Non-specificity of a colorimetric method for the estimation of N-acetyl-d-glucosamine

The colorimetric method of Reissig et al. for the estimation of N-acetylamino sugars, is often used as a specific method for the quantification of the N-acetyl-d-glucosamine. Although this assay is more sensitive to the monomer, it recognizes all soluble N-acetyl-d-glucosamine oligomers. This result is very important because this method is extensively used in biology for the estimation of chitinolytic activity.     

On-line estimation of kLa by an analog computer

A new method was developed for estimating the volumetric oxygen transfer coefficient, k_La, in a fermentor. Various methods were investigated for the on-line estimation of k_La with an analog computer employing a steepest-descent calculation technique. The method by which k_La is estimated (by minimizing the error residue of the model) was found to be very applicable. A method for the simultaneous estimation of the volumetric oxygen transfer coefficient and respiration rate in biological systems is also presented.     

Comparison of swabbing and destructive methods for microbiological pig carcass sampling

Aims: To compare the Belgian swabbing sampling method for pig carcasses with the reference destructive method with regard to Escherichia coli and aerobic plate counts, Salmonella and Campylobacter prevalence and their relationship. Methods and Results: Recovery was significantly lower for the swabbing method and corresponded to a recovery of 36% for E. coli counts and 81% for aerobic plate counts in comparison with the destructive method. There was no significant difference between the swabbing and destructive sampling methods for the prevalence of Salmonella or Campylobacter. A higher median for E. coli counts was detected for samples where Salmonella or Campylobacter were detected. The same association was also observed between the median for aerobic plate counts and the presence of Campylobacter. Conclusions: The method of swabbing used, covering 600 cm² on each half‐pig carcass, is efficient for the sampling of pig carcasses in comparison with the reference destructive method. Significance and Impact of the Study: This study describes an efficient method for microbiological pig carcass sampling. The Belgian swabbing method should continue to be used to allow the follow up of bacterial contamination in the Belgian meat production chain.     

Optimising multi-tier open nucleus breeding schemes

Summary The constant migration (CM) method and the ebv migration (EBVM) method of optimising the design of multi-tier open nucleus breeding schemes are presented and compared. The equation for the equilibrium rate of genetic gain of a three-tier open nucleus scheme is determined using the CM method. The major advantage of the EBVM method is the reduction in the number of parameters which have to be varied in order to locate the maximum equilibrium rate of genetic gain. For the CM method for the number of variable parameters is 5, 14, 27 and (2n + 1) (n - 1) for unrestricted male and female migration in schemes with 2, 3, 4 and n tiers respectively. The corresponding number of variable parameters for the EBVM method is 1, 2, 3 and n-1 respectively. A procedure is given for the EBVM method so as to accomodate variance loss due to selection and variance gain due to the mixing of groups with a different mean breeding value.     

Convergence to Normality of the Asymptotic Quasi‐Score Function on a Linear Model

The asymptotic quasi‐likelihood method is considered for the model y_t = f_t(θ) + M_t, t = 0,1, …,T where f_tθ) is a linear predictable process of the parameter of interest θ, M_t is a martingale difference, and the nature of E(M_t² | ℱ_t–1) is unknown. This paper is concerned with the limiting distribution of the asymptotic quasi‐score function of such a model. Confidence intervals and hypothesis testing of θ is derived from the limiting distribution. Comparison is made between the estimates obtained through this method and those obtained through the least squares method.     

Tests of Independence and Zero Correlations Among P Random Variables

Suppose it is desired to determine whether there is an association between any pair of p random variables. A common approach is to test H₀ : R = I, where R is the usual population correlation matrix. A closely related approach is to test H₀ : R_pb = I, where R_pb is the matrix of percentage bend correlations. In so far as type I errors are a concern, at a minimum any test of H₀ should have a type I error probability close to the nominal level when all pairs of random variables are independent. Currently, the Gupta-Rathie method is relatively successful at controlling the probability of a type I error when testing H₀: R = I, but as illustrated in this paper, it can fail when sampling from nonnormal distributions. The main goal in this paper is to describe a new test of H₀: R_pb = I that continues to give reasonable control over the probability of a type I error in the situations where the Gupta-Rathie method fails. Even under normality, the new method has advantages when the sample size is small relative to p. Moreover, when there is dependence, but all correlations are equal to zero, the new method continues to give good control over the probability of a type I error while the Gupta-Rathie method does not. The paper also reports simulation results on a bootstrap confidence interval for the percentage bend correlation.     

