利用乙腈作为内标气相色谱法快速测定发酵液中的乳酸

建立了一种内标法准确、快速测定发酵液中乳酸含量的方法。色谱柱为2m×3mm(i.d.),内填GDX-103的不锈钢柱,FID检测器。内标为1.0%的乙腈溶液。实验结果表明,该方法的相对标准偏差小于1.0%(n=7),回收率在99%以上。方法具有前处理简单、干扰小、灵敏度高、分析速度快,线性测定范围宽等优点。     

Development of a simplified, economical polyacrylamide gel staining protocol for phosphoproteins

Pro-Q Diamond (Pro-Q DPS) is a commercially available stain that binds the phosphate moiety of phosphoproteins with high sensitivity and linearity. To conserve consumable costs we demonstrate that threefold diluted Pro-Q DPS offers the same sensitivity and linearity of signal to that obtained with undiluted Pro-Q DPS. The optimal conditions for Pro-Q DPS indicate that fixation, staining, and destaining of gels longer than 1 h, 2 h, and four 30-min incubations, respectively, are not required. The fixation and destaining solutions, but not the threefold diluted Pro-Q DPS, can be re-used without compromising the signal intensity or linear dynamic range. This modified protocol of Pro-Q DPS reduces the cost at least by fourfold, making the stain economically attractive for large-scale analysis of phosphoproteins.     

RP-HPLC内标法测定5种中草药的齐墩果酸含量

建立了反相高效液相色谱-内标法测定了齐墩果酸的含量。以苯甲酸为内标物,C18反相柱（250mm×4.6mmi.d.,5μm）为分析柱,甲醇-水-冰醋酸（95：5：0.1）为流动相,流速1.0mL/min;检测波长210nm,柱温35℃。齐墩果酸浓度10.0～500mg/L范围内,对照品与苯甲酸的色谱峰面积比呈良好的线性关系。该方法准确、快速、灵敏度高、重现性好。     

Use of a fluorescent internal protein standard to achieve quantitative two-dimensional gel electrophoresis

2-DE is a powerful separation method for complex protein mixtures. However, large intergel variations in spot intensity limit its use for quantitative proteomics studies. To address this issue, we developed a fluorescent internal protein standard for use in 2-DE analysis. Protein samples are spiked with an Alexa-labeled internal standard (ALIS) prior to separation with 2-DE. Due to the high extinction coefficient of the Alexa-fluor, incorporation of 0.1% of total protein is sufficient to allow visualization of the internal standard yet low enough to avoid interference in subsequent quantification and identification steps. Following 2-DE, total proteins are visualized with fluorescent postelectrophoretic stains spectrally separated from ALIS. Four protein stains, Deep Purple, Sulforhodamine G, ruthenium II-tris(bathophenanthroline disulfonate) (RuTBS), and SYPRO Ruby, including improved purification and staining protocols for RuTBS and ten-fold dilutions of SYPRO Ruby were evaluated. All staining protocols were compatible with the ALIS method and had similar LODs (1-4 ng) and dynamic ranges (10(3)). ALIS is a powerful normalization method for quantitative 2-DE which avoids potential problems associated with dual spot migration patterns observed in the DIGE method. Furthermore, ALIS provides significantly improved normality in the distribution of spot abundance-variance compared to normalization through division by the total spot volume.     

Proteomic analysis of phosphoproteins regulated by abscisic acid in rice leaves

Abscisic acid (ABA) is a hormone that regulates plant development and adaptation to environmental stresses. Protein phosphorylation has been recognized as an important mechanism for ABA signaling. However, the target phosphoproteins regulated by ABA are still largely unknown. Here, we report the identification of ABA-regulated phosphoproteins in rice using proteomic approaches. Six ABA-regulated phosphoproteins were identified as G protein beta subunit-like protein, ascorbate peroxidase, manganese superoxide dismutase, triosephosphate isomerase, putative Ca²⁺/H⁺ antiporter regulator protein, and glyoxysomal malate dehydrogenase. These results provide new insight into the regulatory mechanism for some ABA signaling proteins and implicate several previously unrecognized proteins in ABA action.     

