Metabolisms of Nucleosides in Bacteria

In this study, the authors developed a simplified method for the separation and the quantitative determination of nucleosides and bases, using paper electraophoretic technique. By this method, nucleosides and bases were well separated and determined in a fairly short time. Thus, this method was expected to be as accurate as the published methods and was believed to be a better method for both quantitative determination and detection of the nucleosides and bases in a large number of samples.     

A new X-ray-free measurement method for postoperative 3D-position analysis of navigated inserted implants]

In this article a new X-ray-free measurement method for postoperative 3D-position analysis of dental implants is proposed. The advantage of this new method is the possibility of precise 3D comparison to preoperative planning data without the use of X-rays. Standard methods for postoperative implant analysis are performed manually on the basis of orthopantomography or computer tomography scans. The proposed method uses a navigation system and a specially designed measurement device. Analysis of the measurement data shows a mean position error of 0.2 mm +/-0.1 mm and a mean depth error of 0.4 mm +/- 0.3 mm. Thus, this method is suitable for postoperative analysis of the horizontal implant position and for comparison to preoperative planning     

A hierarchical method for molecular docking using cloud computing

Discovering small molecules that interact with protein targets will be a key part of future drug discovery efforts. Molecular docking of drug-like molecules is likely to be valuable in this field; however, the great number of such molecules makes the potential size of this task enormous. In this paper, a method to screen small molecular databases using cloud computing is proposed. This method is called the hierarchical method for molecular docking and can be completed in a relatively short period of time. In this method, the optimization of molecular docking is divided into two subproblems based on the different effects on the protein–ligand interaction energy. An adaptive genetic algorithm is developed to solve the optimization problem and a new docking program (FlexGAsDock) based on the hierarchical docking method has been developed. The implementation of docking on a cloud computing platform is then discussed. The docking results show that this method can be conveniently used for the efficient molecular design of drugs.     

RAPID DETECTION OF BRUCELLA ABORTUS BY A NOVEL PROXIMITY LIGATION-BASED LOOP-MEDIATED ISOTHERMAL AMPLIFICATION METHOD

Brucellosis is one of the most common zoonotic diseases, and current methods of detecting this pathogen are quite difficult. This work combines the benefits of a proximity ligation assay with those of a loop-mediated isothermal amplification method to develop a novel proximity ligation-based loop-mediated isothermal amplification method useful for Brucella detection. The genomic DNA extraction procedure is not needed. Sensitivity of this assay for detecting Brucella abortus is 1  ×  10⁴ cells/mL in buffer and 1  ×  10⁵ cells/mL in milk. The time to detection is within 2 h of initiating the procedure, and no special equipment is needed. This new method is also suitable for the detection of other pathogens, and as such will be useful in the food safety industry.

PRACTICAL APPLICATIONS

Polymerase chain reaction (PCR) is a sensitivity method for microbe detection, but the complicated genomic DNA extraction procedure and costly equipment needed for this method makes the PCR method unpopular in developing countries. In this study, we present the novel proximity ligation-based loop-mediated isothermal amplification (P-LAMP) method for Brucella detection; this is the first time to combine the monoclonal antibody for identify microbe and LAMP method for high performance amplification DNA. The genomic DNA extraction procedure is not needed and a water-bath boiler is the only equipment required to complete the detection process. The P-LAMP method is useful for food safety pathogen detection in developing countries.     

多QTL定位的压缩估计方法

本文综述了多标记分析和多QTL定位的压缩估计方法。对于前者,Xu（Genetics,2003,163：789—801）首先提出了Bayesian压缩估计方法。其关键在于让每个效应有一个特定的方差参数,而该方差又服从一定的先验分布,以致能从资料中估计之。由此,能够同时估计大量分子标记基因座的遗传效应,即使大多数标记的效应是可忽略的。然而,对于上位性遗传模型,其运算时间还是过长。为此,笔者将上述思想嵌入极大似然法,提出了惩罚最大似然方法。模拟研究显示：该方法能处理变量个数大于样本容量10倍左右的线性遗传模型。对于后者,本文详细介绍了基于固定区间和可变区间的Bayesian压缩估计方法。固定区间方法可处理中等密度的分子标记资料;可变区间方法则可分析高密度分子标记资料,甚至是上位性遗传模型。对于上位性检测,已介绍的惩罚最大似然方法和可变区间Bayesian压缩估计方法可供利用。应当指出,压缩估计方法在今后的eQTL和QTN定位以及基因互作网络分析等研究中也是有应用价值的。     

Shrinkage Estimation Method for Mapping Multiple Quantitative Trait Loci

In this article, shrinkage estimation method for multiple-marker analysis and for mapping multiple quantitative trait loci (QTL) was reviewed. For multiple-marker analysis, Xu (Genetics, 2003, 163:789-801) developed a Bayesian shrinkage estimation (BSE) method. The key to the success of this method is to allow each marker effect have its own variance parameter, which in turn has its own prior distribution so that the variance can be estimated from the data. Under this hierarchical model, a large number of markers can be handled although most of them may have negligible effects. Under epistatic genetic model, however, the running time is very long. To overcome this problem, a novel method of incorporating the idea described above into maximum likelihood, known as penalized likelihood method, was proposed. A simulated study showed that this method can handle a model with multiple effects, which are ten times larger than the sample size. For multiple QTL analysis, two modified versions for the BSE method were introduced: one is the fixed-interval method and another is the variable-interval method. The former deals with markers with intermediate density, and the latter can handle markers with extremely high density as well as model with epistatic effects. For the detection of epistatic effects, penalized likelihood method and the variable-interval approach of the BSE method are available.     

