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槲皮素对星形胶质细胞增殖及细胞周期相关蛋白cdc25A的影响苑召虎;胡子有;姚芳;张兰兰;颜晓慧;李科;曾勇;吴炳义【期刊名称】《中国神经精神疾病杂志》【年(卷),期】2013(039)001【摘要】目的通过体外星形胶质细胞的划痕模型,观察槲皮素对星形胶质细胞增殖和其细胞周期的影响,并探究可能的信号通路,分析cdc25A的表达变化.方法经划痕处理过的原代培养的新生SD大鼠的星形胶质细胞用槲皮素处理后,将其放入37℃ 5％CO2孵箱中进行培养.分别通过Click-iT Edu test,流式细胞术和Western Blotting检测槲皮素对其增殖、细胞周期的影响及细胞周期相关蛋白cdc25A表达的变化.结果与对照组相比,在第24h时就能明显观察到槲皮素抑制划痕的愈合；槲皮素处理24h后,其增殖细胞百分比从20.92％降低到2.74％,G1期星形胶质细胞百分比从82.04％增加到92.06％,其S和G2期有一个相应的减少；50μmol/L槲皮素处理24h以后细胞周期相关蛋白cdc25A的表达强度下调约50％,具有明显的统计学差异(F=40.579,P＜0.01).结论通过划痕模型的研究发现,槲皮素可以通过抑制星形胶质细胞的增殖来抑制划痕的愈合,并通过降低细胞周期相关蛋白cdc25A的表达将其阻断在G1期.%Objective To study the effects of quercetin on the proliferation and cell cycle of astrocytes and expressionof cdc25A in scratch-injured astrocytes. Methods Scratch injured primary cultured neonatal SD rat astrocytes were incubated with or without quercetin at 37℃ 5%CO2,Click-iT Edu test,Flow cytometry and Western Blotting were used to examine the influence of quercetin on theproliferation,cell cycle and the expression of the cell cycle related proteins-cdc25 A of astrocytes,respectively. Results Compared with control group,quercetin significantly inhibited scratch healing. Within 24 h following quercetin treatment,the proliferation ratio of astrocytes decreased from 20.92% to 2.74%. However,quercetin significantly increased the cells population in the G1 phase from 82.04% to 92.06% and caused a decrease in the S and G2 phase at 24 h following quercetin treatment,. After 24 h,50μmol/L quercetin treatment decreased the expression of cell cycle related protein-cdc25A at about 50% compared with control (F=40.579,P<0.01). Conclusions Quercetin can inhibit the scratch healing in scratched-injured astrocytes via inhibiting astrocytes proliferation and induce cell cycle arrest at G1 phase via decreasingcdc25A expression.【总页数】5页(P6-10)【作者】苑召虎;胡子有;姚芳;张兰兰;颜晓慧;李科;曾勇;吴炳义【作者单位】南方医科大学南方医院临床医学实验研究中心,广州510515;南方医科大学南方医院临床医学实验研究中心,广州510515;南方医科大学南方医院临床医学实验研究中心,广州510515;南方医科大学南方医院临床医学实验研究中心,广州510515;南方医科大学南方医院临床医学实验研究中心,广州510515;南方医科大学南方医院临床医学实验研究中心,广州510515;南方医科大学南方医院临床医学实验研究中心,广州510515;南方医科大学南方医院临床医学实验研究中心,广州510515【正文语种】中文【中图分类】R651【相关文献】1.高血压相关基因hrg-1对血管平滑肌细胞周期蛋白E和P27蛋白表达及细胞增殖的影响 [J], 姜广建;温进坤;韩梅;潘磊;黄秀英2.槲皮素对结肠癌细胞SW480增殖、细胞周期和cyclin B1蛋白表达的影响 [J], 李润青;单保恩3.细胞外信号调节激酶的磷酸化水平对星形胶质细胞增殖及其细胞周期的影响 [J], 苑召虎;胡子有;王惠丽;吴炳义4.缺血缺氧对体外培养星形胶质细胞细胞周期和周期相关蛋白的影响 [J], 骆翔;喻志源;冯永东;王伟5.螯合白血病患者骨髓细胞内外钙离子对硫化氢生成影响的实验研究相关检索词免疫组化蛋白表达白血病硫化细胞增殖 cell proliferation bax h2s 骨髓胃癌硫化氢细胞周期 bcl-2 间充质干细胞图像分析单个核细胞电极乳腺癌钙离子leukemia 相关专家李杰张旻李艳平葛楚天相关机构· 中国科学院上海生命科学研究院生物化学与细胞生物学研究所· 中国科学院上海生命科学研究院· 北京师范大学· 浙江大学动物科学学院· 浙江大学螯合白血病患者骨髓细胞内外钙离子对硫化氢生成影响的实验研究 [J], 孙晓红;于志刚;张雪莉;庄宝祥;张圣明因版权原因，仅展示原文概要，查看原文内容请购买。
因果知觉促进视听时间整合——来自心理物理法和alpha瞬时频率的证据摘要跨通道感觉整合又称多感觉整合，指个体将不同感觉通道内的信息有效整合为一个统一、连贯、完整的多感觉事件的加工过程。
这一加工过程能够将不同感觉通道输入中枢神经系统的信息整合起来，形成整体性知觉以帮助个体更好地感知和适应环境。
其中，视听整合一直是跨通道感觉整合的关键研究领域，且以往研究发现，时间上邻近的视听刺激更容易被整合。
因此，本研究重点关注视听刺激的时间关系，探讨跨通道感觉整合中的视听时间整合。
而另一方面，个体倾向于将具有因果关系的客体知觉为一个整体性事件，从而以因果知觉的形式影响个体的判断，比如影响客体之间的时间距离知觉。
然而，客体在感知过程中存在的因果关联（或者说因果知觉）是否会影响视听时间整合呢？以往研究并未对此进行深入探讨。
为此，本研究采用两项实验予以检验。
实验一将因果知觉的经典客体碰撞范式和视听整合中的经典同时性判断任务相结合，以视听整合的时间窗作为行为指标，探讨因果知觉是否会影响个体的视听时间整合。
结果发现：在有因果关系的条件下，被试的时间窗要显著窄于无因果关系的条件，即被试对视听刺激的时间间隔知觉敏感性变高，促进了视听时间整合。
这一结果证明，因果知觉确实会促进个体的视听时间整合。
实验二则基于脑电实验，在实验一的基础上探讨因果知觉对视听时间整合产生影响的内部机制。
鉴于以往研究发现神经振荡的alpha波代表着信息处理的时间单位，它与感知觉过程、跨通道感觉整合的时间信息密切相关，并且与因果判断也存在联系。
因此实验二采用与实验一中相同的范式，以神经振荡的瞬时频率作为指标，重点检验alpha瞬时频率是否能够作为神经指标，反映因果知觉影响视听时间整合的神经加工。
该实验结果进一步表明，相比无因果条件，有因果关系的条件下alpha瞬时频率显著增加。
并且，我们对比了theta、beta瞬时频率及ERSP，结果发现三者在有无因果两种条件下均未出现显著差异。
中西医结合护理Chinese Journal of Integrative Nursing2023 年第 9 卷第 11 期Vol.9， No.11， 2023抑郁和宫颈癌发病风险相关的孟德尔随机化分析刘银侠1， 俞萍2， 徐阳1（1. 扬州大学护理学院·公共卫生学院， 江苏 扬州， 225009；2. 扬州大学附属医院 护理部， 江苏 扬州， 225009）摘要： 目的 通过孟德尔随机化（MR ）方法，探究抑郁与宫颈癌发病风险的因果关联。
方法 以全基因组关联分析研究（GWAS ）数据为基础，筛选出与抑郁的关联单核苷酸多态性（SNP ）作为基因工具变量，利用逆方差加权分析方法（IVW ）、加权中值方法和 MR -Egger 方法三种孟德尔随机化方法探究抑郁与宫颈癌发病风险的因果关联。
采用Radial MR 用来识别异常值。
分析结果以比值比（OR ）和 95%可信区间（CI ）显示，P ＜0.05 为效应具有统计学意义。
结果 共筛选到57个与抑郁相关的SNP 。
IVW 结果显示，抑郁是宫颈癌发病风险的危险因素［OR=1.002， 95%CI （1.0002～1.003）， P =0.03］；加权中值法和 MR -Egger 效应估计值具有相似的统计结果。
敏感性分析发现没有对因果估计结果产生较大的 SNP 。
结论 抑郁是宫颈癌发病风险的危险因素，加强抑郁患者宫颈癌的筛查、预防以及健康宣教，对于降低该人群的宫颈癌发病率具有重要意义。
关键词： 抑郁； 孟德尔随机化； 宫颈癌； 筛查； 健康宣教中图分类号： R 473.71 文献标志码： A 文章编号： 2709-1961（2023）11-0106-08A Mendelian randomization study of depressionand risk of cervical cancerLIU Yinxia 1，YU Ping 2，XU Yang 1（1. School of Nursing and Public Health Yangzhou University ， Yangzhou ， Jiangsu ， 225009； 2. Department of Nursing ， Affiliated Hospital of Yangzhou University ， Yangzhou ， Jiangsu ， 225009）ABSTRACT ： Objective To explore the causal relationship between depression and the risk ofcervical cancer by Mendelian randomization （MR ）.Methods Based on Genome Wide Associa⁃tion Study （GWAS ） data ， single nucleotide polymorphism （SNP ） associated with depression were selected as genetic instrumental variables. Three Mendelian randomization methods ， inverse vari⁃ance weighted analysis （IVW ）， weighted median methods and MR -Egger were used to investigate the causal association between DP and the risk of cervical cancer. Radial MR was used to identify outliers. The analysis results were shown by odds ratio （OR ）and 95% confidence interval （CI ）， and the effect was statistically significant with P ＜0.05.Results Totally 57 SNPs related to de⁃pression were included. The result of IVW show that depression was a risk factor for the risk of cervical cancer （OR=1.002， 95%CI ： 1.0002～1.003， P=0.03）. Weighted median method and MR -Egger provided the similar result. Sensitivity analysis found that no single SNP was strongly or reversely driving the overall effect for causal estimates.Conclusion Depression was a risk fac⁃tor of cervical cancer. Strengthening the screening and prevention of cervical cancer in DP pa⁃tients was of great significance to reduce the incidence of cervical cancer in this population.KEY WORDS ： depression ； Mendelian randomization ； cervical cancer ； screening ； health education作为最常见的妇科恶性肿瘤，宫颈癌是严重危害妇女健康的主要恶性肿瘤之一，居女性恶性肿瘤的第二位［1-2］。
