CPSM_M1_study_notes
cufflinks输入输出文件分析
cufflinks输入输出文件分析Cufflinks输入文件要求：Cufflinks识别对齐的SAM文件，或二进制的BAM文件。
Cufflinks 要求输入文件有正负号XS标签，如果你使用的是Tophat输出的BAM文件可正常识别，如果使用的是其他的工具，在进行分类时要输入以下指令：sort –k 3,3 –k 4,4n hits.sam>hits.sam.sortdcufflinks 输出文件：cufflinks输出三个文件。
1.转录组的组装：transcipts.gtfGTF文件包含组装isoforms。
前7个columns为标准GTF格式。
最后的column包含标准的gene_id和transcript_id. 具体的columns 如下：1. 片段名称chrX染色体名称2. 来源cufflinks 生产此文件的程序3. 结构exon 常为transcript或exon4. 开始125. 结束206 所在链+7 其他2.转录本表达水平：isoforms.fpkm_tracking3.基因表达水平：genes.fpkm_trackingCuffquant输入文件要求：单独的SAM或BAM文件和单独的GTF或GFF基因注释文件。
Cuffquant输出文件：输出唯一的abundances.cxb格式文件，cxb文件为二进制文件可继续使用cuffnorm和cuffdiff进一步处理。
Cufdiff输入文件要求：程序需要一个GTF2或GFF3转录本输入，及两个及以上的SAM文件。
Cuffdiff也可处理BAM文件，但cuffdiff 只接受一种类型的文件，不可将cxb与BAM文件同时处理。
Cuffdiff需要提供GTF注释文件使用已确定的实行来寻找初始表达转录本，剪切，编码输出，启动子等。
这些属性是：Tss_id为transcript start siteP_id转录本中编码序列，需要注释文件中有CDS的记录才可。
染色质免疫沉淀技术在研究DNA与蛋白质相互作用中的应用
遗传HEREDITAS Beijing 27 5 : 801 807 2005 技术与方法染色质免疫沉淀技术在研究DNA 与蛋白质相互作用中的应用1 1 1 2 1 王春雨石建党朱彦张琚 1. 南开大学分子生物学研究所教育部生物活性材料重点实验室天津300071 2. 美国塔芙茨大学医学院圣伊丽莎白医学中心心血管研究所分子生理学实验室波士顿马萨诸塞02135 2997 美国摘要: 在后基因组时代DN A 蛋白质的相互作用是研究基因表达调控的一个重要领域。
与其他方法相比染色质免疫沉淀技术chromat in immuno pr ecipitatio n assay ChIP 是一种在体内研究DN A 蛋白质相互作用的理想的方法。
近年来这种方法与DN A 芯片和分子克隆技术相结合可用于高通量的筛选已知蛋白因子的未知DN A 靶点和研究反式作用因子在整个基因组上的分布情况这将有助于深入理解DN A 蛋白质相互作用的调控网络。
总结了染色质免疫沉淀技术的方法特别介绍了使用这些方法取得的最新进展。
关键词: DN A 蛋白质相互作用染色质免疫沉淀技术ChIP DN A 芯片ChIP 克隆中图分类号: Q 78 文献标识码: A 文章编号: 0253- 9772 2005 05- 0801- 07 Application of Chromatin Immunoprecipitation Assay in Deciphering DNA Protein Interactions WANG Chun Yu1 SH I Jian Dang 1 ZH U Yan1 2 Z H ANG Ju 1 1. The Key La borat ory of Bioactive Mat erials Minist ry of Educat ion Ins titute for Molecular Biology Nankai Universit y Tianjin 300071 China 2. Laboratory of Molecula r Physiology Division of Cardiovas cular Res earch St . Eliza bet h s Medica l Center Tufts Univers ity S chool of Medicine Bost on MA 02135 2997 US A Abst ract : In the post genomic era identifyi ng and characterizing vari ous DNA protein inter actions are a major chal lenge in the research of gene tr anscripti onal regulation. Although many techni ques can be used for thi s purpose chr o matin immunopreci pitation assay ChIP by contrast is ideally suited for studyi ng DNA protein interactions in v ivo . In recent years standard ChIP assay has been modi fied to uncover some known factors unknown target sequences es pecial ly when combined wi th DNA microarray and molecular cloni ng strategi es. These hi gh throughput ChIP assays are more and more used to reveal the di stributi on profile of trans acti ng factor binding si tes throughout the genome which may yield many new insights into the DNA protein interaction network. This article summarized the methods of ChIP as say and highl ighted recent progress in the appli cation of this technique. Key words: DNA pr otein i nteractions chromati n immunoprecipitati on assay ChIP DNA micr oarr ay ChIP cl oning收稿日期: 2004 07 08 修回日期: 2004 08 04基金项目: 国家自然科学基金资助项目编号: 30471733 30271297 和留学人员短期回国工作讲学项目两个基地项目编号: 30410403237 2002 年度高等学校博士学科点专项科研基金编号: 20020055026 Supported by National Natural Sciences Foundat ion of China No. 30471733 30271297 Fund for Chinese Scholars Abroad to Work and Lecture in China Two Base Project No. 30410403237 and Spe o. cialized Research Fund for t he Doctoral Program of Higher Educat ion N 20020055026 作者简介: 王春雨1979男天津市人博士研究生专业方向: 医学分子生物学。
自身免疫性肝炎小鼠肝组织中的膜联蛋白A1表达情况
论著-基础研究自身免疫性肝炎小鼠肝组织中的膜联蛋白A1表达情况么刘耿烽1范俊华1吕晓丹2张怡华1曾睿智1陈如艳1詹灵凌彳吕小平1 (广西医科大学第一附属医院1消化内科;2检验科，南宁市530021,电子邮箱：369215736@)【摘要】目的探讨自身免疫性肝炎(AIH)小鼠肝组织中的膜联蛋白Al(AnxAl)表达情况及意义。
方法选取正常C57BL/6雄性小鼠16只，随机分为正常组和模型组，各8只。
模型组通过尾静脉注射刀豆蛋白A造模,正常组通过尾静脉注射等量的生理盐水。
48h后处死小鼠,取肝组织进行苏木精■伊红染色观察组织病理学变化，比较两组小鼠血清ALT、AST水平，以及肝组织AnxAl原位表达情况、肝组织AnxAl mRNA及蛋白表达水平。
分析AnxAl mRNA及蛋白表达水平与血清ALT.AST水平的相关性。
结果模型组血清ALT、AST水平均高于正常组(均P<0.05)o模型组小鼠肝脏充血肿大，镜下可见明显炎症改变。
模型组肝组织存在AnxAl的表达，且mRNA和蛋白的相对表达量均高于正常组(均P<0.05)o模型组肝组织AnxAl mRNA和蛋白的相对表达量均与血清ALT.AST水平呈正相关(均P<0.05)o结论AIH小鼠肝组织AnxAl表达上调，其可能在AIH发生、发展中起到重要的作用。
【关键词】自身免疫性肝炎;膜联蛋白A1；炎症损伤；相关性；小鼠【中图分类号】R593.2【文献标识码】A【文章编号】0253-4304(2021)04-0455-04DOI：10.11675/j.issn.0253-4304.2021.04.16Expression of annexin Al in liver tissues of autoimmune hepatitis miceUU Geng-feni,FAN Jun-hu^,LYU Xiao-dar^,ZHANG Yi-hua',ZENG Rui-zh沽,CHEN Ru-yan^,ZHAN Ung-Un^,LYU Xiao-ping1 (1Department of Gastroenterology,2Department of Clinical Laboratory,the First Affiliated Hospital ofGuangxi Medical University,Nanning530021,China)[Abstract]Objective To investigate the expression and significance of annexin Al(AnxAl)in liver tissues of autoimmune hepatitis(AIH)mice.Methods Sixteen normal male C57BL/6mice were selected,and were randomly divided into normal group and model group,with8mice in each group.The model group was modeled by tail vein injection of concanavalin A,and the normal group received tail vein injection of isometric normal saline. After48hours,all mice were sacrificed,and liver tissues were removed for observation on histopathological changes after hematoxylin-eosin staining.The two groups were compared in terms of serum ALT and AST levels,as well as the in situ expression of AnxAl in liver tissues and the expression levels of AnxAl mRNA and protein in liver tissues.The correlation of AnxAl mRNA or protein expression level with serum ALT or AST level was analyzed.Results The model group had higher serum ALT and AST levels than the normal group(all P<0.05).The livers of mice in the model group exhibited congestion and edema,and obvious inflammatory changes were observed under microscope. AnxAl was expressed in the liver tissues of the model group,and the relative expression of AnxAl mRNA and protein was higher in the model group than in the normal group(all P<0・05)・In the model group,the relative expression of AnxAl mRNA and protein in liver tissues positively correlated with serum ALT or AST level(all P<0.05).Conclusion AnxAl is highly expressed in liver tissues of AIH mice,and may play an important role in the occurrence and development of AIH.[Key words]Autoimmune hepatitis,Annexin Al,Inflammatory damage,Correlation,Mouse▲基金项目：国家自然科学基金(81860120.81860104)；广西自然科学基金(2017GXNSFBA198134、2017GXNSFAA198299)；广西医疗卫生适宜技术开发与推广应用项目(S2018049)作者简介:刘耿烽(1992~)，男，硕士，住院医师，研究方向：自身免疫性肝炎的病因和发病机制。
Development and Validation of a Liquid Chromatogra
J. Chem. Chem. Eng. 5 (2011) 1-6.Development and Validation of a LiquidChromatography–Tandem Mass Spectrometry Method for Determination of Artemisinin in Rat PlasmaElhassan Gamal1,2, Yuen Kah1, Wong Jiawoei1, Chitneni Mallikarjun1,3, Al-Dahli Samer1, Khan Jiyauddin1 and Javed Qureshi31. School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden 11800, Penang, Malaysia2. Local Pharmaceutical Manufacturing Department, General Pharmacy Directorate, MOH, 11111, Khartoum-Sudan3. School of Pharmacy and Health Sciences, International Medical University, 5700, Kula Lumpur, MalaysiaReceived: September 03, 2010 / Accepted: October 11, 2010 / Published: January 10, 2011.Abstract: Artemisinin is a potent anti-malarial drug isolated from traditional Chinese medicinal herb, Artemisia annua. The objective of this study was to develop and validate a sensitive and specific LC-MS/MS method for the determination of artemisinin in rat plasma using amlodipine as Internal Standard. The method consist of a simple liquid-liquid extraction with methyl tertiary butyl ether (MTBE) with subsequent evaporation of the supernatant to dryness followed by the analysis of the reconstituted sample by LC-MS/MS with a Z-spray atmospheric pressure ionization (API) interface in the positive ion-multiple reaction monitoring mode to monitor precursor→product ions of m/z 282.70→m/z 209.0 for artemisinin and m/z 408.9→m/z 237.0 for amlodipine respectively. The method was linear (0.999) over the concentration range of 7.8–2000 ng/mL in rat plasma. The intra and inter-day accuracy were measured to be within 94-104.2% and precision (CV) were all less than 5%. The extraction recovery means for internal standard and all the artemisinin concentrations used were between 82-85%.Key words: Artemisinin, LC-MS/MS, amlodipine, plasma, accuracy and precision.1. IntroductionArtemsinin is the name given to the active principle of qinghaosu, an extract of the Chinese medicinal plant qinghaosu or green Artemisia (Artemisinin annua L.) which has been used for many years centuries in Chinese traditional medicine for treatment of fever and malaria [1]. In 1972, Chinese researchers isolated artemisinin from Artemisia annua L. sweet wormwood) and its structure was elucidate in 1979 as show in Fig. 1.The determination of artemisinin and its derivatives in biological matrices have previously been characterized using several analytical techniques suchCorresponding author: Gamal Osman Elhassan Ph.D., research field: pharmaceutical technology. E-mail: ******************.as LC, HPLC, GC-MS etc [3-8]. However, some of these methods suffer from few drawbacks. In particulars, interference with endogenous constituents in the plasma at the absorption wave length of the derivatized compounds may render these techniques unsatisfactory and few of them lacked the required sensitivity to be used for measurement of drugFig. 1 The chemical structure of artemisinin [2].ll Rights Reserved.Development and Validation of a Liquid Chromatography–Tandem Mass Spectrometry Method forDetermination of Artemisinin in Rat Plasma2concentration in blood sample obtained from clinical investigation [9].To increase the specificity and sensitivity of HPLC-UV method, some workers combined it with a mass spectrometry (MS) and the total system is described as LC-MS technique [10, 11]. The development of LC-tandem mass spectrometry (LC-MS/MS) has made a more specific and sensitive analysis of artemisinin and its derivatives possible [12, 13]. The objective of this study was to develop a sensitive and specific LC-MS/MS method for the determination of artemisinin in rat plasma by simple liquid-liquid extraction procedure.2. Materials and Methods2.1 MaterialsArtemisinin was purchased from Kunming Pharmaceutical Corporation (Kunming, China). Amlodipine was obtained from Sigma Chemical (Louis, USA). Acetonitrile (ACN), formic acid and methyl tertiary butyl ether (MTBE) were purchased from J.T Baker (USA).3. Methods3.1 Instrumentation and ConditionsThe instrumentation comprised of Quattro-micro tandem mass spectrometer with Z-spray atomospheric pressure ionization (API) source (Micromass, Manchester, UK) using electrospray ionization (ESI) operated at positive mode. Chromatography was performed on an Alliance 2,695 separation module (Waters, M.A, USA). The delivery system consisted of an autosampler and a column heater. The chromatographic separation was obtained using an X Terra MS C8 encapped (5 μm) (150 × 2.1 mm) analytical column (Water, USA).3.2 Sample PreparationA 250 μL aliquot of plasma was pipetted into a screw-capped culture tube, followed by 100 μL of internal standard solution (50 ng/mL). To each tube, 5 mL (MTBE) extraction solvent was then added and the mixture was vortexed for 2.5 minutes followed by centrifuging for 15 minutes at 3,500 rpm. The upper layer was transferred to a reactive vial and dried under nitrogen flow at 40 °C. The residue was then reconstituted with 250 μL of mobile phase and 20 μL was injected into the LC-MS/MS system.3.3 Assay ValidationCalibration curve at a concentration range of 7.8–2,000 ng/mL were constructed by spiking blank human plasma with a known amount of artemisinin. Plasma sample spiked with artemisinin at these concentrations 7.8, 62.5, 250, 2,000 ng/mL were used to determine the within and between-day accuracy and precision. For within-day accuracy and precision, replicates analysis (n = 6) for each concentration were performed in a single day. For between-day evaluation, analysis was carried out with a single sample of each concentration daily over 6 days, with calibration curve constructed on each day of analysis. The extraction recovery of artemisinin was estimated by comparing the peak height obtained after extraction of the samples from plasma with that of aqueous artemisinin solution of the corresponding concentration.4. Results and DiscussionBoth electrospray (TIS) and atmospheric pressure chemical ionisation (APCI) methods have been reported previously for the quantification of artemisinin derivatives in biological fluids [11, 12, 14-16]. According to the previously reported methods TIS was found to be superior to APCI for the quantification of artesunate and dihydroartemisinin (DHA) mainly because of improved linearity [16]. Therefore in this method electrospray ionization was used. When artemisinin and amlodipine were injected directly into the mass spectrometer along with mobile phase in the positive mode, the protonated molecules of artemisinin and amlodipine were set as precursorll Rights Reserved.Development and Validation of a Liquid Chromatography–Tandem Mass Spectrometry Method forDetermination of Artemisinin in Rat Plasma3(a)(b)Fig. 2 (a) Positive-ionization electrospray mass spectra of precursor ion for artemisinin; (b) Positive-ionization electrospray mass spectra of product ion for artemisinin.ions with m/z of 282.7 and 408.7, respectively. The product ion that gave