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土耳其比尔肯特大学研制出一种全硫属化物可变红外滤光片

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图１（）ａ斜蒸发几何结构示意图，（）间可变多ｂ空层滤光片结构，（）０层Ａ２３ＧｌｓＳｉｅＣ２ｓ一ｅｓ２ｅｓ４ＳＡ方法的研究［．外与毫米Ｊ红１
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哥召开的 “ 红外系统与光电子技术” ＳＩＰＥ专题
会议上，土耳其比尔肯特大学的研究人员ＨＥａ．ｓｔ
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图２该红外光谱仪实验装置的草图
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Quality evaluation of Flos Lonicerae through a simultaneous determination of seven saponins by HPLC with ELSDXing-Yun Chai1, Song-Lin Li2, Ping Li1*1Key Laboratory of Modern Chinese Medicines and Department of Pharmacognosy, China Pharmaceutical University, Nanjing, 210009, People’s Republic of China2Institute of Nanjing Military Command for Drug Control, Nanjing, 210002, People’s Republic of China*Corresponding author: Ping LiKey Laboratory of Modern Chinese Medicines and Department of Pharmacognosy, China Pharmaceutical University, Nanjing 210009, People’s Republic of China.E-mail address: lipingli@Tel.: +86-25-8324-2299; 8539-1244; 135********Fax: +86-25-8532-2747AbstractA new HPLC coupled with evaporative light scattering detection (ELSD) method has been developed for the simultaneous quantitative determination of seven major saponins, namely macranthoidinB (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7)in Flos Lonicerae, a commonly used traditional Chinese medicine (TCM) herb.Simultaneous separation of these seven saponins was achieved on a C18 analytical column with a mixed mobile phase consisting of acetonitrile(A)-water(B)(29:71 v/v) acidified with 0.5% acetic acid. The elution was operated from keeping 29%A for 10min, then gradually to 54%B from 10 to 25 min on linear gradient, and then keep isocratic elution with 54%B from 25 to 30min.The drift tube temperature of ELSD was set at 106℃, and with the nitrogen flow-rate of 2.6 l/min. All calibration curves showed good linear regression (r2 0.9922) within test ranges. This method showed good reproducibility for the quantification of these seven saponins in Flos Lonicerae with intra- and inter-day variations of less than 3.0% and 6.0% respectively. The validated method was successfully applied to quantify seven saponins in five sources of Flos Lonicerae, which provides a new basis of overall assessment on quality of Flos Lonicerae.Keywords: HPLC-ELSD; Flos Lonicerae; Saponins; Quantification1. IntroductionFlos Lonicerae (Jinyinhua in Chinese), the dried buds of several species of the genus Lonicera (Caprifoliaceae), is a commonly used traditional Chinese medicine (TCM) herb. It has been used for centuries in TCM practice for the treatment of sores, carbuncles, furuncles, swelling and affections caused by exopathogenic wind-heat or epidemic febrile diseases at the early stage [1]. Though four species of Lonicera are documented as the sources of Flos Lonicerae in China Pharmacopeia (2000 edition), i.e. L. japonica, L. hypoglauca,L. daystyla and L. confusa, other species such as L. similes and L. macranthoides have also been used on the same purpose in some local areas in China [2]. So it is an important issue to comprehensively evaluate the different sources of Flos Lonicerae, so as to ensure the clinical efficacy of this Chinese herbal drug.Chemical and pharmacological investigations on Flos Lonicerae resulted in discovering several kinds of bioactive components, i.e. chlorogenic acid and its analogues, flavonoids, iridoid glucosides and triterpenoid saponins [3]. Previously, chlorogenic acid has been used as the chemical marker for the quality evaluation of Flos Lonicerae,owing to its antipyretic and antibiotic property as well as its high content in the herb. But this compound is not a characteristic component of Flos Lonicerae, as it has also been used as the chemical marker for other Chinese herbal drugs such as Flos Chrysanthemi and so on[4-5]. Moreover, chlorogenic acid alone could not be responsible for the overall pharmacological activities of Flos Lonicerae[6].On the other hand, many studies revealed that triterpenoidal saponins of Flos Lonicerae possess protection effects on hepatic injury caused by Acetaminophen, Cd, and CCl4, and conspicuous depressant effects on swelling of ear croton oil [7-11]. Therefore, saponins should also be considered as one of the markers for quality control of Flos Lonicerae. Consequently, determinations of all types of components such as chlorogenic acid, flavonoids, iridoid glucosides and triterpenoidal saponins in Flos Lonicerae could be a better strategy for the comprehensive quality evaluation of Flos Lonicerae.Recently an HPLC-ELSD method has been established in our laboratory for qualitative and quantitative determination of iridoid glucosides in Flos Lonicerae [12]. But no method was reported for the determination of triterpenoidal saponins in Flos Lonicera. As a series studies on the comprehensive evaluation of Flos Lonicera, we report here, for the first time, the development of an HPLC-ELSD method for simultaneous determination of seven triterpenoidal saponins in the Chinese herbal drug Flos Lonicerae, i.e.macranthoidin B (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7) (Fig. 1).2. Experimental2.1. Samples, chemicals and reagentsFive samples of Lonicera species,L. japonica from Mi county, HeNan province (LJ1999-07), L. hypoglauca from Jiujang county, JiangXi province (LH2001-06), L. similes from Fei county, ShanDong province (LS2001-07), L. confuse from Xupu county, HuNan province (LC2001-07), and L. macranthoides from Longhu county, HuNan province (LM2000-06) respectively, were collected in China. All samples were authenticated by Dr. Ping Li, professor of department of Pharmacognosy, China Pharmaceutical University, Nanjing, China. The voucher specimens were deposited in the department of Pharmacognosy, China Pharmaceutical University, Nanjing, China. Seven saponin reference compounds: macranthoidin B (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7) were isolated previously from the dried buds of L. confusa by repeated silica gel, sephadex LH-20 and Rp-18 silica gel column chromatography, their structures were elucidated by comparison of their spectral data (UV, IR, MS, 1H- NMR and 13C-NMR) with references [13-15]. The purity of these saponins were determined to be more than 98% by normalization of the peak areas detected by HPLC with ELSD, and showed very stable in methanol solution.HPLC-grade acetonitrile from Merck (Darmstadt, Germany), the deionized water from Robust (Guangzhou, China), were purchased. The other solvents, purchased from Nanjing Chemical Factory (Nanjing, China) were of analytical grade.2.2. Apparatus and chromatographic conditionsAglient1100 series HPLC apparatus was used. Chromatography was carried out on an Aglient Zorbax SB-C18 column（250 4.6mm, 5.0µm）at a column temperature of 25℃.A Rheodyne 7125i sampling valve (Cotati, USA) equipped with a sample loop of 20µl was used for sample injection. The analog signal from Alltech ELSD 2000 (Alltech, Deerfield, IL, USA)was transmitted to a HP Chemstation for processing through an Agilent 35900E (Agilent Technologies, USA).The optimum resolution was obtained by using a linear gradient elution. The mobile phase was composed of acetonitrile(A) and water(B) which acidified with 0.5% acetic acid. The elution was operated from keeping 29%A for 10min, then gradually to 54%B from 10 to 25 min in linear gradient, and back to the isocratic elution of 54%B from 25 to 30 min.The drift tube temperature for ELSD was set at 106℃and the nitrogen flow-rate was of 2.6 l/min. The chromatographic peaks were identified by comparing their retention time with that of each reference compound tried under the same chromatographic conditions with a series of mobile phases. In addition, spiking samples with the reference compounds further confirmed the identities of the peaks.2.3. Calibration curvesMethanol stock solutions containing seven analytes were prepared and diluted to appropriate concentration for the construction of calibration curves. Six concentrationof the seven analytes’ solution were injected in triplicate, and then the calibration curves were constructed by plotting the peak areas versus the concentration of each analyte. The results were demonstrated in Table1.2.4. Limits of detection and quantificationMethanol stock solution containing seven reference compounds were diluted to a series of appropriate concentrations with methanol, and an aliquot of the diluted solutions were injected into HPLC for analysis.The limits of detection (LOD) and quantification (LOQ) under the present chromatographic conditions were determined at a signal-to-noise ratio (S/N) of 3 and 10, respectively. LOD and LOQ for each compound were shown in Table1.2.5. Precision and accuracyIntra- and inter-day variations were chosen to determine the precision of the developed assay. Approximate 2.0g of the pulverized samples of L. macranthoides were weighted, extracted and analyzed as described in 2.6 Sample preparation section. For intra-day variability test, the samples were analyzed in triplicate for three times within one day, while for inter-day variability test, the samples were examined in triplicate for consecutive three days. Variations were expressed by the relative standard deviations. The results were given in Table 2.Recovery test was used to evaluate the accuracy of this method. Accurate amounts of seven saponins were added to approximate 1.0g of L. macranthoides,and then extracted and analyzed as described in 2.6 Sample preparation section. The average recoveries were counted by the formula: recovery (%) = (amount found –original amount)/ amount spiked ×100%, and RSD (%) = (SD/mean) ×100%. The results were given in Table 3.2.6. Sample preparationSamples of Flos Lonicerae were dried at 50℃until constant weight. Approximate 2.0g of the pulverized samples, accurately weighed, was extracted with 60% ethanol in a flask for 4h. The ethanol was evaporated to dryness with a rotary evaporator. Residue was dissolved in water, followed by defatting with 60ml of petroleum ether for 2 times, and then the water solution was evaporated, residue was dissolved with methanol into a 25ml flask. One ml of the methanol solution was drawn and transferred to a 5ml flask, diluted to the mark with methanol. The resultant solution was at last filtrated through a 0.45µm syringe filter (Type Millex-HA, Millipore, USA) and 20µl of the filtrate was injected to HPLC system. The contents of the analytes were determined from the corresponding calibration curves.3. Results and discussionsThe temperature of drift tube and the gas flow-rate are two most important adjustable parameters for ELSD, they play a prominent role to an analyte response. In ourprevious work [12], the temperature of drift tube was optimized at 90°C for the determination of iridoids. As the polarity of saponins are higher than that of iridoids, more water was used in the mobile phase for the separation of saponins, therefore the temperature for saponins determination was optimized systematically from 95°C to 110°C, the flow-rate from 2.2 to 3.0 l/min. Dipsacoside B was selected as the testing saponin for optimizing ELSD conditions, as it was contained in all samples. Eventually, the drift tube temperature of 106℃and a gas flow of 2.6 l/min were optimized to detect the analytes. And these two exact experimental parameters should be strictly controlled in the analytical procedure [16].All calibration curves showed good linear regression (r2 0.9922) within test ranges. Validation studies of this method proved that this assay has good reproducibility. As shown in Table 2, the overall intra- and inter-day variations are less than 6% for all seven analytes. As demonstrated in Table 3, the developed analytical method has good accuracy with the overall recovery of high than 96% for the analytes concerned. The limit of detection (S/N=3) and the limit of quantification (S/N=10) are less than 0.26μg and 0.88μg respectively (Table1), indicating that this HPLC-ELSD method is precise, accurate and se nsitive enough for the quantitative evaluation of major non- chromaphoric saponins in Flos Lonicerae.It has been reported that there are two major types of saponins in Flos Lonicerae, i.e. saponins with hederagenin as aglycone and saponins with oleanolic acid as the aglycone [17]. But hederagenin type saponins of the herb were reported to have distinct activities of liver protection and anti-inflammatory [7-11]. So we adoptedseven hederagenin type saponins as representative markers to establish a quality control method.The newly established HPLC-ELSD method was applied to analyze seven analytes in five plant sources of Flos Lonicerae, i.e. L. japonica,L. hypoglauca,L. confusa,L. similes and L. macranthoides(Table 4). It was found that there were remarkable differences of seven saponins contents between different plant sources of Flos Lonicerae. All seven saponins analyzed could be detected in L. confusa and L. hypoglauca, while only dipsacoside B was detected in L. japonica. Among all seven saponins interested, only dipsacoside B was found in all five plant species of Flos Lonicerae analyzed, and this compound was determined as the major saponin with content of 53.7 mg/g in L. hypoglauca. On the other hand, macranthoidin B was found to be the major saponin with the content higher than 41.0mg/g in L. macranthoides,L. confusa, and L. similis, while the contents of other analytes were much lower.In our previous study [12], overall HPLC profiles of iridoid glucosides was used to qualitatively and quantitatively distinguish different origins of Flos Lonicerae. As shown in Fig.2, the chromatogram profiles of L. confusa, L. japonica and L. similes seem to be similar, resulting in the difficulty of clarifying the origins of Flos Lonicerae solely by HPLC profiles of saponins, in addition to the clear difference of the HPLC profiles of saponins from L. macranthoides and L. hypoglauca.Therefore, in addition to the conventional morphological and histological identification methods, the contents and the HPLC profiles of saponins and iridoids could also be used as accessory chemical evidence toclarify the botanical origin and comprehensive quality evaluation of Flos Lonicerae.4. ConclusionsThis is the first report on validation of an analytical method for qualification and quantification of saponins in Flos Lonicerae. This newly established HPLC-ELSD method can be used to simultaneously quantify seven saponins, i.e. macranthoidin B, macranthoidin A, dipsacoside B, hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester, macranthoside B, macranthoside A, and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside in Flos Lonicerae. Together with the HPLC profiles of iridoids, the HPLC-ELSD profiles of saponins could also be used as an accessory chemical evidence to clarify the botanical origin and comprehensive quality evaluation of Flos Lonicerae.AcknowledgementsThis project is financially supported by Fund for Distinguished Chinese Young Scholars of the National Science Foundation of China (30325046) and the National High Tech Program（2003AA2Z2010）.[1]Ministry of Public Health of the People’s Republic of China, Pharmacopoeia ofthe People’s Republic of China, V ol.1, 2000, p. 177.[2]W. Shi, R.B. Shi, Y.R. Lu, Chin. Pharm. J., 34(1999) 724.[3]J.B. Xing, P. Li, D.L. Wen, Chin. Med. Mater., 26(2001) 457.[4]Y.Q. Zhang, L.C. Xu, L.P. Wang, J. Chin. Med. Mater., 21(1996) 204.[5] D. Zhang, Z.W. Li, Y. Jiang, J. Pharm. Anal., 16(1996) 83.[6]T.Z. Wang, Y.M. Li, Huaxiyaoxue Zazhi, 15(2000) 292.[7]J.ZH. Shi, G.T. Liu. Acta Pharm. Sin., 30(1995) 311.[8]Y. P. Liu, J. Liu, X.SH. Jia, et al. Acta Pharmacol. Sin., 13 (1992) 209.[9]Y. P. Liu, J. Liu, X.SH. Jia, et al. Acta Pharmacol. Sin., 13 (1992) 213.[10]J.ZH. Shi, L. Wan, X.F. Chen.ZhongYao YaoLi Yu LinChuang, 6 (1990) 33.[11]J. Liu, L. Xia, X.F. Chen. Acta Pharmacol. Sin., 9 (1988) 395[12]H.J. Li, P. Li, W.C. Ye, J. Chromatogr. A 1008(2003) 167-72.[13]Q. Mao, D. Cao, X.SH. Jia. Acta Pharm. Sin., 28(1993) 273.[14]H. Kizu, S. Hirabayashi, M. Suzuki, et al. Chem. Pharm. Bull., 33(1985) 3473.[15]S. Saito, S. Sumita, N. Tamura, et al. Chem Pharm Bull., 38(1990) 411.[16]Alltech ELSD 2000 Operating Manual, Alltech, 2001, p. 16. In Chinese.[17]J.B. Xing, P. Li, Chin. Med. Mater., 22(1999) 366.Fig. 1 Chemical structures of seven saponins from Lonicera confusa macranthoidin B (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7)Fig. 2Representative HPLC chromatograms of mixed standards and methanol extracts of Flos Lonicerae.Column: Agilent Zorbax SB-C18 column（250 4.6mm, 5.0µm）, temperature of 25℃; Detector: ELSD, drift tube temperature 106℃, nitrogen flow-rate 2.6 l/min.A: Mixed standards, B: L. confusa, C: L. japonica, D: L. macranthoides, E: L. hypoglauca, F: L. similes.Table 1 Calibration curves for seven saponinsAnalytes Calibration curve ar2Test range(μg)LOD(μg)LOQ(μg)1 y=6711.9x－377.6 0.9940 0.56–22.01 0.26 0.882 y=7812.6x－411.9 0.9922 0.54–21.63 0.26 0.843 y=6798.5x－299.0 0.9958 0.46–18.42 0.22 0.724 y=12805x－487.9 0.9961 0.38–15.66 0.10 0.345 y=4143.8x－88.62 0.9989 0.42–16.82 0.18 0.246 y=3946.8x－94.4 0.9977 0.40–16.02 0.16 0.207 y=4287.8x－95.2 0.9982 0.42–16.46 0.12 0.22a y: Peak area; x: concentration (mg/ml)Table 2 Reproducibility of the assayAnalyteIntra-day variability Inter-day variability Content (mg/g) Mean RSD (%) Content (mg/g) Mean RSD (%)1 46.1646.2846.2246.22 0.1346.2245.3647.4226.33 2.232 5.385.385.165.31 2.405.285.345.045.22 3.043 4.374.304.184.28 2.244.284.464.024.255.204 nd1)-- -- nd -- --5 1.761.801.821.79 1.701.801.681.841.77 4.706 1.281.241.221.252.451.241.341.201.26 5.727 tr2)-- -- tr -- -- 1): not detected; 2): trace. RSD (%) = (SD/Mean) ×100%Table 3 Recovery of the seven analytesAnalyteOriginal(mg) Spiked(mg)Found(mg)Recovery(%)Mean(%)RSD(%)1 23.0823.1423.1119.7122.8628.1042.7346.1351.0199.7100.699.399.8 0.722.692.672.582.082.913.164.735.515.7698.197.6100.698.8 1.632.172.152.091.732.182.623.884.404.6598.8103.297.799.9 2.94nd1)1.011.050.980.981.101.0297.0104.8104.1102.0 4.250.880.900.910.700.871.081.561.752.0197.197.7101.898.9 2.660.640.620.610.450.610.751.081.211.3397.796.796.096.8 0.97tr2)1.021.101.081.031.111.07100.9102.799.1100.9 1.81): not detected; 2): trace.a Recovery (%) = (Amount found –Original amount)/ Amount spiked ×100%, RSD (%) = (SD/Mean) ×100%Table 4 Contents of seven saponins in Lonicera spp.Content (mg/g)1 2 3 4 5 6 7 L. confusa45.65±0.32 5.13±0.08 4.45±0.11tr1) 2.04±0.04tr 1.81±0.03 L. japonica nd2)nd 3.44±0.09nd nd nd nd L. macranthoides46.22±0.06 5.31±0.13 4.28±0.10 tr 1.79±0.03 1.25±0.03 tr L. hypoglauca11.17±0.07 nq3)53.78±1.18nd 1.72±0.02 2.23±0.06 2.52±0.04 L. similes41.22±0.25 4.57±0.07 3.79±0.09nd 1.75±0.02tr nd 1): trace; 2): not detected.. 3) not quantified owing to the suspicious purity of the peak.。

实验室层析填料应用指南

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3
层析介质应用
2、分子筛技术：是利用多肽分子大小、形状差异来分离纯化多肽物质。Superdex peptide 专门分离多肽类产品的高分辨率分子筛凝胶，分离的分子量范围为 100-7000 Da。高化学稳定性 , pH 1-14, 兼容有机溶剂 ( 乙腈 +TFA)。
• 如果反相和离子交换技术都效果不好，原因可能是溶解性的问题。
• 使用凝胶过滤如 Superdex peptide，Sephadex LH20 都很好的用在多肽纯化中。
• 需要增加选择性，载量和稳定性：用多维纯化方法，可以将离子交换，分子筛和反相层析等多种技术结合，而代替一步反相或反复用反相柱子纯化。
常见的重组细胞因子有：干扰素（Interferon,IFN）、肿瘤坏死因子（tumor necrosis factor, TNF）、集落刺激因子（colony-stimulating factor）、趋化因子（chemokine）、生长因子（growth factor, GF）、白细胞介素系列（interleukin, IL) 等。
4
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二、多肽分离技术的综合运用
对于大多数的多肽来说，仅仅一步纯化或一种技术应用是不够的，通常会将反相，离子交换和凝胶过滤这三种技术综合起来进行多步纯化，最终达到高的分辨率。例 1 、PY574a 多肽，14aa，pI>10, 在酸性条件下不溶。
一、原核表达细胞因子的纯化工艺
1、重组人干扰素 α2b(rh-IFNα2b) 的纯化：
大肠杆菌发酵

A scale-invariant cohesive crack model

Department of Structural and Geotechnical Engineering, Politecnico di Torino, Corso Duca Degli Abruzzi 24, 10129 Torino, Italy
Abstract The ®ctitious crack model by Hillerborg is the most widely used model to simulate damage and fracture in concrete structures. Its peculiar capability to capture the evolution of the cracking process is accompanied by its simplicity. However, some aspects of the phenomenon are not considered in the model, for instance the size-dependence of the nominal quantities involved in the cohesive law. This aects the predictive capabilities of the model, when it is used to extrapolate results from small laboratory specimens to full-scale structures. In this paper, a scale-independent cohesive law is put forward, which overcomes these drawbacks and permits to obtain a unique constitutive relationship for softening in concrete. By assuming damage occurring in a fractal band inside the specimen, nominal stress, crack opening displacement and nominal fracture energy become scale dependent. Hence they should be substituted by fractal quantities, which are the true material constants. A mutual relation among their fractal physical dimensions puts a strong restriction to disorder. By varying the scaling exponents of the kinematical quantities, a clear transition from discrete to smeared cracking can be obtained. The fractal cohesive law is eventually applied to some tensile test data, showing perfect agreement between theory and experiments. Ó 2001 Elsevier Science Ltd. All rights reserved.

