Purification,biochemical, and thermal properties of fibrinolytic enzyme secreted by Bacillus cereus SRM-001

The discovery of microbial fibrinolytic enzymes is essential to treat cardiovascular diseases. This study reports the discovery of a fibrinolytic enzyme secreted by Bacillus cereus SRM-001, a microorganism isolated from the soil of a chicken waste-dump yard. The B. cereus SRM-001 was cultured and the secreted fibrinolytic enzyme purified to show that it is a ～28 kDa protein. The purified enzyme was characterized for its kinetics, biochemical and thermal properties to show that it possesses properties similar to plasmin. A HPLC-MS/MS analysis of trypsin digested protein indicated that the fibrinolytic enzyme shared close sequence homology with serine proteases reported for other Bacillus sp. The results show that the B. cereus SRM-001 secreted enzyme is a ～28 kDa serine protease that possesses fibrinolytic potential.     

Design considerations for proteomic reference materials

In order to improve the repeatability, comparability, and accuracy of MS-based proteomic measurements, there has been considerable international effort to develop appropriate reference materials. Although the majority of reference materials are developed to support measurement quality of routine assays, the development of reference materials for a diverse and changing research field such as proteomics represents unique challenges. In order to define common measurement components and common features of typical proteomic samples, the metrology underpinning proteomics must be considered due to the diversity and changing nature of the field. Reference materials can then be designed around common aspects in order to produce reference materials with the broadest applicability. Reference materials are needed to support both qualitative and quantitative proteomic measurements, involving different design considerations. Consensus and validated statistical approaches to describe the confidence in qualitative measurement, such as protein identification, needs to be established. Common sources of measurement bias also need to be considered in proteomic reference material design.     

Traceability, reference systems and result comparability

The standardisation of measurements is of high priority in laboratory medicine, its purpose being to achieve closer comparability of results obtained using routine measurement procedures. At present, there is international cooperation in developing reference measurement systems (reference methods, reference materials, and reference laboratory networks) for analytes of clinical significance. These reference systems will reduce, wherever possible, measurement uncertainty and promote the comparability of results. The implementation of measurement traceability through the reference system provides one of the most important tools that supports the standardisation process in laboratory medicine. It aims to achieve result comparability regardless of the measurement procedure (test kit) and the clinical laboratory where analyses are carried out. The aim of this review is to discuss some concepts related to the achievement of standardisation by the implementation of a metrologically-correct measurement system and to provide some examples that illustrate the complexity of this approach and the impact of these activities on patient care.     

Photon activation analysis of several kinds of marine invertebrates for trace elements

The concentrations of trace elements in several mollusk, arthropod, echinoderm, and tunicata species were determined by photon activation analysis. The samples were freeze-dried, pulverized by a mill, and fractionated through a 200-mesh sieve. Three standard materials were used for comparative standards: NIST SRM-1566a Oyster Tissue, NIST SRM-1577b Bovine Liver, and NRCC TORT-1 Lobster. The samples and comparator standards were sealed in silica tubes and irradiated for 3 h by 30-MeV bremsstrahlung from a linear accelerator at Tohoku University. By measuring gamma-ray spectra, the concentrations of 16 elements (As, Br, Ca, Cr, Cu, Fe, I, Mg, Mn, Mo, Na, Ni, Pb, Rb, Sr, and Zn) were determined.     

Seeds consumed by waterfowl in winter: a review of methods and a new web-based photographic atlas for seed identification

Diet quantification has long been and still is one of the first steps undertaken when studying the basic ecology of a species. We conducted a literature review of the techniques used to study and analyse diet among waterfowl. Even though the identification of food items is the most challenging task performed when studying granivorous waterfowl diet, there is a general lack of reference collections to support the taxonomic determination of food resources. We present here a collection of indexed digital pictures of seeds from the reference collections of several French programs of research into waterfowl diet. Photographs are ranked by seed size or by taxonomic key to ease seed identification. This collection is freely accessible at the website , and it can help wildlife researchers studying the diets of granivorous species to determine food items. A complete bibliography is also provided, including recent papers in archeobotany and useful websites.     

