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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］。
08 改善民生1.改善民生，提高生活质量Improving the people's well-being and raising quality of life2. 民谋利是治国理政的根本原则。
为人民谋福祉，是我们党坚持为公益服务、执政为民的重要内容。
Working for the people's wellbeing is an essential part of the Party's commitment to serving the public good and exercising governance for the people.3. 我们必须在发展中保障和改善民生，推动全体人民为实现对美好生活的向往而共同奋斗。
We must ensure and improve the people's well-being in the course of pursuing development and encourage everyone to work hard together to meet the people's aspirations for a better life.For many, moss(苔藓) is nothing but a humble tiny plant growing in some invisible corners, but for villagers in the mountainous town of Maoyang, it is 1 cash cow that has carried them out of poverty and on a path to further affluence.The remote town 2 (locate) in one of the least developed counties of East China's Zhejiang province, a major economic powerhouse and a pilot zone for 3 (achiev) common prosperity.Building on its distinctive 4 (nature) resources, the town has nurtured a burgeoning niche industry of breeding moss and selling related products such as potted plants, 5 are in rising demand in China as people's lives improve. Last year, the moss business generated over 17 million yuan (about $2.4 million) for the 6 (villager)."To make locals rich, you have 7 (foster) an industry. To grow bigger, the industry has to leverage local advantages," 8 (say) Lei Xiaohua, the town's Party chief and a delegate to the ongoing 20th National Congress of the Communist Party of China.Lei now has even 9 (strong) confidence in her town's future, as new arrangements have been made in a report to the congress to attain solid progress in promoting common prosperity10 the country's entire population.【答案】1.a 2．is located 3．achieving 4．natural 5．which6.villagers 7．to foster 8．said 9．stronger 10．forPassage1Measles(麻疹), which once killed 450 children each year and disabled even more, was nearly wiped out in the United States 14 years ago by the universal use of the MMR vaccine(疫苗). But the disease is making a comeback, caused by a growing anti-vaccine movement and misinformation that is spreading quickly. Already this year, 115 measles cases have been reported in the USA, compared with 189 for all of last year.The numbers might sound small, but they are the leading edge of a dangerous trend. When vaccination rates are very high, as they still are in the nation as a whole, everyone is protected. This is called “herd immunity”, which protects the people who get hurt easily, including those who can’t be vaccinated for medical reasons, babies too young to get vaccinated and people on whom the vaccine doesn’t work.But herd immunity works only when nearly the whole herd joins in. When some refuse vaccination and seek a free ride, immunity breaks down and everyone is in even bigger danger.That’s exactly what is happening in small neighborhoods around the country from Orange County, California, where 22 measles cases were reported this month, to Brooklyn, N.Y., where a 17-year-old caused an outbreak last year.The resistance to vaccine has continued for decades, and it is driven by a real but very small risk. Those who refuse to take that risk selfishly make others suffer.Making things worse are state laws that make it too easy to opt out(决定不参加) of what are supposed to be required vaccines for all children entering kindergarten. Seventeen states allow parents to get an exemption(豁免), sometimes just by signing a paper saying they personally object to a vaccine.Now, several states are moving to tighten laws by adding new regulations for opting out. But no one does enough to limit exemptions.Parents ought to be able to opt out only for limited medical or religious reasons. But personal opinions? Not good enough. Everyone enjoys the life-saving benefits vaccines provide, but they’ll exist only as long as everyone shares in the risks.1、The first two paragraphs suggest that ____________.A.a small number of