the highest intensity was m/z of 209.0 for artemisinin and 237.7 for amlodipine. Fig 2(a) shows the spectra precursor ion, 2(b) production for artemisinin.Artemisinin and amlodipine have retention time of approximately 6.9 and 1.65 minutes, respectively (Fig.3). The peak was well resolved and free from interference from endogenous compounds in rat plasma (Fig. 4).ll Rights Reserved.Development and Validation of a Liquid Chromatography–Tandem Mass Spectrometry Method forDetermination of Artemisinin in Rat Plasma4Fig. 3 Plasma spiked with 500 ng/ml artemisinin and amlodipine 50 ng/mL.Fig. 4 Chromatograms for analysis of artemisinin in plasma (Rat blank plasma).Calibration curve was linear over the entire range of calibration curves with a mean correlation coefficient greater than 0.9995 (Fig. 5).The limit of quantification (LOQ) of the assay method was 7.8 ng/mL being the lowest concentration used to construct the calibration curve whereas the limit of detection (LOD) was 3.9 ng/mL at a signal to noise ratio of 3. The validation data demonstrated a good precision, accuracy and recovery. The extraction recovery means for internal standard and all artemisinin concentrations used were 75-85% (Table 1). The within-day and between-day accuracy and precision values are given in Table 2.Neither artemisinin nor the internal standard producedll Rights Reserved.Development and Validation of a Liquid Chromatography–Tandem Mass Spectrometry Method forDetermination of Artemisinin in Rat Plasma5Fig. 5 Mean calibration curve of artemisinin (ng/mL).Table 1 Extraction recovery.Concentration (ng/mL) Mean recovery (%) CV (%)7.81 75.081.5062.50 82.161.94250.00 82.03 2.072000.00 85.23 1.48Table 2 Within-day and between-day precision andaccuracy.Added (ng/mL)Within-day Between-day Accuracy (%) C.V (%) Accuracy (%) C.V (%)7.81 96.00 4.60 104.11 2.30 62.50 98.10 1.60 94.10 2.20 250.00 98.10 1.50 98.10 1.60 2000.00 96.10 2.50 97.10 1.80any detectable carry-over after three injections of upper limit of quantification. Blank rat plasma showed no interference with artemisinin. Interfering signals from blank plasma contributed less than 20% of the artemisinin signal at LOQ. There was no interference of artemisinin on the internal standard or vice versa. A small enhancement for artemisinin and the internal standard could be detected when references in neat injection solvent were compared with references in extracted blank biological matrix. The normalized matrix effects (artemisinin/internal standard) were close to 1 with a low variation in accordance with international guidelines. Post-column infusion experiments confirmed the absence of regions with severe matrix effects (i.e., no sharp drops or increases in the response) for blank human plasma extracted with the developed method.Xing et al. used artmether as an internal standard for the analysis of artemisinin [17]while for the analysis of artemisinin derivatives; artemisinin was used as internal standard [14]. In the present study amlodipine was found to be suitable because it could be separated chromatographically, ionized and fragmented under the conditions that optimized the intensity of artemisinin peak (Fig. 3).The analysis of artemisinin and its derivatives with mass spectrometry are most often performed with a different mode of ionization. Xing et al. used ESI inletin the positive ion-multiple reaction monitoring mode which relatively producing a higher sensitivity than in the SIM mode. Therefore, the mass spectrometry was operated at positive ion-MRM mode.4. ConclusionThe LC-MS/MS method described in this work is suitable for the determination of artemisinin in plasma. The assay procedure is simple with a relatively shortll Rights Reserved.Development and Validation of a Liquid Chromatography–Tandem Mass Spectrometry Method forDetermination of Artemisinin in Rat Plasma6retention time allowing sufficient sample to beprocessed to be applied to pharmacokinetic and bioavailability studies of artemisinin. The accuracy and precision of the assay method, as well as the recovery of extraction procedure were found to be satisfactory.References[1] D.L. Klayman, Qinghasou (Artemisinin): An antimalaria drug from China, Science 228 (1985) 1049-1055.[2] X.D. Luo, C.C. Shen, The chemistry, pharmacology andclinical applications of Qinghaosu (artemisinin) and it’sderivatives, Med. Res. Rev. 7 (1987) 29-52.[3] K.T. Batty, M. Ashton, K.F. Llett, G . Edwards, T.M. Davis,Selective high-performance liquid chromatography ofartesunate and α-and β-dihydroartemisinin in patients withfalciparum malaria, J. Chromatog. B 677 (2-3) (1996)345-350.[4] J. Karbwang, K. Na-Bangchang, P. Molunto, V . Banmairuroi, Determination of artemisinin and its majormetabolite, dihydroartemisinin, in plasma usinghigh-performance liquid chromatography withelectrochemical detector, J. Chromatog. B 7 (1-2) (1997)259-265.[5] K.L. Chan, K.H. Yuen, H. Takayanki, S. Jinandasa, K.K. Peh, Polymorphism of artemisinin from Artemisia annua,Phytochemistry 46 (7) (1997) 1209-1214.[6] G .Q. Li, T.O. Peggins, L.L. Fleckenstein, K. Masonic,M.H. Heiffles, T.G . Brewer, The pharmacokinetics andbiovailability of dihydroartemisinin, arteether, artemether,artesunic acid and artelinic acid in rats, J. Pharm.Pharmacol 5 (1998) 173-182.[7] B.A. Avery, K.K. Venkatesh, M.A. Avery, Rapid determination of artemisinin and related analogues usinghigh-perfomance liquid chromatography and anevaporative light scattering detector, J. Chromat. B 730 (1)(1999) 71-80.[8] S.S. Mohamed, S.A. Khalid, S.A. Ward, T.S.M. Wan,H.P.O. Tang, M. Zheng, R.K. Haynes, G . Edwards,Simultaneous determination of artemether and its majormetabolite dihydroartemisinin in plasma by gaschromatography-mass spectrometry-selected ionmonitoring, J. Chromat. B 731(1999) 251-260.[9] K.T. Batty, M. Ashton, K.F. Llett, G . Edward, T.M. Davis,The pharmacokinetics of artemisinin (ART) and artesunate (ARTS) in healthy volunteers, Am J. Trop Med. Hyg. 58(2) (1998) 125-126.[10] C. Souppart, N. Gouducheau, N. Sandenan, F. Richard,Development and validation of a high-performance liquid chromatography-mass spectrometry assay for the determination of artemisinin and its metabolite dihydraartemisinin in human plasma, J. Chromat. B 774(2002) 195-203.[11] H. Naik, D.J. Murry, L.E. Kirsch, L. Fleckenstein,Development and validation of high-performance liquid chromatography-mass spectroscopy assay for determination of artesunate and dihydrroartemisinin in human plasma, J. Chromat. B 816 (1-2) (2005) 233-242. [12] J. Xing, H. Yan, S. Zhang, G . Ren, Y . Gao, A high-performance liquid chromatography/tandem mass spectrometry method for the determination of artemisinin in rat plasma, Rapid Commun in Mass Spectro. 20 (9) (2006) 1463-1468. [13] J. Xing, H.X. Yan, R.L. Wang, L.F. Zhang, S.Q. Zhang,Liquid chromatography-tandem mass spectrometry assay for the quantitation of β-dihydroartemisinin in rat plasma, J. Chromat. B 852 (1-2) (2007) 202-207. [14] M. Rajanikanth, K.P. Madhusudanan, R.C. Gupta, An HPLC-MS method for simultaneous estimation of alpha, beta-arteether and its metabolite dihydroartemisinin, in rat plasma for application to pharmacokinetic study, J Biomed. Chromat. 17 (7) (2003) 440-446. [15] Y . Gu, Q. Li, M.V . Elendez, P. Weina, Comparison of HPLC with electrochemical detection and LC–MS/for the separation and validation of artesunate and dihydroartemisinin in animal and human plasma, J. Chromatogr B 867 (2008) 213-218. [16] W. Hanpithakpong, B. Kamanikom, A.M. Dondorp, P.Singhasivanon, N.J. White, N.P. Day, N. Lindegardh, A liquid chromatographic-tandem mass spectrometric method for determination of artesunate and its metabolite dihydroartemisinin in human plasma, J. Chromatogr. B 876 (2008) 61-68. [17] Y . Xing, H. Yan, S. Zhang, G . Ren, Y . Gao, A high-performance liquid chromatography/tandem mass spectrometry method for the determination of artemisinin rat plasma, Rapid Communication in Mass Spectrometry 20 (9) (2006) 1463-1468.ll Rights Reserved.。
26448102_氯膦酸二钠脂质体清除巨噬细胞对小鼠造血干祖细胞归巢过程的影响
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细胞自动机化学系统建模教材说明书
Cellular Automata Modeling of Chemical SystemsCellular Automata Modeling of Chemical SystemsA textbook and laboratory manualLemont B.Kier,PhDProfessor of Medicinal ChemistrySenior Fellow,CSBCrVirginia Commonwealth UniversityUSAPaul G.Seybold,PhDProfessor of ChemistryWright State UniversityExternal Fellow,CSBCrVirginia Commonwealth UniversityUSAChao-Kun Cheng,PhDAssociate Professor of Computer ScienceFellow,CSBCFrVirginia Commonwealth UniversityA publication of the Center for the Study of Biological ComplexityrVirginia Commonwealth UniversityRichmond VirginiaUSAA C.I.P.Catalogue record for this book is available from the Library of Congress.ISBN-101-4020-3657-4(HB)ISBN-13978-1-4020-3657-6(HB)ISBN-101-4020-3690-6(e-book)ISBN-13978-1-4020-3690-3(e-book)Published by Springer,P.O.Box17,3300AA Dordrecht,The Netherlands.Printed on acid-free paperAll Rights ReservedC2005SpringerNo part of this work may be reproduced,stored in a retrieval system,or transmittedin any form or by any means,electronic,mechanical,photocopying,microfilming,recording or otherwise,without written permission from the Publisher,with the exceptionof any material supplied specifically for the purpose of being enteredand executed on a computer system,for exclusive use by the purchaser of the work. Printed in the Netherlands.Table of ContentsPreface vii1.Modeling Nature12.Cellular Automata93.Water as a System394.Solution Systems575.Dynamic Aqueous Systems736.Water-Surface Effects877.First-Order Chemical Kinetics1098.Second-Order Chemical Kinetics1259.Additional Applications in Chemical Kinetics139e of the CASim Program157Index169PrefaceOver the past two decades there has been a significant growth in the use of computer-generated models to study dynamic phenomena in the nature.These studies have ranged over many of thefields of human endeavor.For example, insect behavior is a target for dynamic models;automobile traffic is another. The sociologists have picked up on the possibilities afforded by computer mod-els to study dynamic systems.In the physical and biological sciences,dynamic computer models have been used to study a variety of phenomena.Some studies in chemistry have appeared in the literature,but thefield is so vast that only a small area has been considered for computer modeling.In our view chemistry is ripe for studies utilizing this paradigm.The study of chemistry is usually focused on changes;we establish a structure,a form,but it is of real interest wwhen we consider how and to what it is boratory studies in schools introduce the student to simple processes that always work.More com-plex transformations are difficult to set up as experiments;they often do not “work”and so the didactic value of such experiences is marginal.It is our purpose in this book to explore and reveal how some computer mod-els might enrich the practical experiences,traditionally carried out in“wet”labs. We pursue this goal using one of the modeling schemes that was developed a half century ago:cellular automata.The record of cellular automata as a model-ing paradigm is revealed in the literature.We have used cellular automata in our research for a decade,modeling solution and kinetic phenomena of chemical systems.We feel that this approach can bring new meaning to experimental chemistry in the form of in silico experiments.This book is dedicated to that objective.The book is organized into three sections.In thefirst section we introduce the student to some of the concepts that are fundamental to an understandingviii Preface of chemical phenomena.These include a look at the subject of complexity. Imbedded in these concepts are general chemical phenomena such as self-organization,emergent properties,and local interactions.This section sets the stage for a look at some of the modeling techniques used to explore complex systems.In the second section we present a brief overview of some currently used dynamic modeling methods before introducing cellular automata.After a brief history of this method we describe the ingredients that drive the dynamics exhibited by cellular automata.These include the platform on which cellular automata plays out its modeling,the state variables that define the ingredients, and the rules of movement that develop the dynamics.Each step in this section is accompanied by computer simulation programs carried on the CD in the back of the book.WWith this background the student is then equipped to witness what has been done in chemistry using cellular automata models.These studies are accompanied by unfinished studies and challenges,“what if”ideas for the student.The laboratory in a general chemistry course is an ideal place to use this approach since it brings to the student views of many phenomena,previously difficult to visualize.As an adjunct to experimental work in the lab,it opens up a new level of understanding.It may even pique interest in pursuing new theoretical investigations in chemistry.At a nearfinal stage of writing this book,we had a golden opportunity to test the modeling exercises.Seven students in the Integrated Life Sciences graduate program at the Virginia Commonwealth University were asked to read the text and to perform many of the examples and studies.Their experiences were of immense value to us infinalizing the manuscript.We want to acknowledge them and thank them for their efforts.They are Xiangrong Kong,Julie Naumann, Jean Nelson,Antoine Nicolas,Elizabeth Prom,Alexander Tulchinsky,and Carl Zimmerman.We also want to thank Yingjin Cui for her help in creating some of thefigures.The authors thank Marco Tomassini for early,helpful reviews of the manuscript.We thank Enguang Zhao for his help in preparing the Java version of the CA program.Finally we acknowledge the scholarly climate and encour-agement given to us at the Center for the Study of Biological Complexity at the Virginia Commonwealth University.Lemont B.KierPaul G.SeyboldChao-Kun Cheng。
