Genotyping of single nucleotide polymorphism using model-based clustering

MOTIVATION: Single nucleotide polymorphisms have been investigated as biological markers and the representative high-throughput genotyping method is a combination of the Invader assay and a statistical clustering method. A typical statistical clustering method is the k-means method, but it often fails because of the lack of flexibility. An alternative fast and reliable method is therefore desirable. RESULTS: This paper proposes a model-based clustering method using a normal mixture model and a well-conceived penalized likelihood. The proposed method can judge unclear genotypings to be re-examined and also work well even when the number of clusters is unknown. Some results are illustrated and then satisfactory genotypings are shown. Even when the conventional maximum likelihood method and the typical k-means clustering method failed, the proposed method succeeded.     

Comparative studies of polyethylene glycol-modified liposomes prepared using different PEG-modification methods

To address the issue of excess polyethylene glycol (PEG)-lipid degradation observed when PEG-modified liposomes are prepared using the pH-gradient method, a concept using a novel PEG-modification method, called the post-modification method, was proposed and evaluated. To assess the proof concept, a preservation-stability study and a pharmacokinetic study were performed that compared the conventional PEG-modification method, called the pre-modification method, with the post-modification method. The results show that PEG-lipid degradation could be markedly inhibited in the post-modification method. Furthermore, the post-modification method could be used without any manufacturing process difficulties, especially with high PEG-lipid content. In addition, a higher blood circulation capability was observed in the post-modification method. Through comparative studies, it was found that the post-modification method was advantageous compared to the pre-modification method. In conclusion, the post-modification method has the potential to be a novel PEG-modification method that can achieve a higher preservation stability of PEG-lipid, a greater ease of manufacturing, and a higher blood circulation capability, especially in the manufacturing of pH-gradient liposomal products.     

广西金丝桃属植物有效成分金丝桃素的含量测定研究

采用 UV-VIS法和 HPLC法检测了广西产金丝桃属植物及其提取物中有效成分金丝桃素的含量 ,建立了 HPLC测定金丝桃素的新方法 ,该法有良好的重现性及精密度。     

Condition Number Estimation of Preconditioned Matrices

The present paper introduces a condition number estimation method for preconditioned matrices. The newly developed method provides reasonable results, while the conventional method which is based on the Lanczos connection gives meaningless results. The Lanczos connection based method provides the condition numbers of coefficient matrices of systems of linear equations with information obtained through the preconditioned conjugate gradient method. Estimating the condition number of preconditioned matrices is sometimes important when describing the effectiveness of new preconditionerers or selecting adequate preconditioners. Operating a preconditioner on a coefficient matrix is the simplest method of estimation. However, this is not possible for large-scale computing, especially if computation is performed on distributed memory parallel computers. This is because, the preconditioned matrices become dense, even if the original matrices are sparse. Although the Lanczos connection method can be used to calculate the condition number of preconditioned matrices, it is not considered to be applicable to large-scale problems because of its weakness with respect to numerical errors. Therefore, we have developed a robust and parallelizable method based on Hager’s method. The feasibility studies are curried out for the diagonal scaling preconditioner and the SSOR preconditioner with a diagonal matrix, a tri-daigonal matrix and Pei’s matrix. As a result, the Lanczos connection method contains around 10% error in the results even with a simple problem. On the other hand, the new method contains negligible errors. In addition, the newly developed method returns reasonable solutions when the Lanczos connection method fails with Pei’s matrix, and matrices generated with the finite element method.     

Microbiological Aspects of Cold Cleaning with an lodophor of Milk Pipeline Installations

Over a period of three years bacteriological comparisons were made of a method of cleaning pipeline milking machines with (1) cold iodophor solution (cold method), (2) a chlorinated alkaline solution at 55-60°C circulated for 10 min (hot method), and (3) acidified boiling water (ABW method). Rinse samples from the entire installations gave the best results with the ABW method. The cold method favoured a psychrotrophic flora, coliform bacteria or lactic streptococci, depending on the part of the milking machine rinsed; the hot method tended to cause a build-up of the heat-resistant flora. In spite of this, the two methods can give acceptable results provided that the installations are correctly designed and constructed.     

Utilization of mixtures of carbon compounds by yeasts

As a tool for the classification of yeasts a method is presented for determining the aerobic utilization of each of 28 carbon compounds distributed over 4 mixtures containing respectively 10, 7, 7 and 4 of these compounds. Paper chromatography is used to follow the eventual disappearance of each individual carbon compound from the medium. This method is compared to the well-known standard method where a growth response to singly offered compounds in liquid medium is used for biochemical characterization. The proposed method has two advantages: results are obtained more rapidly and with less work, and the paper-chromatographic method may reveal a time sequence of utilization. Both the new method and the established standard method were applied to eight yeasts. Although the backgrounds are different, the experimental results were similar. The method presented is suitable for the differentiation of species as well as of strains within one species. It should be considered for use in taxonomy.     