Genetic Characteristics of the Human Hepatic Stellate Cell Line LX-2

Genetic Characteristics of the Human Hepatic Stellate Cell Line LX-2Ralf Weiskirchen1*.,Jo¨rg Weimer2.,Steffen K.Meurer1,Anja Kron3,Barbara Seipel3,Inga Vater4, Norbert Arnold2,Reiner Siebert4,Lieming Xu5,Scott L.Friedman6,Carsten Bergmann3,7,81Institute of Clinical Chemistry and Pathobiochemistry,RWTH Aachen University,Aachen,Germany,2Department of Gynaecology and Obstetrics,UKSH Campus Kiel, Kiel,Germany,3Center for Human Genetics,Bioscientia,Ingelheim,Germany,4Institute of Human Genetics,University Hospital Schleswig-Holstein&Christian-Albrechts University Kiel,Kiel,Germany,5Institute of Liver Diseases,Shanghai University of Traditional Chinese Medicine Shuguang Hospital,Shanghai,PR China,6Division of Liver Diseases,Mount Sinai School of Medicine,New York,New York,United States of America,7Department of Human Genetics,RWTH Aachen University,Aachen,Germany, 8Center for Clinical Research,University Hospital Freiburg,Freiburg,GermanyAbstractThe human hepatic cell line LX-2has been described as tool to study mechanisms of hepatic fibrogenesis and the testing of antifibrotic compounds.It was originally generated by immortalisation with the Simian Vacuolating Virus40(SV40) transforming(T)antigen and subsequent propagation in low serum conditions.Although this immortalized line is used in an increasing number of studies,detailed genetic characterisation has been lacking.We here have performed genetic characterisation of the LX-2cell line and established a single-locus short tandem repeat(STR)profile for the cell line and characterized the LX-2karyotype by several cytogenetic and molecular cytogenetic techniques.Spectral karyotyping(SKY) revealed a complex karyotype with a set of aberrations consistently present in the metaphases analyses which might serve as cytogenetic markers.In addition,various subclonal and single cell aberrations were detected.Our study provides criteria for genetic authentication of LX-2and offers insights into the genotype changes which might underlie part of its phenotypic features.Citation:Weiskirchen R,Weimer J,Meurer SK,Kron A,Seipel B,et al.(2013)Genetic Characteristics of the Human Hepatic Stellate Cell Line LX-2.PLoS ONE8(10): e75692.doi:10.1371/journal.pone.0075692Editor:Golo Ahlenstiel,University of Sydney,AustraliaReceived May9,2013;Accepted August20,2013;Published October8,2013Copyright:ß2013Weiskirchen et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use,distribution,and reproduction in any medium,provided the original author and source are credited.Funding:This work was supported by the German Research Foundation(DFG,SFB/TRR57)and National Institutes of Health(DK56621to SLF).The funders had no role in study design,data collection and analysis,decision to publish,or preparation of the manuscript.Competing Interests:It is correct that three of the listed authors(Carsten Bergmann,Barbara Seipel,and Anja Kron)are employed by a commercial company (Bioscientia Ingelheim,Ingelheim,Germany).The relationship to this company has however not altered the authors’adherence to all the PLOS ONE policies in sharing data and materials.*E-mail:rweiskirchen@ukaachen.de.These authors contributed equally to this work.IntroductionDuring liver fibrogenesis and the establishment of cirrhosis, contractile myofibroblasts(MFB)that originate from quiescent hepatic stellate cells(HSC)are the major source of extracellular matrix(ECM)[1].HSC/MFB possesses the capacity not only for matrix synthesis,but also for the expression and secretion of pro-and anti-inflammatory cytokines and growth factors[2].Based on their pivotal role in the initiation and progression of liver fibrogenesis,HSC/MFB biology is a major focus of fibrosis investigation.However,the preparation of primary cells from the liver is time-consuming and requires special expertise.To overcome these limitations,several spontaneous or experimental-ly-derived immortalized HSC cell lines from mouse,rat and humans have been established[3].Like other permanent cell lines, immortalized stellate cell lines have the advantage of growing continuously to provide unlimited access.Moreover,their clonal origin usually guarantees a considerably homogeneous phenotype that should allow the performance of reproducible experiments in different laboratories[3].Based on this approach,key aspects of stellate cell biology have been uncovered including advances in retinoid metabolism,extracellular matrix expression and turnover,particular,these cell lines are exploited to develop therapeutic approaches.In this context,there is an increasing need for properly characterized stellate cell lines that preserve phenotypic characteristics of HSC/MFB especially for the human derivatives. The human HSC cell lines Lieming Xu-1(LX-1)and Lieming Xu-2(LX-2)were originally generated by transformation of cultured primary HSC obtained from a male human liver with a plasmid encoding the SV40large T-antigen expressed under the control of a Rous sarcoma virus promoter(LX-1)or by spontaneous immortalization of a subset of early passaged LX-1 cells that were grown in low serum conditions(LX-2)[4].Both cell lines express a-SMA,vimentin,the intermediate filament protein glial fibrillary acidic protein(GFAP),and the type b receptor for platelet-derived growth(PDGFR b)suggesting that both cell lines retain key features of activated/transdifferentiated HSC.Both LX cell lines also secrete pro-collagen,pro-MMP-2,MT1-MMP (MMP-14),TIMP-1and TIMP-2,all features characteristic for activated HSC[5].Based on these properties,both LX cell lines have been widely employed as experimental tools in many laboratories worldwide.In particular,LX-2enjoys great popular-ity among researchers interested in the elucidation of mechanismsby the increasing number of publications in which this cell line was utilized.Since the first report in2003[6]and the more detailed characterisation two years later[4],LX-2cells have been cited nowadays in158peer reviewed publications(Figure S1)not only in the field of gastroenterology and hepatology[7–9]but also in studies focused on cellular and molecular biology[10],pharma-cology/toxicology[11],lipid metabolism[12],tissue engineering [13],oncology[14],endocrinology[15]and other general topics [16].Although widely used,LX-2,like all other immortalized cell lines,might be prone to genotypic/karyotypic and phenotypic drifts due to repeated passaging,which may result in cellular sub-lines that are phenotypically and genetically heterogeneous in character.As a consequence,key results obtained with these or other cell lines should be validated in primary cells if possible.Alternatively, further efforts should be made to understand the diverse and heterogeneous outcomes observed with cell lines by more sophisticated characterization of the respective line.With this in mind,we have characterized the genetic profile of LX-2using a number of cytogenetic and molecular methods,including single-locus short tandem repeat(STR)genotyping,standard karyotyp-ing,spectral karyotyping(SKY),and single nucleotide polymor-phism(SNP)array analytics.Our findings indicate that LX-2cells comprise a complex karyotype with various structural abnormal-ities.However,the invariant STR signature and defined specific translocations could be established for LX-2cells.Our results will facilitate proper identity testing,could lead to further mechanistic insights and improve experimental data in the field of liver fibrosis using this cell line.The work also provides importance guidance for the standardized,rigorous evaluation of other cell lines used in experimental hepatology.Materials and MethodsCell CultureHuman hepatic stellate cell line LX-2[4],Wi-38(clone VA-13, subline2RA)(#CCL-75.1,ATCC,Manassas,VA,USA)[17], HepG2[18],and Hep3B[18]were cultured in HEPES-buffered Dulbecco’s modified Eagle medium(DMEM)(Lonza BioWhit-taker,Verviers,Belgium)supplemented with4mmol/L L-glutamine(Lonza),100IU/ml Penicillin/100m g/ml Streptomy-cin(Lonza),and10%(v/v)(Wi-38,HepG2)or2%(v/v)(LX-2) fetal calf serum(FCS)(PAA Laboratories,Pasching,Austria). Human umbilical vein endothelial cells(HUVEC)were isolated from human umbilical cords by collagenase dissociation and grown medium199(Gibco,Life Technologies,Darmstadt, Germany)with20%heat-inactivated fetal bovine serum(FBS) (Gibco),100m g/ml endothelial cell growth supplement(Sigma, Taufkirchen,Germany),and50U/ml heparin(Sigma).Human MFB(hMFB)were isolated as outgrowth from human liver tissues and sub-cultured for several passages as described before[19].All experiments done with LX-2cells were done with passages number between p5and p7in which these numbers refer to those after selection of an immortalized cell population[4]. ImmunocytochemistryApproximately36105LX-2cells were plated on coverslips mounted in6-well dishes and incubated for24h at37u C. Thereafter,the cells were fixed for15min in4%(w/v) paraformaldehyde buffered in phosphate-buffered saline(PBS) (pH7.4)and permeabilized(2min on ice)in0.1%(w/v)sodium citrate/0.1%(v/v)Triton X-100.Then the cells were incubated mapping near the N-terminus of SV40T Ag of SV40origin(sc-20800,Santa Cruz Biotechnology,Santa Cruz,CA),a monoclonal antibody raised against a-smooth muscle actin(a-SMA)or respective normal rabbit(sc-2027,Santa Cruz)or mouse(sc-2343)control IgGs(obtained from Santa Cruz).The antibodies were applied for1h at37u C in1%(w/v)bovine serum albumin in PBS followed by incubation with a goat anti-rabbit IgG-FITC conjugate(sc-2012,Santa Cruz).Cells were mounted in antifade reagent and analysed in UV light using a standard objective. Filamentous actin of LX-2cells was stained with the Alexa Fluor H 488phalloidin conjugate(A12379,Molecular Probes,Invitrogen, Darmstadt,Germany).Nuclei were counterstained with49,6-diamidino-2-phenylindole(DAPI).Western Blot AnalysisProtein extracts were prepared from primary hMFB,LX-2, HepG2,Hep3B,Wi-38,HepG2and HUVEC cells following standard protocols.Equal amounts of proteins were heated at 80u C for10min and separated in4–12%Bis-Tris gels(Invitrogen) under reducing conditions and electro-blotted on nitrocellulose membranes(Schleicher&Schuell,Dassel,Germany).Equal protein loading was monitored in Ponceau S stain and unspecific binding sites were blocked in TBST[10mM Tris/HCl,150mM NaCl,0.1%(v/v)Tween20,(pH7.6)]containing5%(w/v)non-fat milk powder.The membranes were subsequently probed with the antibodies given in Table S1.Primary antibodies were detected with horseradish-peroxidase(HRP)-conjugated secondary antibodies(Santa Cruz)and the Supersignal TM chemiluminescent substrate(Perbio Science,Bonn,Germany).G-banding,SKY and STR AnalysisLX-2cells were trypsinized using Trypsin/EDTA and trans-ferred in multiple T25flasks and cultured for four days.The flask cultures were harvested following standard protocols including treatment with colcemid(10m g/mL),hypotonic KCl solution (0.4%)and fixative(methanol:acetic acid=3:1).G-banding by Pancreatin staining with Giemsa(GPG)was performed following standard procedures.For conventional chromosome analysis,five cells were analyzed by light microscopy and karyotyped using the CytoVision Software(Leica Microsystems).With this method an average banding resolution of250bands was achieved.Aberra-tions were described according to the International Standard of Cytogenetic Nomenclature(ISCN2013)[20].For SKY analysis, metaphases were hybridized with the24-color SKY Paint kit(ASI Applied Spectral Imaging Ltd.,Migdal HaEmek,Israel)according essentially to the manufacturer’s protocol.Spectral images of hybridized metaphases were acquired using the SkyVision II Cytogenetic workstation and analyzed with the SkyView software version1.6(ASI).In addition,images of the DAPI counter-stain of the respective metaphases were acquired as regular CCD images using the direct mode of the SkyVision II system.Inversed and band-enhanced DAPI images were used for the intrachromosomal localization of aberrant chromosome areas.Data from the SKY analysis of LX-2cells was submitted to the public SKY/M-Fish& CGH database platform of the National Cancer Institute(http:// /sky/)that was established for investigators to share and compare their molecular cytogenetic data.For STR multiplex analysis of LX-2cells,two different multiplex assays were taken.The AmpFlSTR Identifiler H PCR amplification kit that amplifies15tetranucleotide repeat loci and the Amelogenin gender-determining marker was obtained from Life Technologies.The PowerPlex H1.2System that detects nine loci(eight STR loci and Amelogenin)was obtained from PromegaSTR profile for LX-2cells was done using the online STR analysis provided by the DSMZ(http://www.dsmz.de)and the ATCC STR Profile database for human cell lines()that actually(April5,2013)contains2455(DSMZ)or1521(ATCC) human PowerPlex H1.2.system-genotyped cell lines.The STR profile of LX-2cells was submitted and deposited in the DSMZ depository.Microarray AnalysisThe Genome-Wide Human SNP Array6.0(Affymetrix,Santa Clara,CA)has been used according to the protocol provided by the manufacturer().Microarrays were washed and stained with the Fluidics Station450(Affymetrix)and scanned with the GeneChip Scanner3000(Affymetrix)using the Genotyping Console software version 4.1(Affymetrix).The Birdseed v2algorithm was used for genotyping.Copy number analysis,loss of heterozygosity(LOH)analysis and segmentation were calculated using Genotyping Console software version4.1. Segments with aberrant copy number were considered as copy number aberration only if they consisted of at least20consecutive SNPs and comprised a minimal size of100kb.Results and DiscussionLX-2Cells Express the SV40Large T-antigen and Markers Characteristic for Activated/transdifferentiated HSCLX-2cells were first introduced in2003[6]and initially characterized in2005[4]as a low-passaged human cell line derived from normal human stellate cells that are spontaneously immortalized.Cells were originally selected by their ability to grow under low serum conditions.The first preliminary description revealed that LX-2cells exhibit typical markers of stellate cells, which in primary culture express desmin and GFAP.Moreover, LX-2cells are responsive to platelet-derived growth factor BB and transforming growth factor-b1,which are the most prominent mitogenic(PDGF-BB)or pro-fibrogenic(TGF-b)stimuli towardsprimary stellate cells,respectively.The expression of a-SMA under all culture conditions indicates that LX-2cells have a partially activated phenotype[4].This conclusion is further supported by their fibroblast-like shape when grown to higher density and their somewhat star-shaped phenotype at lower cell numbers(Figure S2),as well as their high proliferative activity in culture(data not shown).In addition,when early passages of LX-2 cells were stained with fluorescently-labeled phalloidin that binds F-actin with high selectivity and affinity,all cells analysed showed a uniform staining pattern with some preferential expression in distinct areas of the cell membrane and around the nucleus(Figure S3).A similar staining pattern was observed when cells were stained with an antibody directed against a-SMA,confirming their mesenchymal and contractile(i.e.,smooth muscle cell)origin (Figure S3).In the same set of experiments,we further stained early passages of LX-2cells for SV40T Ag that was used for immortalization and found that the nuclei of all cells stained positive for this viral oncogene product demonstrating that all cells within the analysed LX-2culture are more or less homogeneous and further indicates that the T Ag is stably integrated into the genome(Figure S3).The expression of the SV40large T-antigen was also confirmed in Western blot analysis using a rabbit polyclonal antibody raised against amino acids4–30mapping near the N-terminus of SV40T Ag.In this analysis,we used extracts from Wi-38cells as a positive control representing a SV40-transformed human diploid cell line derived from embryonic lung tissue of a female Caucasian that tation antigen[21,22].In line with our immunocytochemical analysis,the Western blot revealed that LX-2cells express low levels of SV40T-Ag(Figure1),while the two human hepatoma-derived cell lines HepG2and Hep3B as well as HUVEC cells were negative for this oncogenic gene product.LX-2cells also express fibronectin,collagen type IV,collagen type I,endoglin,TGF-b receptors type II(T b RII)and I(T b RI), vimentin,desmin,a-SMA,connective tissue growth factor (CTGF),Id2,and p21(Figure1),all representing known pro-fibrogenic matricellular components or mediators involved in hepatic fibrogenesis[23–25].These results partially confirm and extend previous findings demonstrating that LX-2have attributes of primary human HSC in culture[4].In addition,the expression of the tissue inhibitor of metalloproteinase-1(TIMP-1)in LX-2 cells,as previously reported[4],was confirmed(not shown). Conventional and Molecular KaryotypingTo characterize LX-2genetically,we first performed chromo-some G-banding analysis using different LX-2cell cultures.This analysis revealed that LX-2cells have several numerical and structural chromosomal aberrations(Figure2).The karyotype as assessed by this methodology was described as:55,78,der(-X)add(X)(p22.?1),Y[4],del(1)(q4?1)[2],+del(1)(q4?1)[3], der(2;10)(p10;p10)[4],+der(2;10)(p10;p10)[2],del(3)(q25)[3], +del(3)(q25)x2[2],+der(3)add(3)(q2?7)[3], +der(3)?t(3;6)(p25;p2?3)[2],der(4)add(4)(q31)[4],+de-Figure1.Expression analysis.Cell extracts were prepared from hMFB,LX-2,HepG2,Hep3B,HUVEC and Wi-38VA-13subline2RA and analysed in Western blot for expression of(i)extracellular matrix proteins(fibronectin,collagen IV,collagen I),(ii)receptors of TGF-b (endoglin,T b RII,T b RI),(iii)HSC/MFB markers(vimentin,desmin,a-SMA, CTGF,Id2,p21),and(iv)SV40large T-antigen and b-actin.We used two parenchymatic(HepG2,Hep3B)and other cell entities(hMFB,HUVEC, and WI-38)as controls because they are well characterized for the expression of tested genes and served as positive or negative controls in this analysis.doi:10.1371/journal.pone.0075692.g001der(5)?t(5;9)(q35;q34)?del(5)(p15)[5],der(5)add(5)(p1?3)[4],+de-r(5)add(5)(p1?3)x2[2],+6[3],del(7)(p1?3)[3],+del(7)(p1?3)[2],der(9)(11q23-.11q21::14q32-.14q11.2::9p13-.9qter)[2],+der(9)(11q23-.11q21::14q32-.14q11.2::9p13-.9qter)[2],i(10)(q10)[3],del(11)(p11.2)[3],+del(12)(pter-.q12::q15-.q22::q24.2-.qter)[3],213[4],214[4],+15[2],+15[2],der(16)t(16;17)(q11.1;q11.2)[2],+der(16)t(16;17)(q11.1;q11.2)[2],+der(18)(18pter-.18q11.2::1q25-.1q32::7q22-.7qter)[2],+19[2],+20[4],+20[4],+20[2],+21[3],+8,13mar [5][cp5],respectively.In order to further characterize this complex pattern of alterations in more detail,we next performed spectral karyotyping (SKY)that is much more versatile and demanding than conventional chromosome analysis.We detected more than 160chromosomal rearrangements in eight different chromatograms by SKY indicating great genomic heterogeneity of this cell line (Table S2).However,several rearrangements between nonhomologous chromosomes were identified (Figure 3).Notably,translocation products der(9)t(9;11;14),and der(5)t(5;8;14)were frequently observed and especially der(9)t(9;11;14)was present in almost every analysed metaphase (Figure 4,Table S2).In the final SKYGRAM that displays the SKY data of all eight metaphases in the form of coloured chromosome ideograms,it becomes more clear that these chromosomal alterations mainly affect the loss of arm segments in 1q,2p,7p,10q and 14q or rearrangement of individual chromosomes,i.e.der(9)t(9;11;14)and der(8)t(5;8;11),respectively (Figure 5).Based on their common appearance,it is most likely that these rearrangements have already occurred during onset and/or early propagation of this cell line and that they are relatively stable because we observed them in different cell passages (not shown).Subsequently,we performed whole-genome SNP array analysis to confirm the different chromosomal imbalances observed by SKY.This analysis is a useful tool for studying slight variations between whole genomes and provides a signature that is characteristic for each individual.Overall,the generated copy number profile substantially agrees with the described SKYdataFigure 2.Karyogram of LX-2cell.Representative numbers and appearance of chromosomes that was found in one LX-2cell as obtained after Giemsa stain in lightmicroscopic analysis.The karyotype was determined to 74,XYY,+del(1)(q4?1),der(2;10)(p10;p10),+del(3)(q25)x2,+der(4)add(4)(q31),der(5)?t(5;9)(q35;q34)?del(5)(p15),der(5)add(5)(p1?3),+der(5)add(5)(p1?3)x2,+6,del(7)(p1?3),+del(7)(p1?3),+der(9)(11q23-.11q21::14q32-.14q11.2::9p13-.9qter)x3,+i(10)(q10),211,del(11)(p11.2),+del(12)(pter-.q12::q15-.q22::q24.2-.qter),213,214,+15,+der(16)t(16;17)(q11.1;q11.2),+der(18)(18pter-.18q11.2::1q25-.1q32::7q22-.7qter),+20,+20,+21,+21,der(22)?t(22;?)(q11.2;?),+11mar,respec-tively.Please note that a characteristic derivative of chromosome 18,i.e.+der(18)(18pter-.18q11.2::1q25-.1q32::7q22-.7qter),is marked with an arrow in this figure.A characteristic derivative of chromosome 18that contains part of chromosomes 1and 7was seen in three karyograms and is marked by a black arrow.Figure3.Spectral karyogram(SKY)of the hepatic stellate cell line LX-2.Shown is a representative image of LX-2cells hybridized with a24-color SKY probe(upper left panel).The chromosomes were counterstained with DAPI(upper right panel).The spectral images of hybridized metaphases were acquired using a cytogenetic workstation and grouped with the SkyView software(lower panel).Based on this analysis,the karyotype of this LX-2metaphase is determined to:73,3n+.,XXXYY,ish+der(1)t(1;18)(p11;q21)t(1;7)(q42;p12),der(2)t(2;10)(p11;q11),del(2)(q11), +der(3)t(3;6)(qter;q11),der(3)t(3;5)(p11;q23)t(3;5)(q24;q23)del(5)(q14),25,+del(6)(p11),der(6)t(6;14)(p11;q11),der(6)t(6;11)(q11;q12), der(7)t(7;18)(p11;p11),del(7)(q21),+der(8)t(8;5)(q11;q11)t(5;14)(q31;q23),der(8)t(4;8)(q32;p12),+der(9)t(3;9)(p22;qter)del(9)(p11), der(9)t(9;14)(p12;q11)t(14;11)(q24;q14)del(11)(q12),+der(10)del(10)(p12)del(10)(q23),der(10)t(5;10)(p12;p11),del(11)(p14),der(11)del(11)(p14)-del(11)(q23)x2,der(12)t(12;22)(q13;q12),213,214x2,+i(15)(p10),+der(15)t(15;20)(q15;q11),der(16)t(16;17)(p11;q11),del(17)(p11),219,220,221, der(21)t(21;13)(p11;q11),del(X)(q24),del(X)(q25),del(X)(q21).Please note that the derivative that contains parts of chromosomes1,18and7(cf. Figure1)were also seen in this analysis.doi:10.1371/journal.pone.0075692.g003Figure4.Median appearance of chromosome regions per cell based upon SKY analysis of eight metaphases from human LX-2cell line.The average copy numbers per cell of each human chromosome region are shown in correspondence to their chromosome ideogram based onsummary SKY analysis.(cf.Figure4and6).The comparison of results obtained in SKY (Figure4)and SNP array analysis(Figure6)indicates similar chromosomal gains in3q,5p,5q,11q,14q,17q,20and losses in 1q,7p,8q,9p,and11p.Therefore,the SKY results of the eight metaphases seem to reflect a representative intersection of the complete culture genome used for microarray analysis.It is important to note that the LX-2cell line was originally established from primary HSC that were transfected with the large SV40T-antigen in passage4[4].Subsequently,transfected cells were further passaged seven times before an individual clone(LX-1)was finally established.Afterwards LX-2was expanded as a cellular subline that was able to grow under reduced serum selection conditions induced several independent subclonal rear-rangements that were simultaneously passed during prolonged culturing and passages resulting in cellular heterogeneity.We presently do not known if the observed translocations or imbalances might be of relevance for the fibrogenic activities of LX-2or if they are simply induced by the immortalization using the large T-antigen.In addition,SKY in our analysis does not allow concise molecular definition of the respective breakpoints to the gene level,so that we do not know if these rearrangements introduce alterations in gene expression or are causative for the establishment of the immortalized phenotype of this cell line. STR analysis.Inter-and intraspecies cross-contamination inplete SKYGRAM of LX-2cells based on eight cells.Characters above rearranged chromosome ideograms are for numbers of metaphases with this aberration.Rearrangements recognized only once in SKY analysis are ignored.Based on this analysis,the complete SKYGRAM of LX-2was determined to64,83,n3+/2.,XXY,21,del(1)(q11)[3],del(2)(q11)[3],+del(3)(p11)[2],del(6)(p11)[2],del(7)(p11)[4],del(7)(q11)[2], del(7)(p15)[2],+der(7)t(7;18)(p11;p11)[3],der(8)t(5;8)(q11;q11)t(5;14)(q31;q23)[4],der(9)t(9;14)(p12;q11)t(11;14)(q14;q24)del(11)(q12)[7],del(10)(q22) [4],i(10)(q10)[2],der(11)del(11)(p11)del(11)(q22)x2[2],del(12)(q11)[2],+del(13)(q21)[2],-14,-14,+del(17)(p11)[2],+20[cp8],respectively.More detailed SKYGRAM data for LX-2cells were deposited in the SKY/M-FISH&CGH database located at the National Center for Biotechnology Information that can be found at:/sky/skyquery.cgi.doi:10.1371/journal.pone.0075692.g005and a frequent cause of false experimental outcome and scientific misinterpretation.Although this is already recognized for over60 years[26–31],urgent appeals to scientists to address this issue have not been widely accepted,including studies of the liver[32]. However,most cell lines(i.e.HeLa)that have already contributed for over60,000publications are still incompletely characterized [33].Therefore,it was recently proposed that the unambiguous cell authentication in studies using cell lines should be a consideration during peer review of papers submitted for publication or during review of grants submitted for funding[34]. To address this problem,an international expert team of scientists has therefore prepared a consensus standard for the authentication of human cells using STR profiling strategies[35]. This approach provides a simple method for rapid and cheap cell line identification[35]and further enables the establishment of a public STR profile database under the auspices of the National Center for Biotechnology information[30].Consistent with these recommendations,we next performed STR profiling for LX-2cells and all other cell lines used in this study by using two different commercially available kit systems,As expected,both kits revealed a male genotype in LX-2(Table S3). In addition,LX-2were genotyped to be homozygous for the STR loci D16S539(13),D7S820(11),TH01(9.3),vWA(17),D8S1179 (13),D2S1338(17),D18S51(12)and to be heterozygous for markers D13S317(11,13),D5S818(11,12),CSF1PO(10,12), TPOX(8,9),D21S11(28,31),D3S1358(13,15),D19S433(13, 15.2),and FGA(21,26),respectively(Table S3).A detailed STR provided by the DSMZ or ATCC revealed that this STR profile is unique for LX-2and not deposited yet in these data bases.The STR profiles for HepG2,Hep3B and Wi-38were identical to those that are given in respective ATCC and DSMZ depositories (for details see Material and Methods).Expression analysis further revealed that LX-2cells express typical markers of HSC/MFB including fibronectin,collagen type IV,collagen type I,various TGF-b receptors(i.e.endoglin,T b RII, T b RI),vimentin,desmin,a-smooth muscle actin,CTGF,and p21 suggesting that this cell line is indeed originated from hepatic stellate cells(Figure1).It is presently not possible to predict the impact of the observed genetic alterations for studies performed in LX-2cells.However, the observed chromosomal aberrations and the definition of a unique,characteristic STR profile for LX-2will now help to fulfil the recommendation for cellular authentication required by granting agencies and scientific journals when working with LX-2and to avoid unreliable experimental outcomes and scientific misinterpretation.Hopefully,our study increase the value and suitability of LX-2cells in liver research and provides an important benchmark for proper genetic characterization of widely used cell lines in the field of hepatology.Supporting InformationFigure S1Usage of LX-2cells during2003–2013.StudiesFigure6.Chromosomal imbalances detected in the LX-2cell line using a SNP6.0Array.Gains and losses are shown by red and blue triangles,respectively.doi:10.1371/journal.pone.0075692.g006。