M1和M2型巨噬细胞表型的比较分析_李康
《现代免疫学》 2008 年第 28 卷第 3 期
( eBioscience 公司 ) , 小鼠 IL-12 ELISA 检测试 剂 盒( eBioscience 公司 ) ;FIT C 标记 的大 鼠抗 小 鼠 F4/ 80 抗体( CA LT AG Laborato ries 公司) , PE 标 记的驴抗 大 鼠 IgG ( H +L ) 抗 体( eBio science 公 司) , PE 标记的抗小鼠 CD16/ 32( Fc γ III / Fc γ II 受 体, 2. 4G2) 抗体 ( BD Pharming en 公司) , 未标 记 大鼠抗小鼠 CD206 抗体 ( Serotec 公司 ) , 未标记大
表 1 引物序列 基因 i NO S A rg-1 引物序列( F :正义链 R :反义链) PCR 片段( bp) 311 退火温度( ℃ ) 59
F :5 -C TG C AG CA C T TG G A T CA G G A A CC T G R :5 -G G A G T A G CC TG T G T G CA C CTG G A A F :5 -C AG A A G A A T G G A A G A G TC A G R :5 -CA G A TA TG C A G G G A G TCA CC F :5 -C TG C A C CA C CA A CTG CT T A G R :5 -G T C TGG G A T GG A A A T TG T G A
PDHA1及GBP1蛋白在非小细胞肺癌组织中表达规律的研究
中国实用医闭2020 年11 j〗第47 卷第22 期Chinese JournalofPracticalMedicine.Nov. 2020. Vol. 47, No. 22论著•PDHA1及GBP1蛋白在非小细胞肺癌组织中表达规律的研究曹静曾宪旭雷冬梅班振英张威朱超亚郑州大学第三附属医院病理科450052通信作者：曹静，Email: ******************【摘要】目的探讨丙酮酸脱氢酶Ela亚单位（PDHA1)、鸟苷酸结合蛋白1(GBP1)蛋白在非小细胞肺癌(NSCLC)组织中表达规律。
方法抽取2011年1月至2013年12月郑州大学第三附属医院102例NSCLC患者经手术切除的组织标本，均行免疫组化染色分析PDHA1、GBP1蛋白表达情况，应用多因素Cox回归分析二者表达与患者生存期的关系。
结果102例NSCLC组织中，PDHA1、GBP1蛋白阳性表达与组织分化程度、淋巴结转移、T N M分期有关（X2=12. 788、8. 893、8. 268,尸= 0.002、0. 003、0• 016)，而与患者性别、吸烟史、病理类型未见相关性（P>0.05)。
多因素Cox回归分析显示，PDHA1蛋白阳性者生存期更长（月=2.021，尸=0.001 )，GBP1蛋白阴性者生存期更长(/3 = 1.〇80，尸=0.017)。
结论随着癌症进展，？0只人1表达下降而08?1表达升高，二者表达规律或可用于判断其生物学行为及预后。
【关键词】非小细胞肺癌；免疫组化；丙酮酸脱氢酶E l a亚单位；鸟苷酸结合蛋白1基金项目：河南省医学科技攻关计划（LHGJ20190395)D01：10.3760/cm a. j. cnl 15689 - 20200810 -03918Expression of PDHA1 and GBP1 protein in non-small cell lung cancerCao J in g, Zeng X ia n xu, Lei Dongmei, Ban Zhenying, Zhang Wei, Zhu ChaoyaDepartment o f Pathology, the Third Affiliated Hospital o f Zhengzhou University, Zhengzhou 450052,ChinaCorresponding author：Cao J in g，Email: caojingblk@ 126. com【Abstract】Objective To investigate the pyruvate dehydrogenase El alpha subunit ( PDHA1)and guanylate-binding protein 1( GBP1) protein expression in non-small cell lung cancer ( NSCLC ).Methods A total of 102 tissue specimens of NSCLC patients in the Third Affiliated Hospital ofZhengzhou University from January 2011 to December 2013 were collected. The expressions of PDHA1and GBP 1were analyzed by immunohistochemistry. Multivariate Cox regression analysis was used toanalyze the relationship between their expression and patient survival. Results The positive expressionof PDHA1 and GBP1 protein were related to histological differentiation, lymph node metastasis, and TNMstage in 102 NSCLC tissues ( x2= 12. 788，8. 893 , 8. 268，=0•002，0• 003，0• 016)，but were notrelated to gender, smoking history and pathological type(P >0. 05). Multivariate Cox regression analysisshowed that PDHA1 protein-positive patients had longer survival time ( p = 2. 021 , P = 0. 001 ) , andGBP1 protein-negative patients had longer survival time ( p = 1. 080, P = 0.017). Conclusions As thecancer progresses, the expression of PDHA1 decreases and the expression of GBP1 increases. The twoexpression patterns may be used to judge their biological behavior and prognosis.【Keywords】Non-small cell lung cancer; Immunohistochemistry; Pyruvate dehydrogenase Elalpha subunit；Guanylate-binding protein 1Fund program ：Henan Medical science and Technology Research Project Project ( LHGJ20190395)D O I ： 10. 3760/cma. j. cn 115689 - 20200810-03918中MIL:用眹PI2020 年11 第47 卷第22 期Chinese Journal of Practical Medicine.Nov. 2020. Vol. 47，No. 22• 2 •原发性肺癌发病率较高,男性高于女性，其中非小 细胞肺癌（non small cell lung cancer,NSCLC)占比高于 80% ,有学者认为NSCLC患者术后5年总体生存率一 般不超过40%。
Differentiation of Human Pluripotent Stem Cells into Retinal Cells
87M.A. Hayat (ed.), Stem Cells and Cancer Stem Cells, Volume 6,DOI 10.1007/978-94-007-2993-3_9, © Springer Science+Business Media B.V . 20129A bstractRetinal and macular degeneration disorders are characterized by a progressive loss of photoreceptors, which causes visual impairment and blindness. In some cases, the visual loss is caused by dysfunction, degen-eration and loss of underlying retinal pigment epithelial (RPE) cells and the subsequent death of photoreceptors. The grim reality is that there is no successful treatment for most of these blindness disorders. Cell therapy aimed at replenishing the degenerating cells is considered a potential ther-apeutic approach that may delay, halt or perhaps even reverse degenera-tion, as well as improve retinal function and prevent blindness in the aforementioned conditions. Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSCs) may serve as an unlimited donor source of photoreceptors and RPE cells for transplantation into degenerat-ing retinas and for retinal disease modeling.I ntroductionThe vertebrate eyes form as bilateral evaginations of the forebrain, called optic vesicles (Martínez-Morales et al. 2004 ; Fig. 9.1a ). During develop-ment, the optic vesicles begin to invaginate to form a cup-shaped structure, the optic cup. The inner, thicker neural layer of the optic cup differ-entiates into the neural retina, and the outer, thin-ner pigmented layer forms the retinal pigmentepithelium (RPE). At the early developmental stages, the neuroepithelial cells that compose the optic vesicle are morphologically and molecu-larly identical and are all able to give rise to neu-ral retina and RPE. Exogenous signals coming from the adjacent tissues, including factors from the fi broblast growth factor (FGF) and transform-ing growth factor beta (TGF b ) families, dictate the fate of these cells. The mature vertebrate ret-ina is comprised of six types of neurons and one type of glia (the Müller glia). These seven cell types constitute three nuclear layers: retinal gan-glion cells in the ganglion cell layer (GCL); the horizontal, bipolar and amacrine interneurons, and Müller glial cells in the inner nuclear layer (INL); and rod and cone photoreceptors in the outer nuclear layer (ONL; Harada et al. 2007;M . I delson • B . R eubinoff (*)T he Hadassah Human Embryonic Stem Cell Research Center, The Goldyne Savad Institute of Gene Therapy & The Department of Obstetrics and Gynecology , H adassah University Medical Center ,E in Kerem 12000 ,J erusalem 91120 ,I srael e -mail: b enjaminr@ekmd.huji.ac.il D ifferentiation of HumanPluripotent Stem Cells into Retinal Cells Masha Idelson and Benjamin Reubinoff88M. Idelson and B. ReubinoffFig. 9.1b ). The photoreceptor cells capture lightphotons and transform their energy into electrical signals by a mechanism called phototransduction. The visual pigment which is utilized in this process is located on membranal discs in the outer seg-ments of photoreceptors. The outer segments are continuously renewed: the old discs are shed and new disks form. When the photoreceptors absorb light, they send the signal through the retinal interneurons to the ganglion cells which transmit the electrical impulse to the brain by their axons forming the optic nerve. Rods are responsible for night vision, whereas cones are responsible for color vision and detecting fi ne details. The macula is a small part of the retina which is rich in cones and responsible for detailed central vision.R PE cells that compose the outer layer of the optic cup are pigmented cuboidal cells which lie between the neural retina and the choriocapil-laris, which include the blood vessels supplying the retina. The multiple villi on their apical side are in direct contact with the outer segments ofextraocular mesenchymeabneural retinalensoptic nerveoptic cupsurface ectodermRPEFGFoptic vesiclechoroidBM RPE cone ONLINL GCLlightHC BC MC ACONrod F ig. 9.1 D evelopment and structural arrangement of the retina. ( a ) Schematic representation of retinal development including the transition from optic vesicle to optic cup and retinal patterning. ( b ) Schematic diagram of retinal cells arrangement and connections. A bbreviations :A C amacrinecell, B C bipolar cell, B M Bruch’s membrane, G CL gan-glion cell layer, H C horizontal cell, I NL inner nuclear layer, M C Müller cell, O N optic nerve, O NL outer nuclear layer89 9 Differentiation of Human Pluripotent Stem Cells into Retinal Cellsthe photoreceptor cells; on their basal side, the RPE is in contact with the underlying basal mem-brane, termed Bruch’s membrane that separates the RPE from the choroid. These cells play cru-cial roles in the maintenance and function of the retina and its photoreceptors. As a layer of pig-mented cells, the RPE absorbs the stray light that was not absorbed by the photoreceptors. The RPE cells form a blood–retinal barrier due to decreased permeability of their junctions. The RPE cells transport ions, water, and metabolic end products from the retina to the bloodstream. They are involved in supplying the neural retina with nutrients from the bloodstream, such as glu-cose, retinol, and fatty acids. Another important function of the RPE is the phagocytosis of shed photoreceptor outer segments. After the outer segments are digested, essential substances such as retinal are recycled. Retinal is also recycled and returned to photoreceptors by the process known as the visual cycle. The precise functioning of the RPE is essential for visual performance. Failure of one of these functions can lead to degeneration of the retinal photoreceptors, vision impairment and blindness.T here are many inherited and age-related eye disorders that cause degeneration of the retina as a consequence of loss of photoreceptor cells. Retinal and macular degeneration disorders can be divided into two main groups. The fi rst group primarily affects the photoreceptors and involves the majority of cases of retinitis pigmentosa. In the second group, the primary damage is to the adjacent RPE cells, and as a consequence of this damage, the photoreceptors degenerate. This group includes age-related macular degeneration, Stargardt’s macular dystrophy, a subtype of Leber’s congenital amaurosis in which RPE65 is mutated, Best’s disease and some cases of retini-tis pigmentosa, as well.W ith regard to retinitis pigmentosa (RP), it is a group of inherited retinal degeneration diseases that are caused, as mentioned above, by a primary progressive loss of rod and cone photoreceptors, followed by a subsequent degeneration of RPE (Hartong et al. 2006). The disease affects approxi-mately 1.5 million patients worldwide and is the most common cause of blindness in people under 70 years of age in the western world. The disease can be characterized by retinal pigment deposits visible on the fundus examination. In most cases, the disease primarily affects rods. At later stages of the disease, the degeneration of cones takes place. As a consequence of disease progression, the patients’ night vision is reduced. Patients initially lose peripheral vision while retaining central vision (a visual status termed “tunnel vision”). In advanced cases, central vision is also lost, commonly at about 60 years of age. The disease affects about 1 in 4,000. The inheritance can be autosomal-recessive, autosomal-dominant or X-linked (in ~50–60%, 30–40%, and 5–15% of cases, respectively). Mutations in more than 140 genes have been iden-tifi ed as causing RP (Hartong et al. 2006).Among these genes are those involved in phototransduc-tion, like rhodopsin, the a- and b- subunits of phos-phodiesterase, the a- and b- subunits of Rod cGMP gated channel and arrestin. The additional muta-tions were found in genes encoding structural pro-teins, like peripherin, rod outer segment protein and fascin. They were also found in transcription factors involved in photoreceptors’ development such as Crx and Nrl, and in other genes, whose products are involved in signaling, cell-cell interac-tion and trafficking of intracellular proteins. Currently, there is no effective cure for RP. Treatment with vitamin A palmitate, omega-3 fatty acids and other nutrients may somewhat slow the rate of the disease progression in many cases. Reduction in exposure to light was also shown to decrease the rate of retinal degeneration.A mong the group of retinal degenerations that are caused by primary loss of RPE cells or their function, age-related macular degeneration (AMD) is the most frequent condition and the leading cause of visual disability in the western world (Cook et al. 2008).Among people over 75 years of age, 25–30% are affected by AMD, with progressive central visual loss that leads to blindness in 6–8%. The retinal degeneration pri-marily involves the macula. The dry form of AMD is initiated by