measles cases can start a dangerous trendB.the outbreak of measles attracts the public attentionC.anti-vaccine movement has its medical reasonsrmation about measles spreads quickly2、Herd immunity works well when ____________.A.exemptions are allowedB.several vaccines are used togetherC.the whole neighborhood is involved inD.new regulations are added to the state laws3、What is the main reason for the comeback of measles?A.The overuse of vaccine.B.The lack of medical care.C.The features of measles itself.D.The vaccine opt-outs of some people.4、What is the purpose of the passage?A.T o introduce the idea of exemption.B.T o discuss methods to cure measles.C.T o stress the importance of vaccination.D.T o appeal for equal rights in medical treatment.Passage2If you are taking vitamin supplements to reduce your risk of heart disease or cancer, a group of health experts want you to know that those vitamins may actually increase your risk of cancer. The US Preventive Services T ask Force came to this conclusion after reviewing dozens of studies. Nearly half of adults in the US take at least one vitamin or mineral supplement on a regular basis. These pills are advertised as a way to promote general health. In some cases, manufacturers promote them as cancer fighters and heart protectors.Studies in animals and in laboratory dishes suggest that oxidative (氧化性的) stress contributes to diseases like cancer and heart disease. If so, there is a reason to believe that antioxidants—including beta-carotene, vitamins A, C, and E—could be useful as preventive medicines.But when the Task Force examined the medical evidence on vitamins, it found “inadequate(不充分的) evidence” to support the claims that vitamin and mineral supplements benefit healthy adults.“Cardiovascular (心血管的) disease and cancer have a significant health impact in America, and we all want to find ways to prevent these diseases,” Dr. Virginia Moyer, who heads the T ask Force, said in a statement. But so far, she added, the medical evidence does not show that taking vitamins is helpful in this regard.However, the T ask Force did find “ad equate ev idence” that people with a raised risk for lung cancer actually increase their risk further by taking beta-carotene, a precursor of vitamin A.The T ask Force recommendations of taking vitamins regularly apply to healthy adults aged 50 and older who don’t have “special nutritional needs”. The advice does not apply to children, women who are pregnant or may become pregnant, people with chronic illnesses, or people whohave to take supplements because they can’t get all their essential nutrients from their diet.5、Studies in animals and in laboratory dishes find out________.A.ample evidence that taking vitamins are helpful for treating lung cancerB.cardiovascular disease spreads very fast in AmericaC.oxidative stress can lead to heart disease and cancerD.people must take vitamins on a regular basis6、What can we conclude from T ask Force’s findings?A.Scientists want to control cardiovascular disease.B.In some regard, taking vitamins is not useful.C.Manufacturers cannot produce medical-use vitamins.D.Vitamins must be useful to prevent cancer and heart disease.7、Who can take vitamins regularly according to the advice of the T ask Force?A.A 60-year-old healthy worker.B.A 15-year-old boy with short-sightedness.C.A 34-year-old pregnant lady.D.A 40-year-old man who never eats vegetables or fruits.8、What’s the best title for the text?A.An Inside Look at VitaminsB.T ask Force: Ending to VitaminsC.Vitamins: T o Live or to KillD.T aking Vitamins to Prevent Cancer May FailPassage3In 2015, researchers from Australia's Deakin University published one of the first studies measuring food’s physical effect on the left hippocampus (海马体),a seahorse-shaped brain region crucial for memory, learning and decision making. It is also one of the first areas to shrink in people with dementia (痴呆). 