Dual fluorescence self‐assembled multilayers bearing a fluorescent internal standard for interfacial sensing

A novel dual fluorescence film Quartz/PSS/AO/PSS/CS/CdTe sensor was constructed based on layer‐layer electrostatic attraction using CdTe as a fluorescence probe. Acridine orange is shielded effectively by sodium poly(4‐styrenesulfonate) and chitosan, thus its fluorescence intensity does not change in the presence of the analyte, thereby acting as a fluorescent internal standard providing a reference signal. The ratio of dual fluorescence intensities of the film sensor is not affected by the fluctuation of the external environment factors such as excitation intensity, detector voltage of photo multiplier tube and sensor position in the cell holder. Assays based on this dual fluorescence self‐assembled multilayers sensor afforded an extremely high sensitivity for DNA with greatly accuracy. Copyright © 2010 John Wiley & Sons, Ltd.     

Precise blood lead analysis using a combined internal standard and standard addition approach with disposable screen-printed electrodes

We report here a highly accurate and efficient method for blood lead analysis (BLA) through the use of a disposable electrode. A new type of mercury-plated preanodized screen-printed carbon electrode, together with a thallium(III) internal standard, simplifies calibration and gives easily quantifiable signals for accurate BLA. A preanodization procedure improves the preconcentration ability of the working electrode through the effect of lead (Pb) complexing with an electrogenerated surface functional group, mainly >C=O. Under optimized conditions, the ratio of the anodic stripping peak currents of Pb and Tl (i.e., i(Pb)/i(Tl)) is linear against [Pb] in the window of 1-300 ppb with a correlation coefficient and detection limit (signal-to-noise ratio=3) of 0.999 and 0.23 ppb, respectively. This approach was used to analyze blood samples (n=55) from workers at a local battery factory. The results are consistent with those obtained from graphite furnace atomic absorption spectroscopy and confirm the applicability of the proposed method.     

Development of internal standard for lipoprotein subclass analysis using dual detection gel-permeation high-performance liquid chromatography system

The LipoSEARCH® System is an innovative lipoprotein class analysis method based on gel-permeation high-performance liquid chromatography (HPLC). This system uses a gel permeation column to separate the major lipoprotein subclasses (chylomicron, very low-density lipoprotein, low-density lipoprotein, and high-density lipoprotein) in serum according to particle size and splits them into two pathways to measure total cholesterol (TC; esterified + unesterified cholesterol) and triglyceride (TG) concentrations simultaneously to obtain chromatograms for each.These chromatograms were analyzed based on the results of the calibration serum by fitting Gaussian curves to profile the 20 lipoprotein subclasses defined in detail. An important assumption of this HPLC system is its simultaneous detection of two pathways to guarantee the accuracy of each analysis. Therefore, in the present study, we investigated the development of an internal standard that can guarantee the simultaneous detection of this system by adding a pigment to the serum. We focused on quinone pigments with absorption at 550 nm, which is the wavelength used for the enzymatic assay of TC and TG concentrations in the system. As a result, we succeeded in producing overlapping pigment peaks that appeared after the analytical chromatograms in two pathways. It is also suggested that the pigment solution as an internal standard is stable in freezing storage and has little effect on the analysis. The developed internal standard is expected to contribute to the accuracy assurance of lipoprotein analysis by this dual-detection HPLC system.     

An approach for identification of phosphoproteins using the G-electrode-loading method in two-dimensional gel electrophoresis

We assessed whether the G-electrode-loading method (GELM) was helpful in the protein analysis. GELM in 2-DE was compared with the slip-loading, the in-gel rehydration and the cup-loading in 2-DE. GELM showed the best results for protein separation. A total of 14 spots that showed an increase with GELM were analyzed by MALDI-TOF MS. In GELM, all of these spots were identified with a high score and a high sequence coverage. A membrane-associated protein was identified and determined to have phosphorylated site. These tests show that GELM has several advantages for protein analysis compared with the traditional methods.     

