The sterologic method of determining the surface area of the mammalian neocortex]

A method for calculation of the square surface area of the cerebral cortex is proposed which represents a combination of the method of averaged reconstruction after a continuous series of histological sections and the stereological method of determination of the length of any curve disposed on the surface. A comparative analysis of the proposed method with curvometrical and stereological (after Hennig's formula) method used earlier for this purpose is made taking as an example the calculation of the neocortex square surface area of some mammals. The results of calculation of the surface by the proposed method were found to coincide with curvometrical data within the range of 5% while the data obtained by stereological method of determination of the absolute square surface area differ from curvometrical data by more than 22%. The proposed method is very convenient and allows considerable acceleration in obtaining results.     

A new method for the determination of stability parameters of proteins from their heat-induced denaturation curves

A new method has been developed for determining the stability parameters of proteins from their heat-induced transition curves followed by observation of changes in the far-UV circular dichroism (CD). This method of analysis of the thermal denaturation curve of a protein gave values of stability parameters that not only are identical to those measured by the differential scanning calorimetry (DSC), but also are measured with the same error as that observed with a calorimeter. This conclusion has been reached from our studies of the reversible heat-induced denaturation of lysozyme and ribonuclease A at various pH values. For each protein, the conventional method of analysis of the conformational transition curve, which assumes a linear temperature dependence of the pre- and posttransition baselines, gave the estimate of DeltaH(van)(m) (enthalpy change on denaturation at T(m), the midpoint of denaturation) which is significantly lower than DeltaH(cal)(m), the value obtained from DSC measurements. However, if the analysis of the same denaturation curve assumes that a parabolic function describes the temperature dependence of the pre- and posttransition baselines, there exists an excellent agreement between DeltaH(van)(m) and DeltaH(cal)(m) of the protein. The latter analysis is supported by the far-UV CD measurements of the oxidized ribonuclease A as a function of temperature, for the temperature dependence of this optical property of the protein is indeed nonlinear. Furthermore, it has been observed that, for each protein, the constant-pressure heat capacity change (DeltaC(p)) determined from the plots of DeltaH(van)(m) versus T(m) is independent of the method of analysis of the transition curve.     

Evaluation of the sustainability of contrasted pig farming systems: economy

The aim of this paper is to present an efficient tool for evaluating the economy part of the sustainability of pig farming systems. The selected tool IDEA was tested on a sample of farms from 15 contrasted systems in Europe. A statistical analysis was carried out to check the capacity of the indicators to illustrate the variability of the population and to analyze which of these indicators contributed the most towards it. The scores obtained for the farms were consistent with the reality of pig production; the variable distribution showed an important variability of the sample. The principal component analysis and cluster analysis separated the sample into five subgroups, in which the six main indicators significantly differed, which underlines the robustness of the tool. The IDEA method was proven to be easily comprehensible, requiring few initial variables and with an efficient benchmarking system; all six indicators contributed to fully describe a varied and contrasted population.     

Discriminant analysis of event-related potential curves using smoothed principal components

Principal component analysis enhanced by the use of smoothing is used in conjunction with discriminant analysis techniques to devise a statistical classification method for the analysis of event-related potential data. A training set of premedication potentials collected from adolescents with attention-deficit hyperactive disorder (ADHD) is used to predict whether adolescents from an independent subject group will respond to long-term medication. Comparison of outcome prediction rates demonstrates that this method, which uses information from the whole ERP curve, is superior to the classification technique currently used by clinicians, which is based on a single ERP curve feature. The need to administer an initial dose of medication to classify patients is also eliminated.     

Cloning of the regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans.

The areA gene, which mediates nitrogen metabolite repression in the fungus Aspergillus nidulans, lies sufficiently close to a telomere that no indispensable gene can be distal to it. We were able therefore to exploit the existence of a near terminal pericentric inversion to devise a method for cloning areA plus the region beyond it towards the telomere. In crosses heterozygous for this inversion a class of duplication-deficient progeny lacking areA and the region centromere-distal to it is obtained. We, therefore, sought clones from an A. nidulans gene library in lambda Charon 4 able to hybridize to total genomic DNA from a wild-type strain but not to that from a duplication-deficiency strain. A clone, containing an 11.6-kb insert, which hybridised weakly to duplication-deficiency DNA, overlapped chromosome breakpoints of three different aberration-associated areA alleles and was able to transform an areA mutant to areA+. Southern blotting and genetic analysis established that the transforming sequence had integrated in the region centromere distal to areA. The cloning method yielded other clones from the region centromere-distal to areA which were used to show that the translocation associated with a mutant areA allele is reciprocal rather than non-reciprocal, a fact which could not be established by classical genetics. Finally, analysis of the cloned portion of the dispensable region centromere-distal to areA indicates that this region contains at least 0.5% of the A. nidulans genome.     

