making an accurate diagnosis in many cancers including many pediatric sarcomas. Currently available molecular diagnostic approaches to identifying somatic pathognomonic translocations have limitations such as minimal multiplexing, high cost, complex computational requirements, or slow turnaround times. We sought to develop a new fusion-detection assay optimized to mitigate these challenges. To accomplish this goal, we developed a highly sensitive multiplexed digital PCR-based approach that can identify the gene partners of multiple somatic fusion transcripts. This assay was validated for specificity with cell lines and synthetized DNA fragments. Assay sensitivity was optimized using a tiered amplification approach for fusion detection from low input and/or degraded RNA. The assay was then tested for the potential application of fusion detection from FFPE tissue and liquid biopsy samples. We found that this multiplexed PCR approach was able to accurately identify the presence of seven different targeted fusion transcripts with a turnaround time of 1 to 2 days. The addition of a tiered amplification step allowed the detection of targeted fusions from as little as 1 pg of RNA input. We also identified fusions from as little as two unstained slides of FFPE tumor biopsy tissue, from circulating tumor cells collected from tumor-bearing mice, and from liquid biopsy samples from patients with known fusion-positive cancers. We also demonstrated that the assay could be easily adapted for additional fusion targets. In summary, this novel assay detects multiple somatic fusion partners in biologic samples with low tumor content and low-quality RNA in less than two days. The assay is inexpensive and could be applied to surgical and liquid biopsies, particularly in places with inadequate resources for more expensive and expertise-dependent assays such as next-generation sequencing., (© 2022. The Author(s).)

Under the name of lipophilic marine toxins, there are included more than 1000 toxic secondary metabolites, produced by phytoplankton, with the common chemical property of lipophilicity. Due to toxicological effects and geographical distribution, in European legislation relevant compounds are regulated, and their determination is accomplished with the reference liquid chromatography-tandem mass spectrometry method. In this study a modified ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed for the identification and quantification of EU-regulated lipophilic toxins. The method optimization included a refinement of SPE-C18 clean-up, in order to reduce matrix interferences. Improved LC conditions and upgraded chromatographic ammonia-based gradient ensured the best separation of all analytes and, in particular, of the two structural isomers (OA and DTX2). Also, different MS parameters were tested, and confirmation criteria finally established. The validation studies confirmed that all parameters were satisfactory. The requirements for precision (RSD% < 11.8% for each compound), trueness (recoveries from 73 to 101%) and sensitivity (limits of quantification in the range 3−8 µg kg−1) were fulfilled. The matrix effect, ranging from −9 to 19%, allowed the use of a calibration curve in solvent (3−320 µg kg−1 in matrix) for quantification of real samples. Method relative uncertainty ranged from 12 to 20.3%. Additionally, a total of 1000 shellfish samples was analysed, providing a first preliminary surveillance study that may contribute to the knowledge of lipophilic marine toxins contamination. Increase in algae proliferation events and intoxication cases, EFSA suggestions for modification of maximum permitted levels and toxicity equivalency factors, and new studies of important toxic effects underline that implementation of reference methods still represents an important task for health and food safety laboratories.

