A new method for calculating evolutionary substitution rates

Summary In this paper we present a new method for analysing molecular evolution in homologous genes based on a general stationary Markov process. The elaborate statistical analysis necessary to apply the method effectively has been performed using Monte Carlo technqiues. We have applied our method to the silent third position of the codon of the five mitochondrial genes coding for identified proteins of four mammalian species (rat, mouse, cow and man). We found that the method applies satisfactorily to the three former species, while the last appears to be outside the scope of the present approach. The method allows one to calculate the evolutionarily effective silent substitution rate (v_s) for mitochondrial genes, which in the species mentioned above is 1.4×10^–8 nucleotide substitutions per site per year. We have also determined the divergence time ratios between the couples mousecow/rat-mouse and rat-cow/rat-mouse. In both cases this value is approximately 1.4.     

Regular measurement--a new method for biophysical experiment

A system of quantized relations between the value of the quantity being measured and the width of its experimental scattering was experimentally found in the results of measurements of rates of various processes. For determining D, the square of the ratio of the root-mean-square deviation to the mean value of the quantity being measured, particularly long series of stereotypic measurements under stable conditions, so-called regular measurements, are carried out. Regular changes in biochemical, chemical, and physical quantities show that the D value is related by a simple rational expression to the universal dimensionless constant D0 whose numerical value was measured with required accuracy. The simplicity of the ratios of D and D0 may result from the fact that, during regular measurements, the quantum behavior of the system macrodevice-object can be revealed.     

A new bioassay method for quantitative analysis of tetracyclines

A new method is described for quantitative analysis of tetracycline, based on the decrease in external pH of bacterial suspensions after the addition of a glucose pulse. The decrease in external pH of these suspensions was inversely proportional to the concentration of tetracycline. The correlation coefficient of standard response lines derived from the bioassay was 0.99. Tetracycline potency was determined in six tetracycline HCl samples by the sugar pulse bioassay and a turbidimetric method. The turbidimetric assay result varied from the glucose-pulse data by no more than 7 and 3% at 3 and 7 min, respectively. The procedure is rapid, precise and quantitative, and requires minimal preparation and use of media, with savings in laboratory resources and time.     

Regular measurement: A new method of biophysical experiment

A system of quantized relations between the value of the parameter (quantity) measured and the range of its experimental scattering has been revealed by analyzing the results of experiments on measuring the rates of various processes. Especially long series of stereotyped measurements under stable conditions (called regular measurements) are performed for determining the squared ratio of the standard deviation to the mean value of the quantity measured (D). Regular measurements of biochemical, chemical, and physical parameters show that the D value is related to the universal dimensionless constant D ₀, whose numerical value has been determined with considerable accuracy, and this relation is described by a simple rational equation. The simplicity of the relationship between D and D ₀ may be accounted for by the quantum behavior of the macroinstrument-object system manifested in the course of regular measurement.     

A Bayesian method for calculating real-time quantitative PCR calibration curves using absolute plasmid DNA standards

Background  

In real-time quantitative PCR studies using absolute plasmid DNA standards, a calibration curve is developed to estimate an unknown DNA concentration. However, potential differences in the amplification performance of plasmid DNA compared to genomic DNA standards are often ignored in calibration calculations and in some cases impossible to characterize. A flexible statistical method that can account for uncertainty between plasmid and genomic DNA targets, replicate testing, and experiment-to-experiment variability is needed to estimate calibration curve parameters such as intercept and slope. Here we report the use of a Bayesian approach to generate calibration curves for the enumeration of target DNA from genomic DNA samples using absolute plasmid DNA standards.     

A new method for calculating Risk Tolerance in the assessment of threatened flora

This paper reports on a proposed method for evaluating threat categories for regional flora, based mainly on phytogeographical data. The method involves the creation of current distribution maps, analysis of threat factors and use of the RAMAS Red List software to facilitate rapid and objective classification of taxa in accordance with 2001 IUCN criteria. In order to ensure a more objective approach by the assessor and reduce the range of uncertainty, an index was created for calculating Risk Tolerance (RT) that enabled adjustment of the results obtained and thus supplemented the software used. For trial purposes, the proposed model was applied to Spanish gypsophilous flora currently protected under Spanish regional legislation. When classified using the new model, the taxa in question were placed in very different categories, ranging from least concern (LC) to endangered (EN), indicating the need to redefine both their current legal status and recommended management measures. Use of this method will enable scientists to optimise available information on the geographical distribution of endangered taxa, and help governments both to optimise their financial investment in conservation and to define their priorities. The authors endorse the use of IUCN categories in legal protection texts, and advocate as essential a greater degree of coordination between scientists and governments.     

A new concept for DNA-arrays

We will insert a cleavage site in an oligodeoxynucleotide, which can be used for a selective and quantitative cleavage. For that reason we synthesized the four 5'-S-(4,4'-dimethoxytrityl)-mercapto-2'-deoxynucleotide-3'-O-(2-cyanoethoxydiisopropylamino)-phosphites (5a-d). The cleavage of P-S and C-S bonds is described (Mag, M.; Lücking, S.; Engels, J.W. Synthesis and selective cleavage of an oligodeoxy-nucleotide containing a bridged internucleotide 5'-phosphorthioate linkage. Nucleic Acids Res. 1991, 19 (7), 1437-1441; Marriott, J.H.; Mottahedeh, M.; Reese, C.B. 9-(4-methoxyphenyl)xanthen-9-thiol: A useful reagent for the preparation of thiols. Tetrahedron Lett. 1990, 31 (51), 7485-7488; Divakar, K.J.; Mottoh, A.; Reese, C.B.; Shanghvi, Y.S. Approaches to the synthesis of 2' thio analogues of pyrimidine ribosides. J. Chem. Sc., Perkin Trans. 1 1990, 969-974). The oligodeoxynucleotides with an achiral bridged 5'-phosphorothioate linkage 5'-O-P-S-3' are synthesized by the phosphoramidite procedure.     