磷脂脂肪酸分析中内标物的使用及数据处理建模

【目的】评估未知样品内标进样量的参考范围,优化数据处理方法。【方法】采用C19为内标,不同进样量进行分析;编写数据分析程序优化数据处理模型。【结果】随着内标进样量的增加,检测到的PLFA数量和响应值等指标均有增加,但进样量超过16 nmol/g时反而下降;25组数据显示采用响应值计算PLFA含量比百分比值计算所得含量略高;采用Dot Net C#语言编写数据分析模型。【结论】初步确定了未知样品内标进样量的参考范围,得出最优处理方法;采用响应值计算PLFA含量可避免由于百分比值缺失带来的误差;校准系数的引入可减少仪器参数条件改变等因素带来的误差;通过编写谱图数据分析程序获得一定自动化操作,提高了数据分析效率和准确性。     

Possible involvements of 101 kDa, 90 kDa, and 32 kDa phosphoproteins in the phase-shift of Dictyostelium cells from growth to differentiation

Abstract. As demonstrated previously, the transition of starving Dictyostelium cells from growth to differentiation phase occurs at a particular position (putative shift point; PS-point) in G₂-phase of the cell cycle of Dictyostelium discoideum Ax-2. In this study we examined what proteins are phosphorylated or dephosphorylated at the onset of starvation, with special emphasis on changes of phosphoproteins near the PS-point. When AX-2 cells at any particular phase of the cell cycle were pulse-labeled with inorganic ³²P (³²P_i) in the presence or absence of nutrients, it was found that 101 kDa and 90 kDa phosphoproteins exhibit specific changes around the PS-point. From the chase-experiments of ³²P-labeled cells, the 101 kDa and 90 kDa proteins were found to fail to be phosphorylated at the PS-point under starvation conditions. The protein phosphatase inhibitors such as okadaic acid and calyculin A inhibited completely entry of starving Ax-2 cells to differentiation, and also blocked perfectly dephosphorylation of 32 kDa protein. Taken together it is likely that dephosphorylation of 32 kDa protein as well as low phosphorylation levels of 101 kDa and 90 kDa proteins may be required for the phase-shift of Ax-2 cells from growth to differentiation. Subcellular fractionation showed the 101 kDa phosphoprotein to be located in cytoplasm, while parts, at least, of the 90 kDa and 32 kDa phosproproteins were in the nucleus. In addition, the results of cellulose thin-layer electrophoresis of digested 101 kDa and 90 kDa phosphoproteins show that in both proteins only serine residues are phosphorylated. The significance of phosphorylation states of 101 kDa, 90 kDa, and 32 kDa proteins is discussed in relation to a breakaway of cells from proliferation to differentiation.     

22nd International Conference on Medical Physics 2016, Bangkok,Thailand; Medical physics propelling global health

As medical technology evolves and patient needs increase, the need for well-trained and highly professional medical physicists (MPs) becomes even more urgent. The roles and responsibilities of MPs in various departments within the hospital are diverse and demanding. It is obvious that training, continuing education and professional development of MPs have become essential.One of the ways for an MP to advance his or her knowledge is to participate in conferences and congresses. Last year, the 22nd International Conference of Medical Physics (ICMP 2016) took place in Bangkok, Thailand. The event attracted 584 delegates with most of the participants coming from Asia. It attracted also delegates from 42 countries. The largest delegations were from Thailand, Japan and South Korea. ICMP 2016 included 367 oral presentations and e-posters, most of these being in the fields of Radiation Therapy, Medical Imaging and Radiation Safety. All abstracts were published as an e-book of Abstracts in a supplement to the official IOMP Journal. Many companies had exhibition stands at ICMP2016, thus allowing the participants to see the latest developments in the medical physics-related industry. The conference included 42 mini-symposia, part of the first “IOMP School” activity, covering various topics of importance for the profession and this special issue follows from the success of the conference.     

Changes in protein phosphorylation associated with compaction of the mouse preimplantation embryo

In order to investigate the role of protein phosphorylation in the early differentiative events of mouse preimplantation development, timed groups of embryos of various stages were incubated in medium containing [32P]orthophosphate and harvested immediately after labelling or following a chase period. The phosphoproteins obtained were separated by electrophoresis in one and two dimensions. While some of the phosphoproteins found were common to all the stages examined, the detection of many depended on both the combination of pulse-labelling and chase periods used and on the developmental stage examined. Some phosphoproteins were only found in compacted 8-cell embryos, a correlation which suggests a possible link with the post-translational mechanisms which underlie compaction.     