Aims: Echocardiographic assessment of left ventricular filling pressures is performed using a multi-parametric algorithm. Unselected sample of patients with heart failure with reduced ejection fraction (HFrEF) patients may demonstrate an indeterminate status of diastolic indices making interpretation challenging. We sought to test improvement in the diagnostic accuracy of standard and strain echocardiography of the left ventricle and left atrium (LA) to estimate a pulmonary capillary wedge pressure (PCWP) > 15 mmHg in patients with HFrEF., Methods and Results: Out of 82 consecutive patients, 78 patients were included in the final analysis and right heat catheterization, and echocardiogram was performed simultaneously. According to the univariable analysis, E wave velocity, the ratio between E-wave/A-wave (E/A, area under the curve [AUC] = 0.81, respectively), isovolumic relaxation time (AUC = 0.83), pulmonary vein D wave (AUC = 0.84), pulmonary vein S/D Ratio (AUC = 0.85), early pulmonary regurgitation velocity (AUC = 0.80), and accelerationa time at right ventricular out-flow tract (RVOT AT, AUC = 0.84) identified with the highest accuracy PCWP > 15 mmHg. They were all tested in multivariate analysis, and they were not independently correlated with PCWP. Tricuspid regurgitation (TR) velocity was measurement with the highest predictive value in identifying PCWP > 15 mmHg (AUC = 0.89), compared with other established parameters such as the ratio between e-wave velocity divided by mitral annular e' velocity (E/e'), deceleration time, or LA indexed volume (LAVi), which all reached a lower accuracy level (AUC = 0.75; 0.78; 0.76). Among strain measures, global longitudinal strain in four chamber view (GLS 4ch), the ratio between e-wave velocity divided by mitral annular e' strain rate (E/e'sr), and LA longitudinal strain at the reservoir phase were helpful in estimating elevated PCWP (AUC = 0.77; 0.76; 0.75). According to multivariable analysis, the following two models had the greatest accuracy in detecting PCWP > 15 mmHg: (i) TR velocity, LAVi, and E wave velocity (receiver operating characteristic [ROC]-AUC = 0.98), (ii) AT RVOT, LAVi and GLS 4ch (ROC-AUC = 0.96). Neither E/A (ROC-AUC = 0.81) nor E/e' (ROC-AUC = 0.75) was an independent predictor when included in the model. The two MODELS were applicable to the entire population and demonstrated better agreement with the invasive reference (91% and 88%) than the guidelines algorithm (77%) regardless of the type of rhythm., Conclusions: Our suggested echocardiographic approach could be used to potentially reduce the frequency of "doubtful" classification and increase the accuracy in predicting elevated left ventricular filling pressure leading to a decrease in the number of invasive assessment made by right heart catheterization., (© 2020 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.)

Protein phosphorylation plays an important role during the life cycle of many viruses. Venezuelan equine encephalitis virus (VEEV) capsid protein has recently been shown to be phosphorylated at four residues. Here those studies are extended to determine the kinase responsible for phosphorylation and the importance of capsid phosphorylation during the viral life cycle. Phosphorylation site prediction software suggests that Protein Kinase C (PKC) is responsible for phosphorylation of VEEV capsid. VEEV capsid co-immunoprecipitated with PKCδ, but not other PKC isoforms and siRNA knockdown of PKCδ caused a decrease in viral replication. Furthermore, knockdown of PKCδ by siRNA decreased capsid phosphorylation. A virus with capsid phosphorylation sites mutated to alanine (VEEV CPD) displayed a lower genomic copy to pfu ratio than the parental virus; suggesting more efficient viral assembly and more infectious particles being released. RNA:capsid binding was significantly increased in the mutant virus, confirming these results. Finally, VEEV CPD is attenuated in a mouse model of infection, with mice showing increased survival and decreased clinical signs as compared to mice infected with the parental virus. Collectively our data support a model in which PKCδ mediated capsid phosphorylation regulates viral RNA binding and assembly, significantly impacting viral pathogenesis., Competing Interests: The authors have declared that no competing interests exist.

Background: Single-photon emission computed tomography has shown relevant limitations in the quantification of left ventricular (LV) mass. We sought to compare the estimates of LV mass on Cadmium-Zinc-Telluride (CZT) myocardial perfusion imaging (MPI) as compared to cardiac magnetic resonance (CMR)., Methods and Results: Twenty-five patients underwent MPI on a CZT camera and CMR on a 1.5 T scanner within 12 ± 3 weeks. LV mass was quantified on CZT images using two softwares: 4D-MSPECT (4DM) and Emory Cardiac Toolbox (ECTb). LV mass by CMR was quantified using MASS software (Medis, Leiden, The Netherlands). LV mass values obtained with 4DM and ECTb were highly reproducible [intraclass correlation coefficients .98 (95% CI .97-.99), and .98 (95% CI 0.97-.99), respectively]. The mean LVM mass values were 151 ± 44 g on CMR, 151 ± 43 g with 4DM (P = NS vs CMR), and 157 ± 42 g with ECTb (P < .001 vs CMR; P = .007 vs 4DM) CZT images. There was an excellent correlation between LV mass values between CMR and both 4DM (R 2  = .95; P < .001) and ECTb (R 2  = .98; P < .001) with narrow limits of agreement (- 13.6% to + 13.4% for 4DM, and - 5.6% to + 14.1% for ECTb)., Conclusions: The evaluation of LV mass is feasible on CZT images, showing excellent agreement with CMR.