Genetic reference materials and their application to haematology

Genetic investigations are becoming increasingly useful and widespread in many areas of human health. However, there is a worldwide lack of certified reference materials for use in genetic testing, meaning that tests are being run without well validated controls and new assays are more difficult to develop and validate. We have responded to this challenge by starting a programme of developing genetic reference materials (GRMs) for international accreditation and worldwide distribution. Our approach has been to make materials for disorders where testing is commonplace and genotyping errors have been demonstrated. To ensure a continuing supply of DNA, cell lines are established from consenting, phenotypically well-characterised patients and are then grown up in bulk for genomic DNA extraction to yield up to 100 milligrams of DNA. In most cases the DNA is then formulated, freeze-dried and sealed in glass ampoules to ensure greater stability over time and obviate the need for chilled transportation. In this paper we explore the options and routes available to the production of DNA reference materials and describe the establishment of the first internationally recognised reference materials for human genomic DNA, with particular reference to some genetic tests carried out frequently within haematological and cardiovascular laboratories.     

Analysis of mutagenic activity of airborne particulate matter, standard reference materials and reference compounds using base pair-specific Salmonella typhimurium tester strains

The mutagenicity profiles of organic extracts of airborne dust samples from Mannheim, Germany, and two standard reference materials (SRM) as well as eight compounds with different chemical properties were investigated using tester strains Salmonella typhimurium TA700x (Ames II Assay). Each strain of this series carries a unique missense mutation in the histidine operon and is reverted by only one specific base substitution out of six possible changes. Mutation patterns of eight compounds with different modes of genotoxic action reveal significant differences. Samples of airborne particulate matter (APM) from an industrialized town in Germany (Mannheim) were collected for five consecutive days once a month for 1 year using an automatic high-volume air sampler. Samples taken from Monday to Friday were Soxhlet-extracted and prepared according to standard methods. Although the threshold limit for the least active strains is not triggered by all samples, it can be concluded that mutation patterns of the samples do not vary between different seasons. Standard reference materials (SRMs) were prepared and tested using the same methods. SRMs and APM samples from Mannheim reveal similar mutagenicity profiles in TA700x strains. The comparison of the mutagenicity profiles of air dust extracts from Mannheim and the SRMs, respectively, with reference compounds investigated so far shows some similarities although the patterns do not fit perfectly. Mutagenicity profiles of TA700x-activity of nitro-aromatic compounds published so far are similar to those of APM collected in Mannheim, Germany, as well as to standard reference materials 1648 and 1649.     

WHO technical workshop on stability of reference materials for biological medicines and in vitro diagnostics, Geneva, Switzerland, 28–29 November 2005

In November 2005, the World Health Organization convened an informal technical workshop on the stability of reference materials for biological medicines and in vitro diagnostics. The meeting was attended by experts from WHO collaborating centres in the area of biological standardization, national control laboratories, industries and other relevant organizations. The consultation group discussed current practices and approaches to predicting and monitoring the stability of biological reference materials. The group agreed to the need for establishing a working group (i) to continue dialogue on potential issues encompassing the principles, strategies and practicality for assuring the stability of WHO international reference standards for biological medicines and in vitro diagnostics and (ii) to develop more detailed guidance for assessment of the stability of WHO international biological reference materials.     

Requirements for reference (calibration) laboratories in laboratory medicine

In addition to reference measurement procedures and reference materials, reference or calibration laboratories play an integral role in the implementation of measurement traceability in routine laboratories. They provide results of measurements using higher-order methods, e.g. isotope dilution mass spectrometry and may assign values to materials to be used for external quality assessment programs and to secondary reference materials. The requirements for listing of laboratories that provide reference measurement services include a statement of the metrological level or principle of measurement, accreditation as a calibration laboratory according to ISO 15195 and the participation in a proficiency testing system (regular inter-laboratory comparisons) for reference laboratories. Ring trials are currently conducted for thirty well-defined measurands and the results are made available to all laboratories. Through the use of reference laboratory services that are listed by the Joint Committee for Traceability in Laboratory Medicine there is the opportunity to further promote traceability and standardisation of laboratory measurements.     

