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PubMed1990

Methods for estimating release rates during high frequency quantal secretion and for testing such methods.

The rate of spontaneous quantal release must be estimated in some investigations of synaptic transmission, even when frequencies are so high that individual quanta cannot be distinguished. An obvious method is to measure the time integrals of the summed MEPPs and then dividing this value by the integral of an average MEPP. The method was tested by recording miniature end-plate potentials (MEPPs) at frog neuromuscular junctions, counting the number of MEPPS, and then adding together records from the same junction to simulate high frequencies. The estimates from the integral method agreed well with the actual counts. The method can readily be used with a microcomputer and does not require stationary data. Methods based on fluctuation analysis were also used to estimate quantal frequencies, but they did not always give good estimates. This was not a thorough test of the fluctuation method, but an example of testing with MEPP data. The integral method might be reasonably reliable, but there are further potential complications, like changes in MEPP size and short-circuiting of the end-plate membrane, which may make it difficult to obtain reliable measurements of high frequency quantal secretion without voltage clamping and protocols that permit measurements of individual MEPCs during the course of the frequency measurements.

Animals↗
PubMed2026

An enhanced multisegment RT-PCR method for influenza A virus sequencing: Improved performance and reduced preparation time over traditional methods.

Influenza A viruses (IAVs) remain a major global health threat, affecting both human and animal populations. Whole-genome sequencing is essential for monitoring viral evolution, zoonotic transmission, and emerging variants. However, conventional RT-PCR methods often result in incomplete gene coverage, amplification biases, and reduced sequencing accuracy, particularly in clinical samples. We developed a robust In-house method for IAV full-genome sequencing using the Oxford Nanopore Technologies (ONT) long-read sequencing platform. This method integrates an in-house multisegment Reverse Transcription PCR (RT-PCR) method with a streamlined 2-pool primer design targeting all eight IAV gene segments. RNA extracted from clinical and stock virus samples was reverse-transcribed and amplified using Superscript IV-based chemistry, followed by magnetic bead purification to ensure high-quality amplicons. Sequencing libraries were prepared with the Native Barcoding Kit 24 (SQK-NBD114.24) and sequenced on R10.4.1 flow cells on the MinION MK1C device. Data analysis using the Iterative Refinement Meta-Assembler (IRMA) confirmed improved read depth, uniform coverage, and complete genome recovery. Compared to conventional methods, our In-House Multisegment 2-Pool (IH-MS2P) RT-PCR method generated higher numbers of matched read counts, minimized chimeric artifacts, and delivered superior genome coverage across human, swine, and avian isolates. This optimized RT-PCR method provides a high-performance, time-efficient, and portable solution for influenza genomics, demonstrating robust applicability even with clinical samples of low RNA yield.

Influenza A virus↗
PubMed1985

Light microscopical localization of enzymes by means of cerium-based methods. I. Detection of acid phosphatase by a new cerium-lead-technique (Ce-Pb-method).

Cerium-III-ions are more and more used as capturing reagent and opaque marker for the electron microscopic localization of a number of H2O2-generating enzymes as well as phosphohydrolases. Contrary to its advantages over common lead methods in the histochemical detection of enzyme activities at the electron microscopic level, cerium-based methods proved to be a failure for light microscopic investigations. Therefore, our cerium-based method for the ultrahistochemical detection of acid phosphatase was developed for further observations at the light microscopic level. The principle of that new Ce-Pb-method is the conversion of light microscopic not visible cerium phosphate into lead phosphate by the secondary capture reagent alkaline lead citrate. Finally, the lead phosphate can be visualized as lead sulfide in the section. The Ce-Pb-method in its finally proposed manner was compared with a common lead method and showed a range of advantages. Because of that fact, the new Ce-Pb-method is recommended for a broader use in histochemistry, e.g. for the light microscopic enzyme investigation parallel to ultrahistochemical preparations.

Acid Phosphatase↗
PubMed1986

The "Ring" method: a semi-empirical method to calculate dose distributions of irregularly shaped photon beams.