眼球检测在精神分裂症中的临床应用黄超;陈宪生;胡茂荣【摘要】Schizophrenia is one of the most common psychiatric mental illness,The disease course deferment, the recession and social functions of mental activities varying degrees of defects, brings the serious burden to individuals, families and society.According to related research, abnormal eye movement detection index can be used as biological indicators of schizophrenia, so eye detection for early diagnosis of schizophrenia research become a hot spot. In this paper, the detection of eye movement in the early application of schizophrenia, and the research in this field in the fu-ture was discussed.%精神分裂症是精神科最常见的一种精神疾病，目前该病病因不明，诊断缺乏相应的生物学指标。
据相关研究显示，眼动检测的异常指标可作为精神分裂症的生物学指标和状态学指标，因此眼动检测对精神分裂症的临床应用成为近年来的研究热点。
本文概述了眼动检测在精神分裂症的临床应用，并对今后在这方面的研究进行展望。
【期刊名称】《现代诊断与治疗》【年(卷),期】2013(000)017【总页数】3页(P3905-3907)【关键词】眼球运动;精神分裂症;临床应用【作者】黄超;陈宪生;胡茂荣【作者单位】南昌大学医学院，江西南昌 330006;南昌大学医学院，江西南昌330006; 江西省精神病院，江西南昌 330029;江西省精神病院，江西南昌330029【正文语种】中文【中图分类】R749.3精神分裂症是一组病因未明，多起病于青壮年，临床上以思维、知觉、情感和行为等多方面的障碍和精神活动与环境的不协调为主要表现。
硬核性白内障行超声乳化术与现代囊外摘除术对角膜内皮的影响谢艳利;周克相【摘要】Objective To analyze the effect of phacoemulsification and extracapsular cataract extraction on corneal endothe-lium in patients with hard cataract. Methods 50 cases of patients with hard cataract treated from March 2013 to March 2015 were divided into two groups with 25 cases in each, the control group were treated with extracapsular cataract extrac-tion, the observation group were treated with phacoemulsification, the curative effects of the two groups were compared. Re-sults Patients in the observation group whose naked vision reached 1.0 eye numbers were more than those in the control group; the difference in the edema number was not obvious, and both groups had not severe complications of posterior cap-sular rupture and suspension ligament rupture, the endothelium cell loss ratio in the observation group was higher than that in the control group. Conclusion The patients undergoing phacoemulsification has a better naked vision function recovery effect and can used to treat the patients with hard cataract.%目的分析硬核性白内障行超声乳化术与现代囊外摘除术对角膜内皮的影响.方法随机选取2013年3月—2015年3月该院收治的50例硬核性白内障患者采用现代囊外摘除术治疗(对照组25例)和超声乳化术治疗(观察组25例),对比两组患者的疗效.结果观察组裸眼视力达到1.0眼数多于对照组;水肿人数差异无统计学意义,且均未出现后囊膜破裂、悬韧带断裂等严重并发症;观察组角膜内皮细胞丢失率高于对照组. 结论超声乳化术后患者裸眼视力功能恢复效果更好,可用于硬核性白内障患者治疗.【期刊名称】《中外医疗》【年(卷),期】2016(035)002【总页数】2页(P106-107)【关键词】硬核性白内障;超声乳化术;现代囊外摘除术【作者】谢艳利;周克相【作者单位】鄂州科宏眼科医院,湖北鄂州 436000;鄂州科宏眼科医院,湖北鄂州436000【正文语种】中文【中图分类】R5手术治疗白内障对角膜内皮的保护有很高的要求，术中保护角膜内皮的完整性至关重要[1]。
Measurement of the Phase Diagram of DNA Unzipping in theTemperature- Force Plane.C.Danilowicz, Y. Kafri, R.S. Conroy, V. W. Coljee, J. Weeks, M. PrentissPhysics Department, Harvard University, Cambridge, MA 02138, USA.Contact Details:Tel. +1-617-495-4483 Fax. +1-617-495-0416 e-mail: prentiss@October 27, 2003PACS Codes: 87.14.Gg, 87.15.-v, 82.35.Rs, 82.37.RsAbstractWe separate double stranded lambda phage DNA by applying a fixed force at a constant temperature ranging from 15°C to 50°C, and measure the minimum force required to separate the two strands, providing the first experimental determination of the phase boundary between single stranded DNA and double stranded DNA in the temperature- force plane. The measurements also offer information on the free energy of dsDNA at temperatures where dsDNA does not thermally denature in the absence of force. While parts of the phase diagram can be explained using existing models and free energy parameters, others deviate significantly. Possible reasons for the deviations between theory and experiment are considered.Studies of the mechanical separation of double stranded DNA (dsDNA) enhance the understanding of DNA replication in vivo. Although it is possible to separate dsDNA by heating well above body temperature in procedures such as the polymerase chain reaction (PCR), in living organisms DNA replication is a very complex process assisted by a variety of specialized proteins. DNA unzipping, the separation of dsDNA by applying a force that pulls two strands of DNA apart at one end of the molecule, is similar to some of the steps occurring in vivo. Several techniques such as optical and magnetic tweezers, and atomic force microscopy have been used in single molecule studies of the unzipping of DNA and the unfolding of RNA [1,2,3,4,5,6,7,8,9].Recent theoretical work focused on the unzipping of DNA under a constant applied force, where both the unzipping of homopolymers and heteropolymers has been considered [10,11,12,13,14,15,16]. These models assume thermal equilibrium and predict that unzipping is a first order phase transition with a minimum required force that decreases with increasing temperature, ending at the thermal denaturation point where no force is required to separate the two strands [17]. Theoretical frameworks that successfully encompass both the traditional thermal separation and the recently investigated force-induced separation should enhance the predictive power of models of DNA binding potentials that may improve the design of PCR primers and DNA chips.In such models, the phase diagram in the temperature-force plane is obtained by comparing the free energy of the unzipped molecule under an applied constant force to that of force-free dsDNA. Provided that a good estimate for the free energy of single stranded DNA (ssDNA) under an external force is possible, one can then use the critical force to estimate the free energy of dsDNA.Previous measurements of the unzipping of DNA have been made at room temperature [7,8]. In this paper we evaluate the unzipping of the first 1500 of the 48502 base