Background: To assess the role of the mitral valve apparatus (leaflets, chordae and papillary muscles, (PM)) in left ventricle outflow tract (LVOT) obstruction, and results of the surgical treatment for hypertrophic obstructive cardiomyopathy (HOCM)., Methods: Twenty-eight consecutive patients (58±11years, 53% female) undergoing HOCM surgery from 2007 to 2016 at our institute were retrospectively reviewed. Endpoints included the involvement of the mitral valve in LVOT obstruction, mortality, and changes in clinical and echocardiographic characteristics after HOCM surgery., Results: Secondary chordae tendineae tractioning the anterior mitral leaflet to the interventricular septum, and systolic anterior motion were detected in 78% of the patients. Anomalous, hypertrophied, and fused PM with muscularis trabeculae hypertrophy were found in 50%, 25%, and 35% of the patients, respectively. Four patients had posterior leaflet redundancy. Secondary chordae (92%), PM, and muscularis trabeculae resection (71%), and PM splitting and elongation (28%) were added variably to septal myectomy (100%). Nine procedures (32%) on mitral valve leaflets were performed, involving six posterior and three anterior mitral leaflets. Long-term follow-up was 4±2.8years. There was no hospital mortality, and NYHA was reduced from 3±0.5 to 1±0.7 (p<0.0001), the LVOT gradient from 88±35 to 20±18mmHg (p<0.0001), mitral valve regurgitation from grade 3±1 to 1±0.7 (p<0.0001), and septum thickness from 18±3 to 14±2mm (p<0.0001)., Conclusions: The mitral valve apparatus contributes with all its components variably to LVOT dynamic obstruction thus surgical correction in addition to extended myectomy is recommended to achieve the best outcome., (Copyright © 2018 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ). Published by Elsevier B.V. All rights reserved.)

Cancer is the second leading cause of death, and the most common diagnosis among the general population is breast and colon cancer. Recently, an increasing number of new cases of invasive breast and colon cancer have been estimated, and more people die from these diseases. In addition to the genetic pattern, diet and lifestyle including smoking, alcohol consumption, and sedentary behaviour have also been identified as potential risks factors. Recent studies of cancer survivors have shown the beneficial effects of regular physical activity to reduce the prevalence of comorbidity, muscle atrophy, weight changes, reduced aerobic capacity, fatigue, depression, and reduced quality of life. Dedicated and individual programs are crucial for achieving the goals of improving quality of life and reducing comorbidities. A multidisciplinary approach is fundamental: lifestyle assessment, including estimating the level of physical activity, as well as nutritional habits, may be the first step. A periodic cardiovascular examination is crucial for detecting asymptomatic early myocardial failure. According to current ACSM guidelines, different levels of exercise (low-moderate 40% and moderate up to 60% of the maximal HR) may be prescribed, and patients enrolled may follow the exercise program if in the absence of contraindications. The current paper reports observations from our clinical practice and provides practical strategies that bridge contemporary, published guidelines into practice within a multi-disciplinary team working with cancer survivors in Italy.

Based on choline oxidase immobilized by co-crosslinking on an overoxidised polypyrrole modified platinum electrode, a novel electrochemical assay for cholinesterase activity in human serum was developed. The assay was performed by adding an aliquot of cholinesterase standard solution or serum sample to phosphate buffer containing choline or thiocholine ester and measuring the oxidation current of hydrogen peroxide at the rotating modified electrode polarized at +0.7 V vs. SCE. The influence of some experimental parameters such as pH of the assay, mass transport at the electrode, type and concentration of the cholinesterase substrate was studied and optimised. Reversible inhibition of choline oxidase from cholinesterase substrates was evidenced for the first time, which increases in the order of acetylcholine, butyrylcholine and s-butyrylthiocholine. Wide linear range, fast response time and appreciable long-term stability were assured for both acethyl- and butyrylcholinesterase assays. On allowing the polypyrrole layer to efficiently remove interferences from the electroactive compounds in the sample, the present method revealed to be suitable for the detection of butyrylcholinesterase in human serum at activities as low as 0.5 U L -1 . The validation with a reference spectrophotometric method showed no significant differences when human serum samples were analysed.