The "Ring" method provides a fast dose calculation and isodose presentation for photon beams with blocks. The method takes into account the change in scatter due to the blocks at each calculation point. Firstly, the dose in a point is calculated assuming that no blocks are present. Secondly, the scatter reduction caused by the blocks is calculated and subtracted. To determine the scatter reduction the irradiated surface is divided in concentric rings around a point at the surface at the intersection with a ray line between focus and calculation point. The scatter reduction caused by blocks for each ring is calculated. The effect of scatter for rings with an outer radius greater than 15 cm where the scatter contribution is less than 1.0% is neglected. Results of the method for 4 MV photons using eight rings are presented. Comparison of dose measurements with calculations in an arrow-shaped photon field showed maximum deviations of 4.0%, using the IRREG program of Cunningham, 6.5% using the BLKINP program of Schlegel, which is based on Clarkson's method, 5.0% using the method of Wrede and 2.2% using the "Ring" method. Contrary to the first two calculation programs, the programs using the last two calculation methods provide isodose lines dose values at points.

Radiation Dosage↗
PubMed1978

Comparison of the conventional lecture method and the self-instruction method for teaching clinical pathology.

Two separate courses in clinical pathology were presented to two groups of second-year medical students. The first (large) group was taught by the conventional lecture method; the other (small) group, by a self-instructional method for roughly half of the course, followed by the conventional lecture method for the second half of the course. The effectivenesses of the two methods were compared, based on performances on the final written examination. It was found that the self-instructional method was as effective as the conventional method in teaching clinical pathology, at least in teaching basic principles and interpretive clinical chemistry, as judged by the performances of the two groups. Surprisingly, the two groups differed significantly in performances on the portion of the course taught by similar methods.

Education, Medical, Undergraduate↗
PubMed1976

A comparative study of the Bessey method and Hausamen method for serum alkaline phosphatase determination.

The methods of Bessey et al. and Hausamen et al. for serum alkaline phosphatase determination were compared. Determinations were carried out using sera from healthy blood donors and sera from patients suffering from different hepatobiliary diseases. A high reproducibility of values obtained using both methods was noted. The sensitivity of the method of Hausamen et al. is about four times greater than that of the method of Bessey et al., even though the former method employed a lower reaction temperature. A high degree of correlation exists between the two methods and the data reported indicate that both methods are completely valid.

Adult↗
PubMed1991

Collagen-gel-embedded three-dimensional culture of human thyroid epithelial cells: comparison between the floating sandwich method and the dispersed embedding method.

Human thyroid epithelial cells were isolated from surgically resected human thyroid gland with collagenase and cultured for one week under EGF-supplemented conditions to allow them to proliferate. Then the cells were transferred to the following three-dimensional culture systems. One was a culture of isolated cells between floating double layers of collagen gel, designated the "floating sandwich method." The other was a culture of isolated cells mixed with collagen gel, designated the "dispersed embedding method." Many folliclelike structures with lumina of appreciable size were obtained by the former method. The cells cultured by the floating sandwich method exhibited a distinct polarity shown by the presence of numerous microvilli at the apical surface and close contact with collagen gels at the basal surface. On the other hand, only a few folliclelike structures were obtained by the dispersed embedding method, in which the folliclelike structures were small in size and the cells showed less distinct polarity than those observed in the floating sandwich method. Thus, the floating sandwich method appears to be suitable for studying the process and mechanism of in vitro organization of follicular structures by human thyroid epithelial cells.

Cell Division↗
PubMed1990

Particle design of tolbutamide by the spherical crystallization technique. III. Micromeritic properties and dissolution rate of tolbutamide spherical agglomerates prepared by the quasi-emulsion solvent diffusion method and the solvent change method.