pairs of lambda phage DNA by applying a fixed force at a constant temperature, at temperatures varying between 15°C and 50°C. We show that the force required to unzip the heterogeneous DNA decreases with increasing temperature, in accord with previous predictions [10,11,12,13]. We have also observed the rezipping of dsDNA when the applied force is below 2 pN for an hour or longer. Our results show the first experimental phase diagram for dsDNA separation at constant applied force and suggest that a) there is reasonable agreement with projections from bulk thermodynamics between 25°C and 35°C; b) the occurrence of hairpins in the ssDNA neglected in theoretical models, may play an important role at high temperature; c) the temperature dependence of the free energy of the dsDNA changes in a non trivial manner at low temperatures.The critical force required to unzip dsDNA can be calculated using the expression for the free energy difference per base pair, ∆G, between a base pair in dsDNA and the same base pair in a ssDNA stretched at the ends by an external force, F [11,12]. This free energy difference is given by∆G= g b – 2 g u (F) (1)where g b is the average free energy per base pair of double stranded DNA (at zero force) and g u (F) is the free energy per base pair for each of the stretched single strands. The value of g b depends on the specific sequence of the dsDNA and is usually estimated using a nearest neighbor approximation [18]. The dsDNA will separate into ssDNA when the applied force exceeds the critical force F c such thatg b = 2 g u (F c) (2)Though much useful information can be gained from models that treat dsDNA as a homopolymer [10,11,12,13,14], DNA contains a well-defined sequence of base pairs making it a heteropolymer rather than a homopolymer. For homopolymers, the free energy difference between the stretched ssDNA and the dsDNA is the same for all of the base pairs in the sequence, and the free energy difference is a linear function of the number of unzipped base pairs [19]; therefore, a constant applied force, larger than the critical force,F c (T), will unzip homopolymeric dsDNA at a constant rate. In contrast, in heteropolymeric dsDNA, the free energy difference between the stretched ssDNA and the dsDNA does not have the same value for all base pairs. As a result, the energy landscape for this heteropolymer displays a significant sequence dependent variation as a function of the number of unzipped base pairs [11,12,20]. As discussed in Refs. [11,12,20], for a wide range of forces near the critical force the dsDNA will unzip in a series of different, discrete, sequence dependent steps. The locations in the sequence where the unzipping pauses, have been successfully predicted by Monte-Carlo simulations [21]. The critical force F c for a given sequence of dsDNA is the minimum force required to unzip the entire sequence. This unzipping in discrete jumps near F c makes the determination of the critical force difficult,and forces us to define some procedure for locating it (see below).We measure the unzipping of lambda phage DNA using a massively parallel system [20,22] and DNA samples prepared using the techniques described in detail in Ref. [20]. A schematic representation of the apparatus is shown in Fig. 1. The molecular construction consists of two covalently linked lambda phage dsDNA molecules. The dsDNA attached tothe surface is a spacer that separates the dsDNA to be unzipped from the surface to avoidsurface interactions. The spacer is labeled with digoxigenin and therefore can be specifically attached to a glass surface through an antigen-antibody interaction. The second DNA molecule is closed at one end with a hairpin, and the other end has a biotin label that can be bound to a streptavidin coated superparamagnetic bead. Finally, the spacer attached to the glass surface can be stretched to its contour length of 16.5 µm by applying a small force (2 pN) before the unzipping experiment begins at higher force. Before starting the experiment, the sample containing the DNA and the beads is placed in a 0.8 mm clean square capillary with a 0.55 mm round inner capillary and incubated. The temperature of the square capillary can be varied from 10 to 80°C using a thermoelectric cooler placed on top of the aluminum mount holding the square capillary. A temperature sensor close to the capillary channel provides feedback for the stabilization loop controlling the thermoelectric cooler.The inner round capillary is modified with anti-digoxigenin antibody by an overnight adsorption at room temperature and can therefore specifically interact with the DNA construction. The optical apparatus, which consists of an inverted microscope with a computer-controlled frame grabber, tracks the position of the superparamagnetic beads as a function of time for each different force and temperature condition. The distance D between the bead and the inner glass capillary depends both on the number of base pairs that are unzipped and the extent to which the unzipped ssDNA and the dsDNA linker are stretched. The stretching of both dsDNA and ssDNA depends on the applied force [23,24,25,26], so a distance D will correspond to different numbers of unzipped base pairs when different forces are applied to the magnetic bead [20]. The stretching behavior of dsDNA shows that it is relatively stiff at forces above 10 pN while ssDNA exhibits ahigher flexibility. The average length of a dsDNA spacer under 10 pN of force is 16.2 µm, and the average length under 15 pN of force is 16.3 µm. Similarly, at an applied force of 10 pN the average total length of the dsDNA space plus a completely unzipped lambda phage ssDNA is 65 µm whereas at an applied force of 15 pN the total average length is 77 µm [27]. Thus, we can calculate from