In the present study, a column packed with core-shell particles was used for the separation and the quantification of 13 sulphonamides in milk by conventional high-performance liquid chromatography coupled with diode array ultraviolet detection (HPLC/UV-DAD). Preliminary experiments were carried out to investigate selectivity of different stationary phases. Best results were achieved using a C 18 column packed with 2.6μm core-shell particles (diameter 4.6mm, length 75mm). A binary gradient elution based on acetate buffer solution at pH 4.50 and a mixture of methanol acetonitrile 50:50 (v/v) was employed at the flow rate of 1.2mLmin -1 with an injection volume of 6μL. These chromatographic conditions allowed the efficient separation of 13 sulphonamides in about 8min. To evaluate the suitability of the method for official control analysis, the most important validation parameters were investigated according to the European Decision 657/2002/EC as established for analysis of drug residues in food. Sulphonamides were recovered from milk samples by a simple and quick preparation procedure consisting of an extraction step with chloroform/acetone and a purification step with n-hexane. Mean recoveries from raw milk ranged between 55% and 86% at the Maximum Residual Limit of 100μgkg -1 , and RSDs% resulted lower than Thompson and Horwitz RSD% reference values for all sulphonamides. The LOQ value (2.7-15μgkg -1 ) was low enough to satisfy legal limits suggested by European Regulation 37/2010/EC., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A simple, sensitive and selective multiresidue high-performance liquid chromatography with diode array detection method for determination of 13 sulphonamides in eggs was developed and validated. Sample extraction and clean-up conditions were carefully studied and factors as gradient elution and column temperature were found as key parameters to improve separation efficiency. The method was validated following the European Commission Decision 2002/657/EC criteria adopting spiking levels of 20, 40 and 60 μg kg(-1) "As Low As Reasonably Achievable". The necessary requirements for precision (RSDR% below 23%) and trueness (recovery ranging from 45.2% to 87.5%) were fulfilled. Decision limit (CCα) values below 18.5 μg kg(-1), comparable to those reported in LC-MS detection, demonstrated the suitability of the method in residues surveillance plans for the sulphonamides analysis in eggs at the carry-over level without the use of sophisticated and expensive instrumentation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Sample quality is a fundamental parameter for the successful diagnosis of respiratory viruses. This parameter depends upon the concentration of epithelial cells. Respiratory samples are usually heterogeneous, which makes relative quantification of the viral load, against the quantity of cells, the most suitable measurement. The quantification of viral load in the field of respiratory viruses is a vital piece of information. Quantification is required from RNA or DNA viral genomes extracted., Objectives: To design (RT-)PCR assays for reference genes, which show stable expression during viral infection, to be used as cellular controls and cellular quantification tools., Study Design: Assays were designed for two reference genes: hypoxanthine phosphoribosyltransferase 1 (HPRT1) and ubiquitin C (UBC). The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as a reference for this study. The transcriptional activity of the three genes was studied during infection with respiratory syncytial virus and adenovirus. The HPRT1 q(RT-)PCR assay was used on clinical samples., Results: All the analysis methods concluded that the three reference genes were stably expressed during viral infection. The HPRT1 q(RT-)PCR assay indicated that the majority of clinical samples (n=301, 69%) had a cellular load of between 100 and 10,000 cells/PCR. The data showed that the concentration decreased as the age of patient increased., Conclusions: A new tool has been developed and commercialized for quality control and evaluation of cellular concentration in respiratory samples., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Poisoning of domestic animals is frequently caused by anticoagulant rodenticides. Validation and applications of a rapid and reliable method for the simultaneous determination of 8 anticoagulant rodenticides (bromadiolone, brodifacoum, coumachlor, coumafuryl, coumatetralyl, difenacoum, flocoumafen, and warfarin) in baits and animal livers using high-performance liquid chromatography with fluorescence detection are reported herein. The methodology was validated by an in-house validation model at 2.5 mg/kg, which is the level commonly found in the tissues of poisoned domestic animals. The 8 anticoagulants can be determined at the concentration range of 1.25-100 mg/kg with determination coefficients higher than 0.992. A recovery value from 70% to 109% was observed for all the studied molecules. The results of the validation process demonstrate suitability for application in official analysis and for monitoring purposes of animal poisoning by anticoagulant rodenticides.