With the objective of modifying the micromeritic properties of tolbutamide (i.e., to manufacture a highly functional powder form), particle design was attempted using a quasi-emulsion solvent diffusion (QESD) method, and the micromeritic properties and dissolution rate of the obtained spherical agglomerates were evaluated by comparison with agglomerates prepared by the solvent change (SC) method. For the production of tolbutamide agglomerates by the QESD method, a necessary condition was the addition of a sucrose fatty acid ester to the system as an emulsifying agent. The particle diameter of the agglomerates obtained by the QESD method depended on the size of the initially formed quasi-emulsion droplets, which in turn depended on the viscosity of the solution. In addition, the agglomerates were nearly perfectly spherical in shape. In the QESD method, the quasi-emulsion droplets crystallized instantaneously from the droplet surface inward. The resultant agglomerates were dense, had great mechanical strength and showed excellent flowability due to their perfect spherical shape. On the other hand, the agglomerates produced by the SC method were conglomerates of primary crystals, and fine, needle-like crystals formed on their surface. As a result, these agglomerates had a large specific surface area, and they therefore showed greater solubility than the agglomerates prepared by the QESD method.

Chemistry, Pharmaceutical↗
PubMed1992

[Development of a new method for calibration of pneumotachograph according to flow rate--evaluation of a computer-controlled-system for assessing respiratory gas exchange using this method].

1) We evaluated the method for calibrating the pneumotachograph according to flow rate by the software on personal computer. Flow volume was detected using the approximated equation (y = 1.294x2 + 0.989x + 35.824, r = 0.976, p less than 0.01) from various mean flow rate and integration of output voltage. The error between syringe volume (21) and the estimated value on the equation was 10 +/- 8 ml (0.48 +/- 0.42%). 2) Using the method described in 1), we developed computer-controlled-system for assessing respiratory gas exchange by mixing chamber method. To evaluate the accuracy of the system as compared with the Douglas Bag method, we measured the gas parameters of the both methods for two male subjects at rest(3 and 5min) and steady state exercise (3 min., 60-240watt). The error between the both methods were within +/- 5% in ventilation(VE), mixed expired O2 (FEO2) and CO2 (FECO2) concentration, and were within +/- 8% in O2 uptake(VO2) and CO2 production (VCO2). There were high positive correlation (r greater than 0.99, p less than 0.01) between the both methods in the all parameters. These results indicate that the system in this study would be practical use, that VE can be measured in only one pneumotachograph, even if the range of flow rate was wide(e.g. from rest to heavy exercise).

Adult↗
PubMed1987

Comparative study of colorimetric method using diazotization reaction and high-performance liquid chromatographic method in determination of para-aminohippuric acid.

In this study, colorimetric method and high-performance liquid chromatographic (HPLC) method were improved and established, respectively, in order to minimize analytical errors in determination of para-aminohippuric acid (PAH) in rat urine and plasma. In terms of the colorimetric method, an operative step following addition of Tsuda reagent was modified as follows: after the addition of Tsuda reagent, reaction mixture was kept at 40 degrees C for 70 min before spectrophotometry. Linearities were observed both in the higher range of 0 and 2.5 to 12.5 micrograms and in the lower range of 0 and 100 to 1,000 ng per test tube, and its practical detection limit was 100 ng per test tube. In terms of HPLC method, using a reversed-phase column (Nucleosil 5 C18), PAH was separated by a mobile phase of acetonitrile/50 mM KH2PO4 (pH 2.8) = 9/95. Linearities were observed in the higher range of 0 and 10 ng to 2 micrograms and in the lower range of 0 and 1 to 10 ng per injection, and its practical detection limit was 1 ng per injection. These results denote that the above two methods are applicable to routine PAH determination. In addition, our HPLC method is considered to be applicable to microassay of PAH, because its sensitivity is more sensitive and minimization of volume system is more easily achieved as compared with the colorimetric method.

Aminohippuric Acids↗
PubMedDate not supplied

[Comparative evaluation of 2 methods for the determination of pyruvate dehydrogenase activity in tissues in avitaminosis B 1 induced by various methods].

A method for determination of pyruvate dehydrogenase activity in mitochondria and tissue homogenates was developed. The method was based on a spectrophotometric monitoring of p-nitroaniline acetylation under conditions required to ensure correct stoichiometric course of the reaction. In rat tissues absolute amounts of the enzyme activity, determined by the method, were found to be several-fold lower as compared with the values determined by the conventional ferricyanide method. Within 24 hrs after a single administration of hydroxythiamin into rats (400 mg per 1 kg of body weight) the pyruvate dehydrogenase activity was decreased 1.5-fold in heart (as estimated by the reaction of p-nitroaniline acetylation) and did not alter in liver tissue. While if the determinations were carried out by the method of terricyanide reduction the enzyme activity was decreas 7- and 2-fold, respectively, in heart and liver tissue. The data obtained suggest that hydroxythiamin impaired reactions of electron transport in tissues; on the other hand, the data obtained showed that the method for determination of the pyruvate dehydrogenase activity, based on the acetylation reaction, was more specific than the conventional method which involved measuring of ferricyanide reduction.