hyperplasia of the RPE and formation of drusen deposits, consisting of meta-bolic end products underneath the RPE or within the Bruch’s membrane. It may gradually progress into the advanced stage of geographic atrophy90M. Idelson and B. Reubinoff with degeneration of RPE and photoreceptorsover large areas of the macula causing central visual loss. Ten percent of dry AMD patients will progress to neovascular (wet) AMD, with blood vessels sprouting through the Bruch’s membrane with subsequent intraocular leakage and/or bleed-ing, accelerating the loss of central vision. While the complicating neovascularization can be treated with anti-VEGF agents, currently there is no effective treatment to halt RPE and photore-ceptor degeneration and the grim reality is that many patients eventually lose their sight (Cook et al. 2008).S targardt’s macular dystrophy (SMD) is the most common form of inherited macular dystro-phy affecting children (Walia and Fishman 2009). The disease is symptomatically similar to AMD. The prevalence of SMD is about 1 in 10,000 chil-dren. The disease involves progressive central visual loss and atrophy of the RPE beneath the macula following accumulation of lipofuscin in RPE cells, which is suggested to consist of non-degradable material, derived from ingested pho-toreceptor outer segments. The inheritance is predominantly autosomal recessive, although an autosomal dominant form has also been described. The mutation in the ABCA4 gene was found to be a most common cause of SMD. The product of the ABCA4 gene is involved in energy transport to and from photoreceptors. The mutated protein cannot perform its transport function and, as a result, photoreceptor cells degenerate and vision is impaired. Currently, there is no effective treat-ment for SMD.C ell therapy to replenish the degenerating cells appears as a promising therapeutic modality that may potentially halt disease progression in the various retinal and macular degeneration dis-orders caused by loss and dysfunction of RPE cells and photoreceptors (da Cruz et al. 2007).I n this chapter we will discuss the potential of human pluripotent cells which includes human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSCs), to gen-erate various types of retinal cells that could be used for transplantation therapy of retinal degen-eration disorders and disease modeling for drug discovery. C ell Therapy of Retinal and Macular DegenerationsT he eye is an attractive organ for cell therapy as it is easily accessible for transplantation and for simple monitoring of graft survival and potential complications by direct fundoscopic visualiza-tion. Anatomically, it is a relatively confi ned organ limiting the potential of unwanted extra-ocular ectopic cell distribution, and a low number of cells are required to replenish the damaged cells. The eye is also one of the immune privi-leged sites of the body.T he concept of replacing dysfunctional or degenerated retina by transplantation has been developing ever since the fi rst retina-to-retina transplant in 1986 (Turner and Blair 1986).In most studies, primary retinal immature (fetal) tissue has been used as donor material. It was demonstrated that such transplants can survive, differentiate, and even establish connections with the host retina to a limited degree (Ghosh et al. 1999). The subretinal transplantation of healthy RPE has some advantages over neural retinal transplantation, as it concerns only one cell type that is not involved in neural networking. Transplantation of RPE has been studied exten-sively in animal models (Lund et al. 2001).The most commonly used animal model of retinal degeneration is the Royal College of Surgeons (RCS) rat model, in which primary dysfunction of the RPE occurs as a result of a mutation in the receptor tyrosine kinase gene M ertk(D’Cruz et al. 2000). This leads to impaired phagocytosis of shed photoreceptor outer segments, with sec-ondary degeneration and progressive loss of pho-toreceptors within the fi rst months of life. It was reported that rat and human RPE cells rescued photoreceptor cells from degeneration when transplanted into the subretinal space of RCS rats (Li and Turner 1988; Coffey et al. 2002).The ability of transplanted RPE cells to restore retinal structure and function has been demonstrated in clinical trials. In humans, autologous transplanta-tions of peripheral RPE as well as macular trans-locations onto more peripheral RPE provide a proof that positioning the macula above relatively91 9 Differentiation of Human Pluripotent Stem Cells into Retinal Cellshealthier RPE cells can improve visual functionin AMD patients (Binder et al. 2004; da Cruz et al. 2007). Nevertheless, the surgical procedures for autologous grafting are challenging and are often accompanied by signifi cant complications. In addition, autologous RPE transplants may carry the same genetic background, environmen-tal toxic and aging-related effects that may have led to macular RPE failure and the development of AMD in the patient. It is also problematic to use autologous cells when all the RPE cells are damaged. Cell sources that can be used for such therapy include allogeneic fetal and adult RPE (Weisz et al. 1999; Binder et al. 2004; da Cruz et al. 2007). However, the use of fetal or adult retinal tissues for transplantation is severely lim-ited by ethical considerations and practical prob-lems in obtaining sufficient tissue supply. The search for a cell source to replace autologous RPE such as immortalized cell lines, umbilical cord-derived cells as well as bone marrow-derived stem cells continues.T he derivation of hESCs more than a decade ago has raised immense interest in the potential clinical use of the cells for regeneration (Thomson et al. 1998; Reubinoff et al. 2000).Along the years, signifi cant progress has been made towards the use of hESCs in clinical trials.T he other promising source of cells for transplantation therapy is iPSCs that are simi-lar to hESCs in their stemness characteristics and pluripotency. These cells could be gener-ated from different human somatic cells by transduction of four defi ned transcription fac-tors: Oct3/4, Sox2, Klf4, and c-Myc (Takahashi et al. 2007).G eneration of RPE and neural retina from hESCs and iPSC has numerous advantages, as it can be done from pathogen-free cell lines under good manufacturing practice (GMP) conditions with minimal variation among batches. Such cells can be characterized extensively prior to preclinical studies or for clinical applications, and an unlimited numbers of donor cells can be generated from them. In the following para-graphs, strategies for induction of differentiation of hESCs and iPSCs towards RPE and neural retina fate are reviewed. D ifferentiation into Retinal Pigment EpitheliumI t was reported for the fi rst time in mice and pri-mates that the differentiation of ES cells into RPE could be induced by co-culture with PA6 stromal cells (Kawasaki et al. 2002; Haruta et al. 2004). The resulting cells had polygonal epithelial mor-phology and extensive pigmentation. The cells expressed the markers that are characteristic of RPE. They developed typical ultrastructures and exhibited some functions of RPE. The differenti-ation of hESC into RPE was first reported by Klimanskaya et al. (2004).According to their protocol, hESCs underwent spontaneous differ-entiation by overgrowth on mouse embryonic fibroblasts (MEF), in feeder-free conditions or, alternatively, as embryoid bodies (EBs) in com-bination with withdrawal of bFGF from the medium. The yield of the formation of RPE cells after 4–8 weeks of spontaneous differentiation was relatively low; for example,<1% of EBs con-tained pigmented cells at this stage. However, after 6–9 months in culture, all the EBs contained pigmented cells. The areas of pigmented cells could be further isolated mechanically and prop-agated by passaging as RPE lines. Klimanskaya and colleges characterized the hESC-derived RPE cells by transcriptomics and demonstrated their higher similarity to primary RPE tissue than to human RPE lines D407 and ARPE-19. The low yield of spontaneously differentiating RPE cells was improved by induction of differentia-tion with Wnt and Nodal antagonists, Dkk1 and LeftyA, respectively, the factors that are sug-gested to promote retinal differentiation. This treatment gave rise to pigmented cells within 38% of the hESC colonies after 8 weeks (Osakada et al. 2008). Immunostaining with the ZO-1 anti-body showed that by day 120, hESC-derived pig-mented cells formed tight junctions (about 35% of total cells). We showed that differentiation toward the neural and further toward the RPE fate could be augmented by vitamin B3 (nicotin-amide; Idelson et al. 2009).We further showed that Activin A, in the presence of nicotinamide, effi ciently induces and augments differentiation92M. Idelson and B. Reubinoffinto RPE cells. This is in line with the presumed role of Activin A in RPE development i n vivo .In the embryo, extraocular mesenchyme-secreted members of the TGF b superfamily are thought to direct the differentiation of the optic vesicle into RPE (Fuhrmann et al. 2000).Under our culture conditions, when the cells were grown in suspen-sion as free-fl oating clusters, within 4 weeks of differentiation, 51% of the clusters contained pigmented areas and about 10% of the cells within the clusters were pigmented. When we modifi ed the differentiation conditions to includea stage of monolayer culture growth, the yield of the RPE-like pigmented cells was signifi cantly improved and 33% of the cells were pigmented after 6 weeks of differentiation. The derivation of RPE from hESCs and iPSCs without any external factor supplementation was also demonstrated by other groups (Vugler et al. 2008 ; Meyer et al. 2009 ; Buchholz et al. 2009).T he hESC-derived RPE cells were extensively characterized, including demonstration, both at the mRNA and the protein levels, of the expres-sion of RPE-specifi c markers, such as RPE65, CRALBP, Bestrophin, Tyrosinase, PEDF, PMEL17, LRAT, isoforms of MiTF abundant in RPE, and others. The cells expressed markers of tight junctions that join the adjacent RPE cells: ZO-1, occludin and claudin-1 (Vugler et al. 2008 ) . Electron microscopic analysis revealed that the hESC-derived RPE cells showed features characteristic of RPE. The cells were highly polarized with the nuclei located more basally, and the cytoplasm with the mitochondria and melanin granules of different maturity more api-cally. A formation of basal membrane was observed on the basal surface of the RPE cell. Similar to putative RPE, the hESC-derived RPE basal membrane was shown to be composed of extracellular matrix proteins, collagen IV , lami-nin and fi bronectin (Vugler et al.2008).The appearance of apical microvilli was demonstrated at the apical surface of the RPE. The presence of tight and gap junctions on the apical borders of the RPE cells was also confi rmed by electron microscopy. O ne of the most important functions of RPE cells i n vivo is phagocytosis of shed photoreceptor outer segments, as part of the continuous renewal process of rods and cones. The hESC-derived RPE cells demonstrated the ability to phagocyto-size latex beads or purifi ed photoreceptor outer segments, confi rming that these cells are func-tionali n vitro . It may be concluded from all these studies that human pluripotent stem cells have a potential to give rise to pigmented cells exhibiting the morphology, marker expression and functionof authentic RPE.D ifferentiation into Retinal Progenitors and Photoreceptors O ur group showed, for the fi rst time, the potential of highly enriched cultures of hESC-derived neu-ral precursors (NPs) to differentiate towards the neural retina fate (Banin et al. 2006).We demon-strated that the NPs expressed transcripts of key regulatory genes of anterior brain and retinal development. After spontaneous differentiation i n vitro , the NPs gave rise to progeny expressing markers of retinal progenitors and photoreceptor development, though this was uncommon and cells expressing markers of mature photorecep-tors were not observed. We showed that after transplantation into rat eyes, differentiation into cells expressing specifi c markers of mature photoreceptors occurred only after subretinal transplantation (between the host RPE and pho-toreceptor layer) suggesting that this specifi c microenvironment provided signals, yet unde-fi ned, that were required to support differentia-tion into the photoreceptoral lineage.P rogress towards controlling and inducing the differentiation of hESCs into retinal progenitors and neurons i n vitro was reported in the study of Lamba et al. ( 2006).They treated hESC-derived EBs for 3 days with a combination of factors,including Noggin, an inhibitor of BMP signaling, Dkk1, a secreted antagonist of the Wnt signaling pathway and insulin-like growth factor 1 (IGF-1), which is known to promote retinal progenitor dif-ferentiation. The cultivation of EBs with these factors was followed by differentiation on Matrigel or laminin for an additional 3 weeks in the presence of the combination of the three93 9 Differentiation of Human