The occurrence of the fumonisins B(1) and B(2) in maize-based food products marketed in Italy was examined. A simply and reliable chromatographic method with fluorimetric detection and postcolumn o-phtalaldehyde derivatization was used for a monitoring of 100 samples (8 flours, 21 corn-meal, 16 snacks, 7 maize samples, 13 gluten-free products, and 35 corn-flakes) bought in local supermarkets during the years 2008 and 2009. The presence of both fumonisins B(1) and B(2), at a concentration higher than 15 μg/kg, was observed in all samples of corn-meal and maize-flour, in 75% of snacks, in 57% of maize samples, in 54% of gluten-free products, and in 29% of corn-flakes. A total of 7 samples including 4 corn-meals, 2 maize-flours, and 1 maize showed a value exceeding the maximum level fixed in the Regulation 1126/2007/EC; no positive sample was observed in corn-flakes, snacks, and gluten-free foods. Fumonisins contamination, on the whole range of maize-based food products analyzed, emphasizes the need of improve agricultural practices, and increase official control and monitoring studies.

Reliable real-time quantitative PCR assays to measure Epstein-Barr virus (EBV) DNA load (EBV) are useful for monitoring EBV-associated diseases. We evaluated a new commercial kit, EBV R-gene Quantification kit (Argene, Varilhes, France) to quantify EBV DNA load in whole blood. Assay performance was assessed with two PCR platforms (LightCycler 2.0 and SmartCycler 2.0) and three commercial DNA extraction methods. The assay was compared with our in-house real-time EBV PCR using samples from the Quality Control for Molecular Diagnostics 2006 EBV proficiency program and using 167 whole-blood specimens from individuals with infectious mononucleosis, from transplanted or HIV-infected patients, and from EBV-seropositive healthy carriers. The EBV R-gene assay was sensitive to 500 copies of EBV DNA per milliliter of whole blood with the two PCR platforms and the three extraction methods and was linear across 4 orders of magnitude. Intra- and interassay coefficients of variations were less than 20%. Nine of 10 samples tested with the EBV R-gene were in agreement with the expected qualitative results of the Quality Control for Molecular Diagnostics 2006 EBV proficiency program, and 7 of 10 samples were within +/-0.5 log units of the expected quantitative values, with discrepant results mostly observed for low viral load (ie, <1000 copies/ml). In the clinical specimens, the correlation between the R-gene assay and the in-house PCR was high (r=0.92). In conclusion, the EBV R-gene assay accurately assesses the EBV DNA load in whole blood of patients with various forms of EBV infections.

A high performance liquid chromatographic method with fluorimetric detection for the determination of aflatoxin M1 (AFM1) in milk has been optimized and validated according to Commission Decision 2002/657/EC by using the conventional validation approach. The procedure for determining selectivity, recovery, precision, decision limit (CC(alpha)), detection capability (CC(beta)) and ruggedness of the method has been reported. The results of the validation process demonstrate the agreement of the method with the provisions of Commission Regulation 401/2006/EC. The mean recovery calculated at three levels of fortification (0.5, 1.0, and 1.5-fold the MRL) was 91% and the maximum relative standard deviation value for the within-laboratory reproducibility was 15%. Limit of detection (LOD) and limit of quantitation (LOQ) values were 0.006 microg kg(-1) and 0.015 microg kg(-1) while the CC(alpha) and CC(beta) values were 0.058 microg kg(-1) and 0.065 microg kg(-1), respectively. The relative expanded measurement uncertainty of the method was 7%. The method was not affected by slight variations of some critical factors (ruggedness minor changes) as pre-treatment and clean-up of milk samples, thermal treatment and different storage conditions, as well as by major changes valued in terms of milk produced by different species (buffalo, goat and sheep). The method allowed accurate confirmation analyses of milk samples, resulted positive by the screening method. In fact, the Z-score values attained in a proficiency test round were well below the reference value of 1, proving the excellent laboratory performances.