the stretched length of the ssDNA at 10 pN, an extension per base pair of 0.5 nm, and 0.63 nm at 15 pN. These values have also been confirmed with a separate measurement of lambda phage ssDNA obtained through melting at 99°C in a thermal cycler and cooling down quickly to 5°C; an average of 5 beads showed an extension of 24.2 µm and 27.8 µm at 10 pN and 15 pN, respectively.Initially, when the magnet approaches the square channel and a low force is applied (3-5 pN), the beads separate from the surface becoming tethered at a distance of 16- 17 µm. At the desired temperature (between 18°C and 50°C), the measurement starts when the force is increased to a value between 3 to 25 pN whereas the force range for the experiment at 15°C was 18 to 55 pN. The displacement of the beads is followed in time and the distance to the surface can be measured precisely. Typically, the concentration of DNA is several times smaller than the concentration of the beads so that each bead is connected through one single DNA molecular construction.Fig. 2 shows the measured phase diagram as a function of force and temperature. The red circles in the figure represent data from samples that were initially incubated at 50°C for 30 minutes and were then equilibrated to the final temperature at which the phase diagram was measured. The sample was allowed to equilibrate at each temperature for 20 minutes before the experiment started. Samples that were initially at 50°C will be referred to as hot samples. The green squares show the force required to unzip the dsDNA as afunction of temperature for samples that were left at each unzipping temperature for more than 15 hours before unzipping, so they should be approaching the equilibrium value for the critical force. In order to find the force at which the phase transition occurs for a given temperature, we conducted a series of experiments on the same group of single molecules where we applied an initial force F o to the sample for 15 minutes, and then measured the number of unzipped molecules by the end of that time. A molecule was considered to have begun to unzip if the distance between the bead and the surface increased suddenly by more than 1.5 µm (1500 base pairs) during the time interval, and the bead remained tethered for more than 1 minute after the sudden increase in the distance from the surface. The force was then increased to F1, and the molecules were allowed to unzip for another 15 minutes. We counted the number of molecules that had remained zipped when the applied force was F o, but began to unzip when the force was F1. We then increased the force to F2, and repeated the process. As a final step we increased the force to 25 pN for 30 minutes, and measured the total number of molecules that began to unzip. We assumed that at such a large applied force all molecules that were correctly tethered to the surface would begin to unzip, and that any beads that remained tethered must have been bound to the surface by an incorrect construction. We then calculated the fraction of the correctly bound molecules that unzipped at or below a given average applied force. There is a variation in the magnetization of the beads, so if the average force on the beads is the critical force, half of the molecules should unzip and half should remain zipped; therefore we defined the measured critical force as the value of the average force at which 50% of the correctly bound molecules begin to unzip at a given temperature. The critical forces measured using this method, were quite reproducible from sample to sample; each experiment was done atleast twice, and some experiments were done several times with samples prepared separately. The numbers of correctly tethered dsDNA molecules in a given experiment ranged from 20 to 50, with an average value of approximately 30, so many single molecule measurements were included in each experiment. The variation in average force between different samples under identical conditions was less than 1 pN. The temperature inside the square channel was measured at the end of each experiment. We also conducted a few experiments where we applied a single constant force for an hour, and checked that 50% of the correctly bound molecules did unzip at the critical force determined using the method described above allowing us to verify that the 15 minute -interval was adequate to sample each applied force.The purple diamond in Fig.2 shows the measured melting temperature, T m, for lambda phage dsDNA in the same phosphate saline buffer used in the unzipping experiments, where the melting temperature was determined by a bulk measurement of circular dichroism spectrum as a function of temperature. This denaturation temperature required to unzip the DNA at zero force is in good agreement with theoretical calculations reported for similar ionic conditions [28].It is interesting to compare the experimental results to a simple theory that utilizes free energy estimates for both the unzipped ssDNA, g u, and the bound DNA, g b. At forces above 10 pN, the measured force versus extension curves for ssDNA have been well described by the mFJC model [29,30]. Below these forces hairpins begin to play an important role and the model is no longer valid. If one measures the free energy of the stretched ssDNA with respect to the unstretched ssDNA (so that g F(F=0)=0), then within the mFJC model the free-energy per base pair of the unzipped DNA is:()T k F b l T k Fb Fb T k T k b l g B B B B u 22sinh ln l +⎟⎟⎠⎞⎜⎜⎝⎛⎟⎟⎠⎞⎜⎜⎝⎛−= (3) where l is the distance between base pairs (we use a base pair separation, l = 0.7 nm [1] which predicts an extension per base pair of 0.54 nm at 10 pN and 0.58 nm at 15 pN, (consistent with the length per base pair vs force that we measured for the ssDNA stretched by