A noncomputerized scanning method for determining relative protein quantities and synthesis rates on two-dimensional electrophoretic gels

A densitometric method that permits the determination of relative amounts and synthesis rates of proteins separated by two-dimensional gel electrophoresis is presented. The method is applicable to proteins that have been stained by Coomassie blue or proteins that have been radiolabeled. The analysis is achieved by scanning selected spots with an ordinary densitometer in the horizontal and vertical direction and relating the response to the volume of an ellipsoid. A linear dependence is observed between the amount of protein or incorporated radioactivity and the measured optical density. The advantage of this method is that specialized scanning instruments and computer analysis are not required. The method is most useful for the analysis of a few specific proteins which change in their relative amount or specific activity due to experimental manipulations. Difficulties in the analysis of protein spots derived from the twodimensional gel electrophoresis technique are discussed and compared to an analysis of bands from the one-dimensional electrophoresis technique.     

Gene typing of Bacillus anthracis strains isolated on the territory of the countries of the Confederation of Independent States

Thirty eight B. anthracis strains isolated on the territory of the former USSR from different sources at different periods were studied by the method of the multilocus analysis of 6 chromosomal and 2 plasmid regions of B. anthracis genome with a variable number of tandem repeats. The strains belonged to 18 different genotypes; of these, 14 genotypes were described for the first time. The analysis of the genetic relationship of the strains gave grounds to suggest that on this territory both closely related strains and strains whose genotypes were remote from those peculiar to the greater part of other strains could occur. The strains belonging to subgroup A1a of molecular variability were "endemic" for the European part of the former USSR. A modification of the method of gene typing was proposed, which permitted it to be made without the use of an automatic sequencer; this made it possible to greatly widen the circle of laboratories where this method of research could be used.     

An LC-MS-MS method for the determination of cyclizine in human serum

Cyclizine is a piperazine derivative with anti-emetic activity that is useful in the prevention and treatment of nausea and vomiting associated with motion sickness. A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method is presented for the quantitation of cyclizine in serum. Sample pretreatment involved liquid-liquid extraction of 200 microl of serum with dichloromethane after the addition of 100 microl each of ammonium hydroxide and internal standard solutions. The extracts were analyzed by HPLC on a Luna C18 reversed-phase column and an ion-trap mass spectrometer with an electrospray interface. A limit of detection of 1 ng/ml was determined which allowed for the reliable measurement of cyclizine in the serum of human subjects. The method was found to be linear over the calibration range of 2.5-100 ng/ml. The applicability of this method was demonstrated by the analysis of serum obtained from a human volunteer following administration of a single 50 mg cyclizine hydrochloride tablet. The reported method was observed to have the necessary sensitivity, selectivity, precision and accuracy for monitoring cyclizine concentrations in human subjects following oral administration.     

Assessing the Reliability of Amplified RNA Used in Microarrays

A certain minimal amount of RNA from biological samples is necessary to perform a microarray experiment with suitable replication. In some cases, the amount of RNA available is insufficient, necessitating RNA amplification prior to target synthesis. However, there is some uncertainty about the reliability of targets that have been generated from amplified RNA, because of nonlinearity and preferential amplification. This current work develops a straightforward strategy to assess the reliability of microarray data obtained from amplified RNA. The tabular method we developed, which utilises a Down-Up-Missing-Below (DUMB) classification scheme, shows that microarrays generated with amplified RNA targets are reliable within constraints. There was an increase in false negatives because of the need for increased filtering. Furthermore, this analysis method is generic and can be broadly applied to evaluate all microarray data. A copy of the Microsoft Excel spreadsheet is available upon request from Edward Bearden.     

