Assessment of gamma ray-induced DNA damage in Lasioderma serricorne using the comet assay

We attempted a DNA comet assay under alkaline conditions to verify the irradiation treatment of pests. Lasioderma serricorne (Fabricius) were chosen as test insects and irradiated with gamma rays from a ⁶⁰Co source at 1 kGy. We conducted the comet assay immediately after irradiation and over time for 7 day. Severe DNA fragmentation in L. serricorne cells was observed just after irradiation and the damage was repaired during the post-irradiation period in a time-dependent manner. The parameters of the comet image analysis were calculated, and the degree of DNA damage and repair were evaluated. Values for the Ratio (a percentage determined by fluorescence in the damaged area to overall luminance, including intact DNA and the damaged area of a comet image) of individual cells showed that no cells in the irradiated group were included in the Ratio<0.1 category, the lowest grade. This finding was observed consistently throughout the 7-day post-irradiation period. We suggest that the Ratio values of individual cells can be used as an index of irradiation history and conclude that the DNA comet assay under alkaline conditions, combined with comet image analysis, can be used to identify irradiation history.     

Identification of low level gamma-irradiation of meats by high sensitivity comet assay

The detection of low levels of irradiation in meats (pork, beef, and chicken) using the new comet assay was investigated in order to assess the capability of the procedure. The new assay includes a process that improves its sensitivity to irradiation and a novel evaluation system for each slide (influence score and comet-type distribution). Samples used were purchased at retailers and were irradiated at 0.5 and 2 kGy at 0°C. The samples were processed to obtain comets. Slides were evaluated by typing comets, calculating the influence score and analyzing the comet-type distribution chart of shown on the slide. Influence scores of beef, pork, and chicken at 0 kGy were 287(SD=8.0), 305 (SD=12.9), and 320 (SD=21.0), respectively. Those at 500 Gy, were 305 (SD=5.3), 347 (SD=10.6), and 364 (12.6), respectively. Irradiation levels in food were successfully determined. Sensitivity to irradiation differed among samples (chicken>pork>beef).     

Identification of irradiated wheat by germination test, DNA comet assay and electron spin resonance

In several countries, there has been an increase in the use of radiation for food processing thus improving the quality and sanitary conditions, inhibiting pathogenic microorganisms, delaying the natural aging process and so extending product lifetime. The need to develop analytical methods to detect these irradiated products is also increasing. The goal of this research was to identify wheat irradiated using different radiation doses. Seeds were irradiated with a gamma ⁶⁰Co source (Gammacell 220 GC) in the Centro de Energia Nuclear na Agricultura and the Instituto de Pesquisas Energéticas e Nucleares. Dose rate used were 1.6 and 5.8 kGy/h. Applied doses were 0.0, 0.10, 0.25, 0.50, 0.75, 1.0, and 2.0 kGy. After irradiation, seeds were analysed over a 6 month period. Three different detection methods were employed to determine how irradiation had modified the samples. Screening methods consisted of a germination test measuring the inhibition of shooting and rooting and analysis of DNA fragmentation. The method of electron spin resonance spectroscopy allowed a better dosimetric evaluation. These techniques make the identification of irradiated wheat with different doses possible.     

Kinetics of comet formation in single‐cell gel electrophoresis: Loops and fragments

We investigated the mechanisms of DNA exit during single‐cell gel electrophoresis (the comet assay) by measuring the kinetics of the comet tail formation. In the neutral comet assay, the rate of DNA exit was found to be dependent on the topological state of DNA, which was influenced by either ethidium bromide or a low radiation dose. The results clearly show that the comet tail is formed by extended DNA loops: the loop extension, being reversible when the DNA torsional constraint remains in the loops, is favored when the constraint is relaxed. The kinetics of the comet formation in the case of a high radiation dose points out that accumulation of the single‐strand breaks causes DNA fragmentation. In contrast to the neutral comet assay, the alkaline comet assay is not related to the chromatin loops. Our results imply that the alkaline treatment induces detachment of the loops from the nuclear matrix, and the comet tail is formed by ssDNA fragments, the ends of which are pulled out from the comet head by electric force. We suggest that the kinetic approach can be considered as an important improvement of the comet assay.     

Detection of radiation treatment of beans using DNA comet assay

A simple technique of microgel electrophoresis of single cells (DNA Comet Assay) enabled a quick detection of radiation treatment of several kinds of leguminous beans (azuki, black, black eye, mung, pinto, red kidney and white beans). Each variety was exposed to radiation doses of 0.5, 1 and 5 kGy covering the permissible limits for insect disinfestation. The cells or nuclei from beans were extracted in cold PBS, embedded in agarose on microscope slides, lysed between 15 and 60 min in 2.5% SDS and electrophoresis was carried out at a voltage of 2 V/cm for 2–2.5 min. After silver staining, the slides were evaluated through an ordinary transmission microscope. In irradiated samples, fragmented DNA stretched towards the anode and the damaged cells appeared as a comet. The density of DNA in the tails increased with increasing radiation dose. However, in non-irradiated samples, the large molecules of DNA remained relatively intact and there was only minor or no migration of DNA; the cells were round or had very short tails only. Hence, the DNA comet assay provides an inexpensive, rapid and relatively simple screening method for the detection of irradiated beans.     

