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At least 811 records · Page 45Linked to original sources
PubMed2001

Comparison of MB/Bact alert 3D system with radiometric BACTEC system and Löwenstein-Jensen medium for recovery and identification of mycobacteria from clinical specimens: a multicenter study.

The MB/BacT ALERT 3D System (MB/BacT) (Organon Teknika, Boxtel, The Netherlands) is a fully automated, nonradiometric system with a revised antibiotic supplement kit designed for the recovery of mycobacteria from clinical specimens. In a multicenter study, the recovery rate of acid-fast bacilli (AFB) and the mean time to their detection from clinical specimens was determined by using the MB/BacT system. Data were compared to those assessed by the radiometric BACTEC 460 system (B460) and by culture on Löwenstein-Jensen (L-J) solid medium. A total of 2,859 respiratory and extrapulmonary specimens were processed by the N-acetyl-L-cysteine (NALC)-NaOH method using two different concentrations of sodium hydroxide; 1.5% was adopted in study design A (1,766 specimens), and 1.0% was used in study design B (1,093 specimens). The contamination rates for MB/BacT were 4.6% (study design A) and 7.1% (study design B). One hundred seventy-nine mycobacterial isolates were detected by study design A, with 148 Mycobacterium tuberculosis complex (MTB) isolates and 31 nontuberculous mycobacteria (NTM) isolates. Overall recovery rates were 78.8% for MB/BacT (P = 0.0049), 64.2% for L-J (P < 0.0001), and 87.1% for B460, whereas they were 84.5, 70.9, and 91.2%, respectively, for MTB alone. A total of 125 mycobacteria were detected by study design B, with 46 MTB and 79 NTM. Overall recovery rates by the individual systems were 57.6% (P = 0.0002), 56.8% (P = 0.0001), and 80% for MB/BacT, L-J, and B460, respectively, whereas the rates were 91.3, 78.3, and 97.8% for MTB alone. By study design A, the mean times to detection of smear-positive MTB, smear-negative MTB, and NTM were 11.5, 19.9, and 19.6 days, respectively, with the MB/BacT; 8.3, 16.8, and 16.6 days, respectively, with the B460; and 20.6, 32.1, and 27.8 days, respectively, with L-J medium. By study design B, the mean times were 15.1, 26.7, and 26 days with the MB/BacT; 11.7, 21.3, and 24.8 days with the B460; and 20.4, 28.7, and 28.4 days with L-J medium. Identification was attempted by probing (Accuprobe) MB/BacT-positive bottles within the first working day following instrument positive flag. Results were compared to those obtained in the B460 positive vials by the p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) test (study design A) or by the Accuprobe assay (study design B). About 90% of MTB and 100% of NTM could be identified, showing turnaround times closely related to those obtained by combining B460 and the NAP test or the Accuprobe assay. In conclusion, even though recovery rates were shown to be lower than B460, especially for NTM, and contaminants were somewhat higher, MB/BacT represents a valuable alternative to the radiometric system, especially in those laboratories where disposal of radioactive waste is restricted. Finally, when AFB are cultured in nonradiometric liquid media, our data (detection times and bacterial overgrowth rates) suggest that decontamination with 1.5% NaOH may be more suitable than the standard NALC-NaOH.

Bacteriological Techniques↗
PubMed2001

Genome of the extremely radiation-resistant bacterium Deinococcus radiodurans viewed from the perspective of comparative genomics.

