Recombinant luminescent bacteria for measuring bioavailable arsenite and antimonite

Luminescent bacterial strains for the measurement of bioavailable arsenite and antimony were constructed, The expression of firefly luciferase was controlled by the regulatory unit of the ars operon of Staphylococcus aureus plasmid pI258 in recombinant plasmid pT0021, with S. aureus RN4220, Bacillus subtilis BR151, and Escherichia coli MC1061 as host strains, Strain RN4220(pT0021) was found to be the most sensitive for metal detection responding to arsenite, antimonite, and cadmium, the lowest detectable concentrations being 100, 33, and 330 nhl, respectively, Strains BR151(pT0021) and MC1061(pT0021) responded to arsenite, arsenate, antimonite, and cadmium, the lowest detectable concentrations being 3.3 and 330 mu M and 330 and 330 nM with BR151(pT0021), respectively, and 3.3, 33, 3.3, and 33 CIM with MC1061(pT0021), respectively, In the absence of the mentioned ions, the expression of luciferase was repressed and only a small amount of background light was emitted, Other ions did not notably interfere with the measurement in any of the strains tested, Freeze-drying of the cells did not decrease the sensitivity of the detection of arsenite; however, the induction coefficients were somewhat lower

Luminescent bacterial sensor for cadmium and lead

A sensor plasmid was constructed by inserting the regulation unit from the cadA determinant of plasmid pI258 to control the expression of firefly luciferase. The resulting sensor plasmid pTOO24 is capable of replicating in Gram-positive and Gram-negative bacteria. The expression of the reporter gene as a function of added extracellular heavy metals was studied in Staphylococcus aureus strain RN4220 and Bacillus subtilis strain BR151. Strain RN4220(pTOO24) mainly responded to cadmium, lead and antimony, the lowest detectable concentrations being 10 nM, 33 nM and 1 nM respectively. Strain BR151(pTOO24) responded to cadmium, antimony, zinc and tin at concentrations starting from 3.3 nM, 33 nM, 1 mu M and 100 mu M, respectively. The luminescence ratios between induced and uninduced cells, the induction coefficients, of strains RN4220(pTOO24) and BR151(pTOO24) were 23-50 and about 5, respectively. These results were obtained with only 2-3 h incubation times. Freeze-drying of the sensor strains had only moderate effects on the performance with respect to sensitivity or induction coefficients. (C) 1998 Elsevier Science S.A. All rights reserved

Amplifying control RNA for RT-PCR applications by nucleic acid sequence based amplification (NASBA)

Control RNA for RT-PCR applications was amplified by nucleic acid sequence based amplification (NASBA) using the NucliSens (R) Basic Kit. This method was used to construct positive control RNA for enterovirus, insulin, and G-protein RT-PCR, and for interferon-alpha real-time RT-PCR. The primers were designed to amplify identical RNA from RNA templates, which differs from the usual NASBA procedure, where opposite strand RNA is amplified from the target. This "inverse NASBA" method is easy to use and it does not require any expensive special equipment. The amplification reaction is done using a water bath and detection of amplified product by agarose gel electrophoresis. Generated RNA fragments were 195-714 bases long, of positive polarity and the amount of RNA was sufficient for thousands of RT-PCR reactions depending on the sensitivity of the RT-PCR. (c) 2005 Elsevier B.V. All rights reserved

Molecular analysis of an echovirus 3 strain isolated from an individual concurrently with appearance of islet cell and IA-2 autoantibodies