252 people filled out diet surveys and then underwent scans that measured their brains. Four years later, they returned for another scan.The study found that the left hippocampus was heftier in the healthy eaters than in the unhealthy ones, regardless of age, sex, weight, exercise habits or general health. The average difference was 203 square millimeters, nearly one third of a square inch. Sounds small, but that's room for a lot of extra brain cells. And strong new evidence showed that eating the right food and skipping the wrong stuff could help protect against declines in thinking and memory that lead to dementia.“Plant-based diets may protect against memory decline and dementia,” says lead researcher Claire McEvoy, RD. How is the power food working with your brain cells? Animal and test-tube experiments suggest that vitamins and fatty acids found in the plant food help new cells make copies of DNA when they divide and multiply. Meanwhile, the high-fat and the high-sugarprocessed food harms brain cells by leaving brain tissue damaged by free radicals (自由基).This may hold back brain plasticity, making the processed food an especially big threat for the developing brains of kids.While food emerges as an important brain protector, experts say brain supplements(补充剂) aren’t all that effective. These pills and capsules may contai n many ingredients. But actually, studies show that they do not activate brain cells in a significantly positive way. “Let the buyer be aware of it,” says David Hogan? MD, a specialist at Calgary University.9、What does the underlined word “heftier” in par agraph 2 mean?A. Larger.B. Darker.C. Smaller.D. Cleverer.10、What is paragraph 3 mainly about?A. The health benefits of plants.B. The effects of food on the brain.C. The key components of healthy diet.D. The harm of the processed food to the brain.11、What should buyers be aware of according to David Hogan?A. Food is an important protector of the brain.B. Pills and capsules contain many ingredients.C. Brain supplements don't really benefit the brain.D. Supplements affect the brain in a significant way.12、What can be concluded from the text?A. Eating smart can benefit our brain.B. The animal-based diet damages free radicals.C. The high-fat food is the direct cause of dementia.D. A balanced diet contributes to kids’ learning ability.Passage4What did you eat for lunch today? Did you choose this dish because it was healthy, cheap or because it was just very delicious? Are you a selective eater or an adventurous foodie?I love exploring trends in food. Fusion cuisine is not for everybody. My Italian grandmother would turn her nose up at the thought of pizzas with mangoes topping but this marriage of tastes is perfectly fine in the 21st century. Chef and food writer Ching-He Huang, who presented a series on Chinese Food for the BBC, is a fan of fusion cuisine. She says: Fusion has been happening for centuries, for as long as people have travelled, but with the internet and global travel, the exchange of ideas makes the process much faster.”Wolfgang Puck is seen by many as one of the chefs who made fusion cuisine elegant. He opened his own restaurant in Los Angeles in the 1970s. This European devoted himself to Asian cuisine and became one of the first in a long line of celebrity chefs, because he adjusts the Asian cuisine to wes tern customers’ appetites by mixing different types of flavors. He said in aninterview with the Wall Street Journal that initially he got negative responses from traditional American-Chinese restaurant owners but he is not bitter. “Cooking is like paintin g or writing a song. Just as there are only so many notes or colors, there are only so many flavors—it’s how you combine them that distinguishes you.” he explains.My granny's cup of tea would be the Slow Food Movement. Founded by Carlo Petrini in the 1980s and still going strong, it seeks to preserve traditional cuisine and the use of ingredients that are grown locally. But Petrini thinks fusion cuisine is trendy.All these trends give us food for thought. We might be wasting an exciting opportunity to wake up our taste buds(味蕾) when we scoff (狼吞虎咽) a sandwich at