The bacterium Deinococcus radiodurans shows remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens. D. radiodurans is best known for its extreme resistance to ionizing radiation; not only can it grow continuously in the presence of chronic radiation (6 kilorads/h), but also it can survive acute exposures to gamma radiation exceeding 1,500 kilorads without dying or undergoing induced mutation. These characteristics were the impetus for sequencing the genome of D. radiodurans and the ongoing development of its use for bioremediation of radioactive wastes. Although it is known that these multiple resistance phenotypes stem from efficient DNA repair processes, the mechanisms underlying these extraordinary repair capabilities remain poorly understood. In this work we present an extensive comparative sequence analysis of the Deinococcus genome. Deinococcus is the first representative with a completely sequenced genome from a distinct bacterial lineage of extremophiles, the Thermus-Deinococcus group. Phylogenetic tree analysis, combined with the identification of several synapomorphies between Thermus and Deinococcus, supports the hypothesis that it is an ancient group with no clear affinities to any of the other known bacterial lineages. Distinctive features of the Deinococcus genome as well as features shared with other free-living bacteria were revealed by comparison of its proteome to the collection of clusters of orthologous groups of proteins. Analysis of paralogs in Deinococcus has revealed several unique protein families. In addition, specific expansions of several other families including phosphatases, proteases, acyltransferases, and Nudix family pyrophosphohydrolases were detected. Genes that potentially affect DNA repair and recombination and stress responses were investigated in detail. Some proteins appear to have been horizontally transferred from eukaryotes and are not present in other bacteria. For example, three proteins homologous to plant desiccation resistance proteins were identified, and these are particularly interesting because of the correlation between desiccation and radiation resistance. Compared to other bacteria, the D. radiodurans genome is enriched in repetitive sequences, namely, IS-like transposons and small intergenic repeats. In combination, these observations suggest that several different biological mechanisms contribute to the multiple DNA repair-dependent phenotypes of this organism.

Amino Acid Sequence↗
PubMed2000

Comparison of three isolation systems for the culture of mycobacteria from respiratory and non-respiratory samples.

AIMS: To compare the recovery of mycobacteria from clinical samples using the MB/BacT rapid culture system with that obtained using egg medium or the Bactec radiometric method. METHODS: The three methods were compared using 681 clinical samples (462 respiratory and 219 non-respiratory samples) and eight external quality control strains. Culture media were incubated at 35-37 degrees C for six weeks in the MB/BacT system and for 12 weeks in the Bactec system and on egg medium. Solid media were examined macroscopically once a week and the Bactec vials were read six times in the first two weeks, and then weekly for the next 10 weeks (a growth index > 50 indicated a positive vial). The MB/BacT system positive vials were unloaded from the machine as soon as possible after detection. Confirmation of growth for all systems was by Ziehl-Neelson stained smears. Isolates were identified by a combination of phenotypic and molecular methods. RESULTS: Of the 681 clinical samples, 59 (8.7%) were positive on culture, including 23 strains of Mycobacterium tuberculosis. None of the three systems recovered all of the isolates, but each recovered mycobacteria not detected by either of the other two systems. After six weeks incubation, isolation rates were 87%, 78%, and 90%, and mean times to detection were 13, 19, and nine days for the MB/BacT, egg medium, and Bactec systems, respectively. Although the MB/BacT system was slightly slower than the Bactec system, the biomass was greater, allowing earlier use of molecular probes and earlier inoculation of susceptibility tests. CONCLUSIONS: The MB/BacT system provides comparable performance to the Bactec radiometric system, without the problems of disposal of radioactive waste. Optimal recovery is obtained when culture on egg medium is used in conjunction with a rapid culture system.

Bacteriological Techniques↗
PubMed2001

Stable labeled microspheres to measure perfusion: validation of a neutron activation assay technique.