羊毛角蛋白的提取及其成膜性

（１．ｃｏｆｌＥ舻旷ｃｈｅｍ话竹，∞伽ｉｃ口ｆＥ增ｉ聊ｅ一昭ｎ虺ｄ
ＡｂｓｔｒａｃｔＩｎｖｉｅｗｏｆｔｈｅｃｕｒｒｅｎｔｐｒｏｂｌｅｍｓａｓｓｏｃｉａＩｅｄｗｉｔｈｗｏｏｌｋｅｍｔｉｎｅｘｔｒａｃｔｉｏｎａｎｄａｐｐｌｉｃａｔｉｏｎａｎｄｆｏｒ
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ｔｈｅｐｕｒｐ０８ｅｓｏｆｉｍｐｒｏｖｉｎｇｔｈｅ
万方数据
第６期
徐恒星等：羊毛角蛋白的提取及其成膜性
・４３・
２结果与分析
２．１
氧化一还原两浴两步法提取羊毛角蛋白
按照１．３的实验步骤，在氧化温度３０℃和还原
温度ｌｏｏ℃下，采用氧化一还原法提取羊毛中的角蛋白。用尿素做羊毛溶解过程中的溶胀剂。２．１．１过硫酸盐用量对羊毛溶解率的影响按照１．３的实验步骤，采用氧化时间为６０
羊毛角蛋白的提取方法众多，常见的有氧化法、还原法（包括Ａ，Ｂ，Ｃ３种）、酸性法ｐ３、碱性法、铜氨溶液法、金属盐法和酶法等。还原Ｃ法（还原溶液中加入十二烷基硫酸钠，保护角蛋白不被氧化的溶解方法）提取率较高，角蛋白分子质量高，实用性强，是目前效果最好的制备角蛋白大分子的方法，有研究者采用该方法得到平均相对分子质量为４２
ｍｉｎ，
３０４５６０７５
帅
氯化时问／ｍｉｎ
图２氧化时问对羊毛溶解率的影响
Ｆｉｇ．２
Ｅｆｋｃｔｏｆｏｘｉｄ越ｉｏｎ６ｍｅ
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亚硫酸氢钠用量为１５％，尿素用鼍为６ｍｏＬ／Ｌ，还原时间为４ｈ的处理条件，使用不同用量的０ｘ０ＮＥ溶解羊毛，测试溶解率，得到的０ｘｏＮＥ用量和羊毛溶解率关系，结果如图１所示。当氧化剂质量分数低于６％时，随着其用量的增大，溶解率逐渐变大；在６％时溶解率达到９０％左右；之后随着用量的继续增大，溶解率反而逐渐减小。这可能是由于随着氧化剂质量分数提高，其与羊毛纤维接触更充分，且由于氧化剂分子质量较小，在氧化处理的过程中易进入羊毛纤维内部，在下一步的水洗过滤中不易被洗净，从而在纤维内部有氧化剂残留。进行后续的还原ｃ法溶解时，残余的氧化剂会消耗一部分还原剂，造成溶解率下降。故氧化剂用量为６％时，氧化一还原两浴两步法提取角蛋白的溶解率最大。

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liquid–liquid phase separation

Biophysical Chemistry 109(2004)105–1120301-4622/04/$-see front matter ᮊ2003Elsevier B.V .All rights reserved.doi:10.1016/j.bpc.2003.10.021Cloud-point temperature and liquid–liquid phase separation ofsupersaturated lysozyme solutionJie Lu *,Keith Carpenter ,Rui-Jiang Li ,Xiu-Juan Wang ,Chi-Bun Ching a ,a a b bInstitute of Chemical and Engineering Sciences,Ayer Rajah Crescent 28,࠻02-08,Singapore 139959,Singapore aChemical and Process Engineering Center,National University of Singapore,Singapore 117576,SingaporebReceived 31July 2003;received in revised form 8October 2003;accepted 16October 2003AbstractThe detailed understanding of the structure of biological macromolecules reveals their functions,and is thus important in the design of new medicines and for engineering molecules with improved properties for industrial applications.Although techniques used for protein crystallization have been progressing greatly,protein crystallization may still be considered an art rather than a science,and successful crystallization remains largely empirical and operator-dependent.In this work,a microcalorimetric technique has been utilized to investigate liquid–liquid phase separation through measuring cloud-point temperature T for supersaturated lysozyme solution.The effects of cloud ionic strength and glycerol on the cloud-point temperature are studied in detail.Over the entire range of salt concentrations studied,the cloud-point temperature increases monotonically with the concentration of sodium chloride.When glycerol is added as additive,the solubility of lysozyme is increased,whereas the cloud-point temperature is decreased.ᮊ2003Elsevier B.V .All rights reserved.Keywords:Biocrystallization;Microcalorimetry;Cloud-point temperature;Liquid–liquid phase separation1.IntroductionKnowledge of detailed protein structure is essen-tial for protein engineering and the design of pharmaceuticals.Production of high-quality pro-tein crystals is required for molecular structure determination by X-ray crystallography.Although considerable effort has been made in recent years,obtaining such crystals is still difficult in general,and predicting the solution conditions where pro-*Corresponding author.Tel.:q 65-6874-4218;fax:q 65-6873-4805.E-mail address:lujie@.sg (J.Lu ).teins successfully crystallize remains a significant obstacle in the advancement of structural molecu-lar biology w 1x .The parameters affecting protein crystallization are typically reagent concentration,pH,tempera-ture,additive,etc.A phase diagram can provide the method for quantifying the influence of solu-tion parameters on the production of crystals w 2,3x .To characterize protein crystallization,it is neces-sary to first obtain detailed information on protein solution phase behavior and phase diagram.Recently physics shows that there is a direct relationship between colloidal interaction energy106J.Lu et al./Biophysical Chemistry109(2004)105–112and phase diagram.Gast and Lekkerkerker w4,5x have indicated that the range of attraction between colloid particles has a significant effect on the qualitative features of phase diagram.A similar relationship should hold for biomacromolecules, i.e.the corresponding interaction potentials govern the macromolecular distribution in solution,the shape of the phase diagram and the crystallization process w6x.Many macromolecular crystallizations appear to be driven by the strength of the attractive interactions,and occur in,or close to,attractive regimes w7,8x.Recent intensive investigation has revealed that protein or colloidal solution possesses a peculiar phase diagram,i.e.liquid–liquid phase separation and sol–gel transition exists in general in addition to crystallization w9,10x.The potential responsible for the liquid–liquid phase separation is a rather short range,possibly van der Waals,attractive potential w11,12x.The measurement of cloud-point temperature T can provide useful informationcloudon the net attractive interaction between protein molecules,namely,the higher the cloud-point tem-perature,the greater the net attractive interaction. Herein Taratuta et al.w13x studied the effects of salts and pH on the cloud-point temperature of lysozyme.Broide et al.w14x subsequently meas-ured the cloud-point temperature and crystalliza-tion temperature for lysozyme as a function of salt type and concentration.From these works the cloud-point temperature was found to be typically 15–458C below the crystallization temperature. Furthermore,Muschol and Rosenberger w15x deter-mined the metastable coexistence curves for lyso-zyme through cloud-point measurements,and suggested a systematic approach to promote pro-tein crystallization.In general,an effective way to determine the strength of protein interactions is to study temperature-induced phase transitions that occur in concentrated protein solutions.Liquid–liquid phase separation can be divided into two stages w11x:(1)the local separation stage at which the separation proceeds in small regions and local equilibrium is achieved rapidly;and(2) the coarsening stage at which condensation of these small domains proceeds slowly to reduce the loss of interface free energy w16x.The coexisting liquid phases both remain supersaturated but differ widely in protein concentration.The effect of a metastable liquid–liquid phase separation on crystallization remains ambiguous w17x.Molecular dynamics simulations and analyt-ical theory predict that the phase separation will affect the kinetics and the mechanisms of protein crystal nucleation w18x.tenWolde and Frenkel w19x have demonstrated that the free energy barrier for crystal nucleation is remarkably reduced at the critical point of liquid–liquid phase separation, thus in general,after liquid–liquid phase separa-tion,crystallization occurs much more rapidly than in the initial solution,which is typically too rapid for the growth of single crystal with low defect densities w15x.The determination of the location of liquid–liquid phase separation curve is thus crucial for efficiently identifying the optimum solution conditions for growing protein crystals. Microcalorimetry has the potential to be a useful tool for determining:(1)the metastable-labile zone boundary;(2)the temperature-dependence of pro-tein solubility in a given solvent;and(3)the crystal-growth rates as a function of supersatura-tion w20x.Microcalorimeters can detect a power signal as low as a few microwatts whereas standard calorimeters detect signals in the milliwatt range. Because of this greater sensitivity,samples with small heat effects can be analyzed.In addition, microcalorimetry has the advantage of being fast, non-destructive to the protein and requiring a relatively small amount of material.The present work is concerned with the analysis of the transient heat signal from microcalorimeter to yield liquid–liquid phase separation information for lysozyme solutions at pH4.8.To further examine the role of salt and additive on interprotein interactions, cloud-point temperature T has been determinedcloudexperimentally as a function of the concentrations of salt,protein and glycerol.2.Materials and methods2.1.MaterialsSix times crystallized lysozyme was purchased from Seikagaku Kogyo,and used without further107J.Lu et al./Biophysical Chemistry 109(2004)105–112purification.All other chemicals used were of reagent grade,from Sigma Chemical Co.2.2.Preparation of solutionsSodium acetate buffer (0.1M )at pH 4.8was prepared with ultrafiltered,deionized water.Sodi-um azide,at a concentration of 0.05%(w y v ),was added to the buffer solution as an antimicrobial agent.Protein stock solution was prepared by dissolving protein powder into buffer.To remove undissolved particles,the solution was centrifuged in a Sigma centrifuge at 12000rev.y min for 5–10min,then filtered through 0.22-m m filters (Mil-lex-VV )into a clean sample vial and stored at 48C for further experiments.The concentration of protein solution was determined by measuring the absorbance at 280nm of UV spectroscopy (Shi-madzu UV-2550),with an extinction coefficient of 2.64ml y (mg cm )w 21x .Precipitant stock solution was prepared by dissolving the required amount of sodium chloride together with additive glycerol into buffer.The pH of solutions was measured by a digital pH meter (Mettler Toledo 320)and adjusted by the addition of small volumes of NaOH or HAc solution.2.3.Measurement of solubilitySolubility of lysozyme at various temperatures and precipitant y additive concentrations was meas-ured at pH 4.8in 0.1M acetate buffer.Solid–liquid equilibrium was approached through both crystallization and dissolution.Dissolving lasted 3days,while the period of crystallization was over 2weeks.The supernatant in equilibrium with a macroscopically observable solid was then filtered through 0.1-m m filters (Millex-VV ).The concen-tration of diluted supernatant was determined spec-troscopically and verified by refractive meter(Kruss)until refractive index remained unchanged ¨at equilibrium state.Solubility of each sample was measured in duplicate.2.4.Differential scanning microcalorimetry Calorimetric experiments were performed with a micro-differential scanning calorimeter with anultra sensitivity,micro-DSC III,from Setaram SA,France.The micro-DSC recorded heat flow in microwatts vs.temperature,thus can detect the heat associated with phase transition during a temperature scan.The sample made up of equal volumes of protein solution and precipitant solu-tion was filtered through 0.1-m m filters to remove dust particles further.To remove the dissolved air,the sample was placed under vacuum for 3min while stirring at 500rev.y min by a magnetic stirrer.The degassed sample was placed into the sample cell of 1.0ml,and a same concentration NaCl solution was placed into the reference cell.The solutions in the micro-DSC were then cooled at the rate of 0.28C y min.After every run,the cells were cleaned by sonicating for 10–15min in several solutions in the following order:deionized water,methanol,ethanol,acetone,1M KOH and finally copious amounts of deionized water.This protocol ensured that lysozyme was completely removed from the cells.The cells were then placed in a drying oven for several hours.The rubber gaskets were cleaned in a similar manner except acetone and 1M KOH were omitted and they were allowed to dry at low temperature.3.Results and discussionA typical micro-DSC scanning experiment is shown in Fig.1.The onset of the clouding phe-nomenon is very dramatic and easily detected.The sharp increase in the heat flow is indicative of a liquid–liquid phase separation process producing a latent heat.This is much consistent with many recent investigations of the liquid–liquid phase separation of lysozyme from solution w 22,23x .In fact,such a liquid–liquid phase separation is a phase transition with an associated latent heat of demixing.In this work,the cloud-point tempera-tures at a variety of lysozyme,NaCl and glycerol concentrations are determined by the micro-DSC at the scan rate of 128C y h.3.1.Effect of protein concentrationIn semilogarithmic Fig.2we plot the solid–liquid and liquid–liquid phase boundaries for lyso-108J.Lu et al./Biophysical Chemistry 109(2004)105–112Fig.1.Heat flow of a typical micro-DSC scan of lysozyme solution,50mg y ml,0.1M acetate buffer,pH 4.8,3%NaCl.The scan rate 128C y h is chosen referenced to the experimental results of Darcy and Wiencek w 23x .Note the large deflection in the curve at approximately 4.38C indicating a latent heat resulting from demixing (i.e.liquid–liquid phase separation )process.Fig.2.Cloud-point temperature and solubility determination for lysozyme in 0.1M acetate buffer,pH 4.8:solubility (5%NaCl )(s );T (5%NaCl,this work )(d );T (5%cloud cloud NaCl,the work of Darcy and Wiencek w 23x )(*);solubility (3%NaCl )(h );T (3%NaCl )(j ).cloud Fig.3.Cloud-point temperature determination for lysozyme as a function of the concentration of sodium chloride,50mg y ml,0.1M acetate buffer,pH 4.8.zyme in 0.1M acetate buffer,pH 4.8,for a range of protein concentrations.It is worth noting that,at 5%NaCl,our experimental data of T from cloud micro-DSC are quite consistent with those from laser light scattering and DSC by Darcy and Wiencek w 23x ,with difference averaging at approx-imately 0.88C.This figure demonstrates that liquid–liquid phase boundary is far below solid–liquid phase boundary,which implies that the liquid–liquid phase separation normally takes place in a highly metastable solution.In addition,cloud-point temperature T increases with the cloud concentration of protein.3.2.Effect of salt concentrationFig.3shows how cloud-point temperature changes as the concentration of NaCl is varied from 2.5to 7%(w y v ).The buffer is 0.1M acetate (pH 4.8);the protein concentration is fixed at 50mg y ml.Over the entire range of salt concentrations studied,the cloud-point temperature strongly depends on the ionic strength and increases monotonically with the concentration of NaCl.Crystallization is driven by the difference in chemical potential of the solute in solution and in the crystal.The driving force can be simplified as w 24xf sy Dm s kT ln C y C (1)Ž.eq109J.Lu et al./Biophysical Chemistry 109(2004)105–112Fig.4.The driving force required by liquid–liquid phase sep-aration as a function of the concentration of sodium chloride,50mg y ml lysozyme solution,0.1M acetate buffer,pH 4.8.In the same way,we plot the driving force,f ,required by liquid–liquid phase separation as a function of the concentration of sodium chloride in Fig.4.At the moderate concentration of sodium chloride,the driving force required by liquid–liquid phase separation is higher than that at low or high salt concentration.As shown in Fig.3,with NaCl concentration increasing,the cloud-point temperature increases,which is in accord with the results of Broide et al.w 14x and Grigsby et al.w 25x .It is known that protein interaction is the sum of different potentials like electrostatic,van der Waals,hydrophobic,hydration,etc.The liquid–liquid phase separation is driven by a net attraction between protein molecules,and the stronger the attraction,the higher the cloud-point temperature.Ionic strength is found to have an effect on the intermolecular forces:attractions increase with ionic strength,solubility decreases with ionic strength,resulting in the cloud-point temperature increases with ionic strength.It is worth noting that,the effect of ionic strength on cloud-point temperature depends strongly on the specific nature of the ions w 13x .Kosmotropic ions bind adjacent water molecules more strongly than water binds itself.When akosmotropic ion is introduced into water,the entro-py of the system decreases due to increased water structuring around the ion.In contrast,chaotropes bind adjacent water molecules less strongly than water binds itself.When a chaotrope is introduced into water,the entropy of the system increases because the water structuring around the ion is less than that in salt-free water.This classification is related to the size and charge of the ion.At high salt concentration ()0.3M ),the specific nature of the ions is much more important w 25x .The charges on a protein are due to discrete positively and negatively charged surface groups.In lysozyme,the average distance between thesecharges is approximately 10Aw 26x .As to the salt ˚NaCl used as precipitant,Na is weakly kosmo-q tropic and Cl is weakly chaotropic w 27x .At low y NaCl concentrations,as the concentration of NaCl increases,the repulsive electrostatic charge–charge interactions between protein molecules decrease because of screening,resulting in the increase of cloud-point temperature.While at high NaCl con-centrations,protein molecules experience an attrac-tion,in which differences can be attributed to repulsive hydration forces w 14,25x .That is,as the ionic strength increases,repulsive electrostatic or hydration forces decrease,protein molecules appear more and more attractive,leading to higher cloud-point temperature.At various salt concentra-tions,the predominant potentials reflecting the driving force for liquid–liquid phase separation are different.Fig.4shows that the driving force,f ,is parabolic with ionic strength,while Grigsby et al.w 25x have reported that f y kT is linear with ionic strength for monovalent salts.The possible reasons for that difference include,their model is based on a fixed protein concentration of 87mg y ml,which is higher than that used in our study,yet f y kT is probably dependent on protein concentration,besides the solutions at high protein and salt concentrations are far from ideal solutions.3.3.Effect of glycerolFig.5compares cloud-point temperature data for 50mg y ml lysozyme solutions in absence of glycerol and in presence of 5%glycerol,respec-110J.Lu et al./Biophysical Chemistry109(2004)105–112parison of cloud-point temperatures for lysozyme at different glycerol concentrations as a function of the con-centration of sodium chloride,50mg y ml,0.1M acetate buffer, pH4.8:0%glycerol(s);5%glycerol(j).Fig.6.Cloud-point temperatures for lysozyme at different glycerol concentrations,50mg y ml lysozyme,5%NaCl,0.1M acetate buffer,pH4.8.Fig.7.Cloud-point temperature and solubility determination for lysozyme at different concentrations of glycerol in0.1M acetate buffer,5%NaCl,pH4.8:solubility(0%glycerol)(s); T(0%glycerol)(d);solubility(5%glycerol)(h);cloudT(5%glycerol)(j).cloudtively.Fig.6shows the cloud-point temperature as a function of the concentration of glycerol.The cloud-point temperature is decreased as the addi-tion of glycerol.In semilogarithmic Fig.7we plot the solid–liquid and liquid–liquid phase boundaries at dif-ferent glycerol concentrations for lysozyme in0.1 M acetate buffer,5%NaCl,pH4.8,for a range of protein concentration.This figure demonstrates that liquid–liquid and solid–liquid phase bounda-ries in the presence of glycerol are below those in absence of glycerol,and the region for growing crystals is narrowed when glycerol is added. Glycerol has the property of stabilizing protein structure.As a result,if crystallization occurs over a long period of time,glycerol is a useful candidate to be part of the crystallization solvent and is often included for this purpose w28x.In addition,glycerol is found to have an effect on the intermolecular forces:repulsions increase with glycerol concentra-tion w29x.Our experiment results of solubility and cloud-point temperature can also confirm the finding.The increased repulsions induced by glycerol can be explained by a number of possible mecha-nisms,all of which require small changes in the protein or the solvent in its immediate vicinity.The addition of glycerol decreases the volume of protein core w30x,increases hydration and the size of hydration layer at the particle surface w31,32x. In this work,we confirm that glycerol shifts the solid–liquid and liquid–liquid phase boundaries. The effect of glycerol on the phase diagram strong-111 J.Lu et al./Biophysical Chemistry109(2004)105–112ly depends on its concentration and this canprovide opportunities for further tuning of nuclea-tion rates.4.ConclusionsGrowing evidence suggests protein crystalliza-tion can be understood in terms of an order ydisorder phase transition between weakly attractiveparticles.Control of these attractions is thus keyto growing crystals.The study of phase transitionsin concentrated protein solutions provides one witha simple means of assessing the effect of solutionconditions on the strength of protein interactions.The cloud-point temperature and solubility datapresented in this paper demonstrate that salt andglycerol have remarkable effects on phase transi-tions.The solid–liquid and liquid–liquid bounda-ries can be shifted to higher or lower temperaturesby varying ionic strength or adding additives.Ourinvestigation provides further information upon therole of glycerol used in protein crystallization.Glycerol can increase the solubility,and decreasethe cloud-point temperature,which is of benefit totuning nucleation and crystal growth.In continuingstudies,we will explore the effects of other kindsof additives like nonionic polymers on phasetransitions and nucleation rates.Much more theo-retical work will be done to fully interpret ourexperimental results.AcknowledgmentsThis work is supported by the grant from theNational Natural Science Foundation of China(No.20106010).The authors also thank Professor J.M.Wiencek(The University of Iowa)for kinddiscussion with us about the thermal phenomenaof liquid–liquid phase separation.Referencesw1x A.McPherson,Current approaches to macromolecular crystallization,Eur.J.Biochem.189(1990)1–23.w2x A.M.Kulkarni, C.F.Zukoski,Nanoparticle crystal nucleation:influence of solution conditions,Langmuir18(2002)3090–3099.w3x E.E.G.Saridakis,P.D.S.Stewart,L.F.Lloyd,et al., Phase diagram and dilution experiments in the crystal-lization of 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Y. Geometric stability switch criteria in delay differential systems with delay dependent p