our desks. Tomorrow, why not find an exotic(异国风味) restaurant and enjoy a feast?13、What does the underlined word in the second paragraph mean?A. combinationB. weddingC. relationshipD. divorce14、What has made fusion cuisine more popular?A. People from all over the world like to travel.B. People become well educated and like to try new things.C. Fusion has been happening for centuries.D. The internet and global travel increase the exchange of ideas.15、What can we conclude from the passage?A. My granny likes drinking a cup of tea.B. My granny likes pizzas with mangoes topping.C. The way a chef combines different flavors is what sets him apart.D. Puck is painful because of people's negative response to his restaurant.16、Which of the following best summarized the passage?A. Fusion cuisine is becoming more and more popular. Why not have a try?B. People attach great significance to Slow Food Movement. Why not call for it?C. Chefs are making fusion cuisine elegant. Why not preserve traditional cuisine?D. Fusion cuisine is criticized by many people. Why not avoid it?Passage5Consumers, as the manufacturers know well, are interested in avoiding fat because it causes heart problems and makes them fat.①_____ Fat-free cookies, chips and dairy products are all bestsellers. In fact, not eating fat seems to cause some health issues.②_____ Fruit and vegetables are obviously on the top of the list. Fruit and vegetables are usually low in calories and are packed with vitamins and minerals.Fat makes food taste good. With all the fat removed, some tasty food tends to taste like cardboard. ③_____ After all these additives like sugar, salt and artificial flavors are pumped in, the body is unable to process certain nutrients, and fat-free food can actually end up containing more calories than full-fat products.④_____ Fat helps regulate your nervous system, aids vitamin absorption and improves your immune system. Fat-free salad dressings can actually prevent your ability to process all the good-nutrients in that salad.When buying a fat-free product, you should take a second to examine the nutritional information on the package. If it's loaded with sugar, salt or artificial additives, put it back on the shelf. ⑤_____A. We know now that all fat is not bad.B. So the non-fat food is a dieter's dream!C. There is, of course, plenty of naturally fat-free food.D. Fat-free food actually has too much to recommend it.E. So, the manufacturers have to add in something to restore the flavor.F. When food manufacturers sell fat-free products, they will achieve great success.G. Sometimes, it is better to eat controlled full-fat products than fat-free junk.Even though it was only October, my students were already whispering about Christmas plans. With each passing day everyone became more 1 waiting for the final school bell. Upon its 2 everyone would run for their coats and go home, everyone except David.David was a small boy in ragged clothes. I had often 3 what kind of home life David had, and what kind of mother could send her son to school dressed so 4 for the cold winter months, without a coat, boots, or gloves. But something made David 5 . I can still remember he was always 6 a smile and willing to help. He always 7 after school to straighten chairs and mop the floor. W e never talked much. He 8 just smile and ask what else he could do, then thank me for letting him stay and slowly 9 home.W eeks passed and the 10 over the coming Christmas grew into restlessness until the last day of 11 before the holiday break. I smiled in 12 as the last of them hurried out the door. Turning around I saw David 13 standing by my desk.“I have something for you” he said and14 from behind his back a small box.15 it to me, he said anxiously. “Open it.” I took the box from h im, thanked him and slowly unwrapped it. I lift the lid and to my 16 I saw nothing. I looked at David’s smiling face add back into the box and said, “The box is nice, David, but it’s 17 ”.