Neutron activation is an accurate analytic method in which trace quantities of isotopes of interest in a sample are activated and the emitted radiation is measured with high-resolution detection equipment. This study demonstrates the application of neutron activation for the measurement of myocardial perfusion using stable isotopically labeled microspheres. Stable labeled and standard radiolabeled microspheres (15 microm) were coinjected in an in vivo rabbit model of myocardial ischemia and reperfusion. Radiolabeled microspheres were detected with a standard gamma-well counter, and stable labeled microspheres were detected with a high-resolution Ge detection after neutron activation of the myocardial and reference blood samples. Regional myocardial blood flow was calculated from the deposition of radiolabeled and stable labeled microspheres. Both sets of microspheres gave similar measurements of regional myocardial blood flow over a wide range of flow with a high linear correlation (r = 0.95-0.99). Neutron activation is capable of detecting a single microsphere in an intact myocardial sample while providing simultaneous quantitative measurements of multiple isotope labels. This high sensitivity and capability for measuring perfusion in intact tissue are advantages over other techniques, such as optical detection of microspheres. Neutron activation also can provide an effective method for reducing the production of low-level radioactive waste generated from biomedical research. Further applications of neutron activation offer the potential for measuring other stable labeled compounds, such as fatty acids and growth factors, in conjunction with microsphere measured flow, providing the capability for simultaneous measurement of regional metabolism and perfusion.

Animals↗
PubMedDate not supplied

Radioguided surgery of breast cancer: radiation protection survey.

The aim of this study was to estimate the radioactive risk for surgical staff performing radioguided sentinel lymph node (SN) biopsy and to calculate the contamination level in the operating room for assessment of the possible need for specific radiation protection procedures. We studied 20 patients who were selected for quadrantectomy and SN biopsy. The day before surgery a volume of 0.15 mL of 99mTc-nanocoll was injected: the activity was 3.11 +/- 0.85 MBq in group A (15 pts) and 11.6 +/- 0.6 MBq in group B (5 pts). External radiation to staff was evaluated by measuring the exposure rate in air one hour after radiopharmaceutical administration. The air KERMA rate during surgery was estimated considering the physical decay of 99mTc. Contamination of disposable materials and surgical instruments in the operating room was measured using a contamination monitor, whereas the residual activity in the SN and the injection site was measured with a gamma probe. The exposure rate at 20 cm from the injection site was 0.75 microSv/h when the most radioactive patients (group B) were treated. Contamination in the operating room proved to be negligible. Considering the number of radioguided treatments carried out by a surgeon in one year, an equivalent effective dose of 0.075 mSv was estimated; the recommended dose limit according to the relevant Italian law, DL 230/95, is 1 mSv/yr. Surgical staff therefore do not require a classification of "exposed workers" and there is no need to supply the operating room with special containers for radioactive waste.

Breast Neoplasms↗
PubMed2002

High-throughput fluorescence polarization method for identification of FKBP12 ligands.

FKBP12 is best known as the target of the widely used immunosuppressive drug FK506 but may also play a role in neuronal survival. Nonimmunosuppressive ligands of FKBP12 have been shown to have neuroprotective and neuroregenerative activity both in vitro and in vivo, stimulating interest in the development of high-throughput screens to rapidly identify novel ligands. FKBP12 was expressed as a His(6)-fusion in bacteria and purified by metal ion affinity and gel filtration chromatography. A high-throughput fluorescence polarization assay was developed to identify novel ligands of FKBP12. Dissociation constant values of known FKBP12 ligands measured by the new method agreed closely with K(i) values obtained by assaying inhibition of the rotamase activity of the enzyme. The fluorescence polarization assay is rapid, robust, and inexpensive and does not generate radioactive waste. It is very well suited for high-throughput screening efforts.

Drug Evaluation, Preclinical↗
PubMed2003

Screening of chemopreventive tea polyphenols against PAH genotoxicity in breast cancer cells by a XRE-luciferase reporter construct.