EDOARDO BERETTA† AND YANG KUANG‡ Abstract. In most applications of delay diﬀerential equations in population dynamics, the need of incorporation of time delays is often the result of the existence of some stage structure. Since the through-stage survival rate is often a function of time delays, it is easy to conceive that these models may involve some delay dependent parameters. The presence of such parameters often greatly complicates the task of an analytical study of such models. The main objective of this paper is to provide practical guidelines that combine graphical information with analytical work to eﬀectively study the local stability of some models involving delay dependent parameters. Speciﬁcally, we shall show that the stability of a given steady state is simply determined by the graphs of some functions of τ which can be expressed explicitly and thus can be easily depicted by Maple and other popular software. In fact, for most application problems, we need only look at one such function and locate its zeros. This function often has only two zeros, providing thresholds for stability switches. The common scenario is that as time delay increases, stability changes from stable to unstable to stable, implying that a large delay can be stabilizing. This scenario often contradicts the one provided by similar models with only delay independent parameters. Key words. delay diﬀerential equations, stability switch, characteristic equations, stage structure, population models AMS subject classiﬁcations. 34K18, 34K20, 92D25 PII. S0036141000376086

calcination of pulverized limestone particles under furnace injection conditions

(Keywords: limestone; furnace injection; calcination kinetics)
Sulfur dioxide emissions from coal-fired boilers continue to be of significant regulatory concern. To achieve a moderate reduction in such emissions at a relatively low cost, dry pulverized sorbent (usually limestone) is often injected into a high-temperature region of the combustor to achieve rapid heating, calcination and sulfation of the particles. The gas temperature at the injection location is usually between 1300 and 1500K in conventional pulverized-coal-fired boilers, but it can be as high as 1700 K in heat engine applications I . To optimize sorbent performance, considerable effort has been directed towards measuring the rates, and understanding the mechanisms, of calcination and sulfation under furnace injection conditions 1-1°. The calcination of a limestone particle involves several steps, each of which is potentially rate-controlling. They are: (1) heat transfer from the bulk gas to the external surface and from the external surface to the reaction interface; (2) thermal decomposition of CaCO 3 at the reaction interface; and (3) mass transfer of CO2 from the reaction interface to the bulk gas. For small limestone particles dispersed in a high-temperature gas, external and internal heat and mass transfer rates are relatively high. For particles in the size range 1-90 #m and gas temperatures between 748 and 1273K, Borgwardt 3 suggested that the calcination process was under chemical kinetic control, and that the calcination rate was proportional to the BET surface area (determined by nitrogen adsorption at 77 K). Since limestones are not fully crystalline and contain varying porosity (but essentially no microporosity), the BET surface area is a measure of the total surface area, including the surface area contained in accessible pores. Under such condi-

Place Marketing as Governance Strategy An Assessment of Obstacles in Place Marketing

Jasper Eshuis is assistant professor inthe Department of Public Administration at Erasmus University Rotterdam. His research focuses on the governance of complex systems, with a special interest in public branding and place marketing. Together with Erik-Hans Klijn, he recently published Branding in Governance and Public Management (Routledge, 2012).E-mail: eshuis@fsw.eur.nlErik Braun is senior researcher and lecturer in urban economics and city mar-keting at the Erasmus School of Economics, Erasmus University Rotterdam. He published his doctoral dissertation on city marketing in 2008. His research interests include the application of marketing and branding concepts by cities and regions, place brand management, place brand perceptions, and the governance of place marketing as well as a broad range of urban economic issues.E-mail: braun@ese.eur.nlErik-Hans Klijn is professor in the Department of Public Administration at Erasmus University Rotterdam. His research activities focus on complex decision making, network management, public–private partnerships, branding, and the impact of media on complex decision making. He has published extensively in international journals and is author of Managing Uncertainties in Networks (with Joop F . M. Koppenjan, Routledge, 2004) and Branding in Governance and Public Management (with Jasper Eshuis, Routledge, 2012).E-mail: klijn@fsw.eur.nlPlace Marketing as Governance Strategy: An Assessment of Obstacles in Place Marketing and Their Effects on Attracting Target Groups 507Public Administration Review , Vol. 73, Iss. 3, pp. 507–516. © 2013 by The American Society for Public Administration. DOI: 10.1111/puar.12044.Jasper EshuisErik Braun Erik-Hans KlijnErasmus University Rotterdam, The NetherlandsPlace marketing is increasingly being used as a g overnance strategy for managing perceptions about regions, cit-ies, and towns. What are the most important obstacles to implementing place marketing? Based on a survey of 274 public managers involved in place marketing in the Netherlands, this article analyzes the main obstacles as perceived by public managers. It also analyzes the eﬀ ects of obstacles on perceived results of place marketing in terms of attracting target groups. A factor analysis of a variety of obstacles investigated in the survey shows three clearly demarcated obstacles: administrative obstacles within municipalities, obstacles in developing the substance of marketing campaigns, and political obstacles. Obstacles in developing the substance of the marketing campaigns have signiﬁ cant eﬀ ects on the results of place marketing in terms of attracting stakeholders, whereas the two other obstacles have no signiﬁ cant inﬂ uence.Place marketing has become a strategy widely deployed by munici-palities and regional authorities in the governance of cities, towns, and regions. It is used to increase the competitive-ness of places and attract target groups such as tourists, new residents, and investors (see, e.g., Bennett and Savani 2003; Braun 2008; Hospers 2006). It may include promotion and creating a positive image, as well as product development in the sense of developing the place in a way thatresponds to the demands of target groups (Greenberg 2008; Kavaratzis 2004; Kotler and Gertner 2002). Although place marketing has been common in the United States for more than three decades (Gold and Ward 1994), it is a relatively new and growing phe-nomenon in Europe and other parts of the world. Municipalities in smaller cities, such as Hasselt in Belgium or Randers in Denmark, have followed the lead of major cities such as London (“We are Londoners”) or Montevideo (“Montevideo for All”) (see Dinnie 2011).Place Marketing as Upcoming GovernanceStrategy Th e upsurge of place marketing can be understood in the context of the wider governance trend of introduc-ing commercial practices and private sector manage-ment styles (cf. Pierre and Peters 2000). Market-based reforms have taken place under the heading of New Public Management (see, e.g., Barzelay 2001; Pollitt, Van Th iel, and Homburg 2007), which stresses the importance of increasing eﬃ ciency in the public sector by introducing competition and approaching citizens more like customers. Customer consciousness and customer care training have become common in pub-lic services, as well as strategic marketing approaches, including business plans, market segmentation, and branding (Eshuis and Klijn 2012; Walsh 1994). In line with these governance trends, municipalities have introduced more marketing-led urban governancestrategies (Greenberg 2008). Th is has led not only to market-ing plans but also to the restruc-turing of spatial, economic, and ﬁ scal policies. Greenberg (2008) describes how the branding of New York was entwined withpro-business restructuring of(ﬁ scal) policies. Place market-ing has evolved from applyingparticular promotional techniques for purposes such as increasing tourism to marketing as an integral part of urban governance.However, the application of place marketing has not always been smooth or successful. Place marketers face multiple constraints or obstacles when trying to apply marketing strategies in a public sector context. Like other forms of governance, place marketing involvesmany diﬀ erent actors who may disagree about, for example, marketing instruments or the brand that best captures the aspired identity of the city. Another issue in place marketing is that a place is a complex “product” that may be diﬃ cult to market. Although the literature mentions quite a few diﬃ culties in placePlace Marketing as Governance Strategy: An Assessmentof Obstacles in Place Marketing and Th eir Eﬀ ectson Attracting Target GroupsPlace marketing has evolved from applying particular pro-motional techniques for pur-poses such as increasing tourism to marketing as an integral part of urban governance.marketing, no comprehensive research has been undertaken on the obstacles that public managers encounter in place marketing. Obstacles in Place Marketing and Their Effects on OutcomesTh is article aims to address this knowledge gap. Th e goal is to empirically determine the most important obstacles in place market-ing, according to professionals and politicians involved in place marketing, and their eﬀ ects on the outcome of place marketing in terms of attracting target groups.T wo main research questions are addressed:1. What are the main obstacles in place marketing?2. What is the relationship between obstacles and outcomes ofplace marketing in terms of attracting target groups?Th e article is structured as follows: Th e next section lays a theoreti-cal basis by deﬁ ning what place marketing is. Th e literature on risks, limits, and obstacles to place marketing is then discussed. Th e next section describes the research design and methodology. Th is is fol-lowed by the empirical ﬁ ndings. Th e article ends with a discussion and conclusions.Place Marketing: What Is It?Place marketing involves the application of marketing instruments to geographic locations, such as nations, cities, regions, and com-munities. Th e place marketing literature mentions a multiplicity of activities, instruments, and strategies under the heading of the mar-keting mix that can be applied to places. Th erefore, most scholars consider that marketing is broader than promotion only (see Braun 2008; Eshuis and Edelenbos 2008; Hospers 2006). Ashworth and Voogd (1990) develop a geographic marketing mix that includes not only promotional measures, but also spatial-functional measures, organizational measures,and ﬁ nancial measures intended to improvethe place and its management. Kotler, Haider,and Rein (1993) include activities aimedat improving design and service delivery, aswell as developing attractions. Th e emphasison multiple activities and strategies in placemarketing reﬂ ects the idea that mere promo-tion, without developing the product and themanagement, is not very useful if one wantsto attract people or organizations to a placeand increase competitiveness.Place Marketing: More than Communicating Favorable Images Place marketing is more than just developing favorable images and communicating them to the diﬀ erent target groups; it is not only about “selling” an image. Kotler emphasizes time and again that marketing is about fulﬁ lling consumer needs (e.g., Kotler et al. 1999). Th us, place marketing is about developing a place that ﬁ ts the needs and wants of citizens, visitors, and investors. Marketing is about responsiveness more than persuasion, although persuasion is an important part of place marketing. Th e idea behind the broader marketing approach is that marketing is much more eﬀ ective if itis targeted at what stakeholders want. Here, marketing is aboutnot only sending messages but also receiving messages. Marketing then becomes a matter of developing the place that people want and applying elements of policy making, urban planning, and place development (or product development in marketing terms). Th is makes place marketing a special governance strategy that explicitly includes the management of wider processes of urban development. Th is view is shared by scholars such as Van den Berg and Braun, who state that “urban place marketing can be seen as a managerial principle in which thinking in terms of customers and the market is central as well as a toolbox with applicable insights and techniques” (1999, 993). Here, place marketing is a way of thinking and doing that emphasizes a consumer orientation or, to put it slightly diﬀ er-ently, a demand-driven orientation. Th is article follows this line.In the words of Braun, place or city marketing is deﬁ ned as “the coordinated use of marketing tools supported by a shared customer-oriented philosophy, for creating, communicating, delivering, and exchanging urban oﬀ erings that have value for the city’s customers and the city’s community at large” (2008, 43). However, place mar-keting as a governance strategy also faces constraints and obstacles, summarized as obstacles in the next section.Exploring Obstacles in Place MarketingObstacles in place marketing arise from the governance environ-ment in which it is employed and from obstacles in place marketing strategies themselves. Th e literature on place marketing and brand-ing mentions a long list of obstacles to undertaking place marketing and achieving good outcomes from it. Th e governance literature provides insight into obstacles relating to the working of public organizations and political processes in the public sector. In this sec-tion, the main obstacles to place marketing are brieﬂ y discussed.Th e ﬁ rst thing stressed in the literature on both governance (Pierre 2000) and place marketing (Klijn, Eshuis, and Braun 2012) is that,in a governance context, multiple public andprivate parties are involved: for example, thetourist board, hotels, museums, some majorcompanies, and the municipality. A character-istic of governance processes is that actors mayhave diﬀ erent or even conﬂ icting preferences(Koppenjan and Klijn 2004; McGuire andAgranoﬀ 2011; Pierre 2000). Diﬀ erent actorsmay have diﬀ erent perceptions about theaims, strategies, and target groups of placemarketing campaigns. Th ere may be diﬀ er-ences between public and private actors, butvarious public departments also often havediﬀ erent interests and favor diﬀ erent solutions. Government institu-tions are themselves fragmented, as much of the literature on gov-ernance emphasizes (e.g., Atkinson and Coleman 1992). Th is may create obstacles. McGuire and Agranoﬀ (2011) state that depart-mental separation is a barrier to becoming a conductive organization that is capable of calibrating to the needs of its customers and the marketplace. For example, decision making can be hampered if the department of housing favors a campaign that positions the city as a nice and peaceful residential area, whereas the economics depart-ment wants to stress industrial investment opportunities.Another major barrier to interorganizational collaboration in govern-ance is what Bardach (1996) calls “turf problems,” where turf refersTh e emphasis on multipleactivities and strategies in placemarketing reﬂ ects the ideathat mere promotion, withoutdeveloping the product and themanagement, is not very usefulif one wants to attract peopleor organizations to a place andincrease competitiveness.508Public Administration Review • May | June 2013to the domain over which an agency is responsible and exercises legitimate authority. When municipal departments such as planning departments are confronted with new place marketing agencies that want to make city development more market oriented, they may be inclined to protect their turf and resist collaboration. Th is problem is more pressing when a governance strategy, as is the case with place marketing, requires coordination and anintegrated approach to be (more) successful(Braun 2008). Place branding is a relativelynew policy ﬁ eld and thus probably suﬀ ersfrom fragmentation and a lack of coordinationwith other policy activities. Th is fragmentationmay hinder eﬀ ective place marketing strate-gies and implementation (Kavaratzis 2009),as is also known from public administrationimplementation studies (Pressman and Wildavsky 1973). Th us, it is expected that one of the obstacles will relate to this fragmentation and lack of coordination within government institutions. Governance is not only about administrators and administrative bod-ies, but as much about politicians and the public. Although scholars have posited shifts of power from representative political bodies to governance networks (Hanf and Scharpf 1978; Koppenjan and Klijn 2004; Pierre 2000), political bodies still exercise a degree of control. In governance, politicians are not political executives at the apex of a government hierarchy, but they do play a role in steering policy development, determining budgets, and overseeing implementation (Scharpf 1997). Policy development in governance, including place marketing policies, requires political support and suﬃ cient approval by the citizenry. A lack of support among political representatives is an important barrier to good results in governance networks (Klijn and Koppenjan 2000; McGuire and Agranoﬀ 2011).Th e issue of lack of political support has been recognized as a prob-lem in the literature on place marketing. Braun (2008) ﬁ nds, in a study comparing four European cities, that a lack of political prior-ity for place marketing can hinder its proper embedding in wider urban governance. Lack of political priority may also lead to a lack of ﬁ nancial resources for place marketing.Apart from obstacles stemming from the governance context of place marketing, there are also constraints rooted in the nature of place marketing itself. Th e literature on place marketing highlights diﬃ culties in relation to the particular nature of places and the users of places. As Ward and Gold argue, “it is not readily apparent what the product actually is, nor how the consumption of place occurs. Th ough marketing practices make places into commodities, they are in reality complex packages of goods, services and experiences that are consumed in many diﬀ erent ways” (1994, 9). For example, a city may be seen as a tourist destination, but at the same time, it is a place of residence for many people or a location through which they need to travel when going elsewhere. In short, a place is a complex product with multiple identities (Kavaratzis and Ashworth 2005). Th e identities of a city involve, for example, the city’s history and its historic center, but also its information and communication technologies and gaming industry. Th us, the city can have diﬀ er-ent identities for diﬀ erent target groups. T ourists, for example, may value the historic center, whereas private companies may value the information and communication technology industry. A city’s multiple coexisting identities make it diﬃ cult to develop appropri-ate place marketing campaigns.Other obstacles arise from the multiplicity of uses of places and the public character of places. Local governments and other stakehold-ers may use brands to appeal to diﬀ erent groups and evoke diﬀ erentassociations with them, but this is not easy.A brand that is suitable for one group (e.g.,tourists) may not suit other groups (e.g.,residents) (Bennett and Savani 2003). In acommercial setting, marketers can choosetarget groups and ignore others, but in a pub-lic context, it may be illegitimate or impos-sible to ignore groups of residents, voters,or businesses (see also Eshuis and Edwards 2012). It often proves diﬃ cult to create marketing plans that ﬁ t the preferences of all stakeholders. Stigel and Frimann (2006) ﬁ nd this limitation of place marketing in their study of the branding of two Danish towns. Th ey conclude that the wish to arrive at a consensus about brand identities can easily lead to brands with only very gen-eral and nondescript values. Th is inhibits the eﬀ ectiveness of place marketing in terms of creating a distinguishable identity and a clear proﬁ le that makes the place stand out among its competitors.So, in the literature, many obstacles regarding place marketing are discussed. However, almost all of the literature cited earlier is based on case study research, so it remains unclear what the most signiﬁ -cant obstacles are and how diﬀ erent obstacles are interrelated. Th is article addresses this issue through quantitative survey research.Th e obstacles mentioned in the literature have been translated into survey items to identify the obstacles considered most important by practitioners, the underlying factors in the obstacles, and how they relate to outcomes of place marketing.Research Design: Methodology and Methods Organization of the SurveyTh is research is based on data collected for the ﬁ rst National City Marketing Monitor in the Netherlands 2010 (see also Braun et al. 2010; Klijn, Eshuis, and Braun 2012). Th is was a Web-based survey sent to professionals and city administrators involved in the market-ing of cities, towns, and villages. To acquire a reliable set of respond-ents actually involved in Dutch place marketing, the research was carried out in close collaboration with three organizations that provided e-mail addresses of (potential) respondents:• Th e main Dutch network for place marketing in the Neth-erlands, Netwerk City Marketing Nederland. Th is nonproﬁ tassociation for professionals working in place marketing was es-tablished in 2009. Th e association has no paid employees (except for support occasionally hired for secretarial work and account-ing). Th e ﬁ ve board members are professional marketers whodo this work on a voluntary basis. Th e association facilitates the development of a network of professionals working in place mar-keting by maintaining a Web site and a LinkedIn group (2,300members) and by regularly organizing seminars and conferences on place marketing. Th ose meetings attracted about 1,400 at-tendants over the past three years. For this research, the network provided the e-mail addresses of people who had participated in these events. Th is provided a large set of respondents.Local governments and otherstakeholders may use brands toappeal to diﬀ erent groups andevoke diﬀ erent associations withthem, but this is not easy.Place Marketing as Governance Strategy: An Assessment of Obstacles in Place Marketing and Their Effects on Attracting Target Groups 509• Th e Dutch organization for local and regional tourism oﬃ ces, VVV Nederland, the umbrella organization for municipaltourism oﬃ ces. VVV aims to further tourism and recreationin the Netherlands since it was founded in 1885. Th e national organization has about 40 employees, but more than 1,000employees work in approximately 200 local oﬃ ces (see http://www.nuzakelijk.nl/werk/2298066/medewerkers-vvv-hebben-125-jaar-cao.html). Th e local oﬃ ces engage in destinationmarketing and provide tourists with information about theplace, especially regarding recreational possibilities. Th e VVVs are often largely subsidized by municipalities. For this research, the VVV provided details of respondents within local tourism oﬃ ces who were involved in place marketing.• Th e Dutch association for local governments, VerenigingNederlandse Gemeenten (VNG). Th is is an umbrella organiza-tion for municipalities in the Netherlands that has existed since 1912 and has about 270 employees. It advocates for the inter-ests of municipalities and served as a platform for consultation and knowledge exchange among municipalities. All 408 Dutch municipalities are voluntary members of the VNG. For thisresearch, the VNG provided additional addresses of munici-palities and contact persons. Th is facilitated further researchinto people involved in place marketing.Drawing on the three diﬀ erent lists of respondents and comple-menting this with an additional search for respondents in munici-palities (using an existing network of people working in place marketing) resulted in a reasonably complete data set of 600 people involved in place marketing in the Netherlands. To be sure, it is hard to assess whether the list is complete because of the lack of oﬃ -cial registration of people working in place marketing. However, this extensive search for respondents gives reasonable conﬁ dence that at least a very large proportion of this group is included.During the ﬁ rst round, the survey was sent to the 600 names on the list. Of these, 541 were reached. In all, 274 answered at least partof the survey, giving a response rate of 51 percent. Th e high level of response can be attributed at least partly to the involvement of the aforementioned three organizations and their support for the survey. Survey RespondentsOf the 274 respondents, 168 worked for a municipality, 68 worked for a tourism oﬃ ce, and 38 worked for an organization at arm’s length—usually a foundation involved in place marketing (tourism oﬃ ces often participate in such organizations). Th e respondents had a variety of functions, varying from communication advisor to neighborhood manager, policy advisor, and city alderman. More than 53 percent of the respondents had more than two years’ experi-ence with place marketing.Larger cities are overrepresented in the survey. Th e proportion of municipalities in the Netherlands with fewer than 50,000 inhabit-ants is almost 60 percent, but the number of respondents fromthis group is only 37 percent. Th e largest cities in the Netherlands (more than 250,000 inhabitants) represent only 1.5 percent of all municipalities, whereas almost 13 percent of our respondents come from this group. Th is may not come as a surprise, however, as large municipalities tend to employ more people, including those in place marketing, than small municipalities. Although the sample may not be representative of all municipalities, it can be conﬁ dently claimed to represent the people involved in place marketing thanks to the broad coverage of professionals through their representative organiza-tions and the good response rate (51 percent).Measuring Obstacles in Place MarketingTh rough a combination of inductive and deductive steps, 11 survey items were developed to measure obstacles in place marketing. First, the literature on governance and place marketing was explored to ﬁ nd obstacles rooted in the governance context, as well as obstacles stem-ming from diﬃ culties in the marketing of places. Th ese obstacles were then organized into categories (interdepartmental issues, impact on product development, political support, budget, reaching and inﬂ uenc-ing citizens, ﬁ t with place identity, and clarity of the brand) and trans-lated into measurable items. Th e list was shown to a place marketer and another professional from the public sector (a policy advisor) to see whether the most important obstacles (as perceived by practitioners and marketers) had been included. Th e items are presented in table 1. Perceptions of the obstacles were measured on a Likert scale (“com-pletely agree,” “agree,” “neither agree nor disagree,” “disagree,” and “completely disagree”).Measuring the Attraction of Target Groups (Dependent Variable)Our dependent variable aims to measure the overall performanceof place marketing in terms of attracting target groups. Overall performance is determined by the degree to which multiple target groups are attracted. Th erefore, we created a composite dependent variable, attracting target groups, which was measured using three items for the three most important target groups in place marketing:1. Place marketing has contributed positively to attractingvisitors.2. Place marketing has contributed positively to attracting newresidents.3. Place marketing has contributed positively to attractingcompanies/ﬁ rms.Each question was measured on a Likert scale ranging from “com-pletely agree” to “completely disagree.”Table 1Items for Measuring ObstaclesItem1The budget for place marketing is too low.2It is difﬁ cult to reach consensus within the municipality about the place marketing content and strategy.3There is insufﬁ cient expertise within the municipality.4Policy departments view place marketing as a threat, they do not want place marketing to inﬂ uence their policy.5Various municipal departments do not give marketing much consideration in their communication.6Place marketing has insufﬁ cient impact on product development.7There is not enough political support.8Place marketing does not really strike a chord with the citizen.9The campaign does not ﬁ t the identity of the municipality.10The campaign does not provide a clear proﬁ le for the municipality.11The intended target groups are not reached sufﬁ ciently.Note: This article uses the term place marketing. The Dutch survey actually used the term city marketing because in the Netherlands, this is the most commonly used term to denote the marketing of places (whether it be cities, towns,v illages, or districts).510Public Administration Review • May | June 2013Factor AnalysisA factor analysis was performed to ﬁ nd the latent variables behind the 11 identiﬁ ed obstacles. In addition, a factor analysis was conducted on the three items listed earlier to derive the dependent variable, attracting target groups. As already mentioned, all 11 items for the obstacles and the three items for the dependent variable were measured on Likert scales. Both factor analyses used the polychoric correlation matrix rather than the standard Pearson correlation matrix to respect the ordinal character of the data, as suggested by Kolenikov and Angeles (2004). Th e literature has for some time been suggesting that it is incorrect to treat ordinal data as interval or ratio variables (Babakus, Ferguson, and Joereskog 1987; Muthen 1984). After estimation of the polychoric correlation matrix,1 a principal component analysis with a varimax rotation was applied to ascertain the factors. Before applying the varimax rotation, an oblique rotation (oblimin) was performed showing that the factors were not substantially correlated.2 Varimax rotation was chosen because it produces easily interpretable results.Th is procedure was ﬁ rst followed to derive the dependent variable, attracting target groups, using the three items listed earlier. To create this variable, the factor scores of the single factor emanating from this analysis were saved (with an eigenvalue of 2.19930). Bartlett’s test of sphericity, with a p-value of .000, indicated no problem of intercorrelation between the items. A Kaiser-Meyer-Olkin (KMO) measure of sampling adequacy was used to assess whether the factor analysis was good enough to proceed. Th e result of the KMO test was .608, which is above the frequently used critical threshold of .5 (Field 2009). Th e procedure and results of the factor analysis of the obstacles are discussed later.Common Source BiasBecause all of the measures in this article are based on respondents’ self-reports, there is a potential problem of common source bias.T o check for common source bias, Harman’s single-factor test (see, e.g., Andersson and Bateman 1997; Podsakoﬀ and Organ 1986)—a factor analysis with all items underlying the independent and dependent variables—was conducted. Th is analysis resulted in four diﬀ erent factors with an eigenvalue above 1 (the factor with the highest eigenvalue explained 36 percent of the variance). Th is indi-cates that common source bias is not likely to explain the research ﬁ ndings (cf. Andersson and Bateman 1997).Regression AnalysisA multivariate hierarchical regression analysis was conducted with the dependent variable, results of place marketing in terms of attracting groups. Regression analysis was chosen because of its capacity to examine the relationships between multiple variables and the possibility to control for confound-ing inﬂ uences on the relationship betweenobstacles and results of place marketing (cf.Graddy 1998). Th ree variables—the sizeof the municipality, the experience of therespondent, and the position of the respond-ent—were controlled for using hierarchicalregression analyses: the ﬁ rst model includedonly the set of three control variables, andsubsequently the set of independent variableswas added.Th e ﬁ rst control variable included was the size of the municipality, in order to control for the possibility that city marketers in larger cities may have to cope with more complex administrative conditions—for example, they have to deal with more departments within the municipality. Another possibility is that larger cities tend to be more active in place marketing than smaller ones, and this may also inﬂ u-ence perceptions of obstacles or outcomes.Th e second control variable, the respondent’s experience in place mar-keting, was included to control for the possibility that the variance in outcomes is not explained by the obstacles but by incorrect esti-mations attributable to limited experience among our respondents. One could expect experience to have an eﬀ ect on the evaluation of the outcomes by the respondents; respondents with more experience might evaluate the outcomes more correctly than those with less experience.Th e third control variable relates to the respondents’ diﬀ erent posi-tions. Th ey work in diﬀ erent organizations, and it was necessary to control for the possibility of their organizational aﬃ liation inﬂ uenc-ing their perception of obstacles or results. For example, respondents from tourism oﬃ ces may have a diﬀ erent outlook on administrative or political bottlenecks because they work in a diﬀ erent organiza-tion than those from municipalities. Th ree diﬀ erent positions match with the main categories of survey respondents: (1) public managers in municipalities (61.3 percent), (2) public managers in tourismoﬃ ces (24.8 percent), and (3) public managers in other organization (13.9 percent). Th e last group consisted largely of people working in independent associations responsible for place marketing. Dummies were created to measure this variable. Th e respondents working in municipalities were the reference group.Some Main Obstacles in Place MarketingTh e obstacles in place marketing were analyzed by ﬁ rst exploring the most important obstacles according to practitioners in place market-ing, the survey respondents. Th e underlying dimensions of obstacles were analyzed by performing factor analysis. Th is section ends by stating three hypotheses on the relation between obstacles and out-comes, and this relation is explored in the following section.What Obstacles Do Practitioners Find Most Important? Presenting the median and the mode of each obstacle, table 2 sum-marizes how respondents perceive the obstacles. Th e median separates the population of respondents into two groups with an equal number of respondents: one group that leans more toward agreeing and one group that leans more toward disagreeing that this is an obstacle. A median of 4 indicates that half of the respondents strongly agree with the proposition that this is an obstacle in their municipality, whereasthe rest of the respondents do not agree thatthis is an obstacle (choosing a value of 3, 2,or 1). Th e higher the value of the median, themore people lean toward (strongly) agreeingthat this is an obstacle. Th e mode shows themost commonly chosen value.Table 2 shows that respondents (strongly)agree most often with three propositions:the budget for place marketing is too low,it is diﬃ cult to reach consensus within theRespondents (strongly) agreemost often with three proposi-tions: the budget for place mar-keting is too low, it is diﬃ cultto reach consensus within themunicipality, and place market-ing has insuﬃ cient impact onproduct development.Place Marketing as Governance Strategy: An Assessment of Obstacles in Place Marketing and Their Effects on Attracting Target Groups 511。

