种植Bt水稻后的土壤对赤子爱胜蚓生长发育及酶活性的影响

【背景】转基因作物种植的安全问题一直备受关注。关于Bt蛋白对地下非靶标生物影响的研究是转基因作物安全评价的重要内容。【方法】在转Bt基因水稻收割后的稻田里分别种植豌豆、紫云英和油菜作为后茬作物。分别于2013年1、3和6月3次采集不同后茬作物田中的土壤作为材料,于室内饲养赤子爱胜蚓,4周和7周后,测定蚯蚓的生长发育指标、存活率以及体内酶活性的变化情况。此外,还测定了不同深度土壤中Bt蛋白的含量以及用Bt蛋白直接饲喂的赤子爱胜蚓的存活率。【结果】与种植过非转基因水稻MH63的土壤相比,分别种植过含cry2A和cry1C基因水稻后的土壤对赤子爱胜蚓的生长发育、存活率及体内酶活性无显著影响。1月份和3月份转cry2A基因水稻田以及1月份转cry1C基因水稻田采集的表层土样中的Bt蛋白含量显著高于地下10 cm和地下20 cm土壤中的含量,地下2层土样中的Cry2A蛋白含量之间无差异。3月份转cry1C基因水稻田以及6月份转cry2A和转cry1C基因水稻田的土壤中Bt蛋白的含量均不受土壤深度的影响。种植的后茬作物对土壤中的Bt蛋白无显著消解作用。室内模拟土壤最高Bt蛋白浓度的条件下,Cry2A蛋白处理的蚯蚓存活率为96.7%,Cry1C蛋白处理的蚯蚓存活率为95.0%,两者与对照相比无显著差异。【结论与意义】转cry2A和cry1C基因Bt水稻的种植对蚯蚓的生长发育和体内酶活性无显著影响。本研究为转基因水稻的安全评价提供了一定的依据。     

Searching for DNA in museum specimens: a comparison of sources in a mammal species

The number of genetic studies that use preserved specimens as sources of DNA has been steadily increasing during the last few years. Therefore, selecting the sources that are more likely to provide a suitable amount of DNA of enough quality to be amplified and at the minimum cost to the original specimen is an important step for future research. We have compared different types of tissue (hides vs. bones) from museum specimens of Iberian lynx and multiple alternative sources within each type (skin, footpad, footpad powder, claw, diaphysis, maxilloturbinal bone, mastoid process and canine) for DNA yield and probability of amplification of both mitochondrial and nuclear targets. Our results show that bone samples yield more and better DNA than hides, particularly from sources from skull, such as mastoid process and canines. However, claws offer an amplification success as high as bone sources, which makes them the preferred DNA source when no skeletal pieces have been preserved. Most importantly, these recommended sources can be sampled incurring minimal damage to the specimens while amplifying at a high success rate for both mitochondrial and microsatellite markers.     

Highly efficient multiplex PCR of noninvasive DNA does not require pre-amplification

Among the key issues determining success of a study employing molecular genetics tools in wildlife monitoring or research is a large enough set of highly informative genetic markers and a reliable, cost effective method for their analysis. While optimized commercial genotyping kits have been developed for humans and domestic animals, such protocols are rare in wildlife research. We developed a highly optimized multiplex PCR that genotypes 12 microsatellite loci and a sex determination locus in brown bear (Ursus arctos) faecal samples in a single multiplex PCR and a single sequencer run. We used this protocol to genotype 1053 faecal samples of bears from the Dinaric population, and obtained useful genotypes for 88% of the samples, a very high success rate. The new protocol outperformed the multiplex pre-amplification strategy used in a previous study of 473 faecal samples with a 78.4% success rate. On a subset of 182 samples we directly compared the performance of both approaches, and found no advantage of the multiplex pre-amplification. While pre-amplification protocols might still improve PCR success and reliability on a small fraction of low-quality samples, the higher costs and workload do not justify their use when analysing reasonably fresh non-invasive material. Moreover, the high number of multiplexed loci in the new protocol makes it comparable to commercially developed genotyping kits developed for domestic animals and humans.     

Evaluation of the usefulness of a new direct immunofluorescence assay (ScanVIT‐Legionella™) for monitoring hospital water systems contaminated with Legionella spp.