forces between 10 and 15 pN and with the spacing between phosphates for C2’-endo pucker in ssDNA), b is the Kuhn length of ssDNA (current estimate b = 1.9 nm) and l = 0.1 nm is a length which characterizes the elasticity of a bond along the ssDNA between bases. The mFJC does not include the possibility of hairpin formation and is not expected to correctly describe the free energy of the ssDNA at forces below 10 pN where hairpins can occur in ssDNA [29].Given that the free energy reference point was chosen so that g u (F=0)=0, the free energy of the bound dsDNA is just the free energy difference between dsDNA and ssDNA and can be expressed in terms of ∆H and ∆S, the difference in enthalpy and entropy between dsDNA and ssDNA. Then:S T H g b ∆−∆= (4)where T is the temperature in degrees Kelvin. Here the dsDNA is assumed to be completely bound with no denaturated loops inside. This assumption is justified to a very good approximation up to the thermal denaturation temperature due to the extremely small Boltzmann weight (or cooperatively parameter) ~10-4- 10-5 [31], associated with initiating such a loop. Previously it has been assumed that ∆H and ∆S are independent of temperature[18]. Given this assumption, at the melting temperature T m , F c =0 and T m =∆H/∆S[11,12,18]. The value of ∆S= -20.6 cal/ ºK.mol (-1.43 x 10-22 J/ ºK. molecule), is obtainedby averaging over the first 1500 base pairs (corresponding to the experimentally measured critical force) using the values of ∆S found in Ref. [18]. The value of ∆H = T m∆S is then calculated using the experimentally measured thermal denaturation temperature. The value ∆H = -7.5 kcal/ mol (-5.22 x 10-20 J/ molecule) agrees with modifications of the values of Ref. [18] due to the ionic concentration as described in Ref. [28]. It was assumed that ∆S was not significantly affected by the change in ionic concentrations.Using the free energy estimates along with Eq.(2), it is then straightforward to calculate numerically the critical force as a function of temperature. The resulting phase diagram is shown as the blue line in Fig. 2. The line is in good agreement with the data in the temperature range from 24°C and 35°C, though there are significant deviations outside this temperature range. The theory dotted does not fit the experimental values at temperatures above 35°C where the critical force is predicted to be below 10 pN. The line is dotted because the mFJC does not accurately predict measured force vs extension curves for ssDNA in this force range, possibly because hairpin formation is neglected [29]. Inclusion of hairpins in the theory would reduce the predicted difference in binding energy between ssDNA and dsDNA, and might bring the theoretical values closer to those observed in the experiments. Finally, recent experiments suggest that bubbles of 2 to 10 bases pairs can form spontaneously in dsDNA at 37°C [32]. Such bubbles would decrease the binding energy of the dsDNA and increase its entropy. Inclusion of these effects would further reduce the predicted critical force making it more consistent with the measured values in the temperature range between 37°C and 50°C.At temperatures from 15°C to 24°C, the predicted and measured values again diverge. Though the theoretical curve has an approximately constant slope in the temperature rangefrom 15 to 40°C the measured critical force does not. At temperatures below 22°C the measured critical force increases much more steeply with decreasing temperature than it does in the temperature range from 24°C to 35°C, where the theory and experiment are in reasonably good agreement. We attribute the observed decrease in slope at low temperatures to a thermally induced change in the dsDNA conformation. Other signatures of this conformational change include variations in the circular dichroism spectrum and the persistence length of the dsDNA [33]. This hypothesis is supported by preliminary data suggesting that the stretching curves for ssDNA have little dependence on temperature at forces greater than 10 pN. Therefore, g u is expected to be weakly influenced by temperature.We have measured the phase diagram for the unzipping of single molecules of lambda phage dsDNA as a function of force and temperature. In the temperature range 24-35o C, the results agree well with projections from bulk thermodynamic data that assume ∆S and ∆H are independent of temperature. Above 35°C and below 24o C, the critical force required to unzip dsDNA at a given temperature deviates significantly from calculations based on simple projections of the bulk thermodynamic measurements. At temperatures above 35o C the measured critical force is much smaller than the predicted critical force. This difference may be partly attributed to the formation of hairpins in the ssDNA when the applied force is less than 10 pN. At temperatures below 24o C, the measured critical force is larger than predicted. Temperature dependent conformational changes in dsDNA may contribute to this discrepancy. Thus, the observed phase diagram for the unzipping of lambda phage dsDNA is much richer than earlier theoretical work had suggested. Adding information on DNA conformational changes and hairpin formation could greatly improvethe predictive power of theoretical treatments allowing more accurate biological predictions.AcknowledgementsWe thank David R. Nelson and Maxim D. Frank-Kamenetskii for valuable conversations. This research was funded by grants: MURI: Dept. of the Navy N00014-01-1-0782; Materials Research Science and Engineering Center (MRSEC): NSF # DMR 0213805 and NSF Award #PHY-9876929; NSF grant DMR-0231631. Y.K. acknowledges support from Fulbright Foundation, Israel.Figure CaptionsFig.1. Schematic of the DNA binding to the inner glass capillary and the magnetic