Background: Fas antigen (Ag) has recently been identified as the putative surface molecule capable of transducing apoptotic signals into cells. Alterations in the expression of proto-oncogene bcl-2 have been implicated in the regulation of apoptosis., Materials and Methods: By employing a monoclonal antibody to bcl-2 protein (124 clone) and to Fas Ag (UB2 clone) the expression of these molecules was analyzed at flow cytometry on bone marrow (BM) and peripheral blood (PB) samples from patients suffering from different lymphoid and myeloid leukemic diseases (27 acute non-lymphocytic leukemia [ANLL]; 14 acute lymphocytic leukemia [ALL]; 19 B-cell chronic lymphocytic leukemia [CLL]; 2 Ph1+ chronic myeloid leukemia [CML]; one CD8+ T-cell chronic lymphoproliferative disorders). Results were compared with those observed on normal PB leukocytes and BM B-cell precursors from patients with non-neoplastic hematological disorders., Results: Fas Ag was constitutively expressed by both monocytes and neutrophils, while lymphocytes expressed bcl-2 with no difference between B and T cell subsets. Interestingly, bcl-2 expression was always absent on neutrophils. When dealing with ANLL patients, a relatively low bcl-2 and high Fas Ag phenotype characterized subtypes with granulocytic (M2) or promyelocytic (M3) differentiation. This observation was confirmed in a small number of patients for whom bcl-2 levels were quantified as antibody binding capacity (ABC) in molecules/cell. Leukemic cells from patients with ALL constitutively expressed bcl-2, the pattern of this expression being quantitatively lower than that of immature B-cell precursors. Finally, high bcl-2 and low Fas Ag expression represented a crucial part of the B-cell CLL immunophenotype., Conclusions: Although based on a small number of patient and control samples, our results suggest that bcl-2 and Fas Ag are coordinately expressed on normal PB leukocytes. Fas Ag is expressed at low levels on B-CLL cells, generally considered long-surviving cells. The relatively lower bcl-2-expression detected in both M2 and M3 subtypes may explain, at least in part, the higher remission rates obtained in these forms of ANLL than in other less differentiated morphological variants.

Using a combination of oligonucleotide probes and restriction endonuclease enzymes, we characterize beta-thalassemic mutations in 91 homozygous patients and 86 unrelated carriers. Overall, 268 beta-thalassemic genes were obtained. Eleven beta-globin mutations were identified, confirming the wide molecular heterogeneity of beta-thalassemia in Calabria. Information from the present study represents the mainstay for the development of a program of early prenatal diagnosis by direct detection of mutations in Calabria.

Pulmonary toxicity developed in 15 (17%) of 89 patients treated with amiodarone during a follow-up period of 2 weeks to 54 (mean 20 +/- 15) months. Prospective evaluation of serial pulmonary function tests in 67 patients demonstrated both a significant decrease from baseline in three of six variables in patients with toxicity at the time of diagnosis and a significant difference compared with the same variables in patients without toxicity. The most significant of these was the diffusing capacity for carbon monoxide (DLCO). An individual decrease in DLCO greater than or equal to 15% gave an optimal sensitivity of 100% and a specificity of 89% for the diagnosis of pulmonary toxicity. However, a decrease in DLCO greater than or equal to 15% did not alone warrant a change in therapy in asymptomatic patients. Although higher maintenance doses of amiodarone appeared to be related to the development of this complication, an abnormal baseline DLCO (less than 60% of predicted) with or without an initial abnormal chest roentgenogram did not predispose to pulmonary toxicity.

Reproduction of spontaneously occurring ventricular tachycardia (VT) and induction of previously undocumented VT were studied prospectively in 98 patients: 48 with documented sustained VT or ventricular fibrillation, 25 with nonsustained or exercise-induced VT, and 25 with no documented VT. Patients received 1 to 4 ventricular extrastimuli and ventricular burst pacing at 2 right ventricular (RV) sites, first at twice late diastolic threshold, and then at 10 mA using a prospective, tandem study design. Spontaneously occurring VT was reproduced in 37 of 48 patients (77%) at twice late diastolic threshold and in 1 other patient (2%) at 10 mA. VT was reproduced at both RV sites in 17 of 48 patients (35%) and at 1 site in 20 of 48 patients (42%) at twice late diastolic threshold. A previously undocumented VT was induced in 7 of 25 patients (28%) with no documented VT at twice diastolic threshold and 14 of 25 patients (56%) at 10 mA. A previously undocumented VT was induced in 33 of 73 patients (45%) with a history of sustained or nonsustained VT at twice late diastolic threshold and in 47 of 73 patients (64%) at 10 mA. In patients with documented sustained VT, the use of up to 4 ventricular extrastimuli at multiple RV sites increases the sensitivity of the test. In patients without documented VT, the induction of previously undocumented VT with more than 3 ventricular extrastimuli limits the specificity of the test. Increased current provides only a slight advantage over 4 ventricular extrastimuli at twice late diastolic threshold in terms of reproduction of spontaneously occurring VT, but leads to a marked increase in induction of previously undocumented VT.