Quantification of trabecular bone microdamage using the virtual internal bond model and the individual trabeculae segmentation technique

Trabecular bone microdamage significantly influences the skeletal integrity and bone remodelling process. In this paper a novel constitutive model, called the virtual internal bond model (VIB), was adopted for simulating the damage behaviour of bone tissue. A unique 3D image analysis technique, named individual trabeculae segmentation, was used to analyse the effects of microarchitectures on the damage behaviours of trabecular bone. We demonstrated that the process of initiation and accumulation of microdamage in trabecular bone samples can be captured by the VIB-embedded finite-element method simulation without a separate fracture criterion. Our simulation results showed that the microdamage can occur at as early as about 0.2–0.4% apparent strain, and a large volume of microdamage was accumulated around the apparent yield strain. In addition we found that the plate-like trabeculae, especially the longitudinal ones, take crucial roles in the microdamage behaviours of trabecular bone.     

On ROC analysis with nonbinary reference standard

Statistical methods for the evaluation of the accuracy of diagnostic tests usually assume a binary true disease status. However, this assumption may not be realistic in practical settings in which “disease” is defined by dichotomizing continuous or ordinal categorical measures using a pre‐specified threshold value. In this paper, we focus on the analysis of studies in which both the diagnostic test and the reference standard are reported as continuous measures. We propose a semiparametric model for estimating the sensitivity, specificity, and the ROC curve as functions of reference standard thresholds. Under suitable order restrictions on the mean of the test result variable, fitting is done via two alternative approaches: isotonic regression and monotone smoothing splines. The model provides the basis to assess the effect of varying reference standard threshold on the performance of a diagnostic test. An example to evaluate the ability of the maximal SUV‐lean (standardized uptake value normalized to lean body mass) in predicting axillary node involvement in women diagnosed with breast cancer is presented.     

Monitoring global change with phenology: The case of the spring green wave

The centuries-old practice of recording plant and animal events that take place at specific times each year (phenology) should play an important role in monitoring mid-latitude global changes. At least three problems related to the detection of biosphere changes could be investigated using this information. Firstly, the technique can be generalized from the local to global scale. Secondly, an integrated approach could be developed to represent biome diversity effectively. Lastly, physical mechanisms responsible for the events can be deduced in order to incorporate the phenological information into global-scale models, and detect changes in related environmental factors. With these goals in mind, regional phenological data collection networks were initiated in eastern North America during the early 1960s, using cloned lilacs and several species of honeysuckle. This paper reviews research projects which address the problems outlined above, using first leaf data (associated with spring green-up or green wave in mid-latitudes) gathered from these networks. The results of such studies in North America have demonstrated the potential of phenology as an efficient monitor of global change throughout mid-latitude regions. Future research efforts will concentrate on the development of a coordinated strategy to link phenological information from satellites, indicator plants (such as the lilac), and representative species from each biome.     

以技术集成促进生物催化技术转移

生物催化研究和产业化都在迅速发展,但两者之间的联系并不紧密,成功的技术转移很少。缺乏集成研发平台成为生物催化技术转移的瓶颈。在国内相关企业的技术力量不足以进行技术集成的现状下,建议中国科学院建设酶工程集成研发平台,依托此平台企业和科研院所建立广泛的技术合作和技术转移关系,建立生物催化产业的产学研价值链。     

达标污水离岸排海末端处置技术研究综述

达标污水离岸排海处置具有低成本、高处理率等优点,是国内外沿海港口和临港工业园区进行污水处置的重要选择。介绍了国内外达标污水离岸排海末端处置技术的发展情况,分析了排污口位置选划、扩散器射流参数及水力结构优化等研究技术上存在的问题。认为排污口位置选划应在水动力、污染物扩散、泥沙冲淤及工程经济因素分析的基础上,还需要重点考虑污水排放对区域生态要素长期的影响;排海末端扩散器射流参数及水力结构优化也尤为关键,不仅直接影响稀释扩散效果,而且涉及到主管、上升管、喷口等多个可控因子,可以采用建立物理模型、数值模型及量纲分析等方法,合理优化扩散器的结构参数和扩散器的型式,对提高达标污水离岸处置的效果具有重要意义。