Light flux density threshold at which protein denaturation is induced by synchrotron radiation circular dichroism beamlines

New high‐flux synchrotron radiation circular dichroism (SRCD) beamlines are providing important information for structural biology, but can potentially cause denaturation of the protein samples under investigation. This effect has been studied at the new CD1 dedicated SRCD beamline at ISA in Denmark, where radiation‐induced thermal damage effects were observed, depending not only on the radiation flux but also on the focal spot size of the light. Comparisons with similar studies at other SRCD facilities worldwide has lead to the estimation of a flux density threshold under which SRCD beamlines should be operated when samples are to be exposed to low‐wavelength vacuum ultraviolet radiation for extended periods of time.     

Circular dichroism beamline B23 at the Diamond Light Source

Synchrotron radiation circular dichroism (SRCD) is a well established technique in structural biology. The first UV‐VIS beamline, dedicated to circular dichroism, at Diamond Light Source Ltd, a third‐generation synchrotron facility in south Oxfordshire, UK, has recently become operational and it is now available for the user community. Herein the main characteristics of the B23 SRCD beamline, the ancillary facilities available for users, and some of the recent advances achieved are summarized.     

应用SRCD和FTIR分析超高压处理对蘑菇多酚氧化酶二级结构的影响

应用同步辐射紫外真空圆二色光谱(SRCD)、傅里叶变换红外光谱(FTIR)和荧光光谱研究了超高压(HHP)处理对蘑菇多酚氧化酶(PPO)二级结构和三级结构的影响。HHP处理使蘑菇PPO的α-螺旋含量明显减少,二级结构发生改变。通过SRCD光谱和FTIR光谱分析得出的未处理或HHP处理蘑菇PPO的二级结构含量均存在一定的差异,这种差异可能是由于测量温度、酶液浓度和分析方法等多种因素造成的。荧光光谱表明,HHP处理后,蘑菇PPO溶液荧光光谱的强度降低,最大发射峰发生了红移,表明HHP处理改变了蘑菇PPO分子的三级结构。     

基于微流控混合器的连续流动态同步辐射圆二色谱发展

基于微流控混合器,采用连续流探测方法,在北京同步辐射装置真空紫外光谱实验站发展了毫秒动态圆二色谱探测方法。石英微流控混合器采用深度离子刻蚀技术加工,通道深度44.5 μm。混合器采用蛇形通道实现溶液的快速混合。通过荧光倒置显微镜,在模拟真实实验条件的高粘度溶液中,观察蛇形通道内溶液混合的荧光图像,进行混合效率测试。500 μL·min-1流量下,目前可实现4.5～270 μs的时间尺度探测。利用微流控混合器进行动态探测,同步辐射紫外光必须聚焦,但由于聚焦透镜波长色散引起的焦点位移,导致圆二色谱发生畸变。通过精确测试不同波长对应焦点的相对位置,然后在圆二色谱扫描中实现波长和焦点位置精确的反馈控制,获得准确的圆二色谱。利用所发展的方法,测试了去折叠状态下的细胞色素c恢复折叠的动态同步辐射圆二色谱,在4.5 μs处折叠恢复54%。这种方法将为生物大分子折叠动力学研究提供新的探测手段。     

Measurement of intense X-Ray spectra: Methods based on CXR and Ross filter

The interaction between X-ray radiation and the material of secondary targets is simulated using GEANT4 for two methods of measurements of intense X-ray spectra. In the first case, characteristic X-ray radiation (CXR) from the secondary target to the back hemisphere is detected; in the second case, primary radiation passing through pairs of foils (a Ross filter) is registered. Using Ross filters and the CXR technique, the primary spectra of X-ray radiation are recovered. Two methods for recovery primary radiation spectra are compared.     

Third International Synchrotron Radiation Circular Dichroism Spectroscopy Meeting

The International Synchrotron Radiation Circular Dichroism (SRCD) Spectroscopy Meeting was held at the Physikzentrum, Bad Honnef, Germany on May 17–20, 2015, as the 590^th WE-Heraeus-Seminar. It was the third in the series of SRCD Workshops, following the first one held at the Daresbury Synchrotron (UK) in 2001, and the second at the Beijing Synchrotron Radiation Facility (BSRF) and the Institute of High Energy Physics (IHEP) in 2009. SRCD2015 was organized by Dr. Jochen Bürck, Prof. Anne Ulrich, and Dr. Dirk Windisch (all of Institute of Biological Interfaces (IBG-2), Karlsruhe Institute of Technology, Germany) and Prof. Bonnie Ann Wallace (Birkbeck College, University of London, UK). It was aimed at both synchrotron CD beamline scientists and scientific users of the beamlines, and included participants from 14 countries. For the first time, representatives of all operational SRCD beamlines worldwide were present at the same meeting, and scientists developing two new SRCD beamlines also participated.     