Statistical thermodynamics of oligomer-polymer interactions

A new method for formulating partition functions for a system of particles interacting on a one-dimensional lattice has recently been developed.^3,4 In this paper the method is applied to oligomer–polymer systems. The details of the connection between this method and the matrix formulation are first established. The method is then used to find expressions for the partition function of an oligomer–polymer system when the oligomers have a distribution of lengths and an alternating sequence.     

Efficient amplification of multiple transposon-flanking sequences

Transposon mutagenesis is a very useful tool for gene identification in bacteria. Once the transposon mutants of interest are isolated, it is often necessary to identify the sequences that flank the transposon insertions. We devised an efficient method for specific amplification of transposon-flanking sequences that requires the sequence information of only transposon-specific sequences. The basic steps for this method consists of (1) digestion with a restriction enzyme, (2) ligation with a Y-shaped linker and (3) polymerase chain reaction amplification using a transposon-specific primer and a primer specific to the Y-shaped linker. The feasibility of this method was demonstrated with mini-Tn5 mutants of Salmonella typhimurium. We also found that this method can be used for simultaneous amplification of multiple transposon-flanking sequences.     

利用玉米苗饲养稻纵卷叶螟的方法

稻纵卷叶螟Cnaphalocrocis medinalis(Guenée)的饲养一直是个难题,目前还没有很好的人工饲料可供利用。本文研究了一种利用玉米苗进行室内大量饲养稻纵卷叶螟的方法。试验证实,该方法可达到连续多代次饲养稻纵卷叶螟的目的。与水稻苗饲养法相比,玉米苗法不仅具有食料种植简单、周期短的优点,而且稻纵卷叶螟的化蛹率、羽化率、卵孵化率和每雌产卵量均高于或相当于水稻苗法,可以利用玉米苗进行室内大批量饲养稻纵卷叶螟。本文还研究出了一套有效的稻纵卷叶螟成虫交配与产卵的装置。     

Real-time fluorescence loop mediated isothermal amplification for the diagnosis of malaria

BACKGROUND: Molecular diagnostic methods can complement existing tools to improve the diagnosis of malaria. However, they require good laboratory infrastructure thereby restricting their use to reference laboratories and research studies. Therefore, adopting molecular tools for routine use in malaria endemic countries will require simpler molecular platforms. The recently developed loop-mediated isothermal amplification (LAMP) method is relatively simple and can be improved for better use in endemic countries. In this study, we attempted to improve this method for malaria diagnosis by using a simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in one platform. We refer to this as the RealAmp method. METHODOLOGY AND SIGNIFICANT FINDINGS: Published genus-specific primers were used to test the utility of this method. DNA derived from different species of malaria parasites was used for the initial characterization. Clinical samples of P. falciparum were used to determine the sensitivity and specificity of this system compared to microscopy and a nested PCR method. Additionally, directly boiled parasite preparations were compared with a conventional DNA isolation method. The RealAmp method was found to be simple and allowed real-time detection of DNA amplification. The time to amplification varied but was generally less than 60 minutes. All human-infecting Plasmodium species were detected. The sensitivity and specificity of RealAmp in detecting P. falciparum was 96.7% and 91.7% respectively, compared to microscopy and 98.9% and 100% respectively, compared to a standard nested PCR method. In addition, this method consistently detected P. falciparum from directly boiled blood samples. CONCLUSION: This RealAmp method has great potential as a field usable molecular tool for diagnosis of malaria. This tool can provide an alternative to conventional PCR based diagnostic methods for field use in clinical and operational programs.     

Glabellar frown lines: direct excision, an evaluation of the scars

The direct excision of glabellar frown lines is a method that is not new, but neither has it been well evaluated. This paper describes the method, discusses the reason for the problem, and suggests why this method should have long-lasting results. Several examples of the scars are demonstrated. In selected patients, this may be a reasonable alternative to the myriad surgical possibilities in our quest for a solution to this problem.     

New Automated Single-Cell Technique for Segmentation and Quantitation of Lipid Droplets

Lipid droplets are the major organelle for intracellular storage of triglycerides and cholesterol esters. Various methods have been attempted for automated quantitation of fluorescently stained lipid droplets using either thresholding or watershed methods. We find that thresholding methods deal poorly with clusters of lipid droplets, whereas watershed methods require a smoothing step that must be optimized to remove image noise. We describe here a novel three-stage hybrid method for automated segmentation and quantitation of lipid droplets. In this method, objects are initially identified by thresholding. They are then tested for circularity to distinguish single lipid droplets from clusters. Clusters are subjected to a secondary watershed segmentation. We provide a characterization of this method in simulated images. Additionally, we apply this method to images of fixed cells containing stained lipid droplets and GFP-tagged proteins to provide a proof-of-principle that this method can be used for colocalization studies. The circularity measure can additionally prove useful for the identification of inappropriate segmentation in an automated way; for example, of non-cellular material. We will make the programs and source code available to the community under the Gnu Public License. We believe this technique will be of interest to cell biologists for light microscopic studies of lipid droplet biology.     