task-technology fit andindividual performanceMISQ

The linkage between information technology and individual performance has been an ongoing concern in IS research. This article presents and tests a new. comprehensive model of this linkage by drawing on insights from two complementary streams of research (user attitudes as predictors of utilization and task-technology fit as a predictor of performance). The essence of this new model. called the Technology-to-Performance ChainσPC). is the assertion that for an information technology to have a positive impact on individual pe斤ormance. the technology must be utilized. and the technology must be a good fit with the tasks it supports. This new model is consistent with one proposed by DeLone and McLean (1992) in that both utilization and user attitudes about the technology lead to individual performance impacts. It goes beyond the DeLone and McLean model in two important ways. First. it highlights the importance of task-technology fit (TTF) in explaining how technology leads to performance impacts. Task-technology fit is a critical construct that was missing or only implicit in many previous models. Second , it is more explicit concerning the links between the constructs. providing a stronger theoretical basis for thinking about a number of issues relating to the impact of IT on performance. These include: making choi臼5 for surrogate measures of MIS suc臼白 I understanding the impact of user involvement on performance. and developing better diagnostics for IS problems.

1-s2.0-S193459091200063X-main

Harnessing the Mesenchymal Stem Cell Secretome for the Treatment of Cardiovascular DiseaseSudhir H.Ranganath,1,2,3,4,5,6Oren Levy,2,3,4,6Maneesha S.Inamdar,1,5,*and Jeffrey M.Karp2,3,4,*1Jawaharlal Nehru Center for Advanced Scientiﬁc Research,Jakkur,Bangalore560064,India2Center for Regenerative Therapeutics&Department of Medicine,Brigham&Women’s Hospital,Harvard Medical School,Cambridge, MA02139,USA3Harvard-MIT Division of Health Sciences and Technology,65Landsdowne Street,Cambridge,MA02139,USA4Harvard Stem Cell Institute,1350Massachusetts Avenue,Cambridge,MA02138,USA5Institute for Stem Cell Biology and Regenerative Medicine,National Center for Biological Sciences,GKVK,Bellary Road,Bangalore560065,India6These authors contributed equally to this work*Correspondence:inamdar@jncasr.ac.in(M.S.I.),jkarp@(J.M.K.)DOI10.1016/j.stem.2012.02.005The broad repertoire of secreted trophic and immunomodulatory cytokines produced by mesenchymal stem cells(MSCs),generally referred to as the MSC secretome,has considerable potential for the treatment of cardiovascular disease.However,harnessing this MSC secretome for meaningful therapeutic outcomes is challenging due to the limited control of cytokine production following their transplantation.This review outlines the current understanding of the MSC secretome as a therapeutic for treatment of ischemic heart disease.We discuss ongoing investigative directions aimed at improving cellular activity and characterizing the secretome and its regulation in greater detail.Finally,we provide insights on and perspectives for future development of the MSC secretome as a therapeutic tool.IntroductionIschemic heart disease is the leading cause of human mortality globally,resulting in about7.25million deaths each year(World Health Organization,2011).Acute myocardial infarction(AMI)is the most common cause of heart failure.AMI triggers a series of cellular and molecular changes leading to apoptosis,necrosis, and hypertrophy of cardiomyocytes;impaired neovasculariza-tion;interstitialﬁbrosis and inﬂammation;reduced contractility; and pathological remodeling.Current therapies have failed to address the devastating aftermath of AMI.Most clinically approved therapeutics focus on modulating hemodynamics to reduce early mortality but do not facilitate cardiac repair in the way that would be needed to reduce the incidence of heart failure(Velagaleti et al.,2008).It is now widely accepted that treatment of the complex pathology resulting from AMI will require taking approaches designed to enhance tissue regener-ation via cell transplantation or co-opting local mechanisms that promote healing and inhibit pathological remodeling(Wollert and Drexler,2010).Regeneration of an infarcted heart necessitates massive cell replenishment,possibly in the order of a billion cardiomyocytes, and functional integration together with supporting cell types (Laﬂamme and Murry,2005).While the search for cardiac-progenitor cells(CPCs)that can readily engraft within damaged tissue and differentiate into functioning cardiomyocytes continues(Xu et al.,2011),regenerative therapy using bone-marrow-derived mononuclear cells(BM-MNCs)and mesen-chymal stem cells(MSCs)has shown considerable promise in preclinical studies(Chavakis et al.,2010;Mirotsou et al.,2011). Theﬁrst stem-cell-based clinical trials for MI(initiated between 2002and2005)used unfractionated,easily accessible,and highly heterogeneous adult BM-MNCs.Despite initial positive results indicating safety of BM-MNC transplantation and improved cardiac function,the differences in trial design,treat-ment methods,outcome evaluation,and cell isolation have prevented general conclusions,and all of these studies require long-term follow-up analysis(Wollert and Drexler,2010). Recent clinical trials have looked at relatively homogenous MSCs expanded in culture after isolation from bone marrow (containing0.001%–0.01%MSCs)as potential cell therapy candidates for AMI owing to their immunomodulatory properties, ready availability,and cardiac stem cell(CSC)niche-regulatory ability.Theﬁrst clinical trial for AMI using human MSCs(hMSCs) demonstrated the safety of hMSC transplantation and provi-sional efﬁcacy(Hare et al.,2009).However,the improved cardiac function observed in preclinical studies is without long-term MSC engraftment(Iso et al.,2007),and,in animal studies, systemically administered MSCs exhibit low( 2%)engraft-ment levels and limited capacity for transdifferentiation into cardiomyocytes posttransplantation(Leiker et al.,2008).Thus, it seems unlikely that MSCs contribute directly to replenishing cardiomyocyte populations in the heart,and this notion moti-vated MSC-induced immunomodulatory and remodeling effects to be proposed as mechanisms of cardiovascular repair. Although the trophic and immunomodulatory properties of MSCs represent a primary mechanism of therapeutic action that is referred to in many current clinical trials(Ankrum and Karp,2010;Wollert and Drexler,2010),it is important to note that these functions of MSCs have not yet been optimized in preclinical models to maximize their therapeutic potential.The spectrum of regulatory and trophic factors secreted by MSCs,including growth factors,cytokines,and chemokines, is broadly deﬁned as the MSC secretome.A thorough in vivo examination of this MSC secretome and strategies to modulate it are still lacking,but seem essential for rational therapy design and improvement of existing therapies.Despite the absence of244Cell Stem Cell10,March2,2012ª2012Elsevier Inc.such in vivo data,current MSC-based approaches have shown some promise in preclinical models.In these cases the secre-tome was modulated by physiological(hypoxic or anoxic), pharmacological(small molecule),cytokine,or growth factor preconditioning and/or genetic manipulations(Afzal et al., 2010;Kamota et al.,2009;Shi et al.,2009;Tang et al.,2010) prior to transplantation.Nevertheless,several questions regarding MSC secretome function and regulation remain un-answered,including the following:(1)what are the most effec-tive approaches to study the MSC secretome in vivo,and are new technologies required to achieve this?(2)How do the properties of the MSC secretome(composition and sustain-ability)change in vitro and after transplantation,and how does it evolve as a function of the dynamic local microenviron-ment?(3)What are the best methods to achieve sustainability of the secretome and control over its composition posttrans-plantation?Here we discuss current understanding of the MSC secretome and put in perspective its application to cardiovascular therapy. We also review tools for MSC secretome proﬁling and current preconditioning strategies that aim to transiently control the secretome posttransplantation.Finally,we suggest approaches that could exploit the MSC secretome for cardiovasculartherapy.Figure1.A Proposed Approach for Small-Molecule-Mediated Regulation of the MSCSecretome(A)Conditioning MSCs with small molecules canstimulate the production of a customized secre-tome that can be optimized and characterizedin vitro.(B)The effect of small molecule conditioning ofMSCs can be tested under highly dynamic,simulated conditions,mimicking microenviron-ments before and after the onset of MI,and thismay include coculture assays with hypoxiccardiomyocytes and inﬂammatory cells.Proin-ﬂammatory cytokines such as TNF-a and IL-6canbe introduced to an MSC culture,for example,andMSC-secreted factors(in blue)such as sTNFR1and IL-10can be tracked,as can their ability tomodulate the release of inﬂammatory cytokinesfrom activated leukocytes.MSCs may secretemolecules such as IDO and PGE2that induceleukocytes to produce anti-inﬂammatory cyto-kines such as IL-10that attenuate the effects ofactivated leukocytes and inhibit the proin-ﬂammatory activity of constitutively secreted IL-6(from MSCs)and/or IL-6already expressed in themyocardium.Likewise,to attenuate pathologicalremodeling,one can test if small molecules canboost MSC secretion of TIMP-1to inhibit ECM-degrading proteases such as MMP-9.In additionto paracrine effects,conditioned MSCs may actthrough autocrine signaling to improve cellsurvival in hypoxic conditions.Proﬁling the MSC Secretome:Toolsand Critical ParametersTo deﬁne the speciﬁc roles of MSC-secreted factors in cardiovascularregeneration,one should start with bio-molecular proﬁling or secretome analysisof cultured primary MSCs(Figure1A).A typical MSC secretion proﬁle comprises growth factors,cyto-kines,extracellular matrix(ECM)proteases,hormones,and lipid mediators(typically in low abundance).Thus,MSC in vitro secre-tome analysis must consider the effect of serum,which contains many overlapping components and can interfere with detection. To circumvent this problem,MSCs can be cultured for a short time frame in serum-free medium or medium with deﬁned serum replacements.It is critical to consider that secretome expression in vitro is likely very different from what would be expected in vivo where cells within different microenvironments would exhibit unique secretome expression proﬁles.Conversely,as microen-vironments are highly dynamic,they would in turn impact the kinetics of secretome expression.Thus,moving forward it will be critical to examine secretome expression in vitro under condi-tions that model several relevant in vivo microenvironments. The tools available for studying secretome expression in vitro include multiplex antibody-based techniques,such as antibody arrays that offer high sensitivity(typically1–10pg/ml)as well as high speciﬁcity,reproducibility across a broad range of concen-trations,and the potential for massively parallel experimentation. High-throughput analysis of the hMSC secretome using a human cytokine antibody array,for example,identiﬁed at least40 proteins with high expression levels varying from10%to110% spot intensity relative to the negative control and normalized to Cell Stem Cell10,March2,2012ª2012Elsevier Inc.245a positive control(Parekkadan et al.,2007).Antibody arrays have also been employed to assess the contribution of MSC-derived factors such as VEGF,TIMP-1,TIMP-2,and TSP-1in cardiac improvement in swine MI models(Nguyen et al.,2010).The impact of hMSC tissue origin on secretome characteristics (bone marrow versus umbilical cord)has also been examined (Park et al.,2009)using antibody arrays.IL-8was secreted at higher concentrations in umbilical-cord-blood-derived-MSCs (UCB-MSCs),while IGBP class cytokines were speciﬁc to UCB-MSCs compared with BM-MSCs,indicating a potential origin-speciﬁc hMSC secretome.In addition to antibody-based approaches,Liquid Chroma-tography with Tandem Mass Spectrometry Detection(LC-MS/ MS)is useful for characterizing the secretome proﬁle.For example,preconditioning of human adipose-tissue-derived MSCs with TNF-a had a profound impact on the secretome de-tected using LC-MS/MS(Lee et al.,2010),and led to increased expression of cytokines and chemokines such as IL-6,IL-8, MCP-1,MMPs,PTX3,and Cathepsin L.However,LC-MS/MS was unable to detect many cytokines and growth factors that were present in low concentrations.A more systematic integrated approach for hMSC secretome analysis included LC-MS/MS detection,antibody arrays,microarrays,and bio-informatics(Sze et al.,2007),and identiﬁed201unique proteins (132using LC-MS/MS and72using antibody arrays).Impor-tantly,Sze et ed computational analysis to predict the roles of the secretome components in metabolism,immune response,and development.While current techniques have been useful to identify factors expressed at high levels such as IL-6,IL-8,TIMP-2,VEGF,and MCP-1,suggesting constitutive secretion from BM-hMSCs (Park et al.,2009),a complete list of constitutively expressed MSC secretome factors remains to be generated.Despite recent advances in the characterization of the MSC secretome,current techniques suffer from multiple deﬁciencies.Gel-based and LC-MS/MS techniques have limited sensitivity to molecules in low concentrations(10–20fmol),and antibody-based tech-niques(e.g.,ELISA and antibody arrays)are limited by the availability of antibodies to detect secreted proteins.Hence, comprehensive in vitro secretome proﬁling requires an inte-grated approach employing multiple techniques.Although determining the mechanism regulating the expression of the secretome is important,the task is made more challenging given that some of the proteins are released during cell death (Skalnikova et al.,2011).Perhaps the most important goal will be to move toward methods to proﬁle the secretome in vivo that can distinguish between factors released from the host versus those secreted by the transplanted MSCs.Reaching this point will require the development of new techniques that can directly quantify the dynamic expression proﬁle of MSC-secreted factors both locally and systemically.Close to the Heart?Relevance of MSC Paracrine Signaling to Cardiovascular TherapyRecent studies have suggested four potential mechanisms for how exogenous-culture-expanded MSCs may contribute to cardiovascular repair:MSC transdifferentiation into cardiomyo-cytes(Hatzistergos et al.,2010),fusion of MSCs with native cells (Noiseux et al.,2006),MSC-induced stimulation of endogenous CSCs via direct cell-cell interaction(Mazhari and Hare,2007), and MSC-paracrine(or endocrine)signaling(Gnecchi et al., 2005;Lee et al.,2009).MSC transdifferentiation into contractile cardiomyocytes is inefﬁcient at best(Toma et al.,2002)and occurs only in the presence of native cardiomyocytes(Hatzister-gos et al.,2010;Loffredo et al.,2011;Mazhari and Hare,2007). Cell fusion is a rare event,which rules out substantial involve-ment in MSC-mediated cardiovascular regeneration(Loffredo et al.,2011).Nevertheless,there is strong evidence emerging that rat BM-derived MSCs(rMSCs)secrete trophic factors that may induce activation and proliferation of endogenous CPCs in vitro(Nakanishi et al.,2008).Although it is possible that resident CSCs may differentiate into mature and functional cardiomyocytes upon interaction with transplanted MSCs (Hatzistergos et al.,2010),evidence suggests that CSCs possess only a limited capacity to differentiate into fully mature cardiomyocytes with an adult phenotype(Beltrami et al.,2003; Urbanek et al.,2005).Despite evidence of preferential accumu-lation of MSCs at sites of myocardial ischemia(Williams and Hare,2011),exogenously administered MSCs show poor survival and do not persist at the site of AMI(Iso et al.,2007; Terrovitis et al.,2010),probably because of the harsh ischemic microenvironment,characterized by oxidative stress,inﬂamma-tion,cytotoxic cytokines,and in some instances an absence of ECM for MSC attachment(Rodrigues et al.,2010;Song et al.,2010).Such a hostile microenvironment could hinder the interaction of MSCs with endogenous CSCs.A more plausible explanation for MSC-mediated cardiovas-cular repair is an effect on host cells and the microenvironment via MSC-secreted growth factors,cytokines,and other signaling molecules.This proposal is supported by recent preclinical studies(Kanki et al.,2011;Timmers et al.,2011)that demon-strated improved cardiac function upon infusion of cytokines or MSC-conditioned medium(without cell transplantation) (Beohar et al.,2010).Therefore,identifying key MSC-secreted factors and their functional roles in cardiovascular therapies seems a useful approach for rational design of next-generation MSC-based therapeutics.Effects of the MSC Secretome on Cardiovascular Repair The functional roles reported for MSC-secreted factors are both impressive and confusing.MSCs are known to be the source of multiple immunomodulatory agents plus trophic factors involved in repair and regenerative processes(Nauta and Fibbe,2007; van Poll et al.,2008).This broad array of secreted factors suggests possible stress response regulatory roles for MSCs, such as homing of c-kit+cells to injured myocardium(Tang et al.,2010).It is not known whether cytokines released from stressed or dying MSCs make a therapeutic contribution.The hMSC secretome includes multiple factors(Lee et al.,2010;Par-ekkadan et al.,2007;Park et al.,2009;Sze et al.,2007)known to promote cardiovascular repair(Table S1available online)and factors that negatively modulate cardiomyocyte apoptosis, inﬂammation,and pathological remodeling(Table S2).Although several factors in the MSC secretome have shown utility for inﬂuencing cardiac repair when delivered exogenously in the absence of MSCs(factors listed in Tables S1and S2not marked by an[*]),it is still critical to demonstrate the direct functionality of such factors when secreted from MSCs and the potential synergy that may exist with other secreted factors.246Cell Stem Cell10,March2,2012ª2012Elsevier Inc.In the context of cardiovascular repair,the array of potential therapeutic mechanisms offered by MSC secretome compo-nents spans tissue preservation(antiapoptotic and promitotic), neovascularization,cardiac remodeling(ECM alteration and strengthening of the infarct scar),anti-inﬂammatory responses (antiﬁbrosis and suppression of inﬂammatory cells),and the highly contentious endogenous regeneration(activation of CPCs and CSCs).MSCs induce myocardial protection by promoting cardiomyocyte survival and preventing apoptosis through activation of PKC,PI3K/Akt,NF-k B,and STAT3sig-naling(Gnecchi et al.,2008;Mirotsou et al.,2011).In ischemic animal models,MSCs mediate neovascularization via paracrine signaling(Kinnaird et al.,2004a,2004b;Matsumoto et al.,2005; Tang et al.,2005)and have antiapoptotic,anti-inﬂammatory, and antiﬁbrotic effects on cardiomyocytes and endothelial cells(Bartosh et al.,2010;Berry et al.,2006;Iso et al.,2007; Lee et al.,2009;Shabbir et al.,2009).MSC-induced immuno-modulation and antiapoptosis of cardiomyocytes that has been observed in inﬂammatory heart diseases such as acute myocarditis in mice(Van Linthout et al.,2011)and sepsis in rats(Weil et al.,2010)are likely mediated via paracrine effects. In addition,MSCs exert immunomodulatory effects by inducing neighboring cells to secrete relevant cytokines(Aggarwal and Pittenger,2005;Franc¸ois et al.,2012;Ne´meth et al.,2009; Prockop and Oh,2012),which may be useful in inhibiting excessive inﬂammation and pathological remodeling under MI settings.MSC Homing to the Injured Myocardium:The Roleof MSC SecretomeThere is signiﬁcant debate about whether MSCs need to engraft at the target site of injury or can exert their effects systemically. Engraftment at the target site would in principle seem beneﬁcial due to the potential for cell-cell contact and increased concen-trations of immunomodulatory and trophic factors.In the context of cell homing following systemic infusion,sites of MI exhibit increased expression and secretion of selective chemokines, cytokines,and cell adhesion molecules,including ICAM-1, IL-6,SDF-1,VCAM-1,and FN-1(Ip et al.,2007).However, culture-expanded MSCs exhibit limited homing capacity,prob-ably because of poor expression of receptors for chemokines and adhesion ligands such as CXCR4and CCR1.As the number of transplanted MSCs homing to the infarcted heart rapidly declines following intravenous infusion(Assis et al.,2010)due to entrapment in the microvasculature,there is a signiﬁcant need to improve circulation times and homing efﬁciency of systemically administered cells(Karp and Leng Teo,2009).For instance,genetic engineering of MSCs has been employed to overexpress key chemokine receptors such as CXCR4(Cheng et al.,2008)and CCR1(Huang et al.,2010),and growth factor preconditioning has been used(Hahn et al.,2008;Son et al., 2006)to increase MSC homing to injured myocardium and improve cardiac performance.In addition,bioengineering approaches offer signiﬁcant potential for chemically modifying the hMSC surface to improve homing to sites of inﬂammation (Sarkar et al.,2011b).Interestingly,MSCs secrete mobilizing factors such as HGF,LIF,SDF-1,SCF,and VE-Cadherin (Table S1)and thus,optimizing the transplanted MSC secre-tome could also be beneﬁcial for mobilization and homing of host MSCs.Striking a Balance between Positive and Negative FactorsSome factors in the MSC secretome,depending on the concen-tration and release kinetics,may exert inhibitory effects on the cardiac microenvironment,such as apoptosis of cardiomyo-cytes,inﬂammation,pathological remodeling,or scar formation. For instance,the TGF-b class of cytokines secreted from poly(I:C)-treated,TLR3-primed hMSCs(Waterman et al.,2010) are known to mediate pathological remodeling during MI and their repressed secretion likely results in decreased collagen deposition.MMP-2,a factor known to mediate ECM degradation during MI(Matsumura et al.,2005)resulting in pathological remodeling via cardiomyocyte anoikis and macrophage inﬁltra-tion,is endogenously secreted by hMSCs,and the activity of hMSC-secreted MMP-2can be inhibited by treating hMSCs with TNF-a or hypoxia(Lozito and Tuan,2011).Additionally, MSC-secreted factors such as MMP-9and IL-6,responsible for pathological remodeling and proinﬂammatory responses, respectively,should ideally be maintained at minimum levels because these factors are upregulated in the myocardium during MI(Biswas et al.,2010;Liu et al.,2011).Inhibition of negative factors using antagonists(produced by MSCs)such as TIMP-1 (for MMP-9)and IL-10(for IL-6)via either intracellular or extracel-lular targets is one possible strategy for alleviating these effects. Hence,it seems important to not only consider upregulating anti-inﬂammatory or proangiogenic factors,but also to strive to achieve an appropriate balance between stimulatory and inhibitory factors produced by MSCs as depicted in Figure1B. In addition to achieving such a balance through iterative in vitro experiments,ultimately the response will need to be preserved following in vivo transplantation,perhaps through bioengi-neering approaches(Sarkar et al.,2011a)and strategies illus-trated in Figure2.Bench to Bedside:Practical Considerations for Harnessing the MSC Secretome in Clinical SettingsTheﬁrst clinical trial for AMI using hMSCs was a randomized, double-blinded,placebo-controlled,dose-escalation study of allogenic hMSCs(Prochymal,Osiris Therapeutics,Inc.,Balti-more,MD)(Hare et al.,2009).This study demonstrated the safety of intravenous hMSC transplantation and provisional efﬁcacy (increased left ventricular ejection fraction[LVEF],reduced cardiac arrhythmias,and reverse remodeling compared to placebo)in AMI patients.Results from a phase II multicenter, randomized,double-blind,placebo-controlled study to evaluate Prochymal for safety and efﬁcacy are anticipated in the near future.The ongoing MSC-based trials for treatment of cardiovas-cular diseases listed in Table1reveal an interesting trend in trial designs,in which MSC paracrine mechanisms for improving angiogenesis,cardio-myogenesis,stimulating endogenous cardiac progenitors,and inhibiting remodeling have been high-lighted as the primary modes of action.The interim follow-up of two ongoing trials(NCT00677222and NCT00721045)has reported signiﬁcant improvement in cardiac functions such as LVEF and stroke volume,and a reduced number of patients with major adverse cardiac events.Nevertheless,the perfor-mance of MSCs in these clinical trials has not uniformly met expectations,because positive results and statistical sig-niﬁcance were not achieved for all output measures,the Cell Stem Cell10,March2,2012ª2012Elsevier Inc.247mechanism of action is not fully understood,and the MSC formulations are not fully optimized in terms of delivery methods,secretome composition,cell survival/persistence,and engraft-ment efﬁciency.Furthermore,little is known regarding the effect of time of MSC administration on the prevention of cardiomyo-cyte necrosis/apoptosis,which develops rapidly within 30min to 12hr following the onset of MI (Schoen,2007).Direct involvement of factors secreted by MSCs in cardiac functional improvement in humans is difﬁcult to demonstrate.Although identiﬁcation and quantiﬁcation of the myocardial tissue concentrations of paracrine factors is not feasible,their plasma levels could be indicative of their presence.Future clin-ical trials should therefore incorporate systematic analysis of the patient plasma not only to elucidate the presence/absence of MSC-secreted paracrine factors but also to investigate whether the impact on host tissue is sustained after elimination of the transplanted MSCs.Although it is challenging to determine whether paracrine factors originate from host cells or trans-planted MSCs and to characterize their impact on regulating cytokine expression from host cells (new techniques may be required),a comparative analysis of the patient plasma before and after MSC treatment may provide some insight (and be useful for establishing biomarkers for MSC therapy).Approaches for upregulating speciﬁc paracrine factors may help to elucidate (indirectly)mechanisms responsible for the MSC-mediated clinical outcomes (Mirotsou et al.,2007).Although some studies have shown that endocrine activity of dying MSCs can promote regeneration of distant ischemic tissues (Lee et al.,2009),the impact of the MSC secretome on cardiovascular repair can likely be improved through enhancing the survival of the transplanted cells and improving their homing to the target site (Karp and Leng Teo,2009).Hence,MSC modiﬁcations that lead to im-proved survival and facilitate a sustained and regulated secre-tome should be considered.‘‘Cell-free’’Therapy:An Alternate to Using MSCs?Several clinical trials have investigated cytokine therapy approaches for treating cardiovascular diseases (Beohar et al.,2010),and this is further motivated by the improvement in cardiac function seen in preclinical studies from administration of MSC-conditioned medium (Gnecchi et al.,2006).For ex-ample,VEGF protein delivery has been shown to improve angiogenesis in coronary artery disease patients (Henry etal.,Figure 2.A Proposed Engineering Solution for Sustaining a Customized MSC Secretome In Vivo and Facilitating Cardiovascular RepairBioengineering strategies may be employed to control and sustain the expression of the customized MSC secretome through smart-biomaterials-based,intra-and/or extra-cellular,controlled release of stimulating molecules.MSCs,either as single cells or aggregates,may be systemically infused or locally transplanted to facilitate cardiovascular repair with greater control over cell fate and function.2003).G-CSF,a cytokine known to mobilize progenitor cells from the bone marrow,was subsequently explored in a series of AMI clinical trials.Despiteevidence of safety and feasibility of G-CSF administration in MI patients (Valgimigli et al.,2005),treatment with G-CSF with 5or 10m g/kg/day via subcutaneous injection 5to 6days after percutaneous coronary intervention (PCI)did not yield any sig-niﬁcant increase in LVEF (Engelmann et al.,2006;Ripa et al.,2006).Other cytokines such as GM-CSF,EPO,and IGF-1have also been tested in clinical trials of cardiovascular diseases (Beohar et al.,2010).To date,single cytokine therapy trials have not met expectations,and there are several possible ex-planations for why this is the case.Multiple cytokines/growth factors may need to be administered simultaneously at different concentrations and time points to act synergistically to achieve a therapeutic effect.Side effects due to high doses of certain cytokine/growth factors,which may be required due to chal-lenges in protein delivery,can lead to the formation of aberrant and leaky vessels (Carmeliet,2005),hypotension (Henry et al.,2001),and tumor angiogenesis (Epstein et al.,2001).Controlling the local levels of exogenously delivered cytokines is critical given limitations of pharmacokinetics and stability of proteins in vivo.For instance,intramyocardially delivered,protease-resistant SDF-1was undetectable after 1day,yet controlled-release,protease-resistant SDF-1tethered to self-assembling peptide nanoﬁbers was retained within the myocardial tissue even at day 7in a rat MI model,and this persistence translated into signiﬁcant improvement in capillary density and LVEF.Controlled local release of SDF-1also led to a substantial increase in c-kit +cell recruitment into the myocardium (Segers et al.,2007).Recombinant periostin exhibited enhanced tissue distribution and persistence via controlled local delivery fromGelfoam patches in a rat MI model (Ku¨hn et al.,2007).Compared to the delivery of single growth factors or cytokines,the use of cells such as MSCs to supply these agents offers signiﬁcant potential for sustained pharmacokinetics,synergy from multiple factors,and an opportunity for systemic infusion,which is less invasive than local injection and thus amenable to repeated dosing.Secrets of the MSC Secretome:Underlying Signaling PathwaysElucidation of the molecular pathways mediating MSC secre-tome expression is a crucial step toward improving our under-standing of the secreted factor proﬁle and its clinical utility.248Cell Stem Cell 10,March 2,2012ª2012Elsevier Inc.。