Pluripotent Stem Cells into Retinal Cellsfactors together with bFGF. Under these culture conditions, the majority of the cells developed the characteristics of retinal progenitors and expressed the specifi c markers Pax6 and Chx10 (82% and 86% of the cells, respectively). The authors showed that after further differentiation, the cells expressed markers of photoreceptor development Crx and Nrl (12% and 5.75%, respectively). About 12% of the cells expressed also HuC/D, the marker of amacrine and ganglion cells. The expression of markers of the other sub-types of retinal neurons was demonstrated, as well. However, only very few cells (<0.01%) expressed markers of mature photoreceptors, blue opsin and rhodopsin. The abundance of cells expressing markers of photoreceptors could be accelerated by co-culture with retinal explants, especially when the explants originated from mice bearing a mutation that causes retinal degeneration.T o better characterize the phenotype of retinal cells obtained with this differentiation protocol, a microarray-based analysis comparing human retina to the hESC-derived retinal cells was per-formed (Lamba and Reh 2011).It was demon-strated that gene expression in hESC-derived retinal cells was highly correlated to that in the human fetal retina. In addition, 1% of the genes that were highly expressed in the hESC-derived cultures could be attributed to RPE and ciliary epithelium differentiation.A n alternative protocol for the derivation of retinal progenitors and photoreceptors was pro-posed by Osakada et al. (2008).Similar to the protocol for the derivation of RPE cells, they used serum-free fl oating cultures in combination with the Dkk1 and LeftyA. After 20 days of cul-ture in suspension, the cells were replated on poly-D-lysine/laminin/fi bronectin-coated slides. Osakada and co-authors demonstrated that on day 35 in culture, about 16% of colonies were positive for retinal progenitor markers Rx and Pax6. Differentiation towards photoreceptor fate was augmented in the presence of N2 by treat-ment with retinoic acid and taurine, which are known inducers of rod fate differentiation. Under these conditions, after an extended culture period of 170 days, about 20% of total cells were positive for Crx, an early photoreceptor marker. On day 200, about 8.5% of the cells expressed the mature rod photoreceptor marker, rhodopsin, as well as cone photoreceptor markers, red/green and blue opsins (8.9% and 9.4%, respectively).A n alternative approach was proposed by the same group based on the use of small molecules. In this method, the chemical inhibitors CKI-7 and SB-431542 that inhibit Wnt and Activin A signaling, respectively, and Y-27632, the Rho-associated kinase inhibitor, which prevents disso-ciation-induced cell death, were used. These molecules were shown to mimic the effects of Dkk1 and LeftyA (Osakada et al. 2009).This strategy, which doesn’t involve the use of recom-binant proteins which are produced in animal or E scherichia coli cells, is more favorable for the gen-eration of cells for future transplantation therapy.I n another study that was published by Meyer et al .(2009), after initial differentiation in sus-pension for 6 days, the aggregates were allowed to attach to laminin–coated culture dishes. After further differentiation as adherent cultures, neu-roepithelial rosettes were formed, which were mechanically isolated and subsequently culti-vated as neurospheres. The authors didn’t use any soluble factors; moreover, they showed that under these conditions, the cells expressed endogenous Dkk1 and Noggin. They also demonstrated that in concordance with the role of bFGF in retinal specifi cation, the inhibition of endogenous FGF-signaling abolished retinal differentiation. Under their differentiation protocol, by day 16, more than 95% of the cells expressed the retinal pro-genitor markers, Pax6 and Rx. The authors dem-onstrated that by day 80 of differentiation, about 19% of all neurospheres contained Crx+ cells and within these Crx+ neurospheres, 63% of all cells express Crx and 46.4% of the cells expressed mature markers, such as recoverin and cone opsin.I n all of the above studies, differentiated cells expressing the retinal markers were obtained; however, the cells were not organized in a three-dimensional retinal structure. In a paper recently published by Eiraku et al. (2011),the authors cul-tured free-fl oating aggregates of mouse ES cells in serum-free medium in the presence of base-ment membrane matrix, Matrigel, that could also94M. Idelson and B. Reubinoffbe substituted with a combination of laminin, entactine and Nodal. Using a mouse reporter ES cell line, in which green fl uorescent protein (GFP) is knocked in at the Rx locus, the authors showed that Rx-GFP+ epithelial vesicles were evaginated from the aggregates after 7 days of differentiation under these conditions. On days 8–10, the Rx-GFP+ vesicles changed their shape and formed optic cup-like structures. The inner layer of these structures expressed markers of the neural retina whereas the outer layer expressed markers of RPE. The authors demonstrated that differen-tiation into RPE required the presence of the adjacent neuroectodermal epithelium as a source of diffusible inducing factors. In contrast, the differentiation into neural retina did not require tissue interactions, possibly because of the intrinsic inhibition of the Wnt-signaling pathway. Eiraku and colleagues showed that the retinal architecture, which was formed within the optic vesicle-like structures, was comparable to the native developing neural retina.R ecently, optic vesicle-like structures were also derived from hESCs and iPSCs using the protocol described above, which is based on iso-lating the neural rosette-containing colonies and culturing them in suspension (Meyer et al. 2011). The cells within the structures expressed the markers of retinal progenitors, and after differen-tiation gave rise to different retinal cell types. It was shown that the ability of optic vesicle-like structures to adopt RPE fate could be modulated by Activin A supplementation. The production of these three-dimensional retinal structures opens new avenues for studying retinal development in normal and pathological conditions.T ransplantation of Pluripotent Stem Cell-Derived Retinal CellsA key step towards future clinical transplanta-tions of hESC-derived RPE and neural retina is to show proof of their therapeutic potential i n vivo. Various animal models of retinal degeneration have been used to evaluate the therapeutic effect of transplanted retinal cells. Human ESC-derived RPE cells were transplanted subretinally to the degenerated eyes of RCS rats. Transplantation of the hESC-derived RPE cells between the RPE and the photoreceptor layer rescued retinal struc-ture and function (Lund et al. 2006; Vugler et al. 2008; Idelson et al. 2009; Lu et al. 2009).The subretinally engrafted hESC-derived RPE cells salvaged photoreceptors in proximity to the grafts as was shown by the measurement of the thick-ness of the ONL, the layer of photoreceptor nuclei, which is an important monitor of photore-ceptor cell survival. The ONL thickness was significantly increased in transplanted eyes in comparison to the degenerated non-treated eyes.I n order to evaluate the functional effect of transplanted cells i n vivo, the electroretinography (ERG) that directly measures the electrical activ-ity of the outer (a-wave) and inner (b-wave) retina in response to light stimulation was used. It was demonstrated that after transplantation of hESC-derived RPE, ERG recordings revealed a signifi -cant preservation of retinal function in the treated eyes as compared to control untreated eyes (Lund et al. 2006; Idelson et al. 2009).The visual func-tion of the animals was also estimated by an optomotor test, which monitors the animal’s refl exive head movements in response to a rotat-ing drum with fi xed stripes. Animals transplanted with hESC-derived RPE showed signifi cantly better visual performance in comparison to con-trol animals (Lund et al. 2006; Lu et al. 2009). The presence of rhodopsin, a major component of photoreceptor outer segments, within the sub-retinaly transplanted pigmented cells suggested that they could perform phagocytosis i n vivo (Vugler et al. 2008; Idelson et al. 2009).B ridging the gap between basic research and initial clinical trials requires immense resources to ensure safety and efficacy. Human ESC-derived RPE cell lines were generated using a current Good Manufacturing Practices (cGMP)-compliant cellular manufacturing process (Lu et al. 2009). Long-term studies analyzing safety and efficacy of transplantation of these GMP-compliant hESC-derived RPE cells revealed that the subretinally transplanted cells survived for a period of up to 220 days and provided prolonged functional improvement for up to 70 days after transplantation. The potential of the hESC-derived。
基于网络药理学分析冬虫夏草防治急性肾损伤的分子机制
基于网络药理学分析冬虫夏草防治急性肾损伤的分子机制①洪涛李晓宇②李尚妹②刘华锋②③（广东医科大学附属医院肾内科，湛江 524000）中图分类号R285.5 文献标志码 A 文章编号1000-484X（2023）11-2299-06［摘要］目的：基于网络药理学分析冬虫夏草（CS）防治急性肾损伤（AKI）的分子作用机制。
方法：应用中药系统药理学数据库（TCMSP）筛选CS的有效成分和作用靶点，从DisGeNET、GeneCards、OMIM、TTD数据库筛选AKI的疾病靶点，运用Cytoscape 3.8.0软件构建“药物-成分-疾病-靶点”可视化调控网络，采用String在线数据库构建CS与AKI共同靶点的蛋白互作网络，DAVID数据库和KEGG数据库对共同靶点进行GO功能富集分析和KEGG通路富集分析，探讨其潜在分子机制。
结果：CS中筛选得到有效成分9种，防治AKI的作用靶点63个。
GO功能富集分析主要包括对药物的反应、信号转导、衰老、细胞增殖调控、细胞凋亡调控等。
CS防治AKI的主要富集通路有PI3K-Akt信号通路、MAPK信号通路、细胞凋亡通路、TNF信号通路、P53信号通路等。
结论：通过网络药理学研究方法预测了CS防治AKI的有效活性成分及作用靶点，并通过PPI网络和KEGG富集分析推测了CS通过多条信号通路调节防治AKI，涉及细胞自噬、凋亡及炎症反应等多环节生物学过程。
［关键词］冬虫夏草；急性肾损伤；网络药理学；分子机制Network pharmacology-based identification of key mechanism of Cordyceps sinensis' protection from acute kidney injuryHONG Tao， LI Xiaoyu， LI Shangmei， LIU Huafeng. Department of Nephrology， Affiliated Hospital of Guangdong Medical University， Zhanjiang 524000， China［Abstract］Objective：To explore potential key mechanism of Cordyceps sinensis´ （CS） protection from acute kidney injury （AKI） through network pharmacological analysis. Methods：All bioactive ingredients and target of CS were obtained from TCMSP data‐base. Targets related to AKI were obtained from DisGeNET， GeneCards， OMIM， TTD databases. Cytoscape 3.8.0 software was used to visualize "Medicine-Component-Disease-Target" networks. String online database was used to construct PPI network of common target between CS and AKI. GO functional enrichment analysis and KEGG pathway enrichment analysis were performed for common target in DAVID database and KEGG database to explore its potential molecular mechanism. Results：A total of 9 active ingredients and 63 potential targets in treatment of AKI have been identified. GO functional enrichment were mainly related to drug response， signal transduction， senescence， cell proliferation regulation， apoptosis regulation， etc. Pathways of CS to control AKI mainly enriched in PI3K-AKT signaling pathway， MAPK signaling pathway， apoptosis signaling pathway， TNF signaling pathway， P53 signaling path‐way，etc. Conclusion：Effective active ingredients and targets of CS for preventing AKI are predicted by network pharmacology. PPI network and KEGG enrichment analysis also speculates that CS regulates prevention and treatment of AKI through multiple signaling pathways， involving multiple biological processes such as autophagy， apoptosis and inflammatory response.［Key words］Cordyceps sinensis；Acute kidney injury；Network pharmacology；Molecular mechanism急性肾损伤（acute kidney injury，AKI）是多种原因造成的肾功能急性下降，是临床常见危重症。
罗哌卡因抑制M1型巨噬细胞极化缓解大鼠脑缺血再灌注损伤引起的免疫紊乱和组织氧化应激损伤
中国免疫学杂志2022年第38卷罗哌卡因抑制M1型巨噬细胞极化缓解大鼠脑缺血再灌注损伤引起的免疫紊乱和组织氧化应激损伤①肖旭李光才国小青况小军（贵州航天医院麻醉科，遵义563000）中图分类号R743文献标志码A文章编号1000-484X （2022）02-0160-05［摘要］目的：探究罗哌卡因对大鼠脑缺血再灌注损伤引起的免疫紊乱和组织氧化应激损伤的影响。
方法：构建脑缺血再灌注损伤大鼠模型，将大鼠随机分为5组：Control 组、CI/R 组、CI/R+ROP 0.5mg/kg 组、CI/R+ROP 1mg/kg 组和CI/R+ROP 2mg/kg 组进行后续实验。
HE 染色检测脑组织病理损伤程度；跳台实验和Y 迷宫实验检测大鼠学习记忆能力；流式分选检测外周血中M1、M2含量；ELISA 检测IL -6、IL -10含量；试剂盒检测SOD 、GSH 、MDA 含量；蛋白免疫印迹检测脑组织中Arg -1、iNOS 、Bax 、Bcl -2、Caspase -3、c -Myc 蛋白表达水平。
结果：与Control 组相比，CI/R 组呈现明显脑缺血再灌注病理损伤，跳台实验犯错次数增加，新异臂进入次数减少，M1、IL -6、MDA 含量和iNOS 、cleaved Caspase -3/Caspase -3蛋白水平升高，M2、IL -10、SOD 、GSH 含量和Arg -1、c -Myc 蛋白水平降低，以上差异均有统计学意义（P <0.05）；与CI/R 组相比，CI/R+ROP 1、2mg/kg 组病理损伤程度明显改善，跳台实验犯错次数减少，新异臂进入次数增加，M1、IL -6、MDA 含量和iNOS 、cleaved Caspase -3/Caspase -3蛋白水平降低，M2、IL -10、SOD 、GSH 含量和Arg -1、c -Myc 蛋白水平升高，以上差异均有统计学意义（P <0.05）；结论：罗哌卡因通过抑制M1型巨噬细胞极化缓解脑缺血再灌注损伤大鼠引起的免疫紊乱和组织氧化应激损伤。
基于Web_of_Science的药品说明书适老化研究文献计量学分析
·药师与药学服务·基于Web of Science 的药品说明书适老化研究文献计量学分析Δ李茜茜 1＊，陆浩 2，武明芬 3，赵志刚 3 #[1.首都医科大学附属北京胸科医院药学部，北京 101149；2.壹正医院管理发展（北京）中心，北京 100020；3.首都医科大学附属北京天坛医院药学部，北京 100070]中图分类号 R 95 文献标志码 A 文章编号 1001-0408（2024）02-0231-06DOI 10.6039/j.issn.1001-0408.2024.02.19摘要 目的 探索药品说明书适老化的研究热点，为我国药品说明书适老化发展提供依据。
方法 在Web of Science 核心合集数据库中检索2012－2022年发表的老年人使用药品说明书的相关英文文献，应用VOSviewer 和CiteSpace 软件进行文献计量学分析，探索该领域的研究热点，并总结该领域发展的障碍和解决措施。
结果与结论 本研究共收集到药品说明书适老化相关文献335篇，来自51个国家（地区）的819个研究机构，涉及2 174位作者。
近10年，药品说明书适老化研究发展趋缓，美国和日本等发达国家占该领域的主导地位，其中美国西北大学Wolf 等学者的发文量最大（12篇）。
老年人用药风险管理、药品说明书中老年用药信息的更新，以及老年人对药品说明书的理解和依从及其影响因素等是该领域的研究热点。
药品说明书适老化发展中相关障碍的解决措施包括提高药品说明书的可视性、可读性，填补药品说明书中老年用药信息等。
我国可借鉴其他国家的经验和方法，针对我国人口特点开展老年药品说明书影响因素的调查及老年用药的药动学研究，多维度提高老年患者用药的安全性。