“Oh no it isn’t” said David “It’s full of love, my mum told me before she died that love was something you couldn’t see or touch unless you know it’s there”T ears filled my eyes 18 I looked at the proud dirty face that I had rarely given 19 to. After that Christmas, David and I became good friends and I never forgot the meaning 20 the little empty box set on my desk.18、A. anxious B. courageous C. serious D. cautious19、A. warning B. ringing C. calling D. yelling20、A. scolded B. wondered C. realized D. learned21、A. modestly B. naturally C. inaccurately D. inappropriately22、A. popular B. upset C. special D. funny23、A. expressing B. delivering C. wearing D. sharing24、A. practiced B. wandered C. studied D. stayed25、A. would B. should C. might D. could26、A. aim at B. turn to C. put off D. head for27、A. argument B. excitement C. movement D. program28、A. school B. year C. education D. program29、A. relief B. return C. vain D. control30、A. weakly B. sadly C. quietly D. helplessly31、A. searched B. found C. raised D. pulled32、A. Holding B. Handing C. Sending D. Leaving33、A. delight B. expectation C. appreciation D. surprise34、A. cheap B. empty C. useless D. improper35、A. as B. until C. because D. though36、A. advice B. support C. attention D. command37、A. from B. behind C. over D. towardsWolong National Nature Reserve is located in Sichuan Province. The soil types within Wolong are especially ①________(remark). Each mountain layer(层) contains a different type of soil, ②________ (range)from mountain black brown soil ③________ mountain brown soil. These soil types have stimulated the development of a rich biodiversity.About 4,000 plant species have been recorded in the various habitats of the reserve, such as evergreen forests and broadleaf forests. Altogether, 96 mammal(哺乳动物) species, 300 bird species ④________1,700 insect species live in Wolong, while 30 animal species ⑤________(be) under legal protection, including many endangered animals. The presence of the world's largest giant panda population was one of the main reasons why the reserve ⑥________(establish).While agriculture remains the major economic activity of local people, touristactivities are becoming a basic source of income(收入来源) However, ⑦________ construction of tourist facilities, as well as pressure from the large human population, is resulting in a number of negative ⑧________(impact). In addition, illegal firewood and medicinal plant collection presents challenges to local governments. Other challenges include the ⑨________(promote) of sustainable tourism and the development of other income opportunities. Research that was conducted within the reserve in the past has focused on the ecology and biology of the giant panda. Today, however, the ecosystem approach and the rehabilitation of bamboo and forest ecosystems are playing a(n) ⑩________ (increase) important role.Passage1上周日你班举办了以“亲近大自然”为主题的“秋游红叶谷”活动。
- 6 -[7] NEERAJA A，PRASAD A H，OOMMEN M，et al.Evaluation and correlation of nociceptive response index and spectral entropy indices as monitors of nociception in anesthetized patients[J].Journal of Neurosciences in Rural Practice，2023，14（3）：440-446.[8]赵曦，金毅，于砾淳，等.熵指数监测在神经内镜垂体瘤手术全身麻醉深度监测中的应用[J].现代肿瘤医学，2021，29（24）：4388-4391.[9]徐海峰，王开俊，黄龙，等.熵指数在腹腔镜手术全身麻醉深度监测中的应用[J].中国现代药物应用，2021，15（10）：78-80.[10]朱玉梅.熵指数监测在老年全身麻醉下行腹部手术患者中的应用[J].中国当代医药，2020，27（13）：121-124.[11]陈玲坤，苏春燕，陈海林.熵指数在老年全身麻醉患者中的应用效果[J].吉林医学，2016，37（3）：542-544.[12]张萱，高静.麻醉深度监测指标在围术期麻醉中的应用研究[J].医学信息，2022，35（14）：168-171.[13]肯内特（Kenneth Zinzal）.熵指数指导下不同剂量的右美托咪定对全凭静脉麻醉苏醒期的影响[D].通辽：内蒙古民族大学，2022.[14]李威，段春艳，高航.熵指数用于老年患者麻醉的临床效果观察[J].中国医药指南，2019，17（10）：90-91.[15]邓大立，孙丽杰，崔艳，等.熵指数监测下不同全身麻醉方法用于腹腔镜胃癌根治术患者的效果比较[J/OL].临床医药文献电子杂志，2020，7（39）：25.https:///kcms2/article/abstract?v=w1Je9LIFm5DsHfE0QofnujxMlnEUg9GzfCAm qGh8S-SdeUHwrMkLX-uswvmzWXSupu0_0dYVL-0zsDWyzZG FRhe1CaPzJZhoPOxAb4XO3lq2BudKvvqpqopZWPtZ64HmH7o DAPP9KYeIb8XjRmJt0Q==&uniplatform=NZKPT&language=C HS.[16]袁亮婧，金梅，张晓光.“三叶草”法超声引导下腰丛阻滞应用于髋关节镜术后镇痛的前瞻性随机对照研究[J].中国微创外科杂志，2019，19（11）：964-968.[17]杨益锋，范智东.麻醉深度监测指标在围手术期麻醉中的应用研究进展[J].现代医药卫生，2021，37（12）：2026-2029.[18]许克路，沈勤，柏耀林，等.腰丛阻滞复合不同麻醉深度喉罩全麻在老年THA 围手术期的应用[J].蚌埠医学院学报，2021，46（8）：1017-1022.[19]谢秀秀，黄常君，赵建勇，等.镇痛指数与手术容积指数指导全麻镇痛药物使用的价值研究[J].浙江医学，2023，45（19）：2080-2083，2088.[20]谢鹏程，李占芳，赵晓红，等.手术体积描计指数指导髋关节置换术中镇痛药使用的效果研究[J].临床麻醉学杂志，2019，35（5）：451-453.（收稿日期：2024-02-02） （本文编辑：何玉勤）①承德医学院 河北 承德 067000②保定市第一中心医院全科医疗科 河北 保定 071000通信作者：杨晰晰二甲双胍恩格列净片治疗2型糖尿病合并HFpEF的临床效果杨玥①② 杨晰晰②【摘要】 目的：探讨二甲双胍恩格列净片治疗2型糖尿病（T2DM）合并射血分数保留性心力衰竭（HFpEF）患者的临床效果和安全性。