Age estimation from the auricular surface of the ilium: a revised method

A revised method for estimating adult age at death using the auricular surface of the ilium has been developed. It is based on the existing auricular surface aging method of Lovejoy et al. ([1985] Am. J. Phys. Anthropol. 68:15-28), but the revised technique is easier to apply, and has low levels of inter- and intraobserver error. The new method records age-related stages for different features of the auricular surface, which are then combined to provide a composite score from which an estimate of age at death is obtained. Blind tests of the method were carried out on a known-age skeletal collection from Christ Church, Spitalfields, London. These tests showed that the dispersion of age at death for a given morphological stage was large, particularly after the first decade of adult life. Statistical analysis showed that the age-related changes in auricular surface are not significantly different for males and females. The scores from the revised method have a slightly higher correlation with age than do the Suchey-Brooks pubic symphysis stages. Considering the higher survival rates of the auricular surface compared with the pubic symphysis, this method promises to be useful for biological anthropology and forensic science.     

Analysis of the ash of the seeds ofAesculus hippocastanum and someAceres

The seeds contain such a high percentage of phosphoric acid that it interferes with the obtaining of correct results in analysis. Its prior removal from the solution of ash is therefore necessary. A new method is suggested for the entire analysis which is a combination of percolation through an ionexchanger column and colorimetric and gravimetric determination. The oxide values and the average content of ash per 100 g. dry weight of seeds ascertained by analysis were used to calculate the weight of each determined element per 100 g. of dried substance. The analysis showed that traces of tin are present in the ash of the seeds tested. Under the given conditions each species maintains its characteristic way of storing ash constituents in the seeds and this is not obviously affected by the affinity of the plants or the influence of the soil.     

Performances of a multidimensional on-line SPE-LC-ECD method for the determination of three major catecholamines in native human urine: validation, risk and uncertainty assessments

A novel, multidimensional on-line SPE-LC method with electrochemical detection is described for the fully automated and direct analysis of the catecholamines norepinephrine, epinephrine and dopamine in urine. The integrated extractive clean-up of the raw biofluid is based on a SPE-column packed with restricted access material (RAM) which is modified with the affinity ligand nitrophenylboronic acid. The method was fully validated according to a recent approach based on an accuracy profile. The acceptance limits were set at +/-15% of the nominal concentration values. The method was found accurate over a concentration range from 15 to 500 microg/l for norepinephrine, from 5 to 500 microg/l for epinephrine and from 50 to 500 microg/l for dopamine. The relative risk for the use of the validated method in routine analysis was also assessed based on this validation strategy. It was found that at most 3.5% of future sample measurements will fall outside the acceptance limits. This demonstrates the high reliability of the analytical method described. Moreover, the measurements uncertainties were deduced from the validation experiments without any additional effort.     

Application of the function of the number of states of dynamic system to investigation of electroencephalographic reaction to photostimulation

A new way of EEG analysis is developed, which makes it possible to reveal a reaction of the brain cortex to external stimulation at a low response level. The method is based on the creation of an image of the EEG curve in the discrete phase space and comparison of a current condition with the conditions from a certain basic set. The strength of reaction to external stimulation is defined by the incidence of conditions novel with respect to the basic ones.     

A new analysis method for the comparison of 2 temporal patterns,with indices of association

A new method for evaluating the degree of association between the behavioural patterns of 2 individuals is described and illustrated. It is proposed discrimination should be made between the unit for sampling (sample interval) and the unit for analysis. The length of the sample interval affects analysis accuracy, while the length of the analysis unit affects the analysis scale. A new “moving analysis unit (MAU) method” is proposed to divide an observation period into analysis units. The MAU method, with various lengths of analysis units, allows for various aspects of interaction between 2 individuals. As a similarity coefficient between the 2 temporal patterns, Iwao's ω (Iwao 1977) is proposed. Investigation of nonparametric confidence intervals of ω is performed with the bootstrap (Efron 1979), which is also applied for obtaining the null hypothesis distribution of ω. The MAU method, ω and the bootstrap are applied to the analysis of both insect and human communication.     