关键词 药品说明书；适老化；文献计量学；用药安全；老年人Bibliometric analysis of drug package insert adaptation for the elderly based on Web of ScienceLI Xixi 1，LU Hao 2，WU Mingfen 3，ZHAO Zhigang 3[1. Dept. of Pharmacy ， Beijing Chest Hospital ， Capital Medical University ， Beijing 101149， China ；2. Yizheng Hospital Management and Development （Beijing ） Center ， Beijing 100020， China ；3. Dept. of Pharmacy ， Beijing Tiantan Hospital ， Capital Medical University ， Beijing 100070， China]ABSTRACTOBJECTIVE To explore the hotspots of aging adaptation of drug package inserts ， and to provide evidence for thedevelopment of aging adaptation of drug package inserts in China. METHODS The relevant English literature on drug package inserts for the elderly published from 2012 to 2022 was retrieved from Web of Science Core Collection ； bibliometric analysis was performed by using VOSviewer and CiteSpace software ， to explore research hotspots in this field ， and summarize obstacles and solutions for the development of this field. RESULTS & CONCLUSIONS This study collected a total of 335 literature related to the aging adaption of drug package inserts ， from 819 research institutions in 51 countries （regions ）， involving 2 174 authors. The research development of drug package insert adaptation for the elderly has slowed down in the past decade ， and developed countries such as the United States and Japan dominate this field. Authors such as Wolf from Northwestern University in the United States ， have the largest number of publications （12 literature ）. The research focuses in this field include the risk management of medication for the elderly ， the updating of medication information for the elderly in drug package inserts ， and the understanding and compliance of the elderly with drug package inserts and their influencing factors. The solutions to related obstacles in the development of aging adaption in drug package inserts include improving the visibility and readability of drug package inserts ， filling in the information on elderly medication in drug package inserts ， and so on. China can learn from the experiences and methods of other countries ， conduct investigations into the influencing factors of elderly package inserts and pharmacokineticstudies based on the characteristics of the Chinese population ， and improve the safety of medication for elderly patients in multiple dimensions.KEYWORDSdrugpackageinsert ； agingadaption ；bibliometrics ； medication safety ；the elderlyΔ 基金项目国家卫健委药具管理中心委托项目（No.ZDC-2022-001）；中国药品监督管理研究会研究课题（No.2023-Y-Y-018）＊第一作者药师，硕士。
【北京市自然科学基金】_结构检测_基金支持热词逐年推荐_【万方软件创新助手】_20140729
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窄谱x射线 神经干细胞 磷化钨 硝基咪唑类化合物 矿化结晶 癌热疗 玉米 炭黑燃烧 温敏性 温度检测 模态参数 桥梁 柔度曲率矩阵 杂交种 服装动画 智能弹性架构 无损检测 新生儿 支架材料 损伤识别 损伤变量 损伤分析 抽验 心肌膜 微血管内皮细胞 异质扩散 嵌入式数据库 山茱萸 小波包分解 大鼠 多糖 壳聚糖水凝胶 基因 噪声消除 咔唑 听性脑干反应 听功能 同步锚 同步 吉林省 可注射性 可扩展标记语言 叠氮钠 反应选择性 双靶x光管 双歧杆菌 双层候选集 原位 卡那霉素 协调控制 协商控制 医疗器械 北京 动物实验
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朱章志运用扶正祛邪法论治糖尿病经验
ʌ临证验案ɔ朱章志运用扶正祛邪法论治糖尿病经验❋曾绘域1,朱章志2ә,周㊀海3,陈㊀珺3,张文婧3(1.深圳市中西医结合医院,广东深圳㊀518104;2.广州中医药大学第一附属医院,广州㊀510405;3.广州中医药大学,广州㊀510405)㊀㊀摘要:糖尿病属于中医学 消渴病 范畴,以往医家多认为其病机为阴虚燥热,治疗以滋阴清热为法㊂朱章志教授通过长期的临床观察与实践,立足于张仲景 保胃气,扶阳气 的理论,认为糖尿病的病机为正虚邪滞,即太阴虚损㊁阳气不足㊁收敛不及,寒㊁水㊁湿之邪阻滞阳气运行通道㊂治疗上不囿陈法,以扶正祛邪为大法,通过固护太阴㊁扶助阳气㊁收敛阳气,祛除寒水湿之邪,恢复阳气运行之通畅,使阳气功能复常㊁运行有序,为糖尿病的治疗提供临床新思路㊂㊀㊀关键词:扶正祛邪;糖尿病;朱章志㊀㊀中图分类号:R587.1㊀㊀文献标识码:A㊀㊀文章编号:1006-3250(2021)01-0149-03Discussion on ZHU Zhang-zhi's Experience in Treating Diabetes Mellitus by Using The Method of Reinforcing The Healthy Qi and Eliminating The Pathogenic FactorsZENG Hui-yu 1,ZHU Zhang-zhi 2ә,ZHOU Hai 3,CHEN Jun 3,ZHANG Wen-jing 3(1.Shenzhen Hospital of Integrated traditional Chinese and Western Medicine,Guangdong,Shenzhen 518104,China;2.The First Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510405,China;3.Guangzhou University of Chinese Medicine,Guangzhou 510405,China)㊀㊀Abstract :Diabetes mellitus belongs to the category of "xiao ke"in traditional Chinese medicine.Doctors used to think that its pathogenesis was Yin deficiency and dryness heat ,and the treatment was nourishing Yin and clearing heat.Through long-term clinical observation and practice ,and based on ZHANG Zhong-jing's theory of protecting stomach Qi and supporting Yng Q ,professor ZHU Zhang-zhi believes that the pathogenesis of diabetes is deficiency of healthy Qi and stagnation of pathogen.Because of the deficiency of greater Yin and Yang Qi ,and the lack of convergence ,the cold ,water and dampness block the operational channel of Yang Qi.The treatment of diabetes mellitus should be based on reinforcing the healthy Qi and eliminating the pathogenic factors.By strengthening Taiyin ,supporting Yang Qi ,astringent Yang Qi ,dispelling the evil of cold ,water and dampness ,we can restore the smooth operation of Yang Qi ,restore the function of Yang Qi to normal and operate orderly ,which provides a new clinical method for the treatment of diabetes mellitus.㊀㊀Key words :Reinforcing the healthy Qi and eliminating the pathogenic factors ;Diabetes mellitus ;ZHU Zhang-zhi❋基金项目:国家自然科学基金资助项目(81873190)-降糖三黄片在糖脂毒性所致胰岛β细胞损伤的自噬调控作用作者简介:曾绘域(1990-),女,广东云浮人,住院医师,硕士研究生,从事六经辨治内分泌疾病的临床与研究㊂ә通讯作者:朱章志(1963-),男,湖南衡阳人,主任医师,博士研究生导师,从事六经辨治内分泌疾病的临床与研究,Tel :************,E-mail :zhuangi@ ㊂㊀㊀随着人口老龄化和生活方式的改变,我国糖尿病的患病率呈上升趋势,2013年我国18岁以上人群糖尿病患病率为10.4%[1]㊂中医药在延缓糖尿病的进展及防治其并发症方面具有一定优势[2-4]㊂糖尿病属于中医学 消渴病 范畴,以往医家多认为其病机为阴虚燥热,治疗以滋阴清热为法,但疗效尚不能令人满意㊂朱章志教授通过长期的临床观察与实践,认为正虚邪滞乃糖尿病病机之核心,采用扶正祛邪法治之屡获奇效㊂1㊀正虚邪滞之糖尿病病机‘素问㊃经脉别论篇“曰: 饮入于胃,游溢精气,上输于脾,脾气散精 水精四布,五经并行㊂食物入胃,经脾胃运化化生精气,然后输布全身㊂糖尿病患者常嗜食肥甘,起居无常,烦劳紧张,致太阴虚损,正气内虚,阳气戕伐,津液代谢异常,而生寒水湿之邪㊂寒㊁水㊁湿之邪气作为阴邪,又可阻滞阳气运行之通道㊂阳气运行通道不畅,不能敷布温煦四肢,可见手足逆冷;阳气运行受阻,又可出现郁而化热之象㊂因此朱章志认为,疗糖尿病的关键在于恢复阳气运行之通畅,根据糖尿病正虚邪滞的病机,治疗以扶正祛邪为法,顾护太阴㊁扶助阳气㊁收敛阳气,祛除寒水湿之邪,使阳气功能复常则行有序㊂2㊀运用扶正祛邪法治疗糖尿病2.1㊀扶正2.1.1㊀固护中气,扶助阳气㊀张仲景遣方用药常体现 保胃气 之思想[5],如桂枝汤中配伍生姜㊁大枣㊁炙甘草,发汗祛邪不忘顾护中气;又如白虎汤中加梗米㊁炙甘草以和中益胃,又可防止石膏㊁知母大寒伤中㊂ 有胃气则生,无胃气则死 ,故扶正之要以保胃气为先㊂朱章志认为,阳气在人体的生命活动中占主导9412021年1月第27卷第1期January 2021Vol.27.No.1㊀㊀㊀㊀㊀㊀中国中医基础医学杂志Journal of Basic Chinese Medicine地位㊂‘素问㊃生气通天论篇“曰: 阳气者若天与日,失其所则折寿而不彰 是故阳因而上,卫外者也㊂ ‘黄帝内经“把阳气比作太阳,阳气运行失常可致短寿㊂阳气具有抵御外邪㊁护卫生命㊁维持机体生命活动的作用,津液的气化㊁血液的运行均需阳气的温煦与推动㊂因此,在人体的阴阳平衡中阳气起着主导作用㊂朱章志认为,正气虚衰㊁太阴虚损㊁阳气不足是糖尿病发生发展之根本原因,因此扶正首当 固护中气,扶助阳气 ,故常以附子理中汤为底方,固护中宫㊂太阴脾土居中央,犹如足球比赛之中场,能联系前锋与后卫进可攻退可守,进可充养肺卫之气抵御外邪,退可顾护少阴以防寒邪内陷㊂‘四圣心源㊃卷二太阴湿土“提到: 湿者,太阴土气之所化也故胃家之燥,不敌脾家之湿,病则土燥者少而土湿者多也㊂[6] 阴脾土易挟寒湿,附子理中汤功善固护中气㊁温补脾阳而散寒湿,为治疗太阴阳虚寒湿之要方㊂方中附子辛温大热,补坎中真阳,又能散寒湿,荡去群阴;干姜去脏腑沉寒痼冷,温暖脾土,复兴火种;人参被誉为 百草之王 能大补元气,为扶正固本之极品;白术味苦性温,功善健脾燥湿,乃扶植太阴之要药;炙甘草善益气补中,调和药性,诸药合用以收培补中阳㊁散寒除湿之效㊂若其人神疲懒言,气虚较甚,在附子理中汤的基础上可重用红参㊁北芪以大补元气,健脾益气;若其人四肢不温㊁肢体困重㊁寒湿较重者,可加重附子㊁干姜之量,并加细辛㊁吴茱萸以散久寒;若其人口干口苦㊁舌苔黄腻㊁大便黏滞不爽兼夹湿热之象,可仿当归拈痛汤之意,加茵陈㊁当归㊁黄芩以利湿清热㊂2.1.2㊀收敛阳气,阳密乃固㊀朱章志认为, 阴 可理解为 阳气 的收敛㊁收藏状态,糖尿病 阴虚燥热 之象乃阳气不足㊁收敛不及㊁升发太过所致[7]㊂‘素问㊃生气通天论篇“提到: 阳气者,烦劳则张 ㊂现代人起居无节,以妄为常,阳气因而不能潜藏,常常浮越于外容易出现假热之象,医者不察,妄投清热泻火之品,实乃雪上加霜㊂ 凡阴阳之要,阳密乃固 ,收敛阳气即是扶正,犹如太极之能收能放,收敛是为了聚集能量,阳气固密,正气才能强盛,方能更好的制敌㊂朱章志常用砂仁㊁肉桂㊁白芍㊁山萸肉㊁泽泻等药物收敛阳气㊂砂仁辛温,既能宣太阴之寒湿,又能纳气归肾,使阳气收敛于少阴,少火生气㊂‘本草经疏“提到: 缩砂蜜,辛能散,又能润 辛以润肾,故使气下行 气下则气得归元㊂[8] 肉桂引火归原,导浮越之阳气归于命门,益火消阴㊂若患者出现咽痛㊁牙龈肿痛㊁痤疮等阳气不敛㊁虚火上冲之象,常用砂仁㊁肉桂以收敛阳气,纳气归肾,引火归原㊂白芍味酸能敛,敛降甲木胆火,使相火归位㊂‘本草求真“曰: 气之盛者,必赖酸为之收,故白芍号为敛肝之液,收肝之气,而令气不妄行也㊂[9] 朱章志常使用白芍以补肝之体㊁助肝之用,收敛肝气,肝平则郁气自除,火热自消㊂山萸肉秘精气㊁敛阳气,使龙雷之火归于水中㊂朱章志常用山萸肉收敛正气,遇汗出多者,常重用以固涩敛汗㊂泽泻能泻能降,能入肾泻浊,开气化之源,泻浊以利扶正,又能降气而引火下行㊂朱章志常用泽泻打通西方潜藏之要塞[10],在温阳之品中加入泽泻,利于阳气潜藏,使孤阳有归㊂2.1.3㊀填补阴精,以滋化源㊀‘素问㊃金匮真言论篇“提到: 夫精者,身之本也㊂ 精 是人体生命活动的物质基础,能化气生髓,濡养脏腑㊂人体之精禀受于父母,又由后天水谷之精不断充养,归藏于肾中㊂ 孤阴不生,独阳不长 ,无阳则阴无以生,无阴则阳无以化㊂肾乃水火之脏,阴精充足才能涵养坎中真火,使真阳固密于内,化生正气㊂朱章志常在秋冬之季嘱糖尿病患者进补阿胶等血肉有情之品填补肾精㊂肾主封藏,秋冬进补使肾精充养,以滋阳气化生之源㊂阿胶用黄酒烊化,既能祛除阿胶之腥,又能借黄酒通行之性解阿胶滋腻碍胃之弊,每日少量服用,以有形之精难以速生,填补肾精以缓补为要㊂除此之外,遣方用药时亦会注意顾护阴精,在使用温阳药的同时常常配伍山萸肉㊁白芍等养阴药,以防温燥伤阴之弊㊂2.2㊀祛邪2.2.1㊀外散寒水以运太阳㊀ 太阳为开 ,太阳乃三阳之表,巨阳也,其性开泄以应天,为祛邪之重要通道㊂在运气里,太阳在天为寒,在地为水,合而为太阳寒水㊂张仲景太阳病篇研究的是水循环过程,治太阳就是治水[11]㊂寒㊁水之邪闭郁在表,气血运行不畅,可见肌肤麻木不仁㊂邪气滞留太阳,阻碍阳气运行,当因势利导㊁开太阳之表以发汗,外散寒㊁水之邪㊂糖尿病患者正气亏虚为本,祛邪不能伤正,朱章志临床常用桂枝麻黄各半汤小发其汗,使玄府开张,邪有出路㊂桂枝麻黄各半汤乃发汗轻剂,为桂枝汤与麻黄汤相合而得,其中麻黄㊁桂枝㊁生姜㊁北杏发散宣肺以开皮毛,芍药㊁大枣㊁炙甘草酸甘化阴以益营,诸药相合,刚柔相济,祛邪而不伤正㊂邪去正安,阳气运行通畅,水液代谢复常则阳气自充,而无寒水之扰㊂若寒邪较重可用三拗汤,此为麻黄汤去桂枝而成,功善开宣肺气,疏散风寒,因去辛温之桂枝发汗力不及麻黄汤,祛邪而不伤正㊂2.2.2㊀下利水湿以健少阴㊀少阴乃水火交会之脏,元气之根,人身立命之本㊂‘医理真传“提到: 坎中真阳,一名龙雷火,发而为病,一名元阳外越,一名孤阳上浮,一名虚火上冲㊂此际之龙,乃初生之龙,不能飞腾而兴云布雨,惟潜于渊中,以水为家,以水为性,遂安其在下之位㊂水盛一分龙亦盛一分,水高一尺龙亦高一尺,是龙之因水盛而游 [12]㊂阴盛051中国中医基础医学杂志Journal of Basic Chinese Medicine㊀㊀㊀㊀㊀㊀2021年1月第27卷第1期January2021Vol.27.No.1则阳衰,水湿之邪泛滥,则龙雷之火因而飞越在外㊂叶天士深谙张仲景之理,提到 通阳不在温,而在利小便 [10,13],通过利小便的方法,使水湿之邪从下而解,阳气运行通道无水湿之邪阻碍,则阳气无需温养而自通,水盛得除则真龙亦安其位㊂朱章志常用五苓散㊁真武汤下利水湿,以复阳气之通达,少阴之健运㊂五苓散具有通阳化气利水之效,治疗膀胱气化不利形成的蓄水证㊂方中猪苓㊁茯苓㊁泽泻导水湿之邪下行;白术健脾燥湿,杜绝生湿之源;桂枝助膀胱气化,通阳化气行水又通气于表,使全身在表之湿邪皆得解,五药合用,膀胱气化复常,水道通调使小便得利,水湿得出㊂真武汤为治疗少阴阳虚㊁水气泛滥之主方,方中附子振奋少阴阳气,使水有所主;白术㊁茯苓健脾制水;生姜助附子敷布阳气,宣散水气;芍药利小便,制附㊁姜之燥,五味相合共奏温阳利水之功㊂2.2.3㊀开郁逐寒以畅厥阴㊀肝为将军之官,肝气主动主升发,能统帅兵马,捍卫君主㊂厥阴肝经,体阴用阳,内寄相火,相火敷布阳气,祛阴除寒,是祛邪的先锋主力军㊂朱章志常用吴茱萸汤祛除厥阴肝经之寒邪,恢复肝经阳气之运行㊂方中吴茱萸辛苦而温,芳香而燥,‘本草汇言“曰: 开郁化滞,逐冷降气之药 [14],能温胃暖肝,降浊阴止呕逆,为治疗肝寒之要药㊂配以生姜温胃散寒,佐以人参㊁大枣健脾益气补虚,全方散寒与降逆并施,共奏暖肝温胃㊁降逆止呕之效㊂‘素问㊃至真要大论篇“说: 帝曰:厥阴何也?岐伯曰:两阴交尽也㊂ 物极必反,重阴必阳㊂厥阴为阴尽阳生之脏,足厥阴肝经与足少阳胆经互为表里,若出现肝气不疏㊁枢机不利㊁气郁化火,朱章志常用小柴胡汤和畅枢机,开郁以复气机调达㊂方中柴胡疏泄肝胆之气;黄芩清泄胆火,一疏一清,气郁通达,火郁得发;生姜㊁半夏和胃降逆;人参㊁大枣㊁炙甘草固护中宫,全方寒温并用㊁补泻兼施,以复厥阴疏泄之职,使气机和畅㊁阳气运行有序㊂3㊀典型病案患者杨某,女,65岁,2017年3月10日初诊:2型糖尿病病史6年余,症见疲乏,双下肢轻度浮肿,下肢冰凉,背部易汗出,口苦口干,偶有腰膝酸软,纳眠可,二便调,舌淡暗,苔黄腻,脉沉细㊂辅助检查示糖化血红蛋白10.8%,空腹血糖14.59mmol/L,总胆固醇6.38mmol/L,甘油三酯3.66mmol/L,低密度脂蛋白胆固醇4.34mmol/L㊂西医诊断2型糖尿病㊁高脂血症,治疗给予门冬胰岛素30(早餐前22u晚餐前20u)控制血糖,阿托伐他汀钙片(20mg, qn)调脂㊂中医诊断消渴病,少阴阳虚寒湿证㊂患者少阴阳气衰微不足以养神,固见疲乏;腰为肾之府,少阴阳虚则见腰膝酸软,阳虚寒盛则见下肢冰凉;背部正中乃督脉运行之所,阳气虚衰无以固摄则见背部汗出;少阴阳虚不能主水,寒水泛滥则见双下肢浮肿;水湿内停有郁而化热之象,则见口苦口干㊁舌苔黄腻㊁舌淡暗,脉沉细为少阴阳虚寒湿之征,治以温阳散寒㊁利水除湿为法㊂方以扶正祛邪方合当归拈痛汤加减:炮附片10g(先煎1h),红参10g (另炖),干姜15g,白术30g,炙甘草15g,桂枝12 g,麻黄8g,生姜30g,猪苓10g,泽泻30g,茯苓30 g,白芍30g,酒萸肉45g,当归15g,茵陈10g,5剂水煎服,2d1剂,水煎至250ml,饭后分2次服用,次日复煎㊂方中以附子理中汤为底方温补中焦,散寒除湿;加桂枝㊁麻黄使寒湿之邪从皮毛而解;加五苓散通阳化气,使湿邪从下而出;生姜散寒除湿;白芍㊁酒萸肉收敛阳气,以助正气祛邪;当归活血利水;茵陈清热利湿㊂2017年3月24日二诊:患者双下肢浮肿减轻,疲乏较前好转,无口干口苦,无腰膝酸软,仍觉下肢冰凉,背部仍有汗出,动则尤甚,大便每日二行,质偏烂,舌淡暗,苔白腻,脉细㊂患者大便质烂,乃邪有出路,导水湿之邪从大便而解㊂患者无口干口苦,舌苔由黄腻转为白腻,知湿郁化热之象已除,遂去茵陈㊂仍觉下肢冰凉乃内有久寒,加制吴茱萸12g以散沉寒痼冷;上方加酒萸肉至60g以加强收敛阳气㊁固摄敛汗之效,加黄芪60g以健脾益气敛汗;加砂仁6g(后下)㊁肉桂3g(焗服)以加强收敛阳气㊁扶助正气之效,7剂水煎服,服法同前㊂2017年4月7日三诊:患者背部汗出减少,下肢转温,余症皆除,大便每日二行质软,舌淡红,苔薄白,脉细较前有力,继续服二诊方药5剂㊂后给予附子理中丸(每次8粒,每日3次)服用1个月巩固疗效㊂2017年11月17日复诊:患者上述症状皆除,纳眠可,二便调㊂复查糖化血红蛋白6.8%,空腹血糖6.5mmol/L,总胆固醇5.14mmol/L,甘油三酯1.65 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基于CCL2
基于CCL2/CCR2信号轴的肿瘤免疫治疗药物的研究进展崔珍珍，赵一凡，孙 玉，孟佳怡，康 迪*，胡立宏**（南京中医药大学药学院, 江苏省中药功效物质重点实验室, 南京 210023）摘 要 趋化因子配体2（C-C motif chemokine ligand 2，CCL2）及其受体CCR2与肿瘤的发生、发展密切相关。
CCL2/CCR2信号轴通过多种机制促进肿瘤进展，一方面CCL2与肿瘤细胞表面的CCR2结合促进肿瘤的生长/存活和转移；更为重要的是CCL2可以招募多种免疫抑制细胞在肿瘤微环境中聚集，抑制免疫细胞的功能和活性，促进肿瘤进展。
本文综述了CCL2/CCR2信号轴以及其在肿瘤及肿瘤微环境中的作用，并重点介绍了靶向CCL2/CCR2信号轴药物的临床研究进展，以期深入并全面了解CCL2/CCR2信号轴在肿瘤进展中的作用机制，开发更有效的肿瘤免疫治疗药物。
关键词 CCL2；CCR2；肿瘤免疫治疗；肿瘤微环境；免疫抑制细胞中图分类号 R979.1 文献标志码 A文章编号 1000−5048(2024)01−0036−09doi ：10.11665/j.issn.1000−5048.2023112904引用本文 崔珍珍，赵一凡，孙玉，等. 基于CCL2/CCR2信号轴的肿瘤免疫治疗药物的研究进展[J]. 中国药科大学学报，2024，55（1）：36 −44.Cite this article as: CUI Zhenzhen, ZHAO Yifan, SUN Yu, et al . Research progress of drugs for cancer immunotherapy based on CCL2/CCR2signaling axis[J]. J China Pharm Univ , 2024, 55(1): 36 − 44.Research progress of drugs for cancer immunotherapy based on CCL2/CCR2signaling axisCUI Zhenzhen, ZHAO Yifan, SUN Yu, Meng Jiayi, KANG Di *, HU Lihong **Jiangsu Provincial Key Laboratory for Functional Substances of Chinese Medicine, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, ChinaAbstract C-C motif chemokine ligand 2 (CCL2) and its receptor CCR2 are closely related to tumorigenesis and tumor progression. The CCL2/CCR2 signaling axis promotes tumor progression through multiple mechanisms:CCL2 binds to CCR2 on the surface of tumor cells, and thus promotes tumor growth/survival and metastasis;more importantly, CCL2 recruits a variety of immunosuppressive cells to aggregate in the tumor microenvironment, and inhibits the function and activity of immune cells, promoting tumor progression. The article reviews the CCL2/CCR2 signaling axis and its role in tumors and tumor microenvironment, with particular focus on the advances in clinical research on drugs targeting CCL2/CCR2 signaling axis, in order to gain an in-depth and overall understanding of the mechanism of action of CCL2/CCR2 axis in tumor progression and develop more effective anti-tumor immunotherapeutic agents.Key words CCL2; CCR2; cancer immunotherapy; tumor microenvironment; immunosuppressive cellsThis work was supported by the National Natural Science Foundation of China (No. 82104270) and the Graduate Research and Innovation Projects of Jiangsu Province (KYCX22_2035)收稿日期 2023-11-29 通信作者 *Tel ：138****4670 E-mail ：****************.cn**Tel ：************ E-mail ：**************.cn基金项目 国家自然科学基金项目（No.82104270）；江苏省研究生实践创新计划项目（KYCX22_2035）学报36 2024, 55(1): 36 − 441 趋化因子配体2/趋化因子受体2信号轴概述趋化因子是一类能够趋化某种类型细胞定向移动的小分子，通过结合并激活细胞表面的趋化因子受体[1]，触发一系列信号转导，导致细胞朝着趋化因子所在的浓度梯度方向移动。
S t e r e o M a t c h i n g 文 献 笔 记
立体匹配综述阅读心得之Classification and evaluation of cost aggregation methods for stereo correspondence学习笔记之基于代价聚合算法的分类，主要针对cost aggregration 分类，20081.?Introduction经典的全局算法有：本文主要内容有：从精度的角度对比各个算法，主要基于文献【23】给出的评估方法，同时也在计算复杂度上进行了比较，最后综合这两方面提出一个trade-off的比较。
2?Classification?of?cost?aggregation?strategies?主要分为两种：1)The?former?generalizes?the?concept?of?variable?support?by? allowing?the?support?to?have?any?shape?instead?of?being?built?u pon?rectangular?windows?only.2)The?latter?assigns?adaptive?-?rather?than?fixed?-?weights?to?th e?points?belonging?to?the?support.大部分的代价聚合都是采用symmetric方案，也就是综合两幅图的信息。