Validation of remote, on-line, near-infrared measurements for the classification of demolition waste

The classification performance, based on measurements obtained by a dedicated remote near-infrared sensor, is validated. Goal is the separation of demolition waste in three fractions: wood, plastic, and stone. In phase one, reference objects are collected and measured in order to develop the classification algorithm and to obtain reference classification results. In phases two and three, the validation performance and robustness are tested under laboratory and industrial conditions. In phase two, preliminary measurements are performed in the laboratory, indicating that some sensor hardware modifications are necessary. In phase three, measurements are performed on a pilot plant according to the following validation design. On the conveyor belt, objects are measured in the middle and at both borders, wet objects are measured in the middle, and a small set of objects is measured during 4 consecutive days. It is checked whether the classification performance obeys the predefined demands. The applied chemometrical techniques are well capable of separating dry demolition waste if the objects are positioned in the middle of the conveyor belt. It is recommended to overcome the sensor miniaturization-scale limitations by applying larger optical parts. The hardware sensor is not robust to wet objects, although this problem was accounted for during the development of the classification procedure. Including wet objects in the training set might overcome this restriction.     

The myth of data acquisition rate

With the need for high-frequency data acquisition, the influence of the data acquisition rate on the quality of the digitized signal is often discussed and also misinterpreted. In this study we show that undersampling of the signal, i.e. low data acquisition rate will not cause band broadening. Users of modern instrumentation and authors are frequently misled by hidden features of the data handling software they use. Very often users are unaware of the noise filtering algorithms that run parallel with data acquisition and that lack of information misleads them. We also demonstrate that undersampled signals can be restored by a proper trigonometric interpolation.     

Validation of the Volhard method for chloride determination in food

The aim of this paper is to compare the performance parameters of the Volhard method for chlorides determination obtained applying the bottom-up approach with those experimentally achieved. The method precision, trueness, detection and quantification limits and ruggedness are determined analysing various foods with different chloride contents. Otherwise, the measurement uncertainty is assessed applying the bottom-up approach using only pen and paper. The comparison between the performances established with both methods shows the validity of the metrological approach for volumetric procedures. Presented at AOAC Europe/Eurachem Symposium March 2005, Brussels, Belgium     

Validation of the Microreader 28A ID System: A 6-dye multiplex amplification assay for forensic application

The Microreader 28A ID System is a new 28-plex genotyping system with 6-dye multiplex amplification, which allows the simultaneous amplification of all 20 Combined DNA Index System (CODIS) core loci (CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX, vWA, D1S1656, D2S441, D2S1338, D10S1248, D12S391, D19S433, D22S1045), plus five extended STRs loci (D6S1043, Penta D, Penta E, DYS391, SE33), 2 Y-Indels (Rs2032678, Rs771783753), and the amelogenin loci. This system can be used for forensic analyses, such as personal identification, kinship testing, scientific research, database applications, and other aspects of human genetic identification. The validation of the Microreader 28A ID System followed the “Validation Guidelines for DNA Analysis Methods (2016)” described by the Scientific Working Group on DNA Analysis Methods and the regulations published by the China Ministry of Public Security. Our tests included PCR-based studies, sensitivity study, precision and accuracy evaluation, stutter percentage and heterozygous peak height ratio, inhibitor tests, species specificity, and population studies. The validation results suggest that the Microreader 28A ID system is a robust and reliable amplification kit for personal identification, kinship testing, and forensic database applications.     

Methodical developments for X-ray diffraction measurements and data analysis on lyotropic liquid crystals applied to K-soap/glycerol systems

 X-ray diffraction (XRD) data acquisition and processing software measurements on long-spacing binary systems, including adapted noise reduction algorithms, has been developed. The computation of XRD patterns has been summarized and the origin of the distinct patterns of long-spacing compounds has been illustrated with the aid of such simulations. This also provided the possibility to evaluate the retrievable amount of information used for graphical or numerical indexing programs. Numerical indexing programs were applied and limits of indexing method have been discussed. Differential scanning calorimetry (DSC) measurements and temperature-dependent XRD measurements have been carried out for the concentrations x _{KC 12} = 0.50 and 0.30 of the K-laurate/glycerol system. The analysis of the small- and wide-angle diffractograms in the various phase regions of the binary system as well as the DSC measurements provided the following results: – The creation of the lamellar phase extends over a temperature range of ΔT≈ 20 K at a concentration of x _{KC 12} = 0.50. The lamellas show a reduced degree of order. – The crystalline-to-gel phase transition is accompanied by a leap in the d values corresponding to the small-angle reflexes. Also a distinct splitting of the small-angle reflexes is observed within the gel phase. Simultaneously a rearrangement and intensity decay of the wide-angle reflexes occurs. Since some wide-angle reflexes are still present, the structure can be considered to have a partially reduced three-dimensional order. – During the creation of lamellar phases the d value decreases sharply with rising temperature. This decrease of d can be interpreted as a further decay of molecular order in the hydrocarbon chains of the K-laurate within the bilayers. – The XRD measurements correlate with the DSC data. According to our measurements a revision of the phase diagram with respect to the actual extension of the gel-phase region was necessary. Received: 3 March 1999 Accepted in revised form: 29 March 1999     