Animals↗
PubMed1992

von Willebrand factor antigen: a radial immunodiffusion method evaluated and compared with an ELISA method.

A new commercial kit method for the quantification of von Willebrand factor antigen (vWFAg) by radial immunodiffusion was compared to an established ELISA technique. Major discrepancies were found between the two methods. The radial immunodiffusion method had poorer intra- and inter-assay coefficients of variation than the ELISA method, although there was adequate inter-method agreement when 100 plasma samples from controls and patients were compared. However, there was a great difference in values obtained for vWFAg in the reference sample supplied with the commercial kit and that obtained directly from the National Institute for Biological Standards and Controls. There were also differences in levels of significance when vWFAg was measured by the two techniques in different clinical groups, and standard deviations were larger when the kit method was used. It is suggested that on scientific and economic grounds, the commercial radial immunodiffusion kit does not offer a competitive advantage over the ELISA method.

Autoantigens↗
PubMedDate not supplied

Comparison of two immunochemical methods with thin-layer chromatographic methods for determination of aflatoxins.

Three different methods were compared for the determination of total flatoxins in corn and peanuts naturally contaminated with aflatoxins and in corn, peanuts, cottonseed, peanut butter, and poultry feed spiked with aflatoxins B1, B2, and G1. The 3 methods were an enzyme-linked immunosorbent assay (ELISA) screening test; a monoclonal antibody-affinity column-solid-phase separation method; and the AOAC official thin-layer chromatography (TLC) methods for all except poultry feed, for which Shannon's TLC method for mixed feed was used. The ELISA test is designed to provide only positive results for total aflatoxins at greater than or equal to 20 ng/g or negative results at less than 20 ng/g. The affinity column separation is coupled with either bromination solution fluorometry to estimate total aflatoxins or liquid chromatography (LC) to quantitate individual aflatoxins. Fluorodensitometry was used to determine aflatoxins in commodities analyzed by the TLC methods. The LC and TLC results were in good agreement for all the analyses. The results for the affinity column using bromination solution fluorometry were similar except those for cottonseed, which were about 60% higher. The ELISA screening method correctly identified naturally contaminated corn and peanut positive samples. No false positives were found for controls. The correct response for spiked corn, raw peanuts, peanut butter, and cottonseed at greater than or equal to 20 ng aflatoxins/g was about 90%. The correct response for spiked poultry feed at greater than or equal to 20 ng aflatoxins/g was about 50%.

Aflatoxins↗
PubMed1989

[Enhancement of enamel crystallinity with gel method during orthodontic treatment. The 2nd report: application of micro-area X-ray diffraction method].

This study is to review an effect on the improvement of enamel crystallinity with gel method in the course of orthodontic treatment by applying gelatin gel method (group II) as well as silica-hydro gel method (group I) to bracket-bonded enamel surfaces of extracted teeth and for a relative review, the chopped cross sections of the above teeth were analyzed by the method of micro area X-ray diffraction. The results obtained were as follows: 1. As a result of identifying by the method of microarea X-ray diffraction, the present of hydroxyapatite, Ca5(PO4)3OH and fluorapatite, Ca5(PO4)3F was recognized at any section of every group. 2. In making a relative study of the variation of line profile and peak shift, an outstanding difference was not acknowledged in line profile, however, peak shift in the direction of high angle side was indicated on the experimental side of each group and a tendency for peak shift was more noticeable near by enamel cuticle. At boundary sites and on the control side, the variation was only shown in one part of group II. Also a tendency for peak shift in the direction of high angle side was recognized in group I rather than in group II. From the foregoing results, it was found to be effective to improve tooth substance of the bracket-bonded enamel and in that event, the silica-hydro gel method was suggested to be superior to the gelatin gel method. Also with the regard to the bracket-bonded sides, tooth substance was noticeably improved at enamel cuticle but not sufficiently improved at sites underneath the brackets and at boundary sites of the both, therefore, one of the future subjects would be to review such areas.