Freezing of bovine embryos: Effects of a one-step addition of 1.4 M glycerol

The effects of a one-step addition of 1.4 M glycerol (method A) upon morphological appearance and developmental capacity of frozen/thawed day 7 bovine embryos were investigated and compared to a standard stepwise addition of 1.0 M glycerol (method B). With method A, the percentage of intact embryos (classified as excellent, good and poor) was 95.3% (61 out of 64) without differences between morulae (96.5%) and blastocysts (94.4%). With method B, the percentage of intact embryos was 83.0% (44 out of 53). The percentage was similar for blastocysts (89.2%) and significantly (p < 0.05) lower for morulae (68.8%) when compared to method A. The percentage of embryos with a damaged zona pellucida was considerably increased with method A (26.6%) when compared to method B (13.2%). The proportion of embryos with excluded blastomeres was similar in both methods (21.9% method A, 17.0% method B). With method A, pregnancy rates after nonsurgical transfer were 51.0% (25 out of 49) and were better than with method B (40.5%; 15 out of 37). Embryos with a damaged zona pellucida resulted in a high pregnancy rate of 66.7% (8 out of 12). A pregnancy rate of 52.9% (10 out of 17) was obtained with embryos showing some excluded blastomeres. Thus, a one-step addition of 1.4 M glycerol facilitates and accelerates the process of embryo cryopreservation and is compatible with high pregnancy rates. Damage of the zona pellucida does not impair further development of frozen/thawed bovine embryos provided blastomeres are intact.     

蚧虫基因组DNA不同提取方法的比较

实验以日本龟蜡蚧CeroplastesjaponicusGreen,白蜡绵粉蚧PhenacoccusfraxinusTang ,朝鲜球蚧DidesmococcuskoreanusBorchseniush和瘤大球坚蚧EulecaniumgiganteaShinji等 4种蚧虫为材料 ,分别用十二烷基硫酸钠 (SDS)法、十六烷基三乙基溴化铵 (CTAB)法、醋酸钾 (KAc)法和氯化钠 (NaCl)法等 4种方法 ,对单只蚧虫进行基因组DNA提取 ,用 0 8%琼脂糖凝胶电泳检测所提DNA。结果表明 ,4种方法都可以提取到基因组DNA ,但是比较而言 ,CTAB法和NaCl法所提取的DNA质量明显优于SDS法和KAc法 ,并适用于PCR。因此认为 ,CTAB法和NaCl法是实验室提取单只蚧虫基因组DNA更有效而实用的方法。     

Use of a new methylene blue derivative for determination of lipid peroxides in foods

To determine lipid peroxides in chloroform-methanol extracts of foods, a simple and sensitive colorimetric method using a new leucomethylene blue derivative was adopted. The amounts obtained by this method coincided well with those by the iodometric method and paralleled those by the thiobarbituric acid method.     

Comparison of two methods for extraction of enchytraeids

A comparison was made of two methods for the extraction of enchytraeids, the O’Connor (1957) method and the Römbke (1995) method; the latter was originally proposed as a standard method for monitoring environmental pollution. The average number of enchytraeids extracted by the Römbke method was significantly greater. This simple and convenient method was shown to be as efficient as the traditional method when applied in both laboratory and field studies.     

Validation of a questionnaire method for estimating extent of menstrual blood loss in young adult women.

The objective of this study was to validate two indirect methods for estimating the extent of menstrual blood loss against a reference method to determine which method would be most appropriate for use in a population of young adult women. Thirty-two women aged 18 to 29 years (mean +/- SD; 22.4 +/- 2.8) were recruited by poster in Dunedin (New Zealand). Data are presented for 29 women. A recall method and a record method for estimating extent of menstrual loss were validated against a weighed reference method. Spearman rank correlation coefficients between blood loss assessed by Weighed Menstrual Loss and Menstrual Record was rs = 0.47 (p = 0.012), and between Weighed Menstrual Loss and Menstrual Recall, was rs = 0.61 (p = 0.001). The Record method correctly classified 66% of participants into the same tertile, grossly misclassifying 14%. The Recall method correctly classified 59% of participants, grossly misclassifying 7%. Reference method menstrual loss calculated for surrogate categories demonstrated a significant difference between the second and third tertiles for the Record method, and between the first and third tertiles for the Recall method. The Menstrual Recall method can differentiate between low and high levels of menstrual blood loss in young adult women, is quick to complete and analyse, and has a low participant burden.     

Genotyping and annotation of Affymetrix SNP arrays

In this paper we develop a new method for genotyping Affymetrix single nucleotide polymorphism (SNP) array. The method is based on (i) using multiple arrays at the same time to determine the genotypes and (ii) a model that relates intensities of individual SNPs to each other. The latter point allows us to annotate SNPs that have poor performance, either because of poor experimental conditions or because for one of the alleles the probes do not behave in a dose–response manner. Generally, our method agrees well with a method developed by Affymetrix. When both methods make a call they agree in 99.25% (using standard settings) of the cases, using a sample of 113 Affymetrix 10k SNP arrays. In the majority of cases where the two methods disagree, our method makes a genotype call, whereas the method by Affymetrix makes a no call, i.e. the genotype of the SNP is not determined. By visualization it is indicated that our method is likely to be correct in majority of these cases. In addition, we demonstrate that our method produces more SNPs that are in concordance with Hardy–Weinberg equilibrium than the method by Affymetrix. Finally, we have validated our method on HapMap data and shown that the performance of our method is comparable to other methods.     