On the relationship between ultrasonic and micromechanical properties of contacting rough surfaces

Journal of the Mechanics and Physics of Solids50(2002)1397–1416/locate/jmpsOn the relationship between ultrasonic and micromechanical properties of contactingrough surfacesArturo Baltazar a,Stanislav I.Rokhlin a;∗,Claudio Pecorari ba The Ohio State University,Nondestructive Evaluation Program,Edison Joining Technology Center,1248Arthur E.Adams Drive,Columbus,OH43221,USAb Institute for Advance Materials,European Commission,PO BOX2,1755ZG Petten,The NetherlandsReceived9June2001;received in revised form17September2001;accepted26September2001 AbstractIn this work,it is suggested that a unique set of the interfacial sti ness constants,K N and K T,is su cient to characterize the macroscopic elastic response of an interface between two rough contacting surfaces regardless of the direction of incidence of the ultrasonic wave.It is also shown that by combining ultrasonic spectroscopy with the theoretical procedures developed for a single imperfect interface,the sti ness constants of a double interface can be successfully recovered.The values of the sti ness constants determined from ultrasonic measurements are related to the micromechanical interaction and topography of the contacting surfaces using a micromechanical model of two rough surfaces in contact.?2002Elsevier Science Ltd.All rights reserved.Keywords:Imperfect interfaces;Contact mechanics;Elastic waves1.IntroductionThe interaction of ultrasonic waves with interfaces formed by two nonconforming, rough surfaces in contact has been the subject of numerous investigations,e.g.Kendall and Tabor(1971),Haines(1980),Arakawa(1983),Thompson and Fiedler(1984),Baik and Thompson(1984),Nagy(1992),Margetan et al.(1992),Pecorari et al.(1995), Drinkwater et al.(1997),Lavrentyev and Rokhlin(1998)and Baltazar et al.(1999). The motivations behind these studies have been various:from the assessment of the real area of contact between two rough surfaces(Kendall and Tabor,1971)to the modeling∗Corresponding author.Tel.:+1-614-292-7823;fax:+1-614-292-3395.E-mail address:rokhlin.2@(S.I.Rokhlin).0022-5096/02/$-see front matter?2002Elsevier Science Ltd.All rights reserved.PII:S0022-5096(01)00119-31398 A.Baltazar et al./J.Mech.Phys.Solids50(2002)1397–1416of crack closure near the tip of a fatigue crack(Thompson and Fiedler,1984);from the identiÿcation of the nature of interfacial imperfections in kissing and partial bonds (Nagy,1992;Margetan et al.,1992),to the generation of ultrasonic waves by contact transducers(Drinkwater et al.,1997)and application of spectroscopic techniques to study double imperfect interfaces(Lavrentyev and Rokhlin,1998;Baltazar et al.,1999). In most of these investigations,the characterization of the properties of a single interface embedded in an inÿnite medium has been attempted by studying the re ec-tion of longitudinal and shear elastic waves at normal incidence.Little attention has been given to the scattering of ultrasonic waves approaching the interface at oblique incidence.In addition,the problem concerning the interaction of ultrasonic waves with realistic complex systems such as that formed by two neighboring imperfect interfaces has been addressed only vrentyev and Rokhlin(1998)and Lavrentyev et al. (1998)used ultrasonic spectroscopy to evaluate the interfacial conditions from the spec-tra of longitudinal and shear waves re ected normally by the neighboring interfaces. They showed that the frequencies at which the minima of the normalized re ected power spectrum occur are sensitive to interfacial conditions,and,therefore,may be used for their monitoring.In most of the studies cited above,the lowfrequency response of tw o contacting surfaces was described within the framework of the quasi-static approximation(QSA) (Baik and Thompson,1984).According to the QSA,the interfacial imperfections cause a discontinuity in the displacement components(u i)that is proportional to the stress ÿeld( ik)at the interface,while for interfaces between contacting surfaces,the com-ponents of the stressÿelds are assumed to be continuous everywhere.Thus,the spring boundary conditions enforced on the plane of an imperfect interface,z=0,are zz(z=0+)= zz(z=0−)=K N[u z(z=0+)−u z(z=0−)];xz(z=0+)= xz(z=0−)=K T[u x(z=0+)−u x(z=0−)]:(1) In Eq.(1),the quantities K N and K T(N=m3)are the normal and transverse interfacial sti ness constants of the imperfect interface,respectively.The QSA does not provide any way to evaluate K N and K T,and,therefore,additional micro-mechanical modeling is needed to properly account for the e ect of the interfacial imperfections on ultrasonic scattering.Di erent micro-mechanical approaches have been developed to model the elasto-plastic behavior of rough surfaces in contact,e.g.Kendall and Tabor(1971),Haines(1980),Brown and Scholz(1985),Boitnott et al.(1992), Yoshioka and Scholz(1989),Nayak(1973),Webster and Sayles(1986),and Yoshioka (1994b).In most models the contacts between the rough surfaces are assumed to occur at the surface asperities,and their interaction is assumed to be purely elastic(Kendall and Tabor,1971;Brown and Scholz,1985;Boitnott et al.,1992;Yoshioka and Scholz, 1989).Attempts to describe plastic deformation at the asperities can also be found in the literature,e.g.Haines(1980),Nayak(1973),Webster and Sayles(1986),and Yoshioka(1994b).Other approaches that are alternative to the QSA have been developed to describe the interaction between ultrasonic waves and imperfect interfaces.Among these,the independent scatterer approximation(Rose,1990)is of particular interest here.In fact, this approach utilizes the scattering cross-section of each individual defect as a build-A.Baltazar et al./J.Mech.Phys.Solids50(2002)1397–14161399 ing block to evaluate the re ection coe cient of the coherent re ectedÿeld.It is well known that the scattering cross-section depends on the propagation direction of the incident wave with respect to the scatterer’s orientation.In this model,therefore,the angular dependence of the re ection coe cient is built into the solution through the scattering cross-section of the individual interfacial imperfections.This picture of the wave-interface interaction provides the background for a legitimate question con-cerning the QSA:do the interfacial sti ness constants used in the QSA depend also on the angle of incidence of the interrogating ultrasonic wave on the imperfect interface,or, rather,can they be considered as intrinsic properties of the interface itself?The objectives of this work are threefold.Theÿrst objective is to verify experimen-tally that the values of the interfacial sti ness constants obtained from data acquired at normal incidence may be used to predict the dynamic response of the interface even when the interrogating wave impinges on the interface at oblique incidence.The second objective concerns the extension of the results obtained on a single imperfect interface to a double interface.The third objective regards the theoretical description of the micromechanics of rough,contacting surfaces under static normal and shear loads.In particular,the aim is set to predict the correct values for the ratio between the normal and transverse interfacial sti ness constants,a goal that seems to have eluded all the models presented so far.2.Experiment2.1.Experimental setupFig.1illustrates the main features of the experimental setup used in this work.The system consists of a semi-cylindrical aluminum block that is loaded against a second block of the same material.In some experiments an aluminum layer having a thickness of the order of0:5mm is interposed between the two,so as to form a system with a double imperfect interface.The semi-cylindrical block hosts one longitudinal wave transducer for normal incidence pulse-echo measurements and two longitudinal wave transducers for oblique measurements in pitch-catch mode.A shear wave transducer for normal incidence pulse-echo measurements is located in the lower section of the experimental setup allowing simultaneous measurements with shear and longitudinal waves.The transducers’signals are recorded via a digital oscilloscope and further processed by a personal computer.The randomly rough surfaces were prepared by using two di erent procedures.Sand-paper grinding—600×grit—was used to obtain surfaces with rms roughness in the sub-micron range,while sandblasting was used to produce surfaces with rms rough-ness of a fewmicrons.C areful alignment of experimental apparatus is needed since the measurements are sensitive to parallelism of the surfaces in contact.To facilitate the correct alignment,the lower part of the system,as illustrated in Fig.1,is provided with two rotational degrees of freedom by a hemispherical contact allowing normal force transfer.The atness of the contacting surfaces is controlled with a tolerance of ±0:01mm.1400 A.Baltazar et al./J.Mech.Phys.Solids50(2002)1397–1416Applied Force(a)(b)Fig.1.Experimental setup for ultrasonic measurements:(a)mechanical system showing the location of transducers.A thin aluminum plate is placed between the two blocks when performing double interface experiments and is removed for single interface experiments,and(b)Block diagram for signal acquisition and post processing.Roughness measurements were carried out using a proÿlometer—Surface Analyzer model4000—with a trace speed of0:24mm=s.The vertical resolution of the instrument was set to be0:00049 m,and a test length of2mm was chosen.A cubic spline interpolation and a Chebyshev high pass digitalÿlter were used to eliminate noise and waviness,respectively.The statistical parameters of the surfaces were recovered by employing the digitallyÿltered data.The statistical randomness of the surface was veriÿed by performing several measurements in di erent directions over the surfaces.2.2.Single imperfect interfaceMeasurements were performed on a single interface between two surfaces having rms roughness =0:68;0:25and0:23 m.The value of the rms roughness was mea-sured prior to and after loading,and no appreciable variation of the rms roughness was recorded.Both longitudinal and shear waves were used to interrogate the interface at normal incidence.The applied pressure was varied from9to80MPa for a sample with roughness0:68 m.Fig.2shows the spectra of re ected longitudinal waves from the single interface.Results are given for longitudinal and shear waves.The symbols repre-sent the experimental values obtained at di erent values of the applied pressure,while the solid lines illustrate the predictions of the QSA model calculated with the interfacialA.Baltazar et al./J.Mech.Phys.Solids 50(2002)1397–14161401(a)-20-18-16-14-12-10A m p l i t u d e ,d BFrequency, MHz(b)A m p l i t u d e ,d BFrequency, MHzFig.2.Spectra of (a)longitudinal,and (b)shear waves re ected at normal incidence from a single interface.The roughness of the contacting surfaces is =0:68 m.sti ness constants obtained through nonlinear optimization of the QSA model with the experimental data.For values of the pressure lower than 9MPa applied to the system,in the frequency range considered in Fig.2,the interface behaves nearly as a perfectly re ecting surface and the re ected ultrasonic amplitude does not vary with frequency.This pressure threshold varies with the roughness,being less for smaller roughness.For values of the applied pressure higher than 80MPa the e ect of the contacts on the re ected spectra shows signs of saturation,i.e.the re ection amplitude becomes independent of further load increase.Following the ÿrst loading cycle,the spectra did not showappreciable variation from cycle to cycle,suggesting that the contacts w ere subjected to a fully elastic deformation (the ÿrst loading exhibited small hysteresis and was not used for data analysis).Simultaneously,experiments were performed utilizing the shear wave to deduce the transverse sti ness constant.Fig.3a reports the values1402 A.Baltazar et al./J.Mech.Phys.Solids 50(2002)1397–1416(a)I n t e r f a c i a l S t i f f n e s s [1014N /m 3]Nominal Pressure [MPa](b)I n t e r f a c i a l s t i f f n e s s [1014N /m 3]Nominal Pressure [MPa]Fig.3.Normal and transverse sti ness constants versus nominal pressure for a single and double interfaces.The data are for normal wave incidence.(a)Single interface with surface rms roughness is =0:68 m,and (b)results for a single interface with rms roughness 0:25 m (crosses)and for double interface (open circles and squares)with roughness 0:23 m.of the longitudinal and shear interfacial sti ness constants (open squares and circles,respectively)versus the applied normal load obtained in such investigations.Note that the ratio between the transverse and the normal interfacial sti ness constants,K T =K N ,increases from 0.4at 9MPa to 0.51at 80MPa as shown in Fig.10.The data for lon-gitudinal and shear sti ness for roughness =0:25 m are shown in Fig.3b together with the data for a single interface.2.3.Double imperfect interfaceFollowing the approach already used by Lavrentyev and Rokhlin (1998)and Lavren-tyev et al.(1998)on a similar system,ultrasonic spectroscopy was used in this investigation to evaluate the interfacial sti ness of a double imperfect interface from the normally re ected power spectra of both longitudinal and shear waves.The rough-ness of the four surfaces used in these measurements was estimated to be about 0:23 m.Fig.4shows three examples of typical spectra of re ected shear waves forA.Baltazar et al./J.Mech.Phys.Solids 50(2002)1397–14161403A m p l i t u d e [dB ]Frequency [MHz]Fig.4.Shear wave spectra re ected at normal incidence from a double imperfect interface.The solid lines are the best ÿtting curves according to the QSA.increasing values of the applied pressure.The solid lines are the QSA model for the optimized values of the sti ness constants.Similar results were obtained using shear waves at normal incidence.The ratio K T =K N is found to increase from 0.44to 0.55with increasing pressure as shown in Fig.10.The values of K N and K T obtained by nonlinear optimization with the QSA model of the experimental spectra are reported in Fig.3b (open circles and squares).Also,the data in Fig.3b support our assertion that the sti nesses obtained from ultrasonic single or double interfaces are the same.The small di erences between K N for a single and double interface are explained by the slightly di erent roughnesses of the two surfaces and the di erent grade of Al for the thin foil used to form the double interface.The values of K N and K T obtained at normal incidence were used to predict the spectral response of the double imperfect interface at oblique incidence.Fig.5shows an example of the measured and predicted spectra of a longitudinal wavere ected at 30◦and 40◦,respectively,from the double interface.The theoretical results reproduce the main features of the experimental spectrum,and,in particular,correctly predict the position of the minima of the spectrum.Similar results were obtained for other values of the angle of incidence.The data provide evidence that for the given surfaces in contact there exists a unique set of transverse and longitudinal sti nesses which describe sound wave interaction at arbitrary incident angle.3.Surfaces in contact:a micromechanical description 3.1.Problem statement and summit distribution functionTo relate the QSA macroscopic description of longitudinal and transverse ultra-sonic wave interactions with two pressed rough surfaces in contact with the interface1404 A.Baltazar et al./J.Mech.Phys.Solids 50(2002)1397–1416(a)A m p l i t u d e [dB ]Frequency [MHz](b)A m p l i t u d e [dB ]Frequency [MHz]Fig.5.Experimental and theoretical spectra of a longitudinal wave at 9and 79MPa for oblique incidenceat (a)30◦,and (b)40◦of incidence.The sti ness constants used in the model to calculate solid lines are those obtained at normal incidence.microscopic behavior,we employed the static model developed by Brown and Scholz (1985),for the normal closure.The model is based on the early work of Greenwood and Williamson (1966)and is used to describe the dependence of the relative approach between the mean planes of the surfaces on the applied external pressure P .For the analysis of tangential interface interaction,we deal with the elastic behavior of two rough surfaces simultaneously subjected to a constant,external normal load and to an inÿnitely small tangential force assuming transverse displacement occurs without slip.We use static micromechanical models to obtain macroscopic interfacial springs mea-sured in our experiment.To model the tangential displacement,we have included a correction factor that accounts for the misalignment between the contacting tips of the asperities,the e ect of which is to sensibly lower the interfacial tangentialA.Baltazar et al./J.Mech.Phys.Solids50(2002)1397–14161405 sti ness.In this we follow Yamada et al.(1978)who introduced a correction factor to account for the geometrical misalignment,which was also used by Yoshioka and Scholz(1989).Both Brown and Scholz’s(1985)model and our tangential loading ultrasonic model account for the topography of the two rough surfaces through aÿctitious composite surface that is deÿned by means of an appropriate algebraic sum of the proÿles of the two contacting surfaces.In this way the contacts of the real interface are trans-formed into the peaks of the composite surface.The local heights of the peaks z of the composite surface are distributed according to a probability density function’(z). Brown and Scholz(1985)used the inverted 2distribution function for the peak height proposed by Adler and Firman(1981);there was a later improvement by Aronowich and Adler(1985).In our work,a simpler expression for’(z),which represents the limiting case of the exact one when the power spectral density of the composite surface contains high frequency components,is used:’(z)=c√2n2n−1=2(z)(n−2)=2(n=2)exp−12zc=√2n;(2)where c is the composite surface rms roughnesses( c=[ 21+ 22]1=2), 1;2is the rms roughness of the two surfaces in contact(below we use the symbol for the value of the measured surface roughness),n is an integer representing the number of degrees of freedom,and z=Z max−z s where Z max is the maximum height of the composite surface and z s is the summit(peak)height.When Eq.(2)is valid,it holds for both the distribution of summits and the distribution of peaks of the surface line proÿles. One should note that the measured ultrasonic response depends mainly on the actual contact area at the summits and is thus associated with the summit distribution.While we measured the surface proÿle to determine the rms roughness we did not use the experimental proÿle peak distribution in our analysis.3.2.Normal sti nessBrown and Scholz’s model provides the dependence of applied normal pressure P on the relative approach between the two surfaces.The advantage of the inverted 2 distribution function(2)is that it is zero at z=0(z s=Z max)(at the maximum height of the composite surface,Fig.6).Thus,by using the variable substitution z=Z max−z s, the relationship between the external pressure and relative approach by Brown and Scholz can be recast in the following form:P( )=43M( −z)3=2’(z)d z:(3)HereM=Á E ÿ1=2 (4)1406 A.Baltazar et al./J.Mech.Phys.Solids50(2002)1397–1416Reference planeFig.6.Coordinates for the composite surface.z s are summit heights Z max−z s=z.The coordinate system z s shown in the left bottom corner is that of Greenwood and Williamson(1966)and Brown and Scholz(1985). The coordinate system shown in the upper right corner is as selected in the current work.Fig.7.Description of oblique contact between two semi-spheres in contact.and is the relative approach,Áis the summit density of the composite surface, ÿ1=2 is the average of the square root of the radius of curvature of a composite peak, E is the average reduced elastic modulus of contacting asperities.Fig.6illustrates the coordinate system and integration range in Eq.(3).In Eq.(3)’(z)d z is the number of summits in the strip of width d z and unit length.( −z)3=2is proportional to the local load needed to produce a deformation( −z)to a summit located between z and(z+d z).The factor is introduced(Brown and Scholz,1985)to account for the e ect of misalignment;it is deÿned by the angle of contact ,between asperity tips in contact as shown in Fig.7(Yamada et al.,1978)and is given by Eq.(5)for non-slip conditions.=cos5=2 +6GEcos1=2 sin2 :(5)Here:G E =(1− )2(2− );1G=2− 1G 1+2− 2G 2;and1E =1− 21E 1+1− 22E 2;(6)where i is the Poisson ratio,E i longitudinal and G i shear modulus.As shown by Brown and Scholz (1985),for a material like aluminum ( ≈0:3),thecorrection factor is close to one for contact angles below50◦assuming non-slip condition at the asperities.This last assumption is valid,since the ultrasonic waves used in our experiments to modulate the applied static pressure,have very small amplitudes and thus the slip does not occur at the contact as a result of ultrasonic vibration.The average values of E and ÿ 1=2in Eq.(4)can be determined only if the real distribution of such quantities is known.Similarly,the value of the summit density Áshould be assessed to obtain the factor M .Under the experimental condition,none of these quantities can be accurately and independently evaluated,especially when the two contacting rough surfaces are not accessible separately.Therefore,to invert the experimentally determined dependence of K N on the pressure P (or similarly on the relative approach ),the constant M is treated as a single parameter when analyzing the experimental data.The normal interfacial sti ness constant,K N ,is found by di erentiating P with respect to ,K N =9P 9(7)or,explicitly,from Eq.(3)K N =2 M( −z )1=2’(z )d z:(8)In Fig.8a the pressure versus the relative approach is plotted using Eq.(3)fordi erent numbers n of degrees of freedom.Plots of the normalized sti ness,K N =2M ,as a function of the applied pressure P are presented in Fig.8b.The data are for various probability density functions characterized by the same value of roughness ,but di erent values of n .It can be seen that not only the interfacial sti ness curves scale as n is increased,but the shape of the stress–sti ness curve is modiÿed as well.3.3.Transverse sti nessLet us assume the two rough surfaces are subjected to a normal loading and an in-ÿnitely small tangential force.Similar to Eq.(3),we can use the following relationship between the applied microscopic shear stress and the relative tangential displacement w x between the two rough surfaces.=Mf x (w x )’(z )d z;(9)P r e s s u r e [M P a ]δ [µm]K N x 1014[N /m 3]Pressure [MPa](b)(a)Fig.8.(a)E ect of the degree of freedom,n ,on the loading curves;(b)dependence of the normal sti ness K N on the applied pressure at di erent values of n (surfaces roughness is 0:23 m).where f x is the tangential force applied on an asperity.The quantities M and ’in Eq.(9)maintain the same meaning as in Eq.(3).In our model the tangential motion occurs due to the ultrasonic wave motion of smallamplitude (the displacement amplitude is on the Ascale).Thus,as an approximation of a single asperity contact,we can consider a tangential contact loading of spheres without slip.For two spherical bodies pressed together by a normal force and under a small tangential force the relative tangential displacement w x is proportional to the tangential force f x (Johnson,1985):f x =4aG (2− )w x ;(10)where a is the contact area radius,G is the shear modulus and is the Poisson’s ratio.Expressing the contact radius through the approach due to normal compression01020304050607080900.50.60.70.80.91.0F a c t o r ξAngle of misalignment, χ [degrees]Fig.9.E ect of the angle of misalignment ( ).of asperities we have:f x =4(1− )2−E ÿ1=2( −z )1=2w x ;(11)where E and ÿ1=2are deÿned in Eqs.(4)and (6).The other parameters are the same as in Eq.(3).The shear interaction depends on the tangential relative displacement w x ,micromechanical and topographical parameters of the interface,and,via the relative approach ,on the normal pressure P .Eqs.(10)and (11)assume that the line connecting the centers of the spheres that approximate the real asperities coincides with the direction of the normal load,and that the tangential force (f x )is parallel to the plane tangent to the spheres at the point of initial contact.In general this is not the case and a correction factor is required to account for the oblique contact due to misalignment between the shear force f x and the plane containing the contact area.Thus,we introduce a multiplier into Eq.(11),as suggested by Yamada et al.(1978),=1 =20p (x )d x =2− =2(1−sin 2 cos 2Â)d Â:(12)In Eq.(12),Âand are the angles by which the laboratory frame of reference hasto be rotated to obtain the local orthogonal frame of reference relative to a contact as depicted in Fig.7.Both Âand should be regarded as independent random variables.The function p ( )d gives the probability to ÿnd a contact between and +d 0.Fig.9shows a plot of the correction factor evaluated by using a uniform distribution of Âover the interval (− =2; =2),and p ( )= ( − 0).Both distributions are assumed to be independent of z .As shown above,however,in a real system the distribution of is expected to be shifted toward the highest values exhibited by the slope of a surface.In fact,as Brown and Scholz (1985)reported,the slope of a surface withaverage slope value as lowas13◦may reach values of angle of contact as high as 45◦.Considering that such an observation was made on data that were acquired from a one-dimensional proÿle,it is reasonable to assume that the highest values of the slope of the real surface may exceed50◦.Noting that the actual surface of contact does not exceed a fewpercent of the nominal surface,that is,only the highest peaks produce load-bearing contacts(Yoshioka,1994a),the angle may be reasonably considered to be independent of the surface height,z.Introducing Eq.(11)and the multiplier (Eq.(12))into Eq.(9),and performing all the necessary averages over the distributions of the stochastic variables,we obtain:=2 M 2(1− )2−w x( −z)1=2’(z)d z;(13)where =(1− sin2 =2).The initial transverse interfacial sti ness K T(that is,the value of K T as w x approaches zero)is the proportionality factor in the linear relation between applied stress in Eq.(13)and the relative displacement w x:K T=2 M 2(1− )2−( −z)1=2’(z)d z:(14)The initial transverse sti ness,which is measured ultrasonically,is independent of the constitutive lawused to describe the shear interaction betw een the contacts.This implies that the model’s ability to reproduce the experimental results strongly depends on the assumptions made about the probability density function’(z).Therefore,a comparison between experimental and theoretical predictions of the shear interfacial sti ness can be considered as a signiÿcant test of the validity of the assumption made about’(z).3.4.Determination of the model parameters from the ultrasonic dataFig.3presents a comparison between the values of the interfacial sti ness constants obtained from ultrasonic data(points)and the behavior of the same quantities predicted by the contact micromechanics models.The theoretical curves were obtained using the experimental rms roughness .The values of the remaining parameters,n and M, were found by using nonlinear least squared minimization in the space of the unknown model parameters:e=min∈R212n1(K i( )mod el−K exp i)2:(15)In the minimization algorithm,oneÿrst selects the set of initial guesses(M;n)and using Eq.(3)computes relative approach i=f(P i exp)for each experimental value of P i exp.This is done by solving numerically the nonlinear equation P i exp−P( i)=0 for i as a root.Next the normal sti ness K N( i)mod el is computed using Eq.(8) and the error function e is determined.The optimization continues until the error。

