Fabrication of carbon films with ∼500 nm holes for cryo-EM with a direct detector device

Single particle electron cryomicroscopy (cryo-EM) is often performed using EM grids coated with a perforated or holey layer of amorphous carbon. Regular arrays of holes enable efficient cryo-EM data collection and several methods for the production of micropatterned holey-carbon film coated grids have been described. However, a new generation of direct detector device (DDD) electron microscope cameras can benefit from hole diameters that are smaller than currently available. Here we extend a previously proposed method involving soft lithography with a poly(dimethylsiloxane) (PDMS) stamp for the production of holey-carbon film coated EM grids. By incorporating electron-beam (e-beam) lithography and modifying the procedure, we are able to produce low-cost high-quality holey-carbon film coated EM grids with ∼500 nm holes spaced 4 μm apart centre-to-centre. We demonstrate that these grids can be used for cryo-EM. Furthermore, we show that by applying image shifts to obtain movies of the carbon regions beside the holes after imaging the holes, the contrast transfer function (CTF) parameters needed for calculation of high-resolution cryo-EM maps with a DDD can be obtained efficiently.     

The collection and database of Birds of Angola hosted at IICT (Instituto de Investiga??o Científica Tropical), Lisboa,Portugal

The bird collection of the Instituto de Investigação Cientítica Tropical (Lisbon, Portugal) holds 5598 preserved specimens (skins), mainly from Angola, Mozambique, Guinea-Bissau, São Tomé and Principe, and Cape Verde. The subset collection from Angola includes 1560 specimens, which were taxonomically revised and georeferenced for the publication of this data paper. The collection contains a total of 522 taxa, including 161 species and 361 subspecies. Two species are classified by the IUCN Red List as Endangered - the wattled crane (Grus carunculata) and the Gabela bush-shrike (Laniarius amboimensis) - and two are classified as vulnerable - African penguin (Spheniscus demersus) and the white-headed vulture (Trigonoceps occipitalis). The temporal span of the database ranges between 1943 and 1979, but 32% are from years 1958–1959, and 25% from years 1968–1969. The spatial coverage of the collection is uneven, with 2/3 of the records representing only four of the eighteen provinces of the country, namely Huíla, Moxico, Namibe and Cuanza Sul. It adds, however, valuable information for the Huíla area of the Angolan Scarp, which is probably a biodiversity hotspot of global conservation priority. Furthermore, this georeferenced database adds invaluable bird information to the GBIF network, for one of the countries with highest but less known biodiversity in Africa.     

Testing compatibility between molecular and morphological techniques for arthropod systematics: a minimally destructive DNA extraction method that preserves morphological integrity, and the effect of lactic acid on DNA quality

Three practical aspects related to the preservation and destruction of DNA and/or morphological characters of spiders were examined: potential morphological damage during non-destructive DNA extraction was assessed by counting trichobothria, a fragile sensorial feature found on spider legs; the effect on yield of non-destructive DNA extraction; and whether possible DNA degradation is caused by residues of lactic acid, which is used as a temporary mounting medium for the study of morphological structures in spiders and insects. Destructive extractions yielded higher amounts of DNA than non-destructive methods. However, non-destructive methods yielded usable amounts of DNA while leaving delicate trichobothria intact. Of the non-destructive extractions, a longer digestion period (36 h vs. 12) yielded higher amounts of DNA and did not damage trichobothria. Lactic acid did not induce short-term DNA degradation or inhibit PCR reactions, even at high concentrations. These results show compatibility between molecular and morphological requirements without compromising DNA quality or specimen integrity.     

中国兽类标本馆藏数量

我国兽类资源丰富,兽类物种共计12目59科254属686种。近40年来我国的兽类标本采集和馆藏数量快速增加。根据最新发表的兽类名录对19家博物馆（标本馆）兽类标本馆藏的调查结果表明,我国至少保藏兽类标本166 178号,馆藏数量排名前五位的单位分别是中国科学院昆明动物研究所、中国科学院动物研究所、四川省林业科学研究院、中国科学院西北高原生物研究所和西华师范大学,馆藏数量占比84.9%。在馆藏标本中,小型兽类占91.5%,而大中型兽类标本特别是鲸豚类很少,有待加大力度对这些类群标本进行收集与保藏。     

临床常规生化检验结果的影响因素分析

目的:探讨临床常规生化检验结果的影响因素,并提出相应的预防对策。方法:回顾性分析2012年2月~2013年2月我院检验科进行的生化常规检测10256例次的检查结果,采用美国BIORAD公司生化定值多项3水平质控品于检测前、中、后进行质控分析,对产生偏差的原因按实验前、实验中、实验后进行归类总结。结果:84例经复核出现超过10%以上偏差,占总分析例数的0.82%。实验前不良事件是产生实验结果偏差的主要因素,占总体产生偏差例数的78.57%,其中患者因素与标本处理不当构成了实验前干扰因素的主体,分别占实验前因素的39.39%和36.36%。不明原因溶血与标本采集方法不当也是实验前的实验干扰因素。实验中影响检测结果的因素主要为仪器故障、试剂过期和失效、定标品或定标曲线过期,分别占实验中因素的46.67%、33.33%、20%。实验后干扰因素均为审核不及时或不细致导致的检测报告不合格。结论:在进行临床常规生化检查的过程中,任何一个环节出现失误均有可能对检查结果造成影响,特别是患者因素、标本采集方法不当及处理不当,均应该引起医护人员的重视。     