Polycyclic aromatic hydrocarbons (PAHs) are established cancer initiators that can be found in our food and environment. Some dietary plant polyphenols are strong inhibitors to PAH-induced mutagenesis, whereas others may not be as effective. To identify the chemopreventive compounds from a huge volume of dietary components, the development of an efficient screening method is required. In this study, a xenobiotic response element (XRE)-luciferase reporter plasmid was constructed to screen for some potential chemopreventive agents in tea against PAH-induced DNA damage. Tea is one of the most consumed beverages worldwide, and its beneficial effects on health have been documented. Previous studies have claimed that tea polyphenols could be protective against various cancers, and the rich database can be a source for comparison. Among the green and black tea polyphenols, the XRE-luciferase reporter assays suggested that only epigallocatechin gallate (EGCG) was effective in reducing XRE-driven luciferase assay in MCF-7 cells at the concentrations tested. Further study indicated EGCG could reduce CYP1A1 and CYP1B1 mRNA abundances and decrease the DMBA-DNA lesions. The results of DNA covalent binding of all tea polyphenols tested were consistent with the XRE-reporter assays. This study illustrated that the XRE-reporter assay was a viable screening test for dietary chemopreventive agents against PAH-initiated breast mutagenesis. It has the advantages of shorter sample processing time and producing no radioactive waste over directly measuring the CYP1A1/1B1 expressions, DNA lesion, or gel mobility shift assay.

Analysis of Variance↗
PubMed2004

Parametric study of pellets for elemental analysis with laser-induced breakdown spectroscopy.

The effect of various parameters on the accuracy of the laser-induced breakdown spectroscopy (LIBS) data taken from pellet samples has been investigated. The dependence of the standard deviation of the LIBS data on the amount and nature of the binder used, pressure used to press the powder into a pellet, and the position of the focal spot on the pellet has been investigated. Pellets made from industrially important materials such as silica, alumina, and lime with polyvinyl alcohol, sucrose, and starch as binders have been studied. The results thus obtained are tested by preparation of the calibration curves for Si, Fe, and B in the pellets made from the powder glass batch used as a surrogate for the batch employed for the vitrification of radioactive waste.

Journal Article↗
PubMed2000

Transcriptional activation analysis using bioluminescent reporter assays.

Analysis of promoter and enhancer DNA sequences provides the researcher with valuable information regarding the expression patterns of genes. Insertion of small DNA fragments containing the regulatory sequence of interest into vectors carrying reporter genes allows for the accurate quantitative analysis of the gene's expression patterns and responses to various stimuli. The use of bioluminescent reporter genes provides a simple, rapid, and inexpensive system that generates virtually no toxic or radioactive waste products. In addition, bioluminescent reporter vectors are more sensitive than previous methods such as the chloramphenicol acetyl transferase (CAT) systems, that require the use of hazardous chemicals and isotopically labeled reagents.

Animals↗
PubMed1997

Detection of serum insulin-like growth factor binding proteins on western ligand blots by biotinylated IGF and enhanced chemiluminescence.

A novel procedure for the detection of IGF binding capacity of IGFBPs on Western ligand blots (WLB) was developed using biotinylated IGFs as probes. The biotinylated IGF-IGFBP complexes were visualized by streptavidin-horseradish peroxidase and enhanced chemiluminescence (ECL). The procedure was found to be faster and more efficient than the conventional method with iodinated IGFs. In normal human serum a predominant doublet at 38-42 kDa and five smaller bands at 35, 34, 30, 28 and 24 kDa were detected by both methods, whereas two additional bands at 26 and 16 kDa became visible with the ECL method. In pregnancy serum only one single faint band at 30 kDa could be detected by the iodinated method. In contrast, the ECL method revealed five other bands at 42, 34, 28, 26 and 16 kDa. Besides the 38-42 kDa doublet, the 30 and 16 kDa bands reacted strongly with anti-IGFBP-3 antibodies in Western immunoblotting (WIB) and therefore were related to IGFBP-3 fragments. The technical advantages of this ECL method include an extremely short exposure time to the radiographic film and a long stability of the probe. In addition, the ECL method is a non-radioactive method, making radioprotection and radioactive waste removal unnecessary.

Biotin↗
PubMed2002

High-sensitivity hybridization assay for quantitation of residual E. coli DNA.