（实际上在后面的博客中也可以发现，不一定要采用symmetric的形式，而可以采用asymmetric+TAC的形式，效果反而更好）。
采用的匹配函数为（matching?(or?error)?function?）：Lp distance between two vectors包括SAD、Truncated SAD [30,25]、SSD、M-estimator [12]、similarity?function?based?on?point?distinctiveness[32] 最后要指出的是，本文基于平行平面（fronto-parallel）support。
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Module 11 Solicitation/Negotiation/ContractingTask 1.1 Solicitation1. SpecsSpecs:●1st component is “look like or do” (material,performance, SOW, blueprint).●2nd component is quantitative (PPM, MTBF, CpK,default frequency)Acceptance criteria:●Product: measurements during incoming inspection ●Equipment: level of performance, how long at thatlevel●Service: definition of Deliverables1) Design specs: look like●Product: material●Service: step-by-step instruction●Buyer: max control over final result, assume risk2) Performance specs: do●Used to equipment and service●Supplier: max latitude, assume risk3) Internal vs external specs:●Same: by industry or government●Not same: cost more●Good practice: correlate quality measurements4) Supplier samples5) SOW: service2. Solicitation types1) Offer to buy/sellBuyer: solicit supplier’s offer to sell, retain power of acceptance2) Informal bid/quotations●1st way: telephone, written, electronic2nd way: budgetary info 3) Electronic solicitation and e-auctionImportant: Terms and conditions in advance4) Competitive proposals●Send out RFP●Contemplate submission by max no. of possiblesuppliers●Discuss with competitive suppliers●Award5) Sealed bids/formal advertisingFormal advertising used to: building, maintenance, construction, equipment, selling scrap and surplus6) Restricted competitionReasons: specialization of resources, lack of competition in geographic region, regulatory restrictions7) Non-competitive negotiationReason: one supplier is superior8) Two-step bidding●Scenario: specs are not clear●1st step: technical proposals●2nd step: with pricing9) Pre-qualified biddingSupplies qualified to meet min requirements10) Alternative/innovative proposal●Not allowed in Invitation forBid (IFB)●Acceptable in RFP11) Pricing models/market baskets3 dimensions to determine price by supplier:●Market structure:✓Monopoly& Monopsony✓Oligopoly & Oligopsony✓Perfect●Cost-based pricing●Value-based pricing12) Lotting strategies13) Invitation for Bid (IFB)14) RFPSeek input into specs15) RFQPrice offers and execution method16) RFI3. Solicitation procedures●Bidder’s conference✓Written amendment✓All potential suppliers and affected departments ✓Receive bid packages in advance✓Potential problem: time & cost, not comfortable with competitors●Supplier receive same info●Qualified to bid●Be notified whether successful●Include Terms & Conditions✓Standard: cancelation, delivery, inspection,payment terms, packaging, warranties✓Custom: acceptance testing, emergency services, financing, installation, training, initialprovisioning, maintenance, spare parts, contractrenewal, environmental & social sustainability4. Bonds and surety1) Bid bonds●Guarantee bidder acceptsthe contract●If refuse, extra costs of going to alternative source areborne by insurer2) Performance bondsGuarantee supplier performance3) Payment bondsProtect buyer from liens (留置权) if supplier don’t pay its suppliers or employees4) DepositCover the amount in liquidated damages (违约金) if supplier doesn’t perform the contract5) Letter of CreditAssure supplier that the payment will be made by bank 6) Real estate●Put real estate into an escrow account●Forfeit (没收) if supplier doesn’t perform7) CashSet cash aside to guarantee performance8) Earnest money (定金)●Buyer sets deposit to a trust/escrow account●Forfeited by supplier if fail5. Problems related to solicitation●Grant same time extension●Refuse late bid unopened●Allow withdrawal of bid:✓Mistake is mechanical/clerical✓Not blame-deserving error or delayingnotification to buyer●Conflict of interest●File a protest●Confidentiality●Alternate proposals●Debriefing: notify all bidders and internal customer ofsuccessful supplier●Solicitation cancelation ………………………………………………………………………Task 1.2 Prepare negotiation1.Preparation●Porter’s five forces:✓Direct competition✓Threat of entrants✓Substitute products✓Power of buyer✓Power of supplier●SWOT: strength/weakness/opportunity/threat●1st: analyze supplier’s proposal●2nd: set objectives1) Objectives●Fair & reasonable price●Timely performance (JIT ideal)●Meet essential needs●Must-haves and wants●List issues & responsibilitiesMax supplier cooperation●Mutual satisfaction2) SiteBest at buyer’s location3) Team●Understand individual roles●Caucus: break in negotiation, use routinely4) Relative info5) Analysis of seller/buyer position●Seller’s desire●Seller’s certainty●Seller’s financial condition●Seller’scompetitive position (sole source)●Buyer’s financial condition●Available time for negotiation●Price analysis (benchmark)●Cost analysis (breakdown)●Best Alternative to a Negotiated Agreement (BATNA) ●Negotiator skills (planning, tolerance of ambiguity,desire to achieve)●Planning: most important●Long-term objectives6) Market/product conditions●Seller’s/buyer’s market●Long-term relationship7) Strategies & tactics●Strategic: long-term goal●Administrative: logistics●Tactical: analysis, plan timing8) Culture9) Fallback alternativesOptimistic/target/pessimistic position of each issue2. PhilosophiesDual-concern model (concern outcomes):●Win-win or cooperational (high-high)●Win-lose or adversarial/competitive (high-low,moderate-moderate)●Lose-lose or confrontational (low-low) ………………………………………………………………………Task 1.3 Conduct negotiation1. Negotiation strategies1) Depend on:●Philosophies●Power of buyer/supplier●Personalities2) Three strategies:●Reveal no position, when: supplier eager to reachagreement, lack info, complicated proposal●Reveal optimistic position, when: has proposal,resolve each issue●Reveal optimistic and immediately offer targetposition, when: sell offer, pessimistic position ifrefuse2. Negotiation tactics (U.S. culture)●Prioritize issues●Caucus●Missing person●Take it or leave it●Bogey (头疼的问题)3. Considerations●Final proposal revision (Federal AcquisitionRegulations)●Negotiate with sole source●Documentation●E-auction (at least three bidders, standard products,clear specs)●Stakeholder communication●………………………………………………………………………Task 1.4 Prepare contract/PO1. Contract elements●Offer and acceptance●Consideration (约因)●Competent parties●Legality of purpose (合法)2. Contract form1) Written●U.S. Statute of Frauds (反欺诈法) requires writtencontracts:✓Sales > $5,000✓Lease >$1,000✓Service > 1 year✓Real estate●Fixed price●Cost reimbursable●Indefinite delivery●Letter●Dealer’s agreements●Service agreements●Licensing agreements●Master purchase agreements2) OralAcceptable for goods>$500●Writing copy, 10 days to object●Custom-made or labeled goods●Perform contract●Admit under legal oath3) ElectronicU.S. E-Sign Act3. Letter instruments1) Letter of intent●Used in situations:✓Reserve when formal approval requires time✓Base volume discounts on future business✓Encourage seller to stock✓Secure bonding●Binding/Non-binding2) Letter of authorization4. Contract type1) Fixed price●Firmed:Risk borne by supplier, incentive to cost down ●With escalation:Rates of material/labor are unknown,upward/downward price changes●With redetermination:Rates and amount of material/labor are unknown ●With incentive:Profit formula, share supplier savings atpre-negotiated rates●Level of effort:Fixed price per unit level of effort●With downward price:Only be adjusted downward●With remedies:Obtain extraordinary expenses2) Cost reimbursable●Cost plus a fixed fee●Cost plus percentage of costNo incentive to control costs●Cost plus incentive feeShare savings if below target cost, reduce feeaccordingly if above●Cost plus award fee●Cost without fee●Cost sharing3) Indefinite delivery contract●Indefinite delivery/quantity●Task/delivery orderReference Indefinite delivery contract, w/ostandard T&C as PO●Time and material contract●Blanket agreement●Requirements contractRequirements (quantity) is unknown4) Letter contractPreliminary written instrument5) Dealer’s agreementDealer is authorized by supplier6) Service agreement●Master●Professional●MaintenancePerformance-based7) Licensing agreement●Software●General (logo, brand)●Technology8) Master purchase agreementOrder as required9) Specialty contractConstruction, public sector5. Purchase Order●Constitute a contract only after acceptance●Unilateral: accepted through performance by supplier ●Bilateral: a signed PO acknowledgement by supplier ●Exceptions to T&C6. Statement of Work●Work breakdown structure●Hold points/milestones●Performance evaluation factors●Performance-based SOW7. Considerations1) Intellectual property●Patent, copyright (limited time, royalty)●Trademark (indefinite time, register 10 yrs, 10 yrsrenewal)●Licensing●Confidentiality2) Domestic/international laws●United Nations Convention on CISG (ContractsforInternational Sales of Goods)●Common law (precedence)●Codified law (statutes)3) Subcontract/Assignability●Reserve the right to approve4) Notice of awards●Public sector award is public●U.S. federal Freedom of Information5) InsuranceNamed as additional insured on insurance policy ●Coverage: commercial general liability, automobileinsurance, worker’s compensation, employer s’liability6) Indemnification●Liable for injured contractor depends on independentor employee. Considered as employee:✓Has right to control its work✓Furnish tools✓Has right to terminate at any time✓Work is part of regular business✓Contractor’s workers paid by time✓Not hold independent to public●Indemnification✓Hold harmless/Indemnify: reimburse forpenalties or liabilities✓Defend: defend other in legal action●Limitation of liability:✓Product safety (recall or defects)✓Product liability (indemnification)✓Patent infringement7) Liquidated damage (违约金)● A sum agreed upon contract formation●Paid when breach of contract8) Force majeureNot automatic by law9) Restraint of trade issues (antitrust)●U.S.: Sherman, Clayton, Federal Trade Commission,Robinson-Patman acts●Japan: Japanese Antimonopoly Law●EU: articles 101 and 102 of TFEU (Treaty on theFunctioning of EU)10) Security issues●U.S. FAR (Federal Acquisition Regulation) requiregovernment contracts to regard HSPD-12 (HomelandSecurity Presidential Directive)●U.S. GSA (General Services Administration) requires ITSecurity Policy11) Source code escrow accounts12) Collusive offers13) Estoppel (允诺禁反)Unwritten contract enforceableif sufficient evidence exists14) Prime contractor flowdownsRegulations added to subcontractor contracts15) Warranty●Express warranty✓Written/oral in the form offact/description/sample by supplier✓Buyer must refer in contract writing✓Parol evidence rule (口头证据规则：之前的协议不能作为证据来改变现在的书面合同)●Implied warranty✓U.S. provided by UCC (Uniform CommercialCode)✓Title and authority to sell✓Merchantability (适销性)✓Fitness for intended purpose●Supplier can disclaim implied warranty ofMerchantability and Fitness, and limit expresswarranty●Latent conditions (潜在的)✓Not identifiable under normal inspection✓Use replace-or-replace clause to limit thiswarranty16) Remedies●Cure (correct product defects)●Cover (obtain goods in open market)●General damages✓Forseeable✓Incidental damages: reasonably incurred ininspection, receipt, transportation●Special damage✓Unique to a situation, forseeability must beproven✓Consequential and actual damages: lost profit, lost revenue, personal injury, property damage ……………………………………………………………………………….