ICP-MS Method Validation for the Analysis of Trace Elements in Illicit Heroin

The analysis of the trace elements present in the street doses of heroin has currently been undertaken with a dual aim: to estimate the elemental compositions and to cluster the case samples. Inductively coupled plasma-mass spectrometry was optimized to quantify 18 trace elements simultaneously. The method presented high repeatability and reproducibility for the target elements. The linear concentration ranges achieved linearity with r² > 0.9975. The limits of detection (LOD) were sufficiently low for almost all the elements except for Ca (LOD = 100 ppb). The limit of quantification (LOQ) for each element was also found acceptable. All target elements show mean recoveries between 92%–108%. Other major findings of this study including intra-sample (RSD < 5%) and inter-sample (RSD < 14%) precisions, sample weight test (≥30 mg), dissolution vessels, and sample filtration are also reported. The capability of the optimized method was assessed by using forty case samples. With their elemental compositions statistical classification of these case samples is also discussed herein.     

Application of a rapid screening method to detect irradiated meat in Brazil

Based on the enormous potential for food irradiation in Brazil, and to ensure free consumer choice, there is a need to find a convenient and rapid method for detection of irradiated food. Since treatment with ionising radiation causes DNA fragmentation, the analysis of DNA damage might be promising. In this paper, the DNA Comet Assay was used to identify exotic meat (boar, jacaré and capybara), irradiated with ⁶⁰Co gamma rays. The applied radiation doses were 0, 1.5, 3.0 and 4.5 kGy. Analysis of the DNA migration enabled a rapid identification of the radiation treatment.     

Validation of the HS-GC-FID method for the determination of ethanol residue in tablets

This paper describes the validation of a HS-GC-FID method (based on the Pharmacopeia’s method) for the determination of ethanol content in tablets. A general view of the procedure development/optimization process is presented. The main point of this study is the calculation of validation parameters. Selectivity of the method was determined. Linearity (r > 0.997) was observed in the range from 9.0 to 3,040 μg of ethanol per sample (because the mass of the tablets used was around 200 mg, this corresponds to 45–15,200 μg g⁻¹). The method showed good recoveries (average 99.0%), and a relative standard deviation for repeatability and intermediate precision of 4.5% and 5.5% respectively. The limit of detection was calculated to be 3.0 μg of ethanol per sample (15 μg g⁻¹). The uncertainty budget was done according to the "Guide to the Expression of Uncertainty in Measurement" (GUM)[1], and a relative expanded uncertainty was estimated as 4.8%.     

Validation of thin layer and high performance thin layer chromatographic methods

Analytical validation is a key requirement to asses and to prove a method's reliability and suitability for an intended use. Planar chromatographic procedures are used in different applications ranging from simple screening tests to sophisticated instrumental quantitative assays of analytes in complex matrices. This paper intends to give guidance on how to adopt international accepted formal requirements and guidelines for validation of these different TLC/HPTLC procedures. In addition, some selected parameters for robustness testing and for on going quality assurance of analytical performance based on control charts are reported.     

The use of APPLE II microcomputer as kinetic laboratory data acquisition system

A laboratory microcomputer system based on an APPLE II microcomputer is presented. Data transfer from a temperature jump equipment or a stopped flow apparatus can be performed via a serial interface, the data can be stored on a memory expansion card or on the disk drives. Data transfer to a central host computer can also be done. Application of the laboratory data system on kinetic and spectroscopic measurements are shown.Dedicated to Prof. Dr.K. L. Komarek on the occasion of his 60th birthday.     

Validation of analytical methods used for determination of nitrate in soil

The standard method (ISO/DIS 14255) and the quick test were used for determination of nitrate in soil. We validated both methods using parameters such as accuracy, reproducibility within 1 day and between days, limit of detection and limit of quantification. The accuracy of the results was determined using the analysis of samples from the international interlaboratory scheme WEPAL. The accuracy of the standard method was good, while for the quick test the results were not accurate. The standard method showed a solid reproducibility of measurement results (in 1 day, relative standard deviation, RSD=0.2%; between days, RSD=0.8%). The quick test gave poorer results (in 1 day, RSD=6%; between days, RSD=7%). We tried to established the compatibility of both methods on real soil samples and we were satisfied to obtain the correlation coefficient 0.98 using the regression straight line. The analyses with the quick field instrument are much simpler and cheaper than the standard laboratory analyses and can be used for advising on nitrogen fertilisation.     

紫外分光光度法下直接测定蛋白质溶液的浓度

Lowry法是目前蛋白质测定的常用方法[1]。它一般以牛血清蛋白作为标准蛋白来检测其它纯蛋白和混合蛋白质溶液的浓度。但是Lowry法检测过程复杂、检测时间长、检测过程中蛋白质发生不可逆变性,而且硫酸铵、鸟嘌呤、甘氨酸以及其它生物物质[2]对检测结果有明显的影响。紫外分光光