IAEA internet database of natural matrix reference materials

The International Atomic Energy Agency maintains a database on internationally available certified reference materials of natural origin. The database was updated in 1998 and prepared for an Internet implementation. A user-friendly structure was created, providing two main pathways for browsing, either according to the matrix classification or the producer’s name. The database presently contains over 20,000 values for 480 measurands and 1085 reference materials from 43 different producers. Most of the materials entered contain values for trace and minor elements (66%).     

Spatial variability in macroinvertebrate assemblages: comparing regional and multivariate approaches for classifying reference sites in South Africa

Spatial variability in macroinvertebrate assemblages was examined with the aim of evaluating the utility of regional classification systems in aquatic bioassessment. Sampling was undertaken at reference sites in the Western Cape and Mpumalanga, South Africa, using the rapid bioassessment method SASS4 (South African Scoring System Version 4). Multivariate analysis of macroinvertebrate assemblages showed that assemblages varied regionally with differences most apparent in upland areas, i.e. mountain streams and foothill-cobble beds, with lowland areas less regionally distinct. Within regions, longitudinal zonation into upland and lowland areas was important, with sites grouping on the basis of broad geomorphological zones or subregions. Of the upland sites, differentiation into mountain streams and foothill-cobble beds was not evident, although overall variability of assemblages within upland areas, in particular the Western Cape, was very high. In general, a priori regional classification of sites using the hierarchical spatial framework developed in South Africa provided a useful framework for preliminary classification of reference sites. Groups of sites based on a posteriori analysis of macroinvertebrate data, however, provided a more robust classification than any regional classification. Spatial classifications therefore offer geographic partitions within which to expect somewhat similar conditions, and regional reference sites selected within the context of the spatial framework are likely to be more representative of specific river types than those selected without using the framework. Classification of sites needs to be an iterative process that allows for subjective a priori regional classifications to be modified on the basis of independent, objective a posteriori classification of biological assemblages.     

Establishing reference conditions for European streams

The European Water Framework Directive stipulates that Member States have to assess the ecological status of a water body by comparing the present to the expected reference condition. In the AQEM project participating countries used the criteria from the Water Framework Directive to select reference sites for each stream type. The purpose of this study was to evaluate the suitability of these criteria and to validate the reference conditions chosen, by comparing the classification of sites before analysis with the final assessment of the sites using the assessment system which was developed within the AQEM project. Our study showed that not all criteria for reference conditions could be met for all stream types. This implies that `true' reference sites could not be selected for all stream types within the AQEM project. In our study, the differences between ecoregions, countries, and stream types were interpreted in terms of human impact. Validation of reference conditions showed differences between classification of sites as reference sites based on criteria to be used in the field and directly interpreting environmental or biological data and the result of the final assessment system. In some countries it was not possible to select reference conditions, because most of the a priori criteria were met. For example, reference conditions for Dutch stream types were established using historical data and predictions based on data from other geographical regions. Using data from adjacent countries appeared to be a feasible method for establishing reference conditions. Reference sites taken from other geographical areas represented a higher ecological quality than the Dutch sites of good ecological quality. However, metric results showed a large overlap between good and high ecological status. Historical information, on the other hand, was not found to be useful in metric calculations because of the confounding differences in sampling methods. One strong advantage of using historical information is that rare species that are locally extinct but still occur in the same stream type in other geographical areas can be added to the expected reference conditions. When and which methods can be used for establishing reference conditions is illustrated in a decision tree.     