Comparison of Zinc Reduction with Platinum Reduction for Analysis of Deuterium‐Enriched Water Samples for the Doubly Labeled Water Technique

Objective: Isotope ratio mass spectrometry of hydrogen and oxygen is frequently used to determine total energy expenditure (TEE) using doubly labeled water. Conventionally, hydrogen isotope ratio is determined in hydrogen gas generated from water samples using zinc reduction. We compare this with a new automated platinum method to determine the ratios of hydrogen isotopes in deuterium‐enriched water samples. Research Methods and Procedures: The platinum method of sample preparation was compared with the zinc method in three ways: analytical variation in deuterium enrichment (within sample; n = 51), analytical variation in TEE estimates (within sample set; n = 10), and level of agreement of TEE estimates between both methods (n = 14). Results: For the zinc method, the standard deviation for multiple sets of triplicate ²H₂O sample analysis was ±4.36‰ and ±2.07‰ for platinum. The correlation between TEE estimates when sample sets were analyzed in duplicate was r = 0.89 for zinc and r = 0.83 for platinum. The intercept and slope of the regression line were significantly different from the line of identity for duplicate TEE estimates by zinc but were not different from the line of identity for platinum. After correction for the intra‐assay variation of each method, the correlation between zinc and platinum for TEE was 0.77, and the intercept, but not the slope, of the regression was significantly different from the line of identity. The mean difference between the zinc method and the platinum method was 56 kcal/day, and the 95% confidence interval was ?438 to 550 kcal/day. Discussion: These data suggest that the platinum method is at least as reliable as the zinc method as a sample preparation technique for isotope ratio mass spectrometry of deuterium‐enriched water samples. The platinum method is also less costly and less labor‐intensive than the zinc method.     

A convenient method for multiple insertions of desired genes into target loci on the <Emphasis Type="Italic">Escherichia coli</Emphasis> chromosome

We developed a method to insert multiple desired genes into target loci on the Escherichia coli chromosome. The method was based on Red-mediated recombination, flippase and the flippase recognition target recombination, and P1 transduction. Using this method, six copies of the lacZ gene could be simultaneously inserted into different loci on the E. coli chromosome. The inserted lacZ genes were functionally expressed, and β-galactosidase activity increased in proportion to the number of inserted lacZ genes. This method was also used for metabolic engineering to generate overproducers of aromatic compounds. Important genes of the shikimate pathway (aroF ^fbr and tyrA ^fbr or aroF ^fbr and pheA ^fbr ) were introduced into the chromosome to generate a tyrosine or a phenylalanine overproducer. Moreover, a heterologous decarboxylase gene was introduced into the chromosome of the tyrosine or phenylalanine overproducer to generate a tyramine or a phenethylamine overproducer, respectively. The resultant strains selectively overproduced the target aromatic compounds. Thus, the developed method is a convenient tool for the metabolic engineering of E. coli for the production of valuable compounds.     

Consistency of the Asymptotic Quasi-Likelihood Estimate on Linear Models

Consider a model y_t = f_t(θ) + M_t, 0 ⩽ t ⩽ T where θ∈ Θ in an unknown parameter, f_t(θ) is a linear predictable process, M_t is a martingale difference, and the nature of E(M²_t/ℱ_t—1) is unknown. This paper presents an estimating procedure for θ based on the asymptotic quasi-likelihood methodology. Conditions under which the asymptotic quasi-likelihood estimate converges to the true parameter θ₀ are discussed. This method is applied to several simulated examples, and estimates of the unknown parameter are obtained by means of a two-stage technique. Comparison is made between the estimates obtained via this method and those obtained via the ordinary least squares method. Discussion is provided on the application of the model.     

一种适用于链霉菌异源合成基因簇同框缺失的高效方法

【背景】对抗生素生物合成途径的阐明有助于提高目标化合物的产量并开发具有更高活性的新化合物。基因的同框缺失是天然产物生物合成研究的常规手段,通过分析突变菌株积累的中间产物,可以帮助推导天然产物的合成途径及相关基因的功能。天然产物生物合成基因簇的大小一般在20 kb以上,对每个基因进行同框缺失耗时耗力,因此,优化链霉菌来源的基因同框缺失的方法有重要的意义。【目的】基于PCR-targeting重新设计了一套在链霉菌柯斯文库质粒上进行基因同框缺失的方法,实现链霉菌基因在大肠杆菌中快速、高效的基因同框缺失的技术体系。【方法】使用氨苄青霉素抗性基因bla作为PCR-targeting DNA片段的筛选标记,同时使用体外的Pac I酶切和酶连系统代替体内的Flp/FRT系统来介导同框缺失的构建。【结果】利用这种方法,在6 d内完成了米多霉素生物合成基因簇中14个基因的同框缺失。【结论】此方法与传统的PCR-targeting方法相比,构建同框缺失载体的效率明显提高;Pac I识别序列在链霉菌基因组上的稀有性使得此方法在构建抗生素生物合成基因簇必需基因的同框缺失载体上具有普适性。     