Impurity assays for recombinant protein therapeutics are essential to ensure batch-to-batch consistency and to meet the FDA's criteria for a well-characterized biopharmaceutical. For determination of residual host cell DNA, membrane hybridization assays utilizing radiolabeled DNA probes prepared from the host cell's genomic DNA have traditionally been used for products derivedfrom bacterial expression systems to obtain the required low picogram sensitivity. Nonradioactive methods, while desirable to eliminate radioactive waste disposal and safety issues, typically suffer from poor sensitivity and high backgrounds. We report the development of a suitably sensitive, nonradioactive assay to quantitate residual E. coli DNA levels in purified protein drugs by means of a slot-blot hybridization method. The assay utilizes digoxigenin-labeled E. coli DNA probes and SuperSignal chemiluminescent substrate. The optimized chemiluminescent hybridization assay has both low background and high sensitivity, allowing routine detection of 2.5 pg E. coli DNA. The method can be tailored for detection/quantitation of DNA contamination in recombinant protein products expressed in E. coli or other bacterial expression systems.

Antibodies↗
PubMedDate not supplied

ELISA screening tests for specific IgE and IgG antibody in employees exposed to trimellitic anhydride (TMA).

The objective of the study was to determine whether ELISA screening tests could be developed for specific IgE and IgG antibody in surveillance studies of employees exposed to trimellitic anhydride (TMA). Such tests would obviate the need for radiolabeling and disposal of radioactive wastes. The design of the study was a masked comparison of serologic results of ELISA screening tests with the criterion standard radioimmunoassay (RIA) currently used. The participants were 233 employees of a chemical plant that manufactures TMA. When the screening ELISA serologic results for IgG against TM-human serum albumin (TM-HSA) were compared with those of the RIA, the sensitivity was 92%, the specificity was 93%, the positive predictive value was 62%, and the negative predictive value was 99%. When the screening ELISA serologic results for IgE against TM-HSA were compared with those of the RIA, the sensitivity was 100%, the specificity was 91%, the positive predictive value was 31%, and the negative predictive value was 100%. We conclude that ELISA screening tests for antibody to TM-HSA have sufficient sensitivity, specificity, and predictive value to be useful in surveillance studies of employees exposed to TMA.

Chemical Industry↗
PubMed1987

[Determination of residual activities in 99mTc generators and 81mKr generators].

The residual activities present in spent 99Mo-99mTc generators and 81Rb-81mKr generators were determined from a view point of the disposal of radiopharmaceuticals. The results showed that seven kinds of residual nuclides such as 95Zr-95Nb, 103Ru, 90Sr, etc. were found in 99mTc generators with maximum level of 740 Bq/3.7 GBq (0.02 muCi/100 mCi) at one year after the reference date. In 81mKr generators one year old, six nuclides such as 57Co, 83Rb, 84Rb and others remained and these activities were less than 3.7 kBq/370 MBq (0.1 muCi/10 mCi). On the basis of these data, the disposal of 99mTc generators and 81mKr generators is discussed.

Humans↗
PubMed2006

[Cancer incidence and mortality among persons having been exposed to ionizing waves in a school in Val-de-Marne].

The Marie Curie School of Nogent-Sur-Mame (Val-de-Marne, France) was built in 1969 on the site of a former radium extracting plant. Due to remaining radioactive waste in the subsoil, school staff and students who attended the school have been exposed to radiation. A retrospective cohort study was conducted on the 3,403 persons who had attended the school regularly until it closed down in 1998. The national health insurance register was used to trace people. Incidence of cancers, leukaemia and mortality were analysed. In the population of the pupils a significant excess risk for leukaemia was observed (Standardized Incidence Ratio = 4.6 IC 95% [1.66 - 9.89]). These results are not conclusive because of the high proportion of those who could not be traced and were lost to any opportunity for follow-up (42%), and because of preferential recruitment due to a bias generated by the query of the records being centred on searching specifically for those who were sick. The difficulties met by the authors justify that when faced with similar problems in the future, greater attention should be paid to the feasibility study before any involvement or action.

Adult↗