桉蝙蛾幼虫实时荧光定量PCR内参基因的筛选

本研究通过筛选桉蝙蛾Endoclita signifer Walker幼虫不同龄期与不同体节中稳定表达的内参基因,为桉蝙蛾基因表达研究提供参考。通过实时荧光定量PCR技术测定5个候选内参基因（ACTIN、GAPDH、TUB、RIB、EF）在桉蝙蛾幼虫不同龄期（3龄、5龄、9龄、12龄）与不同体节（头部、胸部、腹部）及全样品（由所有样品组成）处理中的表达量,后利用GeNorm、NormFinder、BestKeeper对5个候选基因的稳定性进行评估,最后由RefFinder综合分析结果,选出最佳的内参基因。基于4种分析方法的评估可知,桉蝙蛾5龄和9龄幼虫的不同体节、不同龄期幼虫的头部和胸部中内参基因的最佳数目为2,而3龄和12龄幼虫的不同体节、不同龄期幼虫的腹部及全样本中内参基因的最佳数目为3。3龄、5龄、9龄和12龄幼虫的不同体节中可分别选择ACTIN+RIB+GAPDH、EF+RIB、GAPDH+EF和ACTIN+RIB+GAPDH作为最稳定的内参基因组合;EF+RIB、RIB+GAPDH和EF+GAPDH+ACTIN可分别作为不同龄期幼虫头部、胸部和腹部的最佳内参基因组合;综合考虑桉蝙蛾幼虫不同龄期与不同体节的影响时,可选择RIB、ACTIN和GAPDH作为内参基因。     

RNA-Seq is not required to determine stable reference genes for qPCR normalization

Assessment of differential gene expression by qPCR is heavily influenced by the choice of reference genes. Although numerous statistical approaches have been proposed to determine the best reference genes, they can give rise to conflicting results depending on experimental conditions. Hence, recent studies propose the use of RNA-Seq to identify stable genes followed by the application of different statistical approaches to determine the best set of reference genes for qPCR data normalization. In this study, however, we demonstrate that the statistical approach to determine the best reference genes from commonly used conventional candidates is more important than the preselection of ‘stable’ candidates from RNA-Seq data. Using a qPCR data normalization workflow that we have previously established; we show that qPCR data normalization using conventional reference genes render the same results as stable reference genes selected from RNA-Seq data. We validated these observations in two distinct cross-sectional experimental conditions involving human iPSC derived microglial cells and mouse sciatic nerves. These results taken together show that given a robust statistical approach for reference gene selection, stable genes selected from RNA-Seq data do not offer any significant advantage over commonly used reference genes for normalizing qPCR assays.     

Cry1Ah蛋白标准物质候选物的制备与纯化（英文）

随着转基因技术的迅猛发展,转基因产品的安全性受到了广泛关注。转基因检测用有证标准物质在确保转基因产品定性、定量检测结果的可比性和可追溯性方面发挥着重要作用。但转基因蛋白质标准物质的开发相对缓慢,其中一个难点是制备高纯度的转基因蛋白质候选物。苏云金芽胞杆菌Bacillus thuringiensis cry1Ah1基因因其对亚洲玉米螟等鳞翅目害虫有很好的杀虫活性,已用于转基因抗虫作物的研制,并获得具有较好抗虫性状的转基因株系。为了研发Cry1Ah蛋白有证标准物质,亟需建立其制备及纯化体系。文中优化了利用Bt表达系统制备Cry1Ah蛋白的体系,利用离子交换色谱法和排阻色谱法逐级纯化的方法,获得了高纯度的Cry1Ah蛋白(排阻色谱纯度:99.6%)。生物活性测定结果表明,纯化的Cry1Ah蛋白与原毒素对小菜蛾Plutella xylostella的杀虫活性没有显著差异。最后使用Edman降解法和质谱法确定了Cry1Ah蛋白活化后的氨基酸序列。综上所述,获得的Cry1Ah纯蛋白可用于蛋白质标准物质的研制。     

