A novel multiplex PCR-RFLP method for simultaneous detection of the MTHFR 677 C > T, eNOS +894 G > T and - eNOS -786 T > C variants among Malaysian Malays

Background Hyperhomocysteinemia as a consequence of the MTHFR 677 C > T variant is associated with cardiovascular disease and stroke. Another factor that can potentially contribute to these disorders is a depleted nitric oxide level, which can be due to the presence of eNOS +894 G > T and eNOS −786 T > C variants that make an individual more susceptible to endothelial dysfunction. A number of genotyping methods have been developed to investigate these variants. However, simultaneous detection methods using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis are still lacking. In this study, a novel multiplex PCR-RFLP method for the simultaneous detection of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants was developed. A total of 114 healthy Malay subjects were recruited. The MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants were genotyped using the novel multiplex PCR-RFLP and confirmed by DNA sequencing as well as snpBLAST. Allele frequencies of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C were calculated using the Hardy Weinberg equation. Methods The 114 healthy volunteers were recruited for this study, and their DNA was extracted. Primer pair was designed using Primer 3 Software version 0.4.0 and validated against the BLAST database. The primer specificity, functionality and annealing temperature were tested using uniplex PCR methods that were later combined into a single multiplex PCR. Restriction Fragment Length Polymorphism (RFLP) was performed in three separate tubes followed by agarose gel electrophoresis. PCR product residual was purified and sent for DNA sequencing. Results The allele frequencies for MTHFR 677 C > T were 0.89 (C allele) and 0.11 (T allele); for eNOS +894 G > T, the allele frequencies were 0.58 (G allele) and 0.43 (T allele); and for eNOS −786 T > C, the allele frequencies were 0.87 (T allele) and 0.13 (C allele). Conclusions Our PCR-RFLP method is a simple, cost-effective and time-saving method. It can be used to successfully genotype subjects for the MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants simultaneously with 100% concordance from DNA sequencing data. This method can be routinely used for rapid investigation of the MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants.

Information organisation behaviours within everyday life : an exploration using the diary method

This paper presents the results from a study of information behaviours (IB), with specific focus on information- organisation-related behaviours conducted as part of a larger daily diary study with 40 participants. The findings indicate that organisation of information in everyday life is problematic due to various factors, specifically temporal and spatial ones, i.e., the passage of time, along with the difference in the physical environment between when the information was organised and when the information is retrieved. These have implications for information organisation schemas, and point toward the need for a context-sensitive model of information organisation that reflects the relation between information objects, information representation, and information users.

An ontology-based method for sparsity problem in tag recommendation

Tags or personal metadata for annotating web resources have been widely adopted in Web 2.0 sites. However, as tags are freely chosen by users, the vocabularies are diverse, ambiguous and sometimes only meaningful to individuals. Tag recommenders may assist users during tagging process. Its objective is to suggest relevant tags to use as well as to help consolidating vocabulary in the systems. In this paper we discuss our approach for providing personalized tag recommendation by making use of existing domain ontology generated from folksonomy. Specifically we evaluated the approach in sparse situation. The evaluation shows that the proposed ontology-based method has improved the accuracy of tag recommendation in this situation.

A new method to detect loss of heterozygosity using cohort heterozygosity comparisons

Background Loss of heterozygosity (LOH) is an important marker for one of the 'two-hits' required for tumor suppressor gene inactivation. Traditional methods for mapping LOH regions require the comparison of both tumor and patient-matched normal DNA samples. However, for many archival samples, patient-matched normal DNA is not available leading to the under-utilization of this important resource in LOH studies. Here we describe a new method for LOH analysis that relies on the genome-wide comparison of heterozygosity of single nucleotide polymorphisms (SNPs) between cohorts of cases and un-matched healthy control samples. Regions of LOH are defined by consistent decreases in heterozygosity across a genetic region in the case cohort compared to the control cohort. Methods DNA was collected from 20 Follicular Lymphoma (FL) tumor samples, 20 Diffuse Large B-cell Lymphoma (DLBCL) tumor samples, neoplastic B-cells of 10 B-cell Chronic Lymphocytic Leukemia (B-CLL) patients and Buccal cell samples matched to 4 of these B-CLL patients. The cohort heterozygosity comparison method was developed and validated using LOH derived in a small cohort of B-CLL by traditional comparisons of tumor and normal DNA samples, and compared to the only alternative method for LOH analysis without patient matched controls. LOH candidate regions were then generated for enlarged cohorts of B-CLL, FL and DLBCL samples using our cohort heterozygosity comparison method in order to evaluate potential LOH candidate regions in these non-Hodgkin's lymphoma tumor subtypes. Results Using a small cohort of B-CLL samples with patient-matched normal DNA we have validated the utility of this method and shown that it displays more accuracy and sensitivity in detecting LOH candidate regions compared to the only alternative method, the Hidden Markov Model (HMM) method. Subsequently, using B-CLL, FL and DLBCL tumor samples we have utilised cohort heterozygosity comparisons to localise LOH candidate regions in these subtypes of non-Hodgkin's lymphoma. Detected LOH regions included both previously described regions of LOH as well as novel genomic candidate regions. Conclusions We have proven the efficacy of the use of cohort heterozygosity comparisons for genome-wide mapping of LOH and shown it to be in many ways superior to the HMM method. Additionally, the use of this method to analyse SNP microarray data from 3 common forms of non-Hodgkin's lymphoma yielded interesting tumor suppressor gene candidates, including the ETV3 gene that was highlighted in both B-CLL and FL.