Meeting Reports: International Workshop on the Protein Circular Dichroism Data Bank

The International Workshop on the Protein Circular Dichroism Data Bank (PCDDB) was held on April 11–13, 2005, at Birkbeck College, University of London, UK, under the sponsorship of the UK Biotechnology and Biological Sciences Research Council. The meeting was chaired by Bonnie Ann Wallace, Professor in the Department of Crystallography at Birkbeck College. The workshop organizing committee consisted of Dr. Robert W. Janes, Queen Mary, University of London, and Dr. Lee Whitmore, also of Birkbeck College.

Participants included experts in both conventional circular dichroism (CD) spectroscopy and representatives from synchrotron radiation circular dichroism (SRCD) beamlines: Prof. J. Sutherland of the NSLS SRCD beamlines U9b and U11 and East Carolina University (USA);     

DISCO synchrotron‐radiation circular‐dichroism endstation at SOLEIL

The new synchrotron‐radiation circular‐dichroism (SRCD) endstation on the UV‐visible synchrotron beamline DISCO has been commissioned at the SOLEIL synchrotron. The design has been focused on preservation of a high degree of linear polarization at high flux and moderate resolving power covering the vacuum ultraviolet to visible spectral range (125–600 nm). The beam dimensions have been set to 4 mm × 4 mm at 1 nm bandwidth for lower sample degradation. The nitrogen‐purged sample chamber fits three types of sample holders accommodating conventional round cell mounting, automated rotation of the samples, as well as a microfluidic set‐up. Automated temperature‐controlled data collection on microvolumes is now available to the biology and chemistry communities. Macromolecules including membrane proteins, soluble proteins, bio‐nanotubes, sugars, DNA and RNAs are now routinely investigated.     

Impaired pulmonary blood flow distribution in congestive heart failure assessed using synchrotron radiation microangiography

Synchrotron radiation microangiography is a powerful tool for assessing adverse changes in pulmonary vessel density associated with primary pulmonary hypertension (PH). Congestive heart failure (CHF) leads to a `secondary' onset of PH, yet it is unknown whether secondary PH is also associated with reduced vessel density. This study utilized synchrotron radiation to assess both pulmonary vessel density and endothelial function in a Dahl rat model of CHF with secondary PH. High salt‐fed Dahl salt‐sensitive (Dahl‐S) and salt‐resistant (Dahl‐R) rats were anesthetized and microangiography was performed to assess the pulmonary vessel density and vascular responses to (i) sodium nitroprusside (5.0 µg kg^?1 min^?1), (ii) acetylcholine (3.0 µg kg^?1 min^?1) and (iii) ET‐1_A receptor blockade, BQ‐123 (1 mg kg^?1). Dahl‐S rats developed CHF and secondary PH as evident by endothelial dysfunction, impaired vasodilatory responses to acetylcholine, enhanced vasodilatory responses to BQ‐123 and extensive pulmonary vascular remodeling. Consequently, the pulmonary vessel density was adversely reduced. Interestingly, the etiology of secondary PH manifests with structural and functional changes that are comparable with that previously reported for primary PH. One important discrepancy, however, is that ET‐1 modulation of pulmonary vessels is most striking in vessels with a diameter range of 100–200 µm in secondary PH, in contrast to a range of 200–300 µm in primary PH. Such discrepancies should be considered in future studies investigating primary and secondary forms of PH.     

Neue Meßgeräte unter Einsatz von Mikroprozessoren

A system with 3 new types of radiation meters for intensity measurement, spectrometry, activity measurement and dosimetry is described. The application of microcomputer technique as well as the utilization of data prozessing via personal computers confer to the user extensive rationalization effects in the accomplishment and evaluation of these measurements.     