bead. The magnet exerts an attractive force on the superparamagnetic beads pulling them away from the surface at low force. At forces above the critical force the double stranded DNA shown on the left side of the diagram can be separated into two single DNA strands and complete separation is avoided by including a hairpin at the end of the DNA to be unzipped.Figure 2. Measured phase diagram in the temperature-force plane. The red circles correspond to samples previously incubated at 50°C (hot samples) and the green triangles correspond to samples that were left at each unzipping temperature for more than 15 hours before unzipping. The blue line is calculated according with theory. The purple symbol corresponds to the experimental value for lambda phage DNA melting using circular dichroism in phosphate saline buffer pH 7.4.a) No Applied Forceb) Force SufficientUnzipReferences:[1] S. B. Smith, Y. Cui, and C. Bustamante, Science 271, 795 (1996).[2] S. R. Quake, H. Babcock, and S. 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食蟹猴纹状体内注射α-突触核蛋白预制原纤维对嗅球病理改变的影响丁雨潇1，粟璟曦1，宋琼1，王丽惠1，吴日宝1，况昕宇1，苏迎1，邹春林1，2△摘要：目的探讨α-突触核蛋白预制原纤维（α-Syn PFF）纹状体注射后能否生成病理性α-突触核蛋白（α-Syn），并从纹状体传播至嗅球，从而引起嗅球中神经元损伤。
方法3只健康雌性食蟹猴双侧纹状体注射α-Syn PFF 作为实验组，以脑立体定向注射手术，将300µgα-Syn PFF（7g/L）注射到双侧纹状体的6个位点，即每侧纹状体壳核头部注射60µg，体部注射60µg，尾部注射30µg。
另2只健康雌性食蟹猴相同部位注射同等剂量磷酸盐缓冲液（PBS）作为对照组。
注射2年后，实验猴行安乐死，取出嗅球经固定和切片，然后经尼氏染色检测组织病理改变，免疫组织化学染色检测磷酸化α-Syn（pS129）、酪氨酸羟化酶（TH）、双皮质素（DCX）的表达情况。
结果实验组与对照组比较，嗅球中存在pS129阳性聚集体；实验组TH和DCX阳性神经元数量较对照组均显著减少［TH：（27.00±11.22）个vs.（65.80±36.54）个；DCX：（67.60±17.23）个vs.（88.30±19.89）个］。
结论食蟹猴纹状体注射α-Syn PFF产生的病理性α-Syn可从纹状体传播至嗅球造成嗅球神经元损伤，诱导嗅球中多巴胺能神经元丢失，并抑制新生神经元生成。
关键词：帕金森病；α突触核蛋白；嗅球；成束猴；免疫组织化学中图分类号：R742.5文献标志码：A DOI：10.11958/20221892Effect of intra-striatal injection ofα-Synuclein PFF on pathological changes ofolfactory bulb in macaca fascicularisDING Yuxiao1,SU Jingxi1,SONG Qiong1,WANG Lihui1,WU Ribao1,KUANG Xinyu1,SU Ying1,ZOU Chunlin1,2△1Center for Translational Medicine,Key Laboratory of Longevity and Aging-related Diseases,Ministry of Education, Institute of Neuroscience and Guangxi Key Laboratory of Brain Science,School of Basic Medical Sciences,Guangxi Medical University,Nanning530021,China;2Collaborative Innovation Centre of Regenerative Medicine and Medical BioResource Development and Application Co-constructed by the Province and Ministry,Guangxi Key Laboratory of Regenerative Medicine△Corresponding Author E-mail:Abstract:Objective To investigate whether pathologicalα-Synuclein(α-Syn)can be generated after striatal injection ofα-Syn preformed fibrils(PFF),and then spread from striatum to olfactory bulb,thereby causing neuronal damage in olfactory bulb.Methods Three healthy female macaca fascicularis injected withα-Syn PFF into the bilateral striatum were used as the experimental group.300µgα-Syn PFF(7g/L)was injected into each side of the striatal putaminal nucleus with60µg in head,60µg in body and30µg in tail.The other two healthy female macaca fascicularis were injected with PBS as the control group.Two years after the injection,macaca fascicularis were killed.Pathological changes were detected by Nissl staining,and immunohistochemistry was performed to examine the expression levels of phosphorylatedα-Synuclein (pS129),tyrosine hydroxylase(TH)and doublecortin(DCX)in olfactory bulb.Results Compared to the control group, pS129positive aggregates were found in olfactory bulb of the experimental group.Besides,the number of TH-positive and DCX-positive neurons were significantly decreased in the experimental group[TH:(27.00±11.22)vs.(65.80±36.54);DCX: (67.60±17.23)vs.(88.30±19.89)].Conclusion Pathologicalα-Syn can spread from striatum to olfactory bulb and induce the loss of dopaminergic neurons,inhibiting the generation of newborn neurons in olfactory bulb of macaca fascicularis.Key words:Parkinson disease;alpha-synuclein;olfactory bulb;macaca fascicularis;immunohistochemistry帕金森病（Parkinson，PD）又名震颤性麻痹，是一种多发于老年人群的神经退行性疾病，在65岁以上老年人群中患病率达2%~3%，受到人口老龄化及环境等因素影响，预计到2040年全球患病人数将达到1420万［1］。
a r X i v :0704.3175v 1 [c o n d -m a t .s o f t ] 24 A p r 2007Effects of Eye-phase in DNA unzippingDebaprasad Giri †and Sanjay KumarDepartment of Physics,Banaras Hindu University,Varanasi 221005,IndiaMax-Planck Institute for the Physics of Complex Systems,Noethnitzer,01187Dresden,Germany †Physics Section,MMV,Banaras Hindu University,Varanasi 221005,India Abstract The onset of an “eye-phase”and its role during the DNA unzipping is studied when a force is applied to the interior of the chain.The directionality of the hydrogen bond introduced here shows oscillations in force-extension curve similar to a ”saw-tooth”kind of oscillations seen in the protein unfolding experiments.The effects of intermediates (hairpins)and stacking energies on the melting profile have also been discussed.PACS numbers:64.90.+b,36.20.Ey,82.35.Jk,87.14.GgMolecular interactions play a key role in living organisms.Recent advances in experimental techniques have allowed nanomanipulation in single biological molecule and made possible to mea-sure these interactions[1].The aim is to exert a force in the pN range by optical tweezers,atomic force microscopy,etc and characterize the molecular,elastic,structural and functional properties of bio-molecules[2,3].In typical experiments of double stranded DNA(dsDNA)unzipping,a force is applied to the ends of the chain(Fig.1a)and one studies the force-extension curve which shows the elastic properties and the gross features that reflect the local‘G-C’vs‘A-T’content along the sequence[4,5,6,7,8].Moreover,the force-temperature diagram shows,below the melting temperature,that the over-stretching force[8]decreases nearly linearly with temperature.Theoretically,DNA unzipping may be studied in different ensembles[9]depending on the ex-periments.For example,Atomic