Statistical method for estimating the standard errors of branch lengths in a phylogenetic tree reconstructed without assuming equal rates of nucleotide substitution among different lineages.

A statistical method is developed for estimating the standard errors of branch lengths in a phylogenetic tree reconstructed without assuming equal rates of nucleotide substitution among different lineages. This method can be easily used for testing whether the length of an interior branch in a reconstructed tree is positive, i.e., whether the topology of the tree is correct. Computer simulations indicate that this method is appropriate for a statistical test. As an example, this method is applied to phylogenetic trees reconstructed for the four hominoid species: human, chimpanzee, gorilla, and orangutan. The results obtained show that the present method provides a powerful statistical test.     

A highly sensitive assay method for human placental alkaline phosphatase involving a monoclonal antibody bound to a paper disk

A monoclonal antibody which is specific for human placental alkaline phosphatase and does not cross-react at all with intestinal alkaline phosphatase was prepared, and a procedure for the determination of placental alkaline phosphatase activity in serum was developed involving this monoclonal antibody bound to a paper disk. The minimum amount of placental alkaline phosphatase detectable by this method is 0.0025 King-Armstrong unit. Good correlation with the heat-treatment method was obtained. Therefore this proposed method can be used as a routine clinical test for the determination of serum placental alkaline phosphatase.     

Confidence interval construction for relative toxicity endpoints such as LD50 or LD90 ratios

A common measure of the relative toxicity is the ratio of median lethal doses for responses estimated in two bioassays. Robertson and Preisler previously proposed a method for constructing a confidence interval for the ratio. The applicability of this technique in common experimental situations, especially those involving small samples, may be questionable because the sampling distribution of this ratio estimator may be highly skewed. To examine this possibility, we did a computer simulation experiment to evaluate the coverage properties of the Robertson and Preisler method. The simulation showed that the method provided confidence intervals that performed at the nominal confidence level for the range of responses often observed in pesticide bioassays. Results of this study provide empirical support for the continued use this technique.     

Isolation of compound microsatellite loci in the herbaceous perennial Cirsium purpuratum (Maxim.) Matsum

The herbaceous perennial Cirsium purpuratum is a pioneer on the southeast side of Mount Fuji in Japan. For genetic analysis of reproduction in this species, we developed polymorphic compound microsatellite markers using an adaptor-ligated library method and a simpler method called the intercompound microsatellite method. The latter method was an effective method for developing compound simple sequence repeat markers. In total, 11 polymorphic, codominant microsatellite markers were developed and characterized for this species. These polymorphic markers had three to 20 alleles per locus, a range of observed heterozygosity from 0.25 to 0.90, and were considered effective for genetic analysis.     

Directed next generation sequencing for phylogenetics: An example using Decapoda (Crustacea)

We propose a method using next generation sequencing technology for phylogenetics. The method is PCR based, requires little training beyond basic lab skills and is both cost and time effective. With this method we generated data for and produced a phylogeny of Decapoda that demonstrates this method's potential, the quality of the data, and the ability for the method to fit or even replace current Sanger based methods of generating DNA data for phylogenetic reconstruction. Finally, we discuss advantages and current challenges of the directed next generation sequencing approach.     

Rapid high-performance liquid chromatographic method for Vitamin C determination in human milk versus an enzymatic method

Vitamin C is an antioxidant that can be considered a possible biomarker of oxidative stability in human milk. A high-performance liquid chromatographic method was developed and validated for determining the total Vitamin C (ascorbic acid and dehydroascorbic acid) and ascorbic acid levels in human milk. This method was then compared with an enzymatic method (a Colorimetric technique) for quantifying ascorbic acid levels. Repeatability and reproducibility were acceptable for all methods. However, the high-performance liquid chromatography (HPLC) technique provided more satisfactory results than the enzymatic method due to this last method detected 37% less ascorbic acid and does not determine the total Vitamin C because of the enzymatic method cannot reduce the dehydroascorbic acid (DHA) to ascorbic acid. Furthermore, the HPLC method has the added advantages that it requires less reagents and material, and is simpler and less time consuming than the enzymatic method. In conclusion, the drawbacks of this enzymatic method would justify its substitution for a HPLC method.     

Efficient Cooperative Restraint Training With Rhesus Macaques

It is sometimes necessary for nonhuman primates to be restrained during biomedical and psychosocial research. Such restraint is often accomplished using a “primate chair.” This article details a method for training adult rhesus macaques to cooperate with a chair restraint procedure using positive and negative reinforcement. Successful training was accomplished rapidly in approximately 14 training days. The success of this training technique suggests that this method represents a refinement to traditional techniques. Further, this method worked effectively for animals previously deemed unfit for traditional pole-and-collar training.