Comparison of three methods for measuring electrical resistances of plant cell membranes

The reliability of two different membrane resistance-measuring methods that use a single intracellular microelectrode was tested against a conventional method that uses two intracellular microelectrodes. The first single-electrode method used a single square current pulse and required a constant microelectrode resistance. This method was unreliable because the electrode resistance changed markedly on cell penetration and changed with time within the cell. The second method used a high frequency square wave for injecting current into the cell and depended upon the membrane having a much longer RC (resistance × capacitance)-time constant than the microelectrode. The resistance values obtained by this latter method were usually different from membrane resistances obtained at the same time on the same cells using two intracellular microelectrodes. Therefore, neither single intracellular microelectrode method was as reliable as the conventional method. All tests were with coleoptile cells of Avena sativa var. Victory.     

Results of phalloplasty.

We present our experience with a method of phalloplasty which can be applied by reconstructive surgeons to cases of penile loss from diverse etiologies. The complications with this method have been frequent, but not life-threatening. Achievement of the stated objectives is obtained in a sufficiently high proportion of cases to make this a method whorthwhile to use until a clearly superior method can be demonstrated.     

A simple modification of the Baermann method for diagnosis of strongyloidiasis

The diagnosis of Strongyloides stercoralis infections is routinely made by microscopic observation of larvae in stool samples, a low sensitivity method, or by other, most effective methods, such as the Baermann or agar culture plate methods. We propose in this paper a practical modification of Baermann method. One hundred and six stool samples from alcoholic patients were analyzed using the direct smear test, agar culture plate method, the standard Baermann method, and its proposed modification. For this modification the funnel used in the original version of the method is substituted by a test tube with a rubber stopper, perforated to allow insertion of a pipette tip. The tube with a fecal suspension is inverted over another tube containing 6 ml of saline solution and incubated at 37 degrees C for at least 2 h. The saline solution from the second tube is centrifuged and the pellet is observed microscopically. Larva of S. stercoralis were detected in six samples (5.7%) by the two versions of the Baermann method. Five samples were positive using the agar culture plate method, and only in two samples the larva were observed using direct microscopic observation of fecal smears. Cysts of Endolimax nana and Entamoeba histolytica/dyspar were also detected in the modification of Baermann method. Data obtained by the modified Baermann method suggest that this methodology may helps concentrate larvae of S. stercoralis as efficiently as the original method.     

Analysis of exponential decay curves: a three-step scheme for computing exponents

A study of four techniques is examined to determine which method has the greater overall utility. An iterative least-squares error minimization method provides the greatest accuracy, while a linear algebraic method is accurate only for two-exponential data with small error. Consequently, a three-method sequential identification scheme is devised utilizing a graphical method, a transform method, and an iterative method. This scheme is successfully applied to physiological data containing three and four exponents.     

Bias compensated least squares estimate of the center of rotation

It is shown that a recently published least squares method for the estimation of the average center of rotation is biased. Consequently, a correction term is proposed, and an iterative algorithm is derived for finding a bias compensated solution to the least squares problem.The accuracy of the proposed bias compensated least squares method is compared to the previously proposed least squares method by Monte-Carlo simulations. The tests show that the new method gives a substantial improvement in accuracy.     

Simple preparation method of PCR fragments for automated DNA sequencing

In an effort to find a simple and inexpensive purification method of polymerase chain reaction (PCR) reaction before cycle sequencing reaction, we compared a commercial system with a precipitation protocol performed in our laboratory. We found that, particularly with small PCR products, our method works with greater success than the method compared. Our precipitation method may be used on a larger PCR fragment before cycle sequencing reaction as well. Furthermore, it has the advantage of being simple as the well-known dilution method; in contrast to the dilution method, the precipitation method removes excess primers as well as possible primer dimers.     

指甲游离缘的核DNA分型研究

为探讨指甲游离缘核DNA分型的可行性, 采集无关个体指甲游离缘样本10份, 分别以不同消化体系, 采用有机法、Chelex-100法,有机法结合Chelex-100法等3种方法提取指甲游离缘核DNA, AmpFlSTR IdentifierTM试剂盒复合扩增, ABI PRISM 3130自动遗传分析仪检测分析。结果显示：与对照血样比较,有机法结合Chelex-100法提取的样本核DNA均获得满意的STR分型, 有机法提取的样本核DNA可以进行STR分型, 但部分样本图谱峰值不均衡, Chelex-100法提取的样本核DNA不能分型或出现较多等位基因缺失。提示指甲游离缘可以进行成功地核DNA的分型, 有机法和有机法结合Chelex-100法提取的DNA质量都可成功检测, 其中以有机法结合Chelex-100法提取DNA的检测成功率最高。