Aims: To compare the efficiency of the ScanVIT‐Legionella^? test (Vermicon, Munich, Germany) vs a conventional culture method for the quantification of Legionella spp. in hospital water samples in daily hospital practice. Methods and Results: The detection of Legionella spp. takes place on a cultivated filter brought into contact with dye‐marked gene probes. The results are analysed under fluorescence microscopy. Bacteria that light up green belong to the genus Legionella; those that light up both green and red belong to the species Legionella pneumophila. Our results showed that the ScanVIT test has a sensitivity of 90%; agreement between the two methods was 82%. In the 48 samples that tested positive with both methods, the Legionella concentration detected by the culture method was consistently higher. A statistically significant difference between the results obtained with the two test methods emerged at the Wilcoxon test (P < 0·001). Conclusion: The ScanVIT test may be recommended for investigating the presence of Legionella by qualitative testing. Significance and Impact of the Study: Given the simplicity of colony identification by fluorescence, the ScanVIT test can be used in laboratories where staffs are not experienced in identifying typical colonies of Legionella.     

Microbial signature lipid biomarker analysis – an approach that is still preferred,even amid various method modifications

The lipid composition of microbial communities can indicate their response to changes in the surrounding environment induced by anthropogenic practices, chemical contamination or climatic conditions. A considerable number of analytical techniques exist for the examination of microbial lipids. This article reviews a selection of methods available for environmental samples as applied for lipid extraction, fractionation, derivatization and quantification. The discussion focuses on the origin of the standard methods, the different modified versions developed for investigation of microbial lipids, as well as the advantages and limitations of each. Current modifications to standard methods show a number of improvements for each of the different steps associated with analysis. The advantages and disadvantages of lipid analysis compared to other popular techniques are clarified. Accordingly, the preferential utilization of signature lipid biomarker analysis in current research is considered. It is clear from recent literature that this technique stays relevant – mainly for the variety of microbial properties that can be determined in a single analysis.     

Floral development in Scutellaria pinnatifida (Lamiaceae): the ontogenetic basis for sepal reduction

The Scutellaria is a Labiatae genus (subfamily Scutellarioideae) with a highly specialised floral structure. The genus is characterised by a peculiar two‐lobed calyx with a projecting appendage, named the scutellum. Here, we present a detailed analysis of floral development, using epi‐illumination light microscopy, to clarify open questions about its floral organisation. Floral whorls appeared in an acropetal sequence, with a marked temporal overlap of petal and stamen appearance. Organ appearance in each whorl proceeded unidirectionally from the abaxial to the adaxial side. Significant developmental features included the formation of the scutellum, reduction of sepal lobes and formation of a three‐lobed nectary disc. Our study revealed that both loss of organ initiation and fusion of primordia are responsible for the reduction in sepal members in Scutellaria. The nectary structure was markedly different from most other studied Lamiaceae.     

A novel procedure for protein extraction from formalin-fixed paraffin-embedded tissues

Most of the archived pathological specimens in hospitals are kept as formalin-fixed paraffin-embedded tissues (FFPE) for long-term preservation. Up to now, these samples are only used for immunohistochemistry in a clinical routine as it is difficult to recover intact protein from these FFPE tissues. Here, we report a novel, short time-consuming and cost-effective method to extract full-length, non-degraded proteins from FFPE tissues. This procedure is combined with an effective and non-toxic deparaffinisation process and an extraction method based on antigen-retrieval, high concentration of SDS and high temperature. We have obtained enough intact protein to be detected by Western blotting analysis. This technique will allow utilising these stored FFPE tissues in several applications for protein analysis helping to advance the translational studies in cancer and other diseases.     

Exact tests for one sample correlated binary data

In this paper we developed exact tests for one sample correlated binary data whose cluster sizes are at most two. Although significant progress has been made in the development and implementation of the exact tests for uncorrelated data, exact tests for correlated data are rare. Lack of a tractable likelihood function has made it difficult to develop exact tests for correlated binary data. However, when cluster sizes of binary data are at most two, only three parameters are needed to characterize the problem. One parameter is fixed under the null hypothesis, while the other two parameters can be removed by both conditional and unconditional approaches, respectively, to construct exact tests. We compared the exact and asymptotic p-values in several cases. The proposed method is applied to real-life data.