Apatites↗
PubMed1989

[Confidence factors-method for estimating the confidence limits of standardized population rate of indirect method].

The standardized rate of indirect method is widely used, but no method of interval estimation for its population rate has been reported. The authors have discussed the standard error of standardized rate of indirect method and suggested that a method using this standard error should be used to determine the confidence limits for the population rate. In this paper, the authors put forward another method (Confidence Factors-Method) which can be easily applied to determine the confidence limits. It gives approximately the same result with the method mentioned above.

Mathematics↗
PubMed1985

[Evaluation of bacterial growth rate by the ATP-bioluminescence method (luciferase assay)--comparison with conventional methods and application to clinical studies].

Bacterial growth rate was evaluated by estimating the increase in ATP content of cultures of bacterial suspensions using luciferase assay, and the method was applied to the estimation of bactericidal activity of serum and anti-bacterial activity of antibiotics. The results obtained were as follows: The bacterial numbers or bacterial growth rate determined by the ATP-Bioluminescence method was in accordance with those by CFU assay and spectrometrical assay by optical density readings. Furthermore this method was confirmed to be non-time-consuming and more simple than other methods. Using this method, anti-bacterial activity of fresh serum was evaluated. The serum suppressed the growth rate of E. coli, Str. fecalis, CBS, Kleb. pneumoniae and Staph. aureus at least for 2 hours. Heat-inactivated serum and absorbed serum with bacteria tended to lose such suppressive effect. Thus the main effective factors seem to be complements and specific antibody. The anti-microbial activity of antibiotics was enhanced by addition of fresh serum to bacterial cultures, and the results were not in accordance with those by the conventional Disc method. Thus the sensitivity test under the influence of fresh serum is recommended in order to know the clinical effect of antibiotics. These results suggest the usefulness of ATP-Bioluminescence method in the clinical laboratory examinations or investigations.

Adenosine Triphosphate↗
PubMed1981

Comparison of Eustachian tube function measured by the microflow method and a new quantitative impedance method.

Eustachian tube function in 25 applicants for flight training was tested during simulated flights using two methods alternately; the integrating microflow method with a stationary pressure chamber, and a new quantitative impedance method in combination with a new mobile pressure chamber. All ears were tested by both methods. The results of the various tests did not differ significantly between methods. The reliability of the measurements was good and almost equal in both methods. Several practical advantages with the new impedance method in combination with the mobile pressure chamber make it preferable.

Acoustic Impedance Tests↗
PubMed1981

[Evaluation of methods to determine the vitamin B6 status of humans. 1. alpha-EGOT: methods and validation].

A semiautomatic method to determine the activation coefficient of the glutamic-oxaloacetic-transaminase in the erythrocytes is described. This method is compared with other methods and possible sources of error are discussed. Colorimetric methods show considerable disadvantages, such as lack of precision or unspecific hydrazone formation. The kinetic tests avoid these problems. This is in part due to the fact that enzyme stimulation by excessive PLP-addition as used in our procedure bears practical and theoretical advantages. Nevertheless, comparison with published data on vitamin B6 status remains difficult because of insufficient standardization of the used methods. To validate our method we performed experiments testing the stability of the samples, short-term physiological changes and influence of the chelating agent EDTA on transamination. Heparinised blood samples can be stored only for one day with changing at 4 degrees C or room temperature. Storing of the erythrocytic suspension at deep-freezing temperature (-18 degrees C) leads to considerable changes of results. When kept under liquid nitrogen there is no change of the alpha-values for at least 45 days. The addition of EDTA to the samples is known to exclude the influences of cations, such as non enzymatic transamination of a direct alteration of the enzyme activity and therefore EDTA should be added to improve standardisation of the method. The described procedure is a fast, reliable and precise way to determine the alpha-EGOT.

Aspartate Aminotransferases↗