Real‐space analysis of radiation‐induced specific changes with independent component analysis

A method of analysis is presented that allows for the separation of specific radiation‐induced changes into distinct components in real space. The method relies on independent component analysis (ICA) and can be effectively applied to electron density maps and other types of maps, provided that they can be represented as sets of numbers on a grid. Here, for glucose isomerase crystals, ICA was used in a proof‐of‐concept analysis to separate temperature‐dependent and temperature‐independent components of specific radiation‐induced changes for data sets acquired from multiple crystals across multiple temperatures. ICA identified two components, with the temperature‐independent component being responsible for the majority of specific radiation‐induced changes at temperatures below 130 K. The patterns of specific temperature‐independent radiation‐induced changes suggest a contribution from the tunnelling of electron holes as a possible explanation. In the second case, where a group of 22 data sets was collected on a single thaumatin crystal, ICA was used in another type of analysis to separate specific radiation‐induced effects happening on different exposure‐level scales. Here, ICA identified two components of specific radiation‐induced changes that likely result from radiation‐induced chemical reactions progressing with different rates at different locations in the structure. In addition, ICA unexpectedly identified the radiation‐damage state corresponding to reduced disulfide bridges rather than the zero‐dose extrapolated state as the highest contrast structure. The application of ICA to the analysis of specific radiation‐induced changes in real space and the data pre‐processing for ICA that relies on singular value decomposition, which was used previously in data space to validate a two‐component physical model of X‐ray radiation‐induced changes, are discussed in detail. This work lays a foundation for a better understanding of protein‐specific radiation chemistries and provides a framework for analysing effects of specific radiation damage in crystallographic and cryo‐EM experiments.     

基于THz技术的农产品品质无损检测研究

农产品品质备受关注,而由品质变化引发的农产品安全事故频繁,开发农产品品质的快速无损检测技术成为研究热点。太赫兹波(THz)是介于微波与红外之间的电磁辐射,由于其独特的优势,具有重要的科学研究价值。近年来,随着THz辐射源和探测器研究的不断突破,大大丰富了THz的应用研究。与其他检测技术相比,作为一种新型检测技术,THz辐射具有安全和高信噪比等重要特征,能获得传统检测方法无法获得的物理、化学和生物等信息,在质量安全检测与控制、生物医学和通信等领域的应用前景广阔。首先介绍了THz波的概念和主要性质,接着对THz技术在农产品品质检测应用上的研究进展进行综述,分析THz探测技术存在的问题,对THz技术的应用潜力进行展望,最后提出THz技术在储粮品质检测中新的应用和广阔前景。     

Technique for obtaining and processing spectral information with static fourier spectrometer

We propose a technique and algorithm for processing interferograms obtained with a static Fourier spectrometer. Spectra of secondary radiation from certain compounds are obtained in the visible and near-UV spectral ranges upon excitation by different radiation sources. We present and discuss results of experimental data processing.     

Characterization of TiN coating layers using ultrasonic backward radiation

Since ceramic layers coated on machinery components inevitably experience the changes in their properties it is necessary to evaluate the characteristics of ceramic coating layers nondestructively for the reliable use of coated components and the remaining life prediction. To address such a need, in the present study, the ultrasonic backward radiation technique is applied to examine the very thin TiN ceramic layers coated on AISI 1045 steel or austenitic 304 steel substrate. Specifically, the ultrasonic backward radiation profiles have been measured with variations in specimen preparation conditions such as coating layer thickness and sliding loading. In the experiments performed in the current study, the peak angle and the peak amplitude of ultrasonic backward radiation profile varied sensitively according to two specimen preparation conditions. In fact, this result demonstrates a high possibility of the ultrasonic backward radiation as an effective tool for the nondestructive characterization of the TiN ceramic coating layers even in such a thin regime.     

脉冲微波辐照影响心肌细胞膜蛋白构象及其机制的研究

应用显微傅里叶变换红外光谱技术研究了脉冲微波辐照对心肌细胞膜蛋白质构象、功能的影响和相关分子机制。结果表明,辐照可对心肌细胞的细胞膜蛋白质结构产生明显影响。细胞膜脂质中—CH₂—、磷脂结构中CO、蛋白质酰胺Ⅰ,Ⅱ带的伸缩振动峰消失或位移。辐照后心肌细胞膜蛋白质二级结构也出现明显变化,α-螺旋和β-折叠结构减少,二级结构无序化程度增加。上述变化均与辐照剂量呈正相关。结果提示受脉冲微波辐照后,心肌细胞膜蛋白构象的完整性受损,膜稳定性及流动性下降,膜上多种生物活性结构被破坏,上述变化构成了细胞膜功能丧失、细胞形态和结构损伤、细胞凋亡等病理学效应的生物化学基础。文章首次从蛋白质构象角度阐述了微波辐照对心肌细胞膜损伤的分子病理机制。     