IL-21 production in prokaryotic hosts

专利名称：IL-21 production in prokaryotic hosts发明人：Chung Chan,Bruce L. Zamost,Douglas C.Covert,Hong Y. Liu,Karen S. De Jongh,JeffreyD. Meyer,Susan D. Holderman申请号：US10735149申请日：20031212公开号：US20060134754A1公开日：20060622专利内容由知识产权出版社提供摘要：The expression vectors and methods using an expression system for the large scale production of IL-21 are described. The vectors utilize the IL-21 coding sequence with specific changes in nucleotides in order to optimize codons and mRNA secondary structure for translation in Using the expression vectors, the IL-21 gene was produced in to a level of greater than 1 g/L in fed batch fermentation. Also included are OmpT deficient strains transformed with an IL-21 expression vector.申请人：Chung Chan,Bruce L. Zamost,Douglas C. Covert,Hong Y. Liu,Karen S. De Jongh,Jeffrey D. Meyer,Susan D. Holderman地址：Issaquah WA US,Seattle WA US,Mukilteo WA US,Seattle WA US,Seattle WA US,Lake Forest Park WA US,Seattle WA US国籍：US,US,US,US,US,US,US更多信息请下载全文后查看。

Coliforms—E.coli2

Coliforms—E. coliPage 1 of 6Modified m-TEC prepared Agar Plates for E. coli in recreational watersA revised single-step method uses modified m-TEC Agar to detect E. coli in recreational fresh water samples. The modified m-TEC Agar contains an enzymatic substrate which is cleaved to produce colonies. Red or magenta colored colonies specifically represent the presence of E. coli . Confirmation steps are not required with this method.Coliforms—E. coliDOC316.53.001211USEPA Membrane Filtration MethodMethod 836711USEPA acceptedmodified m-TECScope and Application:For potable water, nonpotable water, recreation water and wastewaterTest preparationBefore starting the test:When the sample is less than 20 mL (diluted or undiluted), add 10 mL of sterile dilution water to the filter funnel before applying the vacuum. This aids in distributing the bacteria evenly across the entire filter surface.The volume of sample to be filtered will vary with the sample type. Select a maximum sample size to give 20 to 200colony-forming units (CFU) per filter. The ideal sample volume of nonpotable water or wastewater for coliform testing yields 20–80 coliform colonies per filter. Generally, for finished, potable water, the volume to be filtered will be 100 mL.Disinfect the work bench with a germicidal cloth, dilute bleach solution, bactericidal spray or dilute iodine solution. Wash hands thoroughly with soap and water.Coliforms—E. coli Page 2 of 6Coliforms—E. coliModified m-TEC for E. coli in recreational waters, method 83671.Set up the Membrane Filter Assembly. Refer to Introduction to Coliforms . Using sterilized forceps, place a membrane filter, grid side up, into the assembly.To sterilize forceps, dip forceps into alcohol and flame in an alcohol or Bunsen burner. Let forceps cool before use.2.Prepare the necessary dilutions to obtain 20–80 E.coli colonies on the membranes.Invert the sample for 30 seconds to mix.Pour the sample into the funnel. Apply vacuum and filter the sample. Rinse the funnel walls with 20 to 30mL of sterile buffered dilution water. Apply vacuum. Repeat rinsing step, two more times.Release the vacuum when the filter is dry to prevent damage to the filter.3.Turn off the vacuum and lift off the funnel top. Using sterilized forceps, transfer the membrane filter to the modified m-TEC prepared agar plate.4.With a slight rolling motion, place the filter, grid side up, on the agar plate. Check for air trapped under the filter and make sure the entire filtertouches the agar. Replace the petri dish lid.5.Invert the petri dish. Incubate at 35±0.5°C for 2hours, then at 44.5±0.2 °C for 22hours.6. Count the number of red or magenta colonies by using a 10 to 15X microscope.7.The presence of these colonies confirms E.coli . Record the results of the test. See Interpreting and reporting results.Coliforms—E. coliColiforms—E. coliPage 3 of 6Interpreting and reporting resultsReport coliform density as the number of colonies per 100 mL of sample. For total coliforms, use samples that produce 20 to 80 coliform colonies, and not more than 200 colonies of all types, per membrane to compute coliform density. For fecal coliform testing, samples should produce 20 to 60 fecal coliform colonies.Use Equation A to calculate coliform density. Note that “mL sample” refers to actual sample volume, and not volume of the dilution.Equation A—Coliform density on a single membrane filter•If growth covers the entire filtration area of the membrane, or a portion of it, and colonies are not discrete, report results as “Confluent Growth With or Without Coliforms.”•If the total number of colonies (coliforms plus non-coliforms) exceeds 200 per membrane or the colonies are too indistinct for accurate counting, report the results as “Too Numerous To Count” (TNTC).In either case, run a new sample using a dilution that will give about 50 coliform colonies and not more than 200 colonies of all types.When testing nonpotable water, if no filter meets the desired minimum colony count, calculate the average coliform density with Equation B.Equation B—Average coliform density for 1) duplicates, 2) multiple dilutions, or 3) more than one filter/sampleControls:Positive and negative controls are important. Pseudomonas aeruginosa is recommended as a negative control and Escherichia coli as a positive control. Use the AQUA QC-STIK™ Device for quality control procedures. Instructions for use come with each AQUA QC-STIK Device.Potable water samples from municipal treatment facilities should be negative for total coliforms and fecal coliforms.Coliform colonies per 100 mL Coliform colonies countedmL of sample filtered--------------------------------------------------------------------100×=Coliform colonies per 100 mL Sum of colonies in all samplesSum of volumes (in mL) of all samples-----------------------------------------------------------------------------------------------------100×=Coliforms—E. coliConsumables and replacement itemsRequired media and reagentsDescription Unit Catalog number m-TEC, modified, prepared agar plates, 47-mm15/pkg2811815Required apparatusDescription Unit Catalog number Counter, hand tally each1469600 Dilution Water, buffered, sterile, 99 mL 25/pkg1430598 Dish, Petri, with pad, 47-mm, sterile, disposable, Gelman100/pkg1471799 Dish, Petri, with pad, 47-mm, sterile, disposable, Millipore150/pkg2936300 Filter Holder, magnetic coupling (use with 24861-00)each1352900 Filters, Membrane, 47-mm, 0.45-µm, gridded, sterile, Gelman200/pkg1353001 Filters, Membrane, 47-mm, 0.45-µm, gridded, sterile, Millipore150/pkg2936100 Filtering Flask, 1000-mL each54653 Forceps, stainless steel each2141100 Incubator, Culture, 120 VAC, 50/60 Hz each2619200 Incubator, Culture, 220 VAC, 50/60 Hz each2619202 Microscope, Compound each2942500 Pump, vacuum/pressure, portable, 115 VAC, 60 Hz each2824800 Pump, vacuum/pressure, portable, 220 VAC, 50 Hz each2824802 Stopper, rubber, one hole, No. 86/pkg211908 Tubing, rubber, 0.8 cm ID 3.7 m (12 ft)56019Optional media, reagents and apparatusDescription Unit Catalog number Adapter for rechargeable battery pack, 230 VAC (for 2580300)each2595902 Alcohol Burner12087742 Aspirator, water each213102 Autoclave, 120 VAC, 50/60 Hz each2898600 Bag, for contaminated items200/pkg2463300 Bags, Whirl-Pak®, without dechlorinating agent, 207 mL100/pkg2233199 Bags, Whirl-Pak®, without dechlorinating agent, 720 mL10/pkg1437297 Battery eliminator each2580400 Battery pack, rechargeable, for portable incubator 12 VDC each2580300 Bottle, sample, sterilized, 100-mL, disposable with dechlorinating agent12/pkg2599112 Bottle, sample, sterilized, 100-mL, disposable with dechlorinating agent50/pkg2599150 Bottle, sample, sterilized, 100-mL, disposable12/pkg2495012 Bottle, sample, sterilized, 100-mL, disposable50/pkg2495050 Bunsen burner with tubing each2162700Coliforms—E. coliPage 4 of 6Coliforms—E. coliOptional media, reagents and apparatus (continued)Description Unit Catalog number Dechlorinating Reagent Powder Pillows100/pkg1436369 Dish, Petri, 47-mm, sterile, disposable100/pkg1485299 Dish, Petri, 47-mm, sterile, disposable500/pkg1485200Filter Funnel Manifold, aluminum, 3-place (use with 1352900)each2486100Filter Unit, sterile, disposable with gridded membrane (use with 2656700)12/pkg2656600 Filtration Support (for field use), stainless steel each2586200 Funnels, Push-Fit and membrane filters (use with 2586200)72/pkg2586300 Germicidal Cloths50/pkg2463200 Incubator, portable, 12 VDC each2569900 Incubator, water bath, 120 VAC, 50/60 Hz each2616300 Isopropyl alcohol500 mL1445949m-ColiBlue24® Broth, 100 mL glass bottle 1 each 2608442 Pad, absorbent, with dispenser1000/pkg1491800 Powder Pillows for buffered dilution water (25 of each)150/pkg2143166 Pump, hand vacuum each1428300 Sterilization Indicator, Sterikon®15/pkg2811115 Sterilization Indicator, Sterikon®100/pkg2811199 Syringe, 140-mL, polypropylene (use with 2586200)each2586100 Wicks, replacement, for alcohol burner 2087742—20978101Add the contents of one potassium dihydrogen phosphate and one magnesium chloride powder pillow to one liter of distilled water and autoclave (sterilize) to prepare American Public Health Association buffered dilution water.Coliforms—E. coliPage5 of 6©Hach Company,2007. All rights reserved. Printed in the U.S.A.Updated February2008,Edition5。