Growing evidence has implicated members of the genus Enterovirus of the family Picornaviridae in the etiology of some cases of type I diabetes (T1D). To contribute to an understanding of the molecular determinants underlying this association, we determined the complete nucleotide sequence of a strain of echovirus 3 (E3), Human enterovirus B (HEV-B) species, isolated from an individual who soon after virus isolation developed autoantibodies characteristic of T1D. The individual has remained positive for over 6 years for tyrosine phosphatase-related IA-2 protein autoantibodies and islet cell autoantibodies, indicating an ongoing autoimmune process, although he has not yet developed clinical T1D. The sequence obtained adds weight to the observation that recent enterovirus isolates differ significantly from prototype strains and provides further evidence of a role for recombination in enterovirus evolution. In common with most HEV-B species members, the isolate exhibits 2C and VP1 sequences suggested as triggers of autoimmunity through molecular mimicry. However, comparisons with the E3 prototype strain and previously reported diabetogenic and nondiabetogenic HEV-B strains do not reveal clear candidates for sequence features of PicoBank/DM1/E3 that could be associated with autoantibody appearance. This is the first time a virus strain isolated at the time of commencement of beta-cell damage has been analyzed and is an invaluable addition to enterovirus strains isolated previously at the onset of T1D in the search for specific molecular features which could be associated with diabetes induction

PCR inhibition in stool samples in relation to age of infants

Background: PCR is rapidly replacing traditional methods in diagnostic virus laboratories. PCR inhibitors,which are often present in clinical samples, may lead to false negative test results.Objectives: The aim was to study the presence of PCR inhibitors in stool samples collected from 3- to24-month old children.Study design: Total RNA fraction extracted from stool samples was spiked with a standardized amount ofSemliki Forest Virus RNA and amplified using specific PCR primers. The presence of PCR inhibitors wasdetected by a decrease in amplification rate compared to spiked water samples. Inhibition in differentage groups and dietary origin of PCR inhibitors were analyzed by comparing the samples taken duringexclusive and non-exclusive breastfeeding periods. The inactivation of PCR inhibitors was also assessed.Results: Complete inhibition was seen in 12% (13/108) and partial inhibition in 19% (21/108) of the samples.Inhibition was seen in none of the stool samples (0/31) taken from infants younger than 6 monthscompared to 17% of samples (13/77) taken from6 to 24 months old infants (p more common in younger age group. Addition of bovine serum albumin (BSA) into the reaction mixtureseliminated the effect of inhibitors leading to all samples being positive.Conclusions: PCR inhibitors are frequent in stool samples. They may originate from dietary componentsand can lead to false negative PCR results. The addition of BSA to the cDNA and PCR reactions proved tobe an easy and effective method for eliminating the inhibitory effect of these compounds

Analysis of pancreas tissue in a child positive for islet cell antibodies

Conclusions/interpretation These observations suggest that positivity for ICA alone, even when lasting for more than 1 year, is not associated with inflammatory changes in the islets. However, it is most likely that the pancreatic islets were infected by an enterovirus in this child

Immunological changes and increased expression of myxovirus resistance protein a in thyroid tissue of patients with recent onset and untreated Graves' disease

Background: Few studies have systematically examined the immune cells that infiltrate thyroid tissue at the time of the onset of Graves&#39; disease (GD). The role of viruses in the pathogenesis of autoimmune thyroid diseases is controversial. The present study analyzed inflammatory responses with respect to signs of virus infection. Methods: Thyroid tissue was obtained from 22 patients with newly diagnosed and untreated GD, 24 patients with chronic GD, and 24 controls. Inflammation was assessed by immunostaining for CD4+ and CD8+ T cells, plasma cells (CD138+), and plasmacytoid dendritic cells (PDCs). The production of interferon-inducible myxovirus resistance protein A (MxA) was analyzed as a sign of virus infection. Results: The degree of thyroid inflammation and fibrosis was significantly higher in both patient groups compared with that in controls. The number of CD4+ T cells and plasma cells (activated B cells) was significantly higher in both patient groups. CD8+ cells were only present in patients with chronic disease. MxA expression and the number of PDCs increased only in patients with newly diagnosed GD. There was a strong positive correlation between the number of PDCs and the number of MxA+ leucocytes. Conclusion: The increase in CD8+ T cells during the chronic stage of GD suggests that they may play a role in progression of the autoimmune process from early to chronic thyroiditis. Upregulation of MxA expression during the early stages of the disease, and the positive correlation between the number of PDCs and the number of MxA+ leucocytes, suggests that activated PDCs secrete type I IFNs at the lesion site, possibly in response to viral infection. &nbsp;</p