A combined method for mitigating sparsity problem in tag recommendation

Tag recommendation is a specific recommendation task for recommending metadata (tag) for a web resource (item) during user annotation process. In this context, sparsity problem refers to situation where tags need to be produced for items with few annotations or for user who tags few items. Most of the state of the art approaches in tag recommendation are rarely evaluated or perform poorly under this situation. This paper presents a combined method for mitigating sparsity problem in tag recommendation by mainly expanding and ranking candidate tags based on similar items’ tags and existing tag ontology. We evaluated the approach on two public social bookmarking datasets. The experiment results show better accuracy for recommendation in sparsity situation over several state of the art methods.

Improved ultra-performance liquid chromatography with electrospray ionization quadrupole-time-of-flight high-definition mass spectrometry method for the rapid analysis of the chemical constituents of a typical medical formula: Liuwei Dihuang Wan

Liuwei Dihuang Wan (LWD), a classic Chinese medicinal formulae, has been used to improve or restore declined functions related to aging and geriatric diseases, such as impaired mobility, vision, hearing, cognition and memory. It has attracted increasingly much attention as one of the most popular and valuable herbal medicines. However, the systematic analysis of the chemical constituents of LDW is difficult and thus has not been well established. In this paper, a rapid, sensitive and reliable ultra-performance liquid chromatography with electrospray ionization quadrupole time-of-flight high-definition mass spectrometry (UPLC-ESI-Q-TOF-MS) method with automated MetaboLynx analysis in positive and negative ion mode was established to characterize the chemical constituents of LDW. The analysis was performed on a Waters UPLCTM HSS T3 using a gradient elution system. MS/MS fragmentation behavior was proposed for aiding the structural identification of the components. Under the optimized conditions, a total of 50 peaks were tentatively characterized by comparing the retention time and MS data. It is concluded that a rapid and robust platform based on UPLC-ESI-Q-TOF-MS has been successfully developed for globally identifying multiple-constituents of traditional Chinese medicine prescriptions. This is the first report on systematic analysis of the chemical constituents of LDW. This article is protected by copyright. All rights reserved.

Elastic buckling analysis of ideal thin-walled structures under combined loading using a finite strip method

The details of an application of the finite strip method to the elastic buckling analysis of thin-walled structures with various boundary conditions and subjected to single or combined loadings of longitudinal compression, transverse compression, bending and shear are presented. The presence of shear loading is accounted for by modifying the displacement functions which are commonly used in cases when shear is absent. A program based on the finite strip method was used to obtain the elastic buckling stress, buckling plot and buckling mode of thin-walled structures and some of these results are presented.

A simple test method for profiled steel cladding systems under wind uplift

A new simple test method using small scale models has been developed for testing profiled steel cladding systems under wind uplift/suction forces. This simple method should replace the large scale test method using two-span claddings used at present. It can be used for roof or wall cladding systems fastened with screw fasteners at crests or valleys.

Identification and simultaneous analysis of harmane, harmol, isovitexin and vitexin in Passiflora incarnata extracts with a novel HLPC method

A high performance liquid chromatographic method for the simultaneous analysis of two flavonoids (iso-vitexin and vitexin), and three indole alkaloids (harmane, harmine, and harmol) was developed. This method was then utilised to quantitate levels of these five constituents in methanolic extracts of Australian Passiflora incarnata. HPLC analysis was performed using a Waters™ Novapak C18 (150 × 4 mm, 4 μm) column, with a gradient solvent system of methanol-water-acetic acid. Detection was achieved by PDA UV (254 nm) and fluorescence (excitation 254 nm, emission 414 nm), utilising the external standard method to obtain quantification.