观赏羽衣甘蓝红叶性状的转录组分析

㊀㊀2021年第62卷第9期1783收稿日期:2021-07-09基金项目:浙江省自然科学基金青年基金(LQ19C150002)㊂作者简介:谭晨(1988 ),男,湖北天门人,助理研究员,博士,从事园林植物遗传与育种研究工作,E-mail:tanch@㊂通信作者:朱开元(1972 ),男,浙江杭州人,副研究员,硕士,从事园林植物栽培与育种研究工作,E-mail:kyzhu1999@㊂文献著录格式:谭晨,刘慧春,张加强,等.观赏羽衣甘蓝红叶性状的转录组分析[J].浙江农业科学,2021,62(9):1783-1788.DOI:10.16178/j.issn.0528-9017.20210933观赏羽衣甘蓝红叶性状的转录组分析谭晨,刘慧春,张加强,周江华,朱开元∗(浙江省农业科学院浙江省园林植物与花卉研究所,浙江杭州㊀311202)㊀㊀摘㊀要:观赏羽衣甘蓝是一种常用的观叶园林植物,但其叶片呈色机理还不清楚㊂本研究通过对羽衣甘蓝红叶和白叶材料不同部位的叶片(内叶和外叶)进行转录组分析,共获得89.58Gb 的有效测序数据,87%以上的序列能够比对到甘蓝参考基因组上㊂通过对红叶和白叶材料基因的差异表达分析,在内叶和外叶中分别鉴定到3726和2851个差异表达基因,其中1665个为共同差异表达基因㊂此外,我们在羽衣甘蓝中鉴定到175个花青素苷合成途径相关的基因,其中有25个为差异表达基因,而基因Bo 3g 024670(C 4H )㊁Bo 6g 100940(MYB 75)㊁Bo 9g 008920(5MAT )㊁Bo 2g 116380(DFR )㊁Bo 6g 112670(MYBL 2)㊁Bo 6g 010860(ACC 1)和novel.283(CHI-L 1)为共同差异表达基因,说明它们可能在羽衣甘蓝红叶性状形成中起到关键作用,为进一步解析羽衣甘蓝叶色形成机理奠定了基础㊂关键词:羽衣甘蓝;红叶;花青素;转录组中图分类号:S681.9;S184㊀㊀㊀文献标志码:A㊀㊀㊀文章编号:0528-9017(2021)09-1783-06㊀㊀羽衣甘蓝(Brassica oleracea var.acephala,2n =18)是十字花科芸薹属2a 生观叶植物,叶形酷似牡丹,又名叶牡丹㊂羽衣甘蓝喜欢冷凉气候,是能够应用于冬季花坛的为数不多的园林绿化材料㊂观赏羽衣甘蓝叶色绚丽,边缘叶有翠绿色㊁深绿色㊁灰绿色㊁黄绿色,中心叶则有纯白㊁淡黄㊁肉色㊁粉红㊁玫瑰红㊁紫红等及复色品种[1]㊂羽衣甘蓝叶色方面的研究还较少,相关色素的积累与代谢平衡的分子机制有待深入研究㊂近年来,国内外研究者对羽衣甘蓝叶色的呈色机理进行了探索研究,主要集中在生理生化测定以及少量的基因定位研究㊂例如,王雅琼[1]对不同品种羽衣甘蓝生长期的叶绿素㊁类胡萝卜素和花青素等色素含量变化进行了系统的研究,从生理学角度阐述了观赏羽衣甘蓝内叶呈现紫色和黄色是叶绿素降解㊁花青素积累㊁类胡萝卜素含量下降的幅度较叶绿素低造成的㊂Zhang 等[2]通过基因表达研究,推测一个花青素代谢途径中MYB 转录调控因子BoPAP 1在低温诱导羽衣甘蓝花青素合成和积累过程中发挥重要作用㊂Liu 等[3]通过构建F 2分离群体,发现羽衣甘蓝紫色性状是由一对显性基因控制的,将紫叶基因BoPr 精细定位到了C09染色体上280kb 的区间,并确认候选基因为Bo 9g 058630(DFR 基因),是一个花青素苷合成途径中的基因㊂Ren 等[4]也通过F 2分离群体将红叶基因Re 定位在1.8cM 区间,候选基因也是DFR 基因㊂这些研究表明,花青素苷的生物合成与积累是羽衣甘蓝红/紫叶色形成的关键㊂花青素苷(anthocyanin)是一种植物体内广泛存在的水溶性类黄酮化合物,多分布于植物的花㊁果实㊁种子㊁叶等组织器官中,赋予植物红色㊁紫色㊁蓝色等不同的色泽[5-6]㊂植物花青素苷的代谢合成途径已较为清楚,花青素苷在细胞质中以苯丙氨酸为底物,再经羧基化㊁糖基化㊁甲基化㊁酰基化修饰后转运至液泡㊂催化这一系列反应的酶类属于结构基因,包括早期基因(PAL ㊁C 4H ㊁4CL ㊁CHS ㊁CHI ㊁F 3H ㊁F 3ᶄH )和晚期基因(DFR ㊁ANS ㊁UFGT )等,这些基因在不同植物种类中较为保守[5]㊂花青素苷的合成还需要转录因子的调控,其中研究最广泛㊁最清楚的主要包括MYB㊁bHLH 和WD40三类转录因子㊂在模式植物拟南芥中,多个调控花青素苷合成的MYB㊁bHLH 和WD40转录因子被鉴定出来:第一类MYB 转录因子包括MYB 75㊁MYB 90㊁MYB 113㊁MYB 114㊁1784㊀㊀2021年第62卷第9期MYB11㊁MYB12㊁MYB111等;第二类bHLH转录因子包括TT8㊁GL3㊁EGL3;第三类WD40转录因子为TTG l㊂大部分花青素苷合成途径的结构基因如DFR㊁ANS㊁UFGT等的转录均是由这三类转录因子组成的蛋白复合体MYB-bHLH-WD40(MBW)调控[6-9]㊂比如在拟南芥幼苗和叶中,在花青素苷含量高的材料中参与花青素苷生物合成的R2R3-MYB蛋白PAP1㊁PAP2㊁MYB113和MYB114,bHLH蛋白TT8㊁EGL3和GL3以及WD40蛋白TTG1均上调表达[10]㊂其他物种中也存在类似的调控机制[6]㊂尽管目前已经有了少量羽衣甘蓝叶色研究的报道[3,11-13],但羽衣甘蓝的花青素苷合成途径的关键基因及相关分子调控网络尚不清楚,有待进一步研究㊂本研究拟通过对羽衣甘蓝生长发育的不同时期花青素苷的积累水平进行比较研究,通过RNA-Seq 的方法从转录水平上揭示羽衣甘蓝叶色形成的基因表达差异,筛选出羽衣甘蓝花青素苷生物合成与积累的关键基因及调控因子,以期在分子水平上解析羽衣甘蓝叶色形成及调控的机制,为羽衣甘蓝叶色遗传改良提供理论依据㊂1㊀材料与方法1.1㊀材料㊀㊀本研究选取2种不同叶色的羽衣甘蓝品种为研究对象:红叶品种(内叶红色㊁外叶绿色)和白叶品种(内叶白色㊁外叶绿色),每个品种30株(设置3个重复,每个重复10株),在观赏期(播种后约90d)随机选取2个品种各3~5个单株,分别剪取内㊁外叶片用锡纸包裹后迅速投入液氮中速冻,于-80ħ冰箱中保存备用,这些材料用于后续生理指标测定和总RNA提取㊂1.2㊀花青素苷含量测定㊀㊀采用UV分光光度计在536nm和700nm下测吸光值,根据pH示差法计算花青素苷总含量㊂1.3㊀高通量测序及数据分析㊀㊀对2个羽衣甘蓝品种材料的内叶和外叶的4个样品(3个生物学重复/样)共计12份样,分别进行转录组文库构建和高通量测序㊂通过FastQC等分析软件对原始数据文件进行质量控制,获得高质量的有效序列(clean reads)㊂将得到的有效序列通过HISAT2软件与甘蓝参考基因组进行比对,采用Subread软件获得基因的表达量数据,归一化后的表达量数据使用DEGSeq2软件包对不同材料㊁不同组织部位的组合进行基因差异表达分析,对差异表达基因进行GO/KEGG注释㊂2㊀结果与分析2.1㊀花青素苷在红叶材料中大量积累㊀㊀本研究选取叶色不同的2个观赏羽衣甘蓝材料作为研究对象:红叶材料(A为红色内叶,B为绿色外叶)和白叶材料(C为白色内叶,D为绿色外叶)(图1中a)㊂各样品中的总花青素苷含量也明显不同(图1中b),色素含量测定结果显示,4个样品中样品A的总花青素苷含量最高,其次是B 和D,而样品C的含量最低;进一步分析表明红叶材料中内叶(A)的花青素苷含量比白叶材料内叶(C)高出20倍以上,差异极显著;此外,红叶材料外叶(B)的花青素苷含量也显著高于白叶材料外叶(D)㊂因此,红叶材料的花青素苷含量显著高于白叶材料,也就是说花青素苷的积累是羽衣甘蓝红叶产生的主要原因,这与前人的研究结论一致[1,11-12]㊂a 红叶和白叶材料观赏期表型,标尺=5cm;b 各样品总花青素苷含量,∗∗表示与A样品比较,P<0.01㊂图1㊀表型及花青素苷含量测定2.2㊀转录组测序与数据分析㊀㊀本研究构建了12个转录组文库并在Illumina HiSeq2500平台上测序,各样品获得了41~56百万个有效序列,一共获得89.58Gb的有效测序数据;测序数据的质量分析结果显示,Q30碱基百分比在92%以上,平均GC含量为46.82%,表明本研究中测序数据质量可靠,为后续分析的准确性提供了保障(表1)㊂随后,采用HISAT2软件将有效序列与甘蓝参考基因组进行比对,发现本研究中87%以上的序列能够比对到甘蓝参考基因组上㊂此外,使用StringTie软件进行了新转录本组装,并对这些新转录本进行了Pfam㊁SUPERFAMILY㊁GO㊁KEGG等数据库注释,最终预测了3067个新基因㊂比对完成后,采用Subread软件对基因进行了表达水平的定量,获得了基因的表达水平数据用于后续分析㊂表1㊀测序数据情况材料样品编号有效序列数有效数据量/Gb Q30/%GC含量/%比对上的序列总数(占比/%)红叶A1543408948.1593.2246.8147991531(88.32) A241201636 6.1893.1946.9236341965(88.21)A3539621728.0992.9046.8447466429(87.96)B1523828307.8693.2446.8146151377(88.10)B2490725087.3693.2146.8343296262(88.23)B3560639228.4192.9846.7549449733(88.20)白叶C1496833507.4593.0146.7243461237(87.48) C2517153627.7692.8746.8145426935(87.84)C3479877427.2092.7646.8842269553(88.08)D1482567147.2492.9346.5442221143(87.49)D2467880007.0293.7547.0541452281(88.60)D345719430 6.8693.2546.8940470692(88.52)2.3㊀差异基因表达分析㊀㊀本研究以∣log2(FoldChange)∣>1且FDR< 0.01为差异基因筛选标准,通过对红叶材料和白叶材料的内叶(A vs C)和外叶(B vs D)的基因分别比较发现,内叶有3726个差异基因,占基因总数的5.85%(3726/63653),其中上调基因1912个,下调基因1814个;外叶有2851个差异基因,占基因总数的4.48%,其中上调为1263个,下调1588个(图2中a)㊂经过共差异表达Veen图分析,A vs C和B vs D两个差异基因集间有共同差异基因1665个,其中绝大多数基因上调或下调表达方向一致,即共同上调表达基因725个,共同下调表达基因920个(图2中b)㊂这些基因极有可能包含影响红叶性状形成的关键候选基因㊂a 差异基因数目;b 差异基因维恩图分析图2㊀差异基因表达情况㊀㊀对共同差异表达的基因进行GO富集分析,显著性富集的GO条目分为生物学过程(Biological Process)㊁细胞组分(Cellular Component)和分子功能(Molecular Function)3大类,其中在细胞组分中共同差异表达的基因富集最显著的条目集中在细胞质(cytoplasm)㊁核糖体(ribosome)㊁质体1786㊀㊀2021年第62卷第9期(plastid)等组分;在分子功能中主要的是结合(Binding);在生物学过程中富集最显著的条目集中在防御反应(defense response)相关条目,包括对光㊁低温㊁金属离子㊁水等的反应(response to light stimulus,response to cold,response to cadmium ion,response to water deprivation),值得特别关注的是花青素类化合物生物合成过程(anthocyanin-containing compound biosynthetic process)也出现在显著性富集条目中(图3),进一步证实了花青素苷在红叶性状的形成中起着关键作用㊂图3㊀共同差异表达基因GO富集分析2.4㊀羽衣甘蓝花青素苷途径相关基因差异分析㊀㊀植物花青素苷途径相关基因在模式植物拟南芥中的研究已经较为清楚,使用同源比对的方法对羽衣甘蓝中花青素苷途径相关基因进行了鉴定[12,14]㊂结果表明,在本研究中共鉴定出175个涉及花青素苷途径的基因,其中上调表达基因有Bo3g024670 (C4H)㊁Bo6g100940(MYB75)和Bo9g008920 (5MAT),下调表达基因包括Bo2g116380(DFR)㊁Bo6g112670(MYBL2)㊁Bo6g010860(ACC1)和novel.283(CHI-L1)(表2)㊂这些基因极可能是羽衣甘蓝红叶性状形成的关键基因,有待深入研究㊂3　讨论羽衣甘蓝等彩叶植物叶色的形成相当复杂,主要受内部遗传因子及外界环境因子共同调控,植物所含色素(主要有叶绿素㊁类胡萝卜素㊁类黄酮㊀㊀表2㊀花青素苷途径相关基因差异表达情况A vs CB vs D基因ID基因表达情况基因ID基因表达情况Bo1g031790ANS(LDOX)上调Bo3g024670C4H上调Bo2g070770MYBL2上调Bo6g100940MYB75上调Bo3g024670C4H上调Bo9g0089205MAT上调Bo3g042330SAT上调Bo00835s060MYB114下调Bo3g0774304CL5上调Bo1g105850UGT84A2下调Bo4g030720TTG2上调Bo2g116380DFR下调Bo4g186590PAL1上调Bo4g092850COI1下调Bo6g100940MYB75上调Bo5g137560PAL4下调Bo7g108300ANS(LDOX)上调Bo5g137600PAL4下调Bo8g002690A3GlcCouT上调Bo6g010860ACC1下调Bo9g004350CHS上调Bo6g099880MYB75下调Bo9g0089205MAT上调Bo6g112670MYBL2下调Bo2g116380DFR下调Bo7g092680MYB111下调Bo6g010860ACC1下调Bo8g088480CHI下调Bo6g112670MYBL2下调novel.283CHI-L1下调novel.283CHI-L1下调novel.436UGT78D2下调类等)的种类㊁分布及含量等的差异均可导致不同叶色的形成[15-16]㊂研究[1,3,12]表明,羽衣甘蓝红/紫叶性状是花青素苷大量积累产生的㊂本研究通过对红/白2种不同叶色观赏羽衣甘蓝内外叶片花青素苷含量的测定也验证了这一观点,本研究发现在红叶材料中有大量花青素苷积累,而白叶材料中却极少积累(图1中b)㊂此外,本研究通过对红叶材料和白叶材料差异基因的GO富集分析发现,花青素苷类化合物合成相关条目被显著性富集(图3),也证实了花青素苷对两者叶色差异起到重要作用㊂因此,羽衣甘蓝红叶性状的研究应该着眼于花青素苷的生物合成调控与积累上㊂模式植物拟南芥中花青素苷的代谢合成途径已较为清楚,同属十字花科的甘蓝与拟南芥亲缘关系非常接近,基因组进化分析表明芸薹属甘蓝相比于拟南芥经历了基因组三倍化事件[17-19]㊂因此,采用同源比对的方法可以利用拟南芥已知的花青素苷途径基因鉴定出甘蓝的相应基因,这一方法在芸薹属物种中的应用也比较成功㊂如Guo等[14]通过同源比对的方法鉴定了芸薹属白菜(Brassica rapa)的花青素苷途径基因73个,Ren等[12,20]分别鉴定了芸薹属甘蓝的花青素苷途径基因84和81个㊂本研究综合不同文献中拟南芥的花青素苷途径相关基因,采用同源比对的方法找到了它们在羽衣甘蓝中的同源基因,最终也鉴定到了175个花青素苷途径相关基因㊂相比前人的研究,本研究中鉴定的基因数目更多㊁更全面,将更加有利于后续研究者筛选关键基因㊂本研究最终鉴定到7个候选基因,这些基因包括结构基因(C4H㊁CHI㊁DFR)㊁调控基因(MYB75㊁MYBL2)和一些其他基因(5MAT㊁ACC)(表2)㊂结构基因C4H㊁CHI㊁DFR等的高转录是花青素苷合成从而显现紫/红色的直接原因[21]㊂肉桂酸羟化酶(C4H)是植物苯丙烷代谢通路中重要的羟基化酶,反肉桂酸被C4H羟基化生成对-香豆酸,再进一步转化为类黄酮㊂研究[22]表明,C4H的表达量能有效影响植物中黄酮类化合物的生物合成量㊂本研究中Bo3g024670(C4H)在红叶材料中的表达量相比白叶材料显著性上调表达(表2),这与Ren等的研究结果一致㊂Jin等在差异基因中鉴定到一个C4H基因(Bo l033349),在紫叶材料的内叶中的表达量远高于白叶材料㊂DFR蛋白特异地催化二氢黄酮醇还原成无色的花色素,是花青素苷合成途径中的的重要节点之一,很多植物紫色性状的形成与其有关[21],在一些羽衣甘蓝叶色基因的定位研究中DFR基因也被认为是候选基因[3-4]㊂本研究中还鉴定到了一个新基因novel.283,功能注释为一个具有CHI基因类似功能的基因,CHI在类黄酮类物质的生物合成途径的早期阶段起关键作用[22-23],而作为上游底物的类黄酮物质对花青素苷的合成也影响甚大㊂转录调控是花青素苷合成积累的关键因素之一,而作为调控枢纽的MBW复合体及其成员在此起到主要作用㊂MYB75(PAP1)是一种MYB类转录因子,最早在拟南芥中证实它参与花青苷生物合成的调控[24],在很多物种中都有报道,例如番茄内源转录因子MYB75的超量表达就能够大量积累花青素苷并获得显著紫色的番茄果实[25];Zhang等[2]也认为, BoPAP1在低温诱导羽衣甘蓝花青素苷合成和积累过程中发挥重要作用㊂MYBL2作为花青素苷合成的一个负调控因子最早是在模式植物拟南芥中发现的,AtMYBL2负调控花青素苷的合成[26]㊂在花青素苷的生物合成途径中,前体物质之一的丙二酰辅酶A是由乙酰辅酶A在乙酰辅酶A羧化酶(Acetyl-CoA Carboxylase,ACC)的催化下羧化形成的[27],因此,ACC在花青素的合成过程中可能发挥着重要的作用;刘和平[28]研究表明, PhACC1㊁PhACC2和PhAAE13作为合成丙二酰辅酶A的关键酶,在矮牵牛花的花青素生物合成过程中起着重要的作用㊂花青素苷生物合成途径的下游部分是不稳定的花青素在一系列酶的催化作用下(诸如糖基化㊁酰基化㊁甲基化等)经过修饰转变为稳定的花青苷的过程㊂拟南芥花青素苷5-O-葡萄糖苷-6ᵡ-O-丙二酰转移酶(At5MAT)编码丙二酰-CoA,花青素5-O-葡糖苷-6ᵡ-O-丙二酸转移酶,参与花青素衍生物的酰基化修饰[29]㊂高通量测序技术的迅猛发展为羽衣甘蓝叶色方面的研究提供了良好的技术手段㊂近年来,基于比较转录组分析的羽衣甘蓝叶片呈色的研究报道不断出现,研究多以红叶/紫叶材料与白叶材料的比较为主,重点关注了花青素苷㊁叶绿素及类胡萝卜素的代谢途径等相关的基因差异表达情况[11-13],这些研究为羽衣甘蓝叶色形成的机理研究提供了许多新的思路,但是解析羽衣甘蓝叶色形成的分子机制仍然任重道远㊂本研究通过对羽衣甘蓝红叶材料和白叶材料不同部位的叶片(内叶和外叶)进行转录组分析,揭示了不同叶色羽衣甘蓝叶片基因表达谱的差异,重点研究了花青素苷途径相关的差异基1788㊀㊀2021年第62卷第9期因,筛选出了一些花青素苷途径相关的关键基因及调控因子,为进一步解析羽衣甘蓝叶色形成机理奠定了良好的基础㊂参考文献:[1]㊀王雅琼.观赏羽衣甘蓝叶片成色机理研究[D].杭州:浙江农林大学,2013.[2]㊀ZHANG B,HU Z L,ZHANG Y J,et al.A putative functionalMYB transcription factor induced by low temperature regulatesanthocyanin biosynthesis in purple kale(Brassica Oleracea var.Acephala f.tricolor)[J].Plant Cell Reports,2012,31(2):281-289.[3]㊀LIU X P,GAO B Z,HAN F Q,et al.Genetics and finemapping of a purple leaf gene,BoPr,in ornamental kale(Brassica oleracea L.var.Acephala)[J].BMC Genomics,2017,18(1):1-9.[4]㊀REN J,FU W,DU J T,et al.Identification of a candidategene for Re,the factor determining the red leaf phenotype inornamental kale using fine mapping and transcriptome analysis[J].Plant Breeding,2017,136(5):738-748. [5]㊀WINKEL-SHIRLEY B.Flavonoid biosynthesis.A colorfulmodel for genetics,biochemistry,cell biology,andbiotechnology[J].Plant Physiology,2001,126(2):485-493.[6]㊀CHAVES-SILVA S,SANTOS A L D,CHALFUN-JÚNIOR JR,et al.Understanding the genetic regulation of anthocyaninbiosynthesis in plants-Tools for breeding purple varieties of fruitsand vegetables[J].Phytochemistry,2018,153:11-27. [7]㊀ZHANG Y,BUTELLI E,MARTIN C.Engineering anthocyaninbiosynthesis in plants[J].Current Opinion in Plant Biology,2014,19:81-90.[8]㊀TANAKA Y,SASAKI N,OHMIYA A.Biosynthesis of plantpigments:anthocyanins,betalains and carotenoids[J].ThePlant Journal,2008,54(4):733-749.[9]㊀BROUN P.Transcriptional control of flavonoid biosynthesis:acomplex network of conserved regulators involved in multipleaspects of differentiation in Arabidopsis[J].Current Opinion inPlant Biology,2005,8(3):272-279.[10]㊀GONZALEZ A,ZHAO M Z,LEAVITT J M,et al.Regulationof the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings[J].ThePlant Journal,2008,53(5):814-827.[11]㊀JIN S W,RAHIM M A,AFRIN K S,et al.Transcriptomeprofiling of two contrasting ornamental cabbage(Brassicaoleracea var.acephala)lines provides insights into purple andwhite inner leaf pigmentation[J].BMC Genomics,2018,19(1):1-21.[12]㊀REN J,LIU Z Y,CHEN W S,et al.Anthocyanin degradingand chlorophyll accumulation lead to the formation of bicolor leafin ornamental kale[J].International Journal of MolecularSciences,2019,20(3):603.[13]㊀LIU X P,ZHANG B,WU J,et al.Pigment variation andtranscriptional response of the pigment synthesis pathway in theS2309triple-color ornamental kale(Brassica oleracea L.var.Acephala)line[J].Genomics,2020,112(3):2658-2665.[14]㊀GUO N,CHENG F,WU J,et al.Anthocyanin biosyntheticgenes in Brassica rapa[J].BMC Genomics,2014,15:426.[15]㊀晁月文,李竞芸,张广辉.彩叶植物呈色机理及其育种研究进展[J].江苏林业科技,2008,35(4):46-48,52. [16]㊀潘志超.羽衣甘蓝花青素苷积累特性及相关调控基因表达分析[D].沈阳:沈阳农业大学,2017.[17]㊀LYSAK M A,KOCH M A,PECINKA A,et al.Chromosometriplication found across the tribe Brassiceae[J].GenomeResearch,2005,15(4):516-525.[18]㊀LIU S Y,LIU Y M,YANG X H,et al.The Brassica oleraceagenome reveals the asymmetrical evolution of polyploid genomes[J].Nature Communications,2014,5:3930. [19]㊀PARKIN I A P,KOH C,TANG H B,et al.Transcriptome andmethylome profiling reveals relics of genome dominance in themesopolyploid Brassica oleracea[J].Genome Biology,2014,15(6):R77.[20]㊀GUO N,HAN S,ZONG M,et al.Identification anddifferential expression analysis of anthocyanin biosynthetic genesin leaf color variants of ornamental kale[J].BMC Genomics,2019,20(1):1-17.[21]㊀李益,张一卉,李化银.紫色芸薹属蔬菜花青素合成调控研究进展[J].山东农业科学,2014,46(11):137-142.[22]㊀MILLAR D J,LONG M,DONOVAN G,et al.Introduction ofsense constructs of cinnamate4-hydroxylase(CYP73A24)intransgenic tomato plants shows opposite effects on flux into stemlignin and fruit flavonoids[J].Phytochemistry,2007,68(11):1497-1509.[23]㊀王文钊.茶树儿茶素生物合成相关结构基因的鉴定㊁调控及CHI基因的功能验证[D].合肥:安徽农业大学,2017.[24]㊀BOREVITZ J O,XIA Y,BLOUNT J,et al.Activation taggingidentifies a conserved MYB regulator of phenylpropanoidbiosynthesis[J].The Plant Cell,2000,12(12):2383-2394.[25]㊀JIAN W,CAO H H,YUAN S,et al.SlMYB75,an MYB-typetranscription factor,promotes anthocyanin accumulation andenhances volatile aroma production in tomato fruits[J].Horticulture Research,2019,6:22.[26]㊀MATSUI K,UMEMURA Y,OHME-TAKAGI M.AtMYBL2,aprotein with a single MYB domain,Acts as a negative regulatorof anthocyanin biosynthesis in Arabidopsis[J].The PlantJournal,2008,55(6):954-967.[27]㊀SASAKI Y,NAGANO Y.Plant acetyl-CoA carboxylase:structure,biosynthesis,regulation,and gene manipulation forplant breeding[J].Bioscience,Biotechnology,andBiochemistry,2004,68(6):1175-1184.[28]㊀刘和平.矮牵牛PhACC1㊁PhACC2和PhAAE13在花青素合成中的作用研究[D].广州:华南农业大学,2016. [29]㊀DᶄAURIA J C,REICHELT M,LUCK K,et al.Identificationand characterization of the BAHD acyltransferase malonyl CoA:Anthocyanidin5-O-glucoside-6ᵡ-O-malonyltransferase(At5MAT)in Arabidopsis thaliana[J].FEBS Letters,2007,581(5):872-878.(责任编辑:王新芳)。

Star CCM+ 风机模拟

9
Rotation of computational domains Moving Reference Frame (MRF) :
Frozen Rotor (in some literatures) Steady-state Position of the cell vertices: Fixed Constant grid flux generation
(with their maximum attainable efficiencies)
Key factors for fan type selection:
◊ Pressure ◊ Flow rate ◊ Efficiency ◊ Noise generation ◊ Space constraints ◊ Drive configuration ◊ Cost ◊ ...
conservation equations
Approximate analysis of Motion (Timeaveraged solution) Time efficient
10
Mesh configurations
Polyhedral Mesh generator Number of Cells (in millions) Trimmer Star-CCM+ PolyhedralTrimmer Structured ANSYS ICEM
4
Sirocco fan specifications
◊ Large blade angles ◊ Small size relative to other fan types ◊ Operation at low speeds low level of noise Flow separation between the blades Low efficiency Scroll housing is required Applications: − Automotive industry − HVAC applications