An iterative method to calculate forces exerted by single cells and multicellular assemblies from the detection of deformations of flexible substrates

We present a new method for quantification of traction forces exerted by migrating single cells and multicellular assemblies from deformations of flexible substrate. It is based on an iterative biconjugate gradient inversion method. We show how the iteration and the solution are influenced by experimental parameters such as the noise on deformations σ _XY , and the mean depth of recorded deformations Z _M. In order to find the validity range of our computational method, we simulated two different patterns of force. The first artificial force pattern mimics the forces exerted by a migrating Dictyostelium slug at a spatial resolution of Δ=20 μm (Rieu et al. in Biophys J 89:3563–3576, 2005) and corresponds to a large and spread force field. The second simulated force pattern mimics forces exerted by a polarized fibroblast at discrete focal adhesion sites separated by Δ=4 μm. Our iterative method allows, without using explicit regularization, the detailed reconstruction of the two investigated patterns when noise is not too high (σ _XY /u _max≤6%, where u _max is the maximal deformation), and when the plane of recorded deformations is close to the surface (Δ/Z _M≥4). The method and the required range of parameters are particularly suitable to study forces over large fields such as those observed in multicellular assemblies. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Direct Calculation of a Tree Length Using a Distance Matrix

Comparative studies of tree-building methods have shown minimum evolution to be in general an accurate criterion for selecting a true tree. To improve the use of this criterion, this paper proposes a method for rapidly and directly calculating a length of a dichotomous tree without having to resort to branch length calculations. This direct calculation (DC) method applies to the complete final topology, giving equal importance to each branch after a dichotomy. According to this method, the tree length S _DC is S _DC =∑ _i ∑ _j (D _ij /2 ^Bij ) = (∑ _i<j ∑D _ij 2 ^Bmax−Bij )/2 ^{Bmax −1} where D _ij is the observed distance between taxa i and j, B _ij is the number of branches connecting i and j, Bmax is the greatest B _ij in the tree, and the powers of two are due to the dichotomy of the tree. This tree length expression may be used as a rapid method for selecting the shortest tree from a set of hypothetical or subobtimal trees. Received: 2 March 2000 / Accepted: 24 March 2000     

Approximate Confidence Interval on Ratio of Two Variances in a Two-way Crossed Model

The model considered is a two-factor cross-classification variance components model with one observation per cell. Let the two factors be A and B, the problem is to obtain an approximate confidence interval for the ratio of variance component A over variance component B. In this paper, a method of solving this problem is established and simulations are performed to check the method.     

SPATIAL DISTRIBUTION PATTERN OF HOPLOPLEURA PACIFICA (ANOPLURA: HOPLOPLEURIDAE) ON ITS DOMINANT RAT HOST, RATTUS FLAVIPECTUS IN YUNNAN, CHINA

Hoplopleura pacifica is a dominant species of ectoparasitic sucking lice on the body surface of a common rat species, Rattus flavipectus in Yunan province of China. To illustrate the spatial distribution pattern of H. pacifica among the individuals of R. flavipectus, Iwao's linear regression method and a significance test of random deviation for the method were used, and a regression equation was established in the light of Iwao's method. The established equation is M*= 12.10 + 4.76M (r= 0.75, P < 0.01) where both α and β are considerably higher than 0 and 1, the border values for determining spatial pattern of populations. The calculated F value is F= 6.07 (P < 0.05) in the significance test of random deviation. The spatial distribution pattern of H. pacifica among the individuals of R. flavipectus is of aggregated distribution. The result suggests that the individuals of H. pacifica have a tendency to congregate together and form different individual groups instead of evenly distributing on the body surface of every rat host.     

Digoxigenin-labeled RNA probes for untranslated regions enable the isoform-specific gene expression analysis of myosin heavy chains in whole-mount in situ hybridization