Simultaneous determination of aldrin, dieldrin, endrin, heptachlor, and p,p'-DDT in medicinal plant extracts using a novel high performance liquid chromatography method

A high performance liquid chromatographic method for the simultaneous determination of five organochlorine pesticides (aldrin, p,p’-DDT, dieldrin, endrin, and heptachlor) was developed. The method was used to determine the levels of these pesticides in medicinal plant samples. Analysis was carried out using a Merck LiChrospher 100 RP C18 (5 μm) column with a gradient solvent system of acetonitrile-water and PDA UV detection (224 nm). Quantification was carried out by the external standard method. The limit of detection for the utilized method was below the local legal limits (ANZFA) for similar plant materials for all 5 pesticides excepting endrin. Medicinal plant extracts were further analyzed by conventional GC-ECD and GC-NPD means using SPE and GPC cleanup as required.

An improved method for the assay of platelet pyruvate dehydrogenase

An improved method for the assay of human platelet pyruvate dehydrogenase is described. By generating the substrate [1-14C]pyruvate in situ from [1-14C]lactate plus l-lactate dehydrogenase, the rate of spontaneous decarboxylation is dramatically reduced, allowing far greater sensitivity in the assay of low activities of pyruvate dehydrogenase. In addition, no special precautions are required for the storage and use of [1-14C]lactate, in contrast to those for [1-14C] pyruvate. These factors allow a 5–10-fold increase in sensitivity compared with current methods. The pyruvate dehydrogenase activity of normal subjects as determined by the [1-14C]lactate system was 215 ± 55 pmol · min−1 · mg−1 protein (n = 18). The advantages of this assay system are discussed.

High resolution melt analysis : a novel method for studying the genetic relatedness of Pseudomonas aeruginosa isolates from clinical and environmental sources

A novel method was developed for studying the genetic relatedness of Pseudomonas aeruginosa isolates from clinical and environmental sources. This bacterium is ubiquitous in the natural environment and is an important pathogen known to infect Cystic Fibrosis (CF) patients. The transmission route of strains has not yet been defined; current theories include acquisition from an environmental source or through patient-to-patient spread. A highly discriminatory, bioinformatics based, DNA typing method was developed to investigate the relatedness of clinical and environmental isolates. This study found a similarity between the environmental and several CF clonal strains and also highlighted occurrence of environmental P. aeruginosa strains in CF infections.

The heterogeneous cluster ensemble method using hubness for clustering text documents

We propose a cluster ensemble method to map the corpus documents into the semantic space embedded in Wikipedia and group them using multiple types of feature space. A heterogeneous cluster ensemble is constructed with multiple types of relations i.e. document-term, document-concept and document-category. A final clustering solution is obtained by exploiting associations between document pairs and hubness of the documents. Empirical analysis with various real data sets reveals that the proposed meth-od outperforms state-of-the-art text clustering approaches.

A concept-based retrieval method for entity-oriented search

Entity-oriented retrieval aims to return a list of relevant entities rather than documents to provide exact answers for user queries. The nature of entity-oriented retrieval requires identifying the semantic intent of user queries, i.e., understanding the semantic role of query terms and determining the semantic categories which indicate the class of target entities. Existing methods are not able to exploit the semantic intent by capturing the semantic relationship between terms in a query and in a document that contains entity related information. To improve the understanding of the semantic intent of user queries, we propose concept-based retrieval method that not only automatically identifies the semantic intent of user queries, i.e., Intent Type and Intent Modifier but introduces concepts represented by Wikipedia articles to user queries. We evaluate our proposed method on entity profile documents annotated by concepts from Wikipedia category and list structure. Empirical analysis reveals that the proposed method outperforms several state-of-the-art approaches.

An innovative method to obtain porous PLLA scaffolds with highly spherical and interconnected pores

Scaffolding is an essential issue in tissue engineering and scaffolds should answer certain essential criteria: biocompatibility, high porosity, and important pore interconnectivity to facilitate cell migration and fluid diffusion. In this work, a modified solvent castingparticulate leaching out method is presented to produce scaffolds with spherical and interconnected pores. Sugar particles (200–300 lm and 300–500 lm) were poured through a horizontal Meker burner flame and collected below the flame. While crossing the high temperature zone, the particles melted and adopted a spherical shape. Spherical particles were compressed in plastic mold. Then, poly-L-lactic acid solution was cast in the sugar assembly. After solvent evaporation, the sugar was removed by immersing the structure into distilled water for 3 days. The obtained scaffolds presented highly spherical interconnected pores, with interconnection pathways from 10 to 100 lm. Pore interconnection was obtained without any additional step. Compression tests were carried out to evaluate the scaffold mechanical performances. Moreover, rabbit bone marrow mesenchymal stem cells were found to adhere and to proliferate in vitro in the scaffold over 21 days. This technique produced scaffold with highly spherical and interconnected pores without the use of additional organic solvents to leach out the porogen.