Initiation of ventricular tachycardia (VT) by right ventricular extrastimulation was analyzed in 142 consecutive patients, 53 with electrocardiographically documented episodes of spontaneous VT or ventricular fibrillation (VF) and 68 with no spontaneous VT or VF; 21 patients with a history of sudden death but no documented arrhythmia were excluded from further analysis. All patients received 1 to 4 extrastimuli (S2, S3, S4 and S5) during pacing at fixed cycle lengths of 600 or 500 msec at 1 or 2 right ventricular sites. Clinical VT was reproduced by extrastimulation in 28 of 43 patients (65%) with sustained VT and in 0 of 10 patients with nonsustained VT. Clinical VT was induced by S2 or S3 in 16 patients and by S4 or S5 in 12 patients. Ventricular burst pacing reproduced clinical VT in 3 other patients. Nonclinical VT, which was most often polymorphic and nonsustained, was induced in 24 of 121 patients (20%), in 11 by S2 or S3 and in 13 by S4 or S5. Ventricular burst pacing induced nonclinical VT in 4 other patients. In patients with spontaneous sustained VT, the use of S4 and S5 in the right ventricle increases the yield of inducible clinical VT compared with use of S2 and S3 alone, but at a cost of increased induction of nonclinical VT. Frequent induction of nonclinical VT limits the interpretation of the results of such stimulation in patients without previously documented VT.

This study investigates the relation of spontaneous ventricular arrhythmia on ambulatory electrocardiographic (ECG) monitoring to the subsequent inducibility of ventricular tachycardia during programmed electrical stimulation. Eighty patients (65 men, 15 women), whose mean age was 58 years, presented with one of the following: sustained ventricular tachycardia (n = 54); sudden death requiring resuscitation (n = 4); ventricular fibrillation (n = 11); or syncope thought to be of cardiac origin (n = 11). All patients had 24 hour ambulatory electrocardiograms and programmed electrical stimulation while receiving no antiarrhythmic therapy. Programmed electrical stimulation resulted in inducible sustained ventricular tachycardia (defined as a rate of greater than or equal to 120 beats/min for greater than or equal to 1 minute or requiring intervention) in 53 of the 80 patients. There was no measure of frequency or complexity of spontaneous arrhythmia detected on ambulatory ECG that could identify the degree of subsequent ventricular tachycardia inducibility during programmed electrical stimulation. In fact, 25% of patients who had inducible sustained ventricular tachycardia had little or no spontaneous arrhythmia on ambulatory ECG. Furthermore, of the 53 patients with inducible sustained ventricular tachycardia, 28 and 55% had no couplets or nonsustained ventricular tachycardia, respectively, during ambulatory monitoring. The combination of a clinical presentation of sustained ventricular tachycardia, confirmed coronary artery disease and a left ventricular ejection fraction of less than 30% had a better positive predictive value than did any ambulatory ECG criterion in predicting the inducibility of sustained ventricular tachycardia.

In a patient with two morphologic forms of ventricular tachycardia, rapid pacing from different ventricular sites produced two distinctively different patterns of entrainment. Pacing from sites contralateral to the site of emergence of ventricular tachycardia caused progressive fusion. Following pacing, the interval (return interval) between the first tachycardia beat (return beat) and the preceding beat was equal to the pacing cycle length. Near the site of emergence, the local electrogram of the return beat was morphologically identical to that of the preceding paced beats. In contrast, pacing from sites ipsilateral to the site of emergence did not cause fusion. The return intervals increased with decreasing pacing cycle lengths. The local electrogram of the return beat was morphologically different from that of the preceding paced beats. Using a model of ventricular reentrance, both patterns of responses can be explained. Pacing impulses arising from sites contralateral to the site of emergence activate the latter orthodromically. Fusion occurs between the emerging tachycardia wavefront and the next pacing wavefront. In contrast, pacing impulses arising from sites ipsilateral to the site of emergence activate the latter antidromically. Fusion is not observed because collision within the area of slow conduction prevents the emergence of the tachycardia during pacing.