处理植物分类学描述语言的国际标准——DELTA系统

DELTA系统是澳大利亚Dallwitz等人经过多年研究发出的一套用于分类学描述语言编码的国际标准。本文对该系统的主要功能,即自然语言描述、检索表、数据矩阵的自动生成、标本鉴定和信息检索以及分支分类运算和表征分类运算所需数据格式的转换等做了综述性介绍,以期促进该软件在我国广大植物分类学工作者中的推广与使用。     

Radial growth responses to gap creation in large,old Sequoiadendron giganteum

Questions: Do large, old Sequoiadondron giganteum trees respond to the creation of adjacent canopy gaps? Do other co‐occurring tree species and younger S. giganteum adjacent to gaps also respond? What are the likely factors affecting growth responses? Location: Mixed‐conifer forests of the southern Sierra Nevada, California, USA. Methods: We measured the growth response of large, old S. giganteum trees (mean DBH=164 cm; ages estimated >1000 yr) to gap creation by coring trees and comparing growth after gap creation to growth before gap creation. We also measured young Abies concolor, Pinus lambertiana, and young S. giganteum. Gap‐adjacent trees were compared with non‐adjacent reference trees. Tree rings were analysed for carbon isotope discrimination and for longer‐term growth trend correlations with climate. Results: Following gap creation gap‐adjacent old S. giganteum grew more than reference trees. Abies concolor trees also exhibited a growth response to gap creation. No response was detected for young S. giganteum or P. lambertiana, although detection power was lower for these groups. There was no difference in carbon isotope discrimination response to gap creation between gap‐adjacent and reference trees for old S. giganteum and radial growth was positively correlated with winter precipitation, but not growing season temperature. Conclusion: It is unclear what caused the growth release in old S. giganteum trees, although liberation of below‐ground resources following removal of competing vegetation appears to be a significant contributor. Sequoiadondron giganteum, the third‐longest lived and the largest of all species, remains sensitive to local environmental changes even after canopy emergence. Management activities that reduce vegetation surrounding individual specimen trees can be expected to result in increased vigor of even these very old and large trees.     

应用DNA条形码对植物标本的核查

DNA条形码主要目的是物种鉴定和新物种或隐存种的发现,而DNA条形码参考数据库是物种快速鉴定的重要基础。目前中国维管植物DNA条形码参考数据库正在建设之中,借助于公共数据库（NCBI）和初步建立的中国植物DNA条形码参考数据库,运用DNA条形码数据开展了植物标本鉴定的核查工作：（1）比较DNA序列信息与标本鉴定信息,从科、属、种级水平查找鉴定错误的标本;（2）基于有较好研究基础的DNA条形码参考数据库,开展未知标本的鉴定;（3）通过对标本核查的总结,提出DNA条形码参考数据库建设过程中的几点建议。     

Clinical analysis of vitamin B(6): determination of pyridoxal 5'-phosphate and 4-pyridoxic acid in human serum by reversed-phase high-performance liquid chromatography with chlorite postcolumn derivatization

A reversed-phase high-performance liquid chromatography (HPLC) method with fluorometric detection was developed for the routine determination of pyridoxal 5'-phosphate (PLP) and 4-pyridoxic acid (4-PA) in serum. Chlorite postcolumn derivatization was used to oxidize PLP to a more fluorescent carboxylic acid form. Sensitivity improved fourfold for PLP using chlorite postcolumn derivatization over traditional bisulfite postcolumn derivatization. The HPLC injection cycle was 15 min, facilitating a throughput of 60 patient samples (72 injections that included standards and quality control (QC) samples) in 18.5h. Method precision was evaluated using three serum QC pools with PLP and 4-PA concentrations of 11.5-34.8 nmol/L and 10.4-21.0 nmol/L, respectively. Within-run (n=7) repeatabilities were 0.6-1.2% for PLP and 0.9-1.8% for 4-PA. Run-to-run (n=23) reproducibilities were 3.6-6.7% for PLP and 3.7-5.6% for 4-PA. Relative detection (3sigma(0)) and quantitation (10sigma(0)) limits were 0.3 and 0.9 nmol/L, respectively, for both PLP and 4-PA using a 10-microl sample injection volume. Analytical recoveries ranged from 97 to 102%. Patient-matched serum and plasma specimens (n=25) were analyzed to evaluate specimen-type bias. Of the plasma types evaluated, heparinized plasma introduced the lowest relative bias for PLP (-5.3%) and minimal bias for 4-PA (-2.3%) compared with serum. Ethylenediaminetetraacetic acid (EDTA) plasma showed the lowest bias for 4-PA (0.7%) but a relatively high bias for PLP (13.0%) due to a chromatographic interference. Human serum samples from a non-representative population subset (n=303) were commensurate with values published for other vitamin B(6) HPLC methods. These values gave geometric means of 42.4 nmol/L for PLP and 27.3 nmol/L for 4-PA. Medians for PLP and 4-PA were 40.1 and 21.8 nmol/L, respectively. The high sensitivity, precision, and throughput of this method, combined with its minimal serum specimen (150 microl) and sample injection (10 microl) volume requirements, make it well suited for routine clinical vitamin B(6) analysis.