A new method for robust quantitative and qualitative analysis of real-time PCR

An automated data analysis method for real-time PCR needs to exhibit robustness to the factors that routinely impact the measurement and analysis of real-time PCR data. Robust analysis is paramount to providing the same interpretation for results regardless of the skill of the operator performing or reviewing the work. We present a new method for analysis of real-time PCR data, the maxRatio method, which identifies a consistent point within or very near the exponential region of the PCR signal without requiring user intervention. Compared to other analytical techniques that generate only a cycle number, maxRatio generates several measurements of amplification including cycle numbers and relative measures of amplification efficiency and curve shape. By using these values, the maxRatio method can make highly reliable reactive/nonreactive determination along with quantitative evaluation. Application of the maxRatio method to the analysis of quantitative and qualitative real-time PCR assays is shown along with examples of method robustness to, and detection of, amplification response anomalies.     

A non-radioactive method for inexpensive quantitative RT-PCR.

We present a novel method for quantitative RT-PCR that involves direct incorporation of digoxigenin-11-dUTP (DIG-dUTP) during amplification of cDNAs, separation of RT-PCR products by agarose gel electrophoresis, Southern transfer to a nylon membrane, and chemiluminescent detection with an anti-DIG antibody. The whole procedure can be done in about a day and has the following advantages: It is highly sensitive, specificity is confirmed by monitoring the size of the RT-PCR product, it is non-radioactive, quantitative, and does not require expensive specialized equipment.     

A new quantitative in vitro microculture method for Giardia duodenalis trophozoites

A reliable, rapid and low-cost method for drug sensitivity determination of Giardia duodenalis trophozoites (WB-strain) was developed in a 96-well plate. Using a standard inoculum of 5 x 10(4) trophozoites per well (300 microl), good growth was obtained after sealing the plate with an air-tight adhesive tape and incubation at 37 degrees C for 72 h in modified TYI-S-33 medium. Viable burdens were quantified using the formazan dyes MTT (100 microg/well) and XTT (20 microg/well) and the fluorescent substrate resazurin (2.5 microg/well). Prior removal of the culture medium is required since it causes spontaneous reduction of the substrate. Resazurin proved to be far superior to MTT and XTT with a level of sensitivity of about 3 x 10(4) trophozoites. Inhibitory concentrations (IC(50)) of several anti-giardial reference drugs were within the range of published values: metronidazole 2.25 microM, tinidazole 1.75 microM, albendazole 0.10 microM, furazolidone 2.00 microM and quinacrine 0.32 microM. The broad-spectrum antibiotics chloramphenicol, rifampicin, penicillin G+streptomycin and gentamycin were devoid of any inhibitory activity and are considered suitable for decontamination during excystation experiments.     

A new method for quantitative estimation of the virus particles number

The virus particles of live mumps virus vaccine widely used for vaccination in Russia have been detected and visualized by the atomic force microscopy. For quantitative estimation of the number of observed virus particles the special method has been developed. The presence of the vaccine virus protein component was tested by ELISA and dot-blot analysis. Using a quantitative real-time PCR assay the number of copies of viral RNA was estimated. The results of the quantitative estimation obtained by real-time PCR corresponded to the atomic force microscopy data.     

A weighted test-area method for calculating surface tension

In this paper, we propose a weighted test-area method to calculate surface tension by incorporating the weighting factor from the Bennett method into the free energy perturbation scheme of the test-area method. This new method was tested by comparing against the results of the Bennett and test-area methods for simulations of square well (SW), Lennard-Jones and point charge fluid models. It is seen that the new method is accurate for all these simulations, giving the same results as the Bennett method, in contrast to the test-area method which cannot calculate the surface tension of a SW fluid. The new method converges as quickly, on the basis of computational time required, as the test-area method and almost twice as quickly as the Bennett method. This combination of speed and accuracy means that the weighted test-area method should be used in preference to the test-area method and Bennett method for surface tension and other macroscopic thermodynamic quantities that can be calculated through perturbation methods.     

A new extraction method for Acinetobacter species ODB-L2 rough form lipopolysaccharide from culture broth.

The Gram-negative bacterium Acinetobacter species ODB-L2 produces lipopolysaccharide (LPS) in culture broth. The LPS could not be purified by conventional extraction methods using 90% phenol/water or 90% phenol/chloroform/petroleum ether mixed solvent. Extraction was achieved employing an admixture of chloroform, ethanol, and 4 M HCI solution. The LPS was purified from dissolving the crude extracts in 90% phenol and LPS sediment formed by addition of methanol. The LPS was characterized by chemical, biochemical, and physicochemical methods as rough form 3-hydroxydodecanoic acid rich LPS.     

A new model for calculating muscle forces from electromyograms

A muscle model is described that uses electromyogram (EMG), muscle length and speed of contraction to predict muscle force. Physiological parameters are the Hill constants and the shape of the twitch response to a single stimulus. The model was incorporated in a jaw model of the rabbit and tested by predicting the bite force produced by the jaw muscles during mastication. The time course of the calculated force appeared to match the bite force, measured in vivo by a strain gauge, applied to the bone below the teeth. The variation in peak strain amplitude from cycle to cycle correlated with the variation predicted by the model. The peak amplitude of the integrated EMGs of individual jaw muscles showed an average correlation with peak strain of 0.41. Use of the sum of the available peak amplitudes, weighted according to their effect upon the bite force increased the correlation to 0.46; the model predicted bite forces showed a correlation of 0.57 with the strain. The increase in correlation was statistically significant. The muscle forces were calculated using a minimum number of easily obtainable constants.