force microscopes(AFM)work in constant distance ensemble (CDE)while magnetic bead uses the concept of constant force ensemble(CFE).The prediction of unzipping transition based on interacting Gaussian chains[10]raised a lot interest and now results are available from dynamical approach[11],exact solutions of lattice models[12,13],simple models of quenched-averaged DNA[14,15],numerical simulations and scaling analysis[16,17].Recently for a model of interacting polymers where any monomer of one chain can interact with any monomer of the other chain(we call it model A),the role of an intermediate entropy-stabilized phase was recognized and a force-induced triple point[18]in a force-temperature plane was established.In most of the models studied for dsDNA[12,13,14,15,16,19],a monomer i of one strand can only interact with the i-th monomer of the other strand,which is similar to the models of DNA(we call it model B)proposed earlier by Poland and Scheraga[20].These models do not take into account the directional nature of the hydrogen bond and underestimate the entropy by restricting the formation of hydrogen bonds.Thus these models may only give a limited picture of the unzipping transition and do not allow to study the effect of intermediate states[21].So far,most of the theories of DNA unzipping have focused on the thermodynamic limit,and therefore,consist of a few parameters typically in the form of an effective base-pair interaction in a simplified way.In all the single molecule experiments,afinite size chain is used and hence no“true phase transition”can in principle be observed.Still,the“phase transition”observed in such experiments may be considered as real if the length of the chain exceeds the characteristic correlation lengths.It is now becoming possible to go to the other limit of studying shorter segments(∼10base pair)at coarse grained level[22,23].The purpose of this letter is to provide exact results of a semi-microscopic model of short chains by incorporating the directional nature of hydrogen bonds and then propose a method to study the effect of molecular interactions right at the individual base pair level and their role on melting profile.In the following,we adopt a more realistic model of DNA,which may be defined in any dimension(a)(c)(d)(e)FIG.1:The schematic representation of DNA unzipping by force(g)applied at(a)one end of the two strands;(b)at the interior of the strands.Figs.(c-e)represent the possible conformations of model C.Here (c)represents the completely zipped state,(d)a non pairing configuration and(e)a partial bound state with hairpin loop.[24](herein after we call it model C).A similar model has been used in Ref.24in the context of relative stabilities of DNA hairpin structures.The model takes care of important shortcomings of model B and also incorporates some additional features like existence of intermediate states, effects of stacking energy,excluded volume properties of nucleotides and the directional nature of hydrogen bonds.Although the importance of bubble formation during thermal melting has been recognized,no attempt has been made so far to experimentally explore the phase diagram when a force is applied to the interior of the chain as shown in Fig1b.Such situations occur in many biological processes, for example,during gene-expression,RNA forms bubbles or“eye-type”conformations on DNA. Therefore,we consider two cases:(i)force has been applied at the end of the chain(Fig.1a,‘END’case or Y case),and(ii)at the middle of the chain(Fig.1b,‘MID’case).The contribution to energy by this force,g,is−2gx,where2x is the absolute distance in the x-direction between the two strands at the point of application of the force.We model the two strands(e.g.A-T)of a homopolymer DNA by two mutually-attracting-self-avoiding walks(MASA Ws)on a square lattice as shown in Figs.1(c-e).The bases are associated with the link between two monomers of a chain as depicted in Fig.1.In one strand the bases point towards the right while on the other they are on the left,as one traverses the chains sequentially.We associate a contact energy−ǫ(effective base pair interaction)with each pairing between complementary strands only if the bases are nearest neighbors(short range nature of the hydrogen bond)and approach each other directly without the strands coming in between[Fig.1c]. Fig.1e shows the possibility of formation of hairpin(which is not possible in model B)in a single strand of DNA.However,in this case,non-native contribution has been taken into account but noFIG.2:Variation of critical force()for model B and C.apparent weight has been assigned to stem as it is made up of same nucleotides.The partitions function(Z N)of the system under consideration can be written as a sum overall possible configurations of MASA W s i.e.m,xC(m,x)exp(βmǫ)exp(βgx),whereβ=1/k B Tis the inverse temperature,k B being the Boltzmann constant.C(m,x)is the number of distinct conformations walks of length2N having m number of intact base pairs whose end(or mid)points are at a distance x apart.We have obtained C(m,x)for N≤16and analyzed the partition function using exact enumeration and series analysis technique[25,26].We prefer this technique because it can predict various phases of the system[27]quite effectively and the scaling corrections can be correctly taken into account by a suitable extrapolation methods[25,26].To achieve the same accuracy in Monte Carlo,a chain of two orders of magnitude larger than in the exact enumeration method is required[28].We setǫ/k B=1and calculate the reduced free energy per base pair from the relation G(T,g)=lim N→∞1∂(1/T)2.A force-temperature(g−T)phase diagram of modelB andC for end and mid case is shown in Fig. 2.The qualitative features of the phase diagram obtained here may be