Influence of X-ray on the autophagic-lysosomal system in rat pancreatic acini

Lysosomes have an important role in radiation injury of cells and tissues. Activation of autophagy is frequently observed in different types of pathological tissue degeneration. In radiation response it increases in some cases, and lysosomes are responsible for regulated degradation of the autophagic vacuoles. Lysosomes are also involved in ionizing radiation induced cell death. In apoptosis lysosomes degrade content of the phagocytotic vacuoles derived from engulfed apoptotic blebs. On the other hand lysosomal enzymes discharged from disintegrated cells have a key role in induction of necrotic changes. In this work we investigate autophagy and lysosomal protein degradation in the relatively radiation insensitive exocrine pancreatic acini in vivo and in vitro. Type of cell death induced by X-ray was also examined in relation to the changes of the lysosomal processes. In 5h after 16 Gy in vivo whole body irradiation we observed significant increase in the cytoplasmic volume fraction of autophagic vacuoles and in the number of apoptotic cells in vivo. But in the acini isolated from irradiated rats we could not detect a change in the lysosomal degradation of intracellular proteins. Therefore irradiation probably influences the autophagy in an earlier step than lysosomal degradation. Extended necrotic lesions were not observed in vivo as long as 48 h. Isolated pancreatic acini usually contain more autophagic vacuoles than in vivo, but we could not observe additional increase in autophagy after 8 Gy, in vitro irradiation. Lysosomal degradation of intracellular proteins was also unaltered after 8 Gy, in vitro irradiation. Other biochemical functional parameters of the isolated pancreatic acini, like protein synthesis and amylase secretion were not changed either after 8 Gy, in vitro X-ray treatment. These results indicate that pancreatic acinar cells in vitro have a high tolerance to irradiation. The observed in vivo radiation induced changes of the exocrine pancreas are possibly indirectly induced by injuries of more sensitive mechanisms.     

Surface modification of the polyethyleneimine layer on silicone oxide film via UV radiation

We herein report a novel method of employing 254 nm of UV radiation (UV) for the modification of a polyethyleneimine (PEI) layer on silicone oxide film. In this study, a PEI layer composed of a 50 mM sodium carbonate solution (pH 8.2) was formed on the surface of a silicone oxide film with spontaneous adsorption. Then, thin film of PEI was patterned by UV radiation. To determine the effect of the UV radiation, fluorescence microscopy, X-ray electron spectroscopy (XPS), and Fourier Transform Infrared spectroscopy (FT-IR) analyses were performed. These results indicated that UV radiation could cause changes in the surface characteristics of the PEI layer. Subsequently, FT-IR analysis showed changes in the chemical composition of the PEI exposed to UV radiation, such as the disappearance of the amine. Based on these results, we can conclude that UV radiation could be used to eliminate the amine group selectively and that this technique could be applied to create a pattern on the surface of a PEI layer.     

ATR-FTIR分析冻结—解冻后的牛肉蛋白二级结构变化

研究了牛肉蛋白质二级结构稳定性在-18,-23及-38 ℃不同温度下的变化情况,明确了肉品工业中不同冷冻温度对肉品品质的作用机制。利用了傅里叶变换衰减全反射红外光谱(attenuated total reflectance Fourier transfer infrared spectroscopy,ATR-FTIR)技术、自动去卷积以及曲线拟合等计算方法,分析了-18,-23及-38 ℃三个温度下,牛肉肌原纤维蛋白(myofibrillar protein,MP)在冻结—解冻过程中的红外光谱图变化及蛋白质二级结构变性程度。ATR-FTIR结果显示,冻结—解冻过程中牛肉MP红外光谱图峰高、峰面积发生变化,且峰波数发生红移或者蓝移;冻结—解冻后牛肉MP红外光谱图中3 500～3 300 cm-1波段的吸收峰强度减弱甚至消失,说明解冻后牛肉MP中的结合水中O—H基团与氨基酸CO基团形成的分子内和分子间氢键断裂;冻结会影响牛肉MP二级结构的稳定性,导致牛肉蛋白质二级结构发生变化,其中除了无规则卷曲比例上升以外,α-螺旋、β-折叠、β-转角含量均下降,造成有序结构向无序结构转变;解冻后,冻结温度为-38 ℃的处理组牛肉β-折叠增加量大于-23和-18 ℃处理组,-38 ℃冻结牛肉MP二级结构的稳定性最好,解冻后蛋白质复性也最好。即蛋白质冷冻变性程度随着冻结温度的降低而减轻,蛋白质二级结构特征保持也越好。该试验研究基于肉品工业生产实际,研究结果从微观层面揭示了冻结温度对牛肉蛋白质变性的影响规律及可能的机制,为冷冻肉冻藏保鲜工艺制定提供了参考。