……Task 1.5 Administer contract1. Contract administration1) Work control●Indefinite delivery contract✓Unit price by quantity ●Material-and-time/labor-hour contract✓SOW with in-house estimate●Cost reimbursement contract2) Contract compliance3) Financial responsibilityPayment to suppliers4) Approving system5) Administrative responsibility●Price adjustment clause✓Supplier’s applicable prices✓Labor and material price indices (actual /published)●Administration of change orders6) Contract T&C7) Documentation8) Contractcloseout2. Supplier management1) SOW2) AWP3) Standards of performance4) Supplier feedback5) Supervision of supplierOn-site, co-location, remote6) Management by exceptionRoutine or special attention7) Progress report9) Customer feedback10) In-plant3. Conflict resolutionNegotiation, arbitration, mediation, litigation●Breach (default) of contract terms●Other conflicts (culture)4. Termination●Rights/obligations on prior performance/breachremain●Cancellation: not excuse “causing” party fromdamages1) CauseDue to some behavior of the other party (breach)2) Convenience●Without fault of the other party●Pay for executed performance and profits for thewhole contract3) Other●Frustration of purpose (commercially impracticable) ●Fairly allocate among involved purchasers●Inexcusable delay2 Cost and FinanceTask 2.1 Budget1. Budget purpose●Two elements:✓Goals✓Comparison of actual results and goals●Purposes:✓Control of expenditure✓Pre-approved funding✓Monitoring of expenditures✓Development of standard costs2. Budget type 1) Zero basedNo predetermined allocations, justify for all proposed expenditures2) Cash flow●Used for tight cash controls●Spend is a function of what revenue is received3) Line item●Individual expenses, incremental●Based on previous budget period4) Program/project●PPBS (Program Planning Budgeting System)●Non-for-profit or government●Use productivity measurements and cost/benefitanalysis5) Capital(plant and equipment)6) FlexibleVariable budget, a function of planned output7) Head count3. Budgeting steps1) Review goal and objective2) Define needed resources●Personnel, equipment, furnishings, training●Forecast of economic trends, purchase prices, salesand profit●Best developed at department level or lower3) Estimate the dollar value of resources4) Present budget/obtain appropriation (拨款)5) variance analysis●Track expenditure against budget………………………………………………………………………………Task 2.2 Cost management1. Cost saving●Cost avoidance/containment (抑制)●Cost reduction2. Value analysis/engineering●Focus on analysis of function, alter specs and qualityrequirements without impairing function●Value analysis: existing products●Value engineering: development phase●Process (工艺), techniques, multidisciplinary team,maximum function value3. Cost modeling●Cost structure of material, direct labor, overhead●Quantitative techniques of regression analysis, scatterplot diagram4. Standard cost setting●Predetermined cost of a single unit●Used as goal or benchmark5. Product cost roll-upERP individual costs roll up to selling price6. Margins●Operating profit/revenue●Cover: admin costs, interest, taxes, profit7. Contribution to profit(contribution margin)●(Price)-(Variable costs)=(Contribution margin)●(Total fixed costs)/(Unit contribution)=(Breakevenvolume)8. Cost of poor qualityTotal costs of quality:●Prevention cost●Appraisal cost●Internal cost●External cost9. Cost of inventory●Opportunity costs of invested funds (finance)●Storage and handling costs (overhead)●Taxes●Insurance (ownership)●Shrinkage, damage, obsolescence (risk)10. Intangible costsLoss of market share, investor confidence, lower share price, interruption of cash flow, erosion of business image11. Total cost of ownership●Acquisition price and before/after deliveryPre-transaction, transaction, post-transaction costs ●Acquisition price and in-house costs12. Other cost management●Improved form, fit and function●Easier use●Administrative savings●Improved quality13. Considerations●Standardization●Time requirements●Market testing●Top management support●Design flexibility●Product longevity●Consumption management (energy) …………………………………………………………………………………. Task 2.3 Spend analysis1. Source●Spend data●Market intelligence●Benchmarking2. Cost baseline●Forecast future use and spend●Cost reduction/avoidance●Budget reduction●Current/historic data as basis for future budgetforecasting3. Implementation& outcomes●Start with category tree●Outcomes:✓Saving opportunities✓Negotiate with internal/external✓Adjust budgets4. Segmentation of spend●Categories●Addressable and non-addressableTaxes or charges, license fees by government●Geography …………………………………………………………………………. Task 2.4 Cost analysis1. Total cost of ownership●Unit total costPrice, policy costs, costs of nonperformance●Life-cycle cost (capital acquisition)✓Operating, maintenance, downtime, energy costs, salvage value (残值)✓Cost per piece by lifetime production●Landed cost (import)Price, freight, handling, duties, customs clearance, storage ●Total cost of performance for services2. Direct/Indirect cost1) DirectDirect materials and labor (variable), as basis for overhead cost allocation2) Indirect(overhead)●Allocation✓X% Indirect Per Direct Labor Dollar:(Total Plant Indirect Costs)/(Total Plant DirectLabor Dollars)✓X% Indirect Per Machine Hour:(Total Plant Indirect Dollar)/(Total Plant directMachine Hours)✓G&A (General & Administrative, SG&A)$ G&A Per Dollar of Sales:(Total Company G&A)/(Total Company Sales)●ABC (Activity-Based Costs)Multiple indirect cost pools3. Fixed/Variable costs●Fixed costsMortgage, rent, depreciation, property taxes●Variable costs●Semivariable costs✓Salaries of supervisors, pension plans, utilities and fuel✓Historical approaches: high and low points, least squares, scattergram4. Carrying costs(inventory)5. Should costA cost modeling technique, based on objective costdata●As benchmark price6. Target cost(Target Price)-(Desired Profit Margin)7. Relevant/Irrelevant costse.g. free up space but still need to pay8. Opportunity cost9. Financial analysis tools1) ROI●(Annual Operating Income)/(Total Capital Invested) ●(Net Present Value of Cash Flows from theProject)/(Total Capital Invested in the Project)2) NPV(Net Present Value)NPV=C0−∑C t−(1+r)tTt=1> 03) IRR(Internal Rate of Return)●r when NPV = 04) ROAE(Return on Assets Employed)●(Net Income + Interest Expense after Tax)/(AverageCapital Employed)5) ROTA(Return on Total Assets)●(Net Income)/(Total Assets)6) Margin●(Net Operating Margin)=(Total OperatingIncome)/(Total Sales)7) Profitability8) Cash flow9) Balance sheet●At a given point, Assets/Liabilities10) P&LGross sales−Returned goodsNet sales− Production costsGross profit− Sales expenses− Interest− TaxesNet income11) TVM(Time Value of Money)10. Lease/buy analysis1) Lease type●Operating leaseRequired for short period/subject to rapid obsolescence ●Financial lease (asset)✓Full payout✓Partial payout(original purchase price)-(resale value) plus (interest charges)✓Purchase option at lease end●Leveraged lease✓The lease provides lessor tax advantages or buying power✓Lessor: insurance organization, pension●Master lease✓Similar to blank order✓Price and length of use✓Extend terms to all equipment●Dry/wet lease✓Dry lease (Straight lease): financing✓Wet lease: financing, fuel, maintenance●Sale and lease back✓Obtain capital while maintaining the use2) Factors in lease/buy decision●Financial factors✓Inflation (cost component of rental payment)✓Capital budget✓Third party reimbursementManufacture sell lease contract to 3rd party,lessee pay to 3rd party✓Balance sheetLease as expense in income statement, notliability in balance sheet, show a greater returnon assets✓Cash flow✓Depreciation✓Tax (lease deductible from taxable income)✓Residual value✓Payment schedules✓IRR●Operational factors✓Obsolescence✓Maintenance services✓Administrative overhead✓Operating costs (utilities, environmental)✓Life of the asset✓Customization✓Term of the lease●Other factors✓Ownership benefits (customize, value afterdepreciation)✓Limitation of sources of supply✓Early termination✓Organization policy✓Emergency situations3) Legal/accounting considerations●UCC Article 2A (lease contract)✓Part 1: General Provisions✓Part 2: Formation and Construction✓Part 3:Effect (enforce, title, sublease, lien)✓Part 4:Performance (repudiate, substitute,excuse)✓Part 5: Default●FASB (Financial Accounting Standards Board)Statement 13✓Define methods of recording transactions✓Capital lease (financial -, leveraged -): ownership transferred to lessee, an acquisition by lessess ✓Operating lease●Sarbanes Oxley Section 401a✓To public listed companies, off-balance sheet transactions and obligations to be listed ✓Require training ethics codes to stakeholder●IASB (International Accounting Standard Board)✓Develop IFRS (International Financial Reporting Standards)✓Lease: report amount, timing, uncertainty of cash flow●Others✓EC (European Commission): InternationalAccounting Standard 17✓New Zealand: Statement of Standard Accounting Practice No. 18✓India: Indian Accounting Standard 17 ……………………………………………………………………………..Task 2.5 Financing for purchases1. Cash flow2. Interest rates3. Payment terms4. Depreciation (reduce tax payments)5. Market conditions●Product life cycle1) Precommercialization:Supplier selection, product design, early supplier involvement2) Introduction:Product failure/success, subcontract, develop preferred and standby suppliers, correct quality problems, engineering changes, lease equipment3) Growth:Continuity of supply, “make”, develop additional sources to lower costs4) Maturity:Reduce lead time, cost down, continuity of supply5) DeclineAsset recovery, subcontract, adaptability●Buyer’s/Seller’s market●Risk/value matrix 6. Commodity markets(competitive)●Method for evaluating risk: rank risks●Method of measuring procurement performance:compare weekly buying price7. Bond & currency markets●Hedge on exchange rate●Contract in purchaser’s currency8. Regulations9. Taxes●Plan acquisitions if tax on inventory●Lease vs. purchase, make vs. buy●Records of all transactions for sales tax liability●Internal sourcing10. Import/export quota11. Supplier financing12. Equity investment(shareholder of supplier organization)13. Centralized/decentralized/hybrid buying14. Consortium/Cooperative purchasing …………………………………………………………………………………3 InternationalTask 3.1 Identify international markets1. Culture awareness●Culture: values (think) and behavior (act)●Barrier:✓Lack understanding of internationalprocurement procedures✓Resistance to changes✓Home market nationalism●Require education and training2. Considerations1) Organizational global strategy2) Global macroeconomic and microeconomic concepts●Introduce competition to domestic supply base●React to the practices of competitors3)Organization structure and dynamics4) Global marketingPhysical, cultural, economic5) Country-specific accounting standards and practices6) Human resource management●Objective: recruit best people, increase effectiveness ●Labor skills, labor practice, human rights, culture7) Corporate governance issues●Market-basedEfficient equity market, dispersed ownership●Family-basedManagement and ownership combined●Bank-basedInfluence in bank lending, lack of transparency, family control●Government-affiliatedState ownership, no minority influence8) Legal implicationsWhich country’s law will govern, location for dispute settlement9) IP protection10) Brand management protection11) Government stability and instability3. Infrastructure●Evaluate: economic, social, financial, marketing●Three elements: technology, transportation &logistics system, utilities4. Risk assessment●Risk score: probability of outcome, severity of impact✓Identification: list all risks✓Measurement: severity of each risk✓Assessment: likelihood to occur✓Evaluation: multiply severity and likelihood, set a threshold below which is acceptable●Four risks of particular scrutiny:✓Geopolitical (war, terrorism)✓Environmental✓Public relations✓Contract enforceability (fraud, duress,undueinfluence)5. Cost/benefit and opportunity analysis………………………………………………………………………..Task 3.2 Develop international sources1. Research potential suppliersIntermediaries, sales team, peers, business libraries, Hoovers (D&B), globalEdge, CAPS2. Financial issues1) Landed cost analysis2) Exchange rates●NOER (Nominal Official) 名义currency prices●ROER (Real Official) 实际purchasing power●SER1 (Shadow)影子WATR (Weighted Average Tariff)●SER2 均衡Balance trade. Equilibrium ER●SER3 经常账户Balance the current account including invisibles.Equilibrium ER●Informal/parallel market ERIllegal market●Affecting factors:✓Inflation rate✓Commercial policies (tariff)✓International movements of capitals & incomes3) Payment mechanisms●Letter of credit 信用证Payment made by bank, fulfill sales agreement●Bill of exchange or draft 汇票Drawn by seller on buyer (check), a trade acceptance by buyer signing it●Open account4) Taxes●Ad valorem 从价税●Specific 从量税●VAT (GST Goods and Services Tax)Consumption: Net tax liability =Tax collected (sales) – Tax paid (purchase)●Supplemental taxes5) FeesCustoms and importservices6) Tariffs●Export dutiesAgricultural, mineral●Import dutiesTariff schedule, revenue, resource protection, MFN (Most Favored Nation)7) Duty drawback(custom duties refund)8) Insurance9) Inventory10) Profit repatriationProfits made in foreign country back to home country3. Develop a business plan●Supplier relationship model●In-country partners (manage international supplier)4. Contingency plan and exit strategy5. Governmental regulations6. Brokers and import merchants7. Trade networks8. Logistics●3PL: tactical services●4PL (strategic partnership)✓Separate entity as joint venture, long-term contract✓Single interface between client and logistics service providers✓All supply chain managed by 4PL9) Quality assurance and regulatory compliance●Quality assuranceEstablish specs, use suppliers, apply control, measure ……………………………………………………………………………Task 3.3 Documentation●CISG (Contracts for International Sale of Goods) byUnited Nation Convention●Incoterms (International Commercial Terms)1. Restricted parties and countries●FCPA (U.S. Foreign Corrupt Practices Act)✓Prohibit corrupt payments (bribes) to foreign officials, require financial reporting✓Not prohibit “grease payment” to expeditegovernment processing●International Anti-Bribery and Fair Competition Act of1998Amendment to FCPA, any entity acting with U.S.●Anti-boycott legislationRequest reporting●Export Administration Act✓Control dual-use goods/technologies withcivilian or military application✓Drawing, specs, prototypes as technology export2. Custom requirements●Maintain documents min. 7 years●Smart seals: not tampered after inspection at originport3. Broker management(tariff classification) …………………………………………………………………………………..4 Social responsibilityTask 4.1 Code of Business ConductThree key areas:●Supply chain safety●Environmental compliance●Ethical standards。