Evaluation of the time course of neurotransmitter release from the measured PSC and MPSC

A method for obtaining delay histograms for the time course of neurotransmitter release is presented. The delay histogram is derived from the measured psc (or the sum of several psc's) and the mpsc (obtained experimentally or otherwise) by means of a simple, quick, mathematical procedure. The procedure may be automated for the greater part. No approximation of the mpsc shape is performed, and the method is applicable to all quantal contents. For low and medium quantal contents, the delay histograms obtained by the method are compared to those obtained by direct analysis. A reasonable agreement is achieved. An experiment of high quantal content, for which direct analysis is impossible, is then analysed using the new method. Difficulties which may arise when applying the procedure and methods to overcome them are discussed at length. Other methods are set forth in the Discussion.     

Application of the finite element simulation method to the adiabatic and potentiometric corrections of calorimetric titration data

A general numerical analysis procedure is described which has been applied to an automated differential pH-thermal titration apparatus operated isoperibolically to obtain thermal corrections for heat loss. It is based on the Direct Byte (D-B) finite element computer simulation technique (FEST) applied to the heat conduction behavior of the instrument with time. Thermal constants of the numerical model are determined, and the results of the correction for titration data obtained from acid-base runs show that a constant upper baseline is achieved using this technique for both fast and slow reactions to an accuracy of 2%. The method is equally valid for endothermic and exothermic reactions.     

On the Three-Taxon Approach to Parsimony Analysis

The following three basic defects for which three-taxon analysis has been rejected as a method for biological systematics are reviewed: (1) character evolution is a priori assumed to be irreversible; (2) basic statements that are not logically independent are treated as if they are; (3) three-taxon statements that are considered as independent support for a given tree may be mutually exclusive on that tree. It is argued that these criticisms only relate to the particular way the three-taxon approach was originally implemented. Four-taxon analysis, an alternative implementation that circumvents these problems, is derived. Four-taxon analysis is identical to standard parsimony analysis except for an unnatural restriction on the maximum amount of homoplasy that may be concentrated in a single character state. This restriction follows directly from the basic tenet of the three-taxon approach, that character state distributions should be decomposed into basic statements that are, in themselves, still informative with respect to relationships. A reconsideration of what constitutes an elementary relevant statement in systematics leads to a reformulation of standard parsimony as two-taxon analysis and to a rejection of four-taxon analysis as a method for biological systematics.     

Chromatography on DEAE-cellulose microcolumns: A quantitative method for the fractionation of small quantities of glycosaminoglycans

A simple, sensitive, and efficient method is described for qualitative and quantitative determination of glycosaminoglycans (GAG) synthesized by embryonic mouse teeth. After release from proteoglycan aggregates by enzymatic treatment, a mixture of different GAG was absorbed on a DEAE-cellulose microcolumn (Whatman DE-52 microgranular) at low salt concentration. The different types of GAG were eluted by stepwise increases in the concentration of NaCl. Glycopeptides, which generally contaminate the extract, can be completely removed prior to the elution of GAG. The eluate fractions were analyzed by rechromatography on the same column, using gradient elution. The stepwise elution is suitable for analysis as well as preparation of labeled GAG, the supply of which is limited in amount. The scale of chromatography can easily be stepped up. Quantitative analysis of GAG from embryonic mouse teeth is presented to demonstrate the usefulness of this method.     

General methods for the analysis of metabolic profiles of bile acids and related compounds in feces

A general method is described for the detailed qualitative and quantitative analysis of bile acids and related compounds from feces. The technique utilizes a novel combination of liquid-gel and liquid-solid extraction, lipophilic ion exchange chromatography, and capillary column gas-liquid chromatography coupled to mass spectrometry, which permits the detailed composition of bile acids in feces in terms of both the individual bile acids present and their mode of conjugation in the original fecal sample. The extraction, purification, and isolation procedures have been evaluated using fecal samples containing endogenous radioactive bile acid metabolites and from the addition of radiolabeled standards to fecal homogenates. The applicability of the general procedure is illustrated with examples from the analysis of bile acids and sterols in the feces collected from normal healthy subjects, patients with chronic diarrhea, and an adult female Sprague-Dawley rat. The flexibility of the method, and the general problems encountered in the extraction, purification, and isolation of bile acids and related classes of compounds from feces for subsequent analysis of gas-liquid chromatography are discussed in detail.