compared with experiments[8].The phase boundary separates the zipped and the unzipped state.At T=0,the critical force can be found from a simple analytic calculation and is equal to0.5[18]that is in agreement with the one from Fig.2.Experimentally,the melting profiles are obtained by monitoring the change in the UV ab-sorbence with temperature which provides the information about the fraction of open base pairs and the melting temperature is defined when half of the total base pairs get open[29].Another quantity of experimental interest is to monitor and measure the end separation by varying the force.We calculate these quantities from the expressions<m>= mC(m,x)exp(βmǫ)exp(βgx)/ZNand<x>= xC(m,x)exp(βmǫ)exp(βgx)/ZN and plot their variations with temperature andforce in Figs.3and4,respectively.It is evident from Fig.3that,with increasing temperatureFIG.3:Variation(MID);(b)withdot andFIG.4:Plot ofconstant g=0.25(or force),thestate to theRemarkably,double the forcewith the exactdouble the forceAnother interesting observation is the variation of average elongation with force which shows a monotonically increasing trend(Fig.4a)at constant temperature and approaches unity.Meanwhile the variation of extension at constant force shows a sharp rise with temperature(Fig.4b)and then a slight decline to approach a value below unity.At constant temperature,there is a transition from the zipped to the unzipped(“rod-like”)state.Keeping force constant,when temperature is varied,there is a transition and the chain acquires conformations close to the rod-like states. As this temperature is still low,with further increase in temperature,the entropy of the system increases and the chain acquires coil-like state at higher temperature and thus average distance decreases.We also study the probability distribution curves P(x)with x and P(m)with m formodel B and C defined by P(x)=mC N(m,x)exp(βmǫ)exp(βgx)/Z N and P(m)=x C N(m,x)exp(βmǫ)exp(βgx)/Z N respectively.In Figs.5(a-f)we have shown P(x)and P(m)FIG.5:Figs.(a-f)at constant T=0.3.Fig.5ethe mid case in model C but absent in model B.for different values of force and afixed temperature T=0.3.The x-component of the distribution function gives information about the states of dsDNA.The maxima of P(x)at x≈0correspond to the zipped state for a given set with g=0.1and T=0.3.For small forces,thermalfluctuations are too weak to unzip the strand and the DNA remains in the zipped state.This is being reflected in the structure-less distribution function with a well defined peak at the most likely value of the extension(Figs.5a and5d).For both the models at higher force and at the same temperature (g=0.6for end and0.95for mid respectively at T=0.3),thermalfluctuations have been sup-pressed by a strong force and strands are found in the segregated“rod-like”state with more or less an identical distribution peaked at maximum extension.However,striking differences are observed in the probability distribution curves for model B and C at intermediate forces below transition line(g−T plane)when the force is applied in the middle.No such differences are observed for the end case.For the mid case,the probability distribution curve of model C shows strong oscil-lations,whereas for model B no such oscillations are observed.This indicates that the model B may be described by two state models.However,model C shows that certain intermediates states (“eye-shape”of even width)are more favorable than others.It is interesting to note that the g−T phase diagram of model B and C are almost identical without any re-entrance at low temperature.This may be because of the fact that the energy of the ground state and the unbound state for both models are the same.At the center point of the‘Y’(end case),the phase boundary is determined by a balance of the net force-2gx and the(a)(b)(c)FIG.6:Schematic diagrams showing the different conformations keeping2x(≤Nν)distance constant.For model B,(b)and(c)has same weight while for model C,(c)has more weight than(b).unzipping potential−ǫm with associated entropy.In the thermodynamic limit,directionality of the hydrogen bonds,which appear in the form of entropy of the partial bound states does not play a crucial role in this balance.The absence of re-entrance is understood with the zero entropy of the ground state for both models.As shown recently by Kapri et al.[13]for mid case,in the CDE there is a possibility of a coexistence region that is better thought of as an“eye-phase”≡two‘Y’joined together.In this case,the separation at the point of application of force is smaller than the fully open case and thus such conformations statistically have more weight than the other conformations.Since the bottom end is keptfixed,the top side of the strand may open due to thermalfluc-tuations(Fig.6b)and form a partial bound state as shown in Fig.6c which is more stable than Fig.6b.Therefore,in model C,the half of the chain undergoes an unzipping transition while other half due to the non-native contacts,shows the combined effects of unzipping and slippage (shearing)transition.Thus in the model C,the transition appears more smoother than the model B which can be seen in Fig.3b.The consecutive peaks in P(x)vs x curve(Fig.5e)represent the“eye-phase”of even widths which contribute most to the partition function.Because of native contacts,the contribution of the“eye-shape”conformation is significantly less and small thermal fluctuations are sufficient to unzip the chain in model B.We substantiate our arguments by extending calculation in CDE also.The partition functionin CDE may be defined as Z N(x,T)=mexp(βmǫ).The two ensemble are related by Z N(T,g)=xZ N(x,T)exp(βgx)[13,14].The free energy is given by the relation F N(x,T)=−T ln Z N(x,T). 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