Myosin heavy chains (MyHCs), which are encoded by myosin heavy chain (Myh) genes, are the most abundant proteins in myofiber. Among the 11 sarcomeric Myh isoform genes in the mammalian genome, seven are mainly expressed in skeletal muscle. Myh genes/MyHC proteins share a common role as force producing units with highly conserved sequences, but have distinct spatio-temporal expression patterns. As such, the expression patterns of Myh genes/MyHC proteins are considered as molecular signatures of specific fiber types or the regenerative status of mammalian skeletal muscles. Immunohistochemistry is widely used for identifying MyHC expression patterns; however, this method is costly and is not ideal for whole-mount samples, such as embryos. In situ hybridization (ISH) is another versatile method for the analysis of gene expression, but is not commonly applied for Myh genes, partly because of the highly homologous sequences of Myh genes. Here we demonstrate that an ISH analysis with the untranslated region (UTR) sequence of Myh genes is cost-effective and specific method for analyzing the Myh gene expression in whole-mount samples. Digoxigenin (DIG)-labeled antisense probes for UTR sequences, but not for protein coding sequences, specifically detected the expression patterns of respective Myh isoform genes in both embryo and adult skeletal muscle tissues. UTR probes also revealed the isoform gene-specific polarized localization of Myh mRNAs in embryonic myofibers, which implied a novel mRNA distribution mechanism. Our data suggested that the DIG-labeled UTR probe is a cost-effective and versatile method to specifically detect skeletal muscle Myh genes in a whole-mount analysis.     

Using a Normal Approximation to Test for the Binomial Distribution

The commonly used method to test for the binomial distribution is the x²-test. In this paper, we introduce an alternative method to test for the binomial distribution. Suppose N is the number of sample groups with n individuals each, x_ij is the jth sample in ith group, a Bernoulli variable with parameter and VVI=s²/[m(1 - m)/n]. Then it is well know that the asymptotic distribution of the statistic (N - 1) VVI is x²(N - 1) under the hypothesis p₁ = p₂ = … = p_N. Here we find that VVI has an asymptotic normal distribution N(1, 2(1 - 1/n)/(N - 1)). Unlike the x²-statistic, the variance of the normal test statistic is a function of n. This method is convenient in detecting spatial patterns and dispersion in the study of diseased organisms (e.g., plants) in field samples.     

Determination of the enantiomeric purity of the selective dopamine transporter inhibitor (+)‐R,R‐4‐(2‐benzhydryloxyethyl)‐1‐(4‐fluorobenzyl)piperidin‐3‐ol

(+)‐R ,R ‐D‐84 ((+)‐R ,R ‐4‐(2‐benzhydryloxyethyl)‐1‐(4‐fluorobenzyl)piperidin‐3‐ol) is a promising pharmacological tool for the dopamine transporter (DAT), due to its high affinity and selectivity for this target. In this study, an analytical method to ascertain the enantiomeric purity of this compound was established. For this purpose, a high‐performance liquid chromatographic (HPLC) method, based on a cellulose derived chiral stationary phase (CSP) was developed. The method was characterized concerning its specificity, linearity, and range. It was shown that the method is suitable to determine an enantiomeric excess of up to 99.8%. With only a few adjustments, this analytical CSP‐HPLC method is also well suited to separate (+)‐R ,R ‐D‐84 from its enantiomer in a semipreparative scale.     

Misconceptions about the comparison of intrinsic rates of natural increase

The intrinsic rate of natural increase (r_m) is a common measurement in entomology to describe and evaluate the growth and adaptation of a population of arthropods to certain environmental conditions. Following the method of Birch, the r_m is the solution of an exponential equation, which depends on the whole life cycle of each female and her survival time. A simplification of this equation was provided by Wyatt and White, which allows the study to be shortened as it does not depend on any survival times and only a part of the life cycle of the females. Therefore, this method has become quite popular among entomologists. As the r_m is a population parameter, it lacks any variance and thus a valid statistical comparison of r_ms for different populations is not straightforward. Hence, many approaches include statistical misconceptions. We discuss those approaches, apply them to real data and demonstrate some drawbacks of them. Furthermore, we present an easy to implement and consistent method for the comparison of r_ms.     

Artefactual responses of mesophyll conductance to CO2 and irradiance estimated with the variable J and online isotope discrimination methods

Studies with the variable J method have reported that mesophyll conductance (g_m) rapidly decreases with increasing intercellular CO₂ partial pressures (C_i) or decreasing irradiance. Similar responses have been suggested with the online isotope discrimination method, although with less consistency. Here we show that even when the true g_m is constant, the variable J method can produce an artefactual dependence of g_m on C_i or irradiance similar to those reported in previous studies for any of the following factors: day respiration and chloroplastic CO₂ photocompensation point are estimated with Laisk method; C_i or electron transport rate is positively biased; net photosynthetic rate is negatively biased; insufficient NADPH is assumed while insufficient ATP limits RuBP regeneration. The isotopic method produces similar artefacts if fractionation of carboxylation or C_i is positively biased or Δ¹³ negatively biased. A non‐zero chloroplastic resistance to CO₂ movement results in a qualitatively different dependence of g_m on C_i or irradiance and this dependence is only sensitive at low C_i. We thus cannot rule out the possibility that previously reported dependence of g_m on C_i or irradiance is a methodological artefact. Recommendations are made to take advantage of sensitivities of the variable J and isotopic methods for estimating g_m.