Cry1Ah蛋白标准物质候选物的制备与纯化

随着转基因技术的迅猛发展,转基因产品的安全性受到了广泛关注。转基因检测用有证标准物质在确保转基因产品定性、定量检测结果的可比性和可追溯性方面发挥着重要作用。但转基因蛋白质标准物质的开发相对缓慢,其中一个难点是制备高纯度的转基因蛋白质候选物。苏云金芽胞杆菌Bacillus thuringiensis cry1Ah1基因因其对亚洲玉米螟等鳞翅目害虫有很好的杀虫活性,已用于转基因抗虫作物的研制,并获得具有较好抗虫性状的转基因株系。为了研发Cry1Ah蛋白有证标准物质,亟需建立其制备及纯化体系。文中优化了利用Bt表达系统制备Cry1Ah蛋白的体系,利用离子交换色谱法和排阻色谱法逐级纯化的方法,获得了高纯度的Cry1Ah蛋白 (排阻色谱纯度：99.6%)。生物活性测定结果表明,纯化的Cry1Ah蛋白与原毒素对小菜蛾Plutella xylostella的杀虫活性没有显著差异。最后使用Edman降解法和质谱法确定了Cry1Ah蛋白活化后的氨基酸序列。综上所述,获得的Cry1Ah纯蛋白可用于蛋白质标准物质的研制。     

The application of preirradiation combustion and neutron activation analysis technique for the determination of iodine in food and environmental reference materials

The pre-irradiation combustion (PC) of samples to liberate iodine, followed by trapping the iodine on charcoal and quantifying the element by neutron activation analysis (NAA), has been used at the National Institute of Standards and Technology for the determination of iodine in biological materials. The applicability of this technique to numerous environmental and dietary matrices is illustrated by analysis of a range of certified reference materials (CRMs) and a powdered grass material that was prepared as an in-house reference material (RM). Because of the combustion step involved, samples with low or no fat content (e.g., cereal products, selected botanical specimens, and nonfat milk powder) and inorganic materials (e.g., coal fly ash and dried sediments) are more suited for analysis by this method. In general, the results for several types of samples obtained by this method agreed with those obtained by a second radiochemical (R) NAA, as well as by a third method using inductively coupled plasma mass spectrometry (ICP-MS). PC-NAA is a useful technique for determining iodine in biological and environmental samples, especially for verification of iodine results obtained from other methods.     

Effects of feeding endophyte-infected tall fescue seed on the reproductive performance in male and female CD-1 mice by combination crosses

The present study was undertaken to assess the effects of endophyte-infected Acremonium coenophialum tall fescue (KY 31) seed (80% infected) on the reproductive performance of male and female CD-1 mice by combination crosses. Forty-eight male and 48 female 30-d-old mice were randomly allocated to two diet treatments. Twenty-four males and 24 females were fed Diet 1, consisting of 50% non-infected fescue and 50% mouse chow; the remaining animals were fed Diet 2, containing 50% infected fescue. At the end of 50 d, the animals were paired in groups (n = 12) as follows: 1) Diet 1 males and females; 2) Diet 2 males and Diet 1 females; 3) Diet 1 males and Diet 2 females; and 4) Diet 2 males and females. The pairs were maintained on the diets of their female partners and were allowed to cohabitate for 96 h. After this period the males were removed and the females were carried to term. Pregnancy rates among the four treatment groups were 100, 83.3, 75.0 and 54.5%, respectively. Similarly, the average number of pups born per litter among the four groups were 11.5, 8.0, 9.7, and 7.5, respectively. Also, the weights (grams) of pups born per litter were 17.5, 12.6, 12.4, and 9.8, respectively. The results point out that the reproductive capacity of both male and female mice are affected (P < 0.05) by the ingestion of endophyte-infected fescue seeds. Such negative effects were more severe in the female than the male (P < 0.05).