Prognostic impact of multiple prior percutaneous coronary interventions in patients undergoing coronary artery bypass grafting

AbstractBackground: Multiple percutaneous coronary interventions (PCIs) are considered determinant of poor outcome in patients undergoing coronary artery bypass grafting (CABG), but scarce data exist to substantiate this.Methods and results: Patients who underwent CABG without history of prior PCI or with PCI performed >30 days before surgery were selected for the present analysis from the prospective, multicenter E‐CABG (European Multicenter Study on Coronary Artery Bypass Grafting) registry. Out of 6563 patients with data on preoperative SYNTAX (Synergy between PCI With Taxus and Cardiac Surgery) score, 1181 patients (18.0%) had undergone PCI >30 days before CABG. Of these, 11.6% underwent a single PCI, 4.4% 2 PCIs, and 2.1% ≥3 PCIs. PCI of a single main coronary vessel was performed in 11.3%, of 2 main vessels in 4.9%, and of 3 main vessels in 1.6% of patients. Multivariable analysis showed that differences in early mortality and other outcomes were not significantly different in the study cohorts. The adjusted hospital/30‐day mortality rate was 1.8% in patients without history of prior PCI, 1.9% in those with a history of 1 PCI, 1.4% after 2 PCIs, and 2.5% after ≥3 PCIs (adjusted P=0.8). The adjusted hospital/30‐day mortality rate was 2.0% in those who had undergone PCI of 1 main coronary vessel, 1.3% after PCI of 2 main vessels, and 3.1% after PCI of 3 main coronary vessels (adjusted P=0.6).Conclusions: Multiple prior PCIs are not associated with increased risk of early adverse events in patients undergoing isolated CABG. The present results are conditional to survival after PCI and should not be viewed as a support for a policy of multiple PCI as opposed to earlier CABG.Clinical trial registration: URL: http://www.Clinicaltrials.gov. Unique identifier: NCT02319083. ( J Am Heart Assoc. 2018;7:e010089. DOI: 10.1161/JAHA.118.010089.)Abstract Background: Multiple percutaneous coronary interventions (PCIs) are considered determinant of poor outcome in patients undergoing coronary artery bypass grafting (CABG), but scarce data exist to substantiate this. Methods and results: Patients who underwent CABG without history of prior PCI or with PCI performed >30 days before surgery were selected for the present analysis from the prospective, multicenter E‐CABG (European Multicenter Study on Coronary Artery Bypass Grafting) registry. Out of 6563 patients with data on preoperative SYNTAX (Synergy between PCI With Taxus and Cardiac Surgery) score, 1181 patients (18.0%) had undergone PCI >30 days before CABG. Of these, 11.6% underwent a single PCI, 4.4% 2 PCIs, and 2.1% ≥3 PCIs. PCI of a single main coronary vessel was performed in 11.3%, of 2 main vessels in 4.9%, and of 3 main vessels in 1.6% of patients. Multivariable analysis showed that differences in early mortality and other outcomes were not significantly different in the study cohorts. The adjusted hospital/30‐day mortality rate was 1.8% in patients without history of prior PCI, 1.9% in those with a history of 1 PCI, 1.4% after 2 PCIs, and 2.5% after ≥3 PCIs (adjusted P=0.8). The adjusted hospital/30‐day mortality rate was 2.0% in those who had undergone PCI of 1 main coronary vessel, 1.3% after PCI of 2 main vessels, and 3.1% after PCI of 3 main coronary vessels (adjusted P=0.6). Conclusions: Multiple prior PCIs are not associated with increased risk of early adverse events in patients undergoing isolated CABG. The present results are conditional to survival after PCI and should not be viewed as a support for a policy of multiple PCI as opposed to earlier CABG. Clinical trial registration: URL: http://www.Clinicaltrials.gov. Unique identifier: NCT02319083. ( J Am Heart Assoc. 2018;7:e010089. DOI: 10.1161/JAHA.118.010089.