高一科学探索英语阅读理解25题1<背景文章>The Big Bang Theory is one of the most important scientific theories in modern cosmology. It attempts to explain the origin and evolution of the universe. According to the Big Bang theory, the universe began as an extremely hot and dense singularity. Then, a tremendous explosion occurred, releasing an enormous amount of energy and matter. This event marked the beginning of time and space.In the early moments after the Big Bang, the universe was filled with a hot, dense plasma of subatomic particles. As the universe expanded and cooled, these particles began to combine and form atoms. The first atoms to form were hydrogen and helium. Over time, gravity caused these atoms to clump together to form stars and galaxies.The discovery of the cosmic microwave background radiation in 1964 provided strong evidence for the Big Bang theory. This radiation is thought to be the residual heat from the Big Bang and is uniformly distributed throughout the universe.The Big Bang theory has had a profound impact on modern science. It has helped us understand the origin and evolution of the universe, as well as the formation of stars and galaxies. It has also led to the development ofnew technologies, such as telescopes and satellites, that have allowed us to study the universe in greater detail.1. According to the Big Bang theory, the universe began as ___.A. a cold and empty spaceB. an extremely hot and dense singularityC. a collection of stars and galaxiesD. a large cloud of gas and dust答案：B。
中国科学技术大学研究生学位论文撰写规范中国科学技术大学学位办编二○○七年五月中国科学技术大学研究生学位论文撰写规范研究生学位论文集中反映研究生在研究工作中所取得的成果，代表研究生研究工作的水平,也是申请和授予相应学位的主要依据。
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论㊀㊀著ʌ文章编号ɔ1006-6233(2024)01-0001-08咪达唑仑调节YAP /TAZ 信号通路对骨肉瘤细胞增殖凋亡和侵袭的影响奥婷婷,㊀涂梦佳,㊀张㊀熙(湖北省十堰市人民医院麻醉科,㊀湖北㊀十堰㊀442099)ʌ摘㊀要ɔ目的:探究咪达唑仑(MDZ )调节Yes 相关蛋白(YAP )/PDZ 结合基序转录共激活因子(TAZ )信号通路对骨肉瘤(OS )细胞增殖㊁凋亡和侵袭的影响㊂方法:qRT -PCR ㊁Western blot 检测人正常成骨细胞系㊁OS 细胞系YAP ㊁TAZ mRNA 和蛋白的表达,筛选最佳干预细胞系;以不同浓度的MDZ(0㊁12.5㊁25㊁50㊁100㊁200μmoL /L )干预OS 细胞,MTT 法检测细胞增殖活性,筛选最佳干预浓度;将OS细胞随机分成control 组㊁MDZ 组㊁pcDNA3.1组㊁pcDNA3.1-YAP /TAZ 组,qRT -PCR 法检测转染效率;EdU 染色㊁流式细胞仪和Transwell 小室分别检测细胞增殖㊁凋亡和侵袭;Western blot 方法检测增殖细胞核抗原(PCNA )㊁Bcl -2相关X 蛋白(Bax )㊁N -钙黏蛋白(N -cadherin )㊁E -钙黏蛋白(E -cadherin )及YAP ㊁TAZ 蛋白表达;构建OS 裸鼠模型,分为对照组和MDZ 组,免疫组化法检测移植瘤组织Ki -67㊁YAP ㊁TAZ 蛋白表达,TUNEL 染色检测凋亡㊂结果:OS 细胞系中YAP ㊁TAZ mRNA 及蛋白表达水平显著升高(P <0.05);MDZ 显著降低PCNA ㊁N -cadherin ㊁YAP ㊁TAZ 表达,抑制MG63㊁U20S 细胞增殖和侵袭,升高Bax ㊁E -cadherin 表达,促进细胞凋亡㊂过表达YAP /TAZ 可逆转MDZ 对MG63㊁U20S 细胞增殖和侵袭的抑制作用,对MG63㊁U20S 细胞凋亡的促进作用(P <0.05);体内实验表明,MDZ 显著降低移植瘤质量㊁体积及Ki -67㊁YAP ㊁TAZ 表达水平,促进移植瘤细胞凋亡(P <0.05)㊂结论:MDZ 可能通过抑制YAP /TAZ 信号通路,抑制OS 细胞增殖和侵袭,促进凋亡㊂ʌ关键词ɔ㊀咪达唑仑;㊀YAP /TAZ ;㊀骨肉瘤;㊀增㊀殖;㊀凋㊀亡;㊀侵㊀袭ʌ文献标识码ɔ㊀A㊀㊀㊀㊀㊀ʌdoi ɔ10.3969/j.issn.1006-6233.2024.01.01Effects of Midazolam on Proliferation Apoptosis and Invasion of Osteosarcoma Cells by Regulating the YAP /TAZ Signaling PathwayAO Tingting ,TU Mengjia ,ZHANG Xi(Shiyan People 's Hospital ,Hubei Shiyan 442099,China )ʌAbstract ɔObjective :To investigate the effects of midazolam (MDZ )on the proliferation ,apoptosisand invasion of osteosarcoma (OS )cells by regulating the Yes associated protein (YAP )/PDZ transcriptional activator of transcription (TAZ )signaling pathway.Methods :qRT -PCR and western blot were applied to de-tect the expression of YAP ,TAZ mRNA ,and proteins in normal osteoblast and OS cell lines ,and to screenthe optimal intervention cell line.OS cells were intervened with different concentrations of MDZ (0,12.5,25,50,100,200μmoL /L ),MTT method was applied to detect cell proliferation activity and screen the opti-mal intervention concentration ;OS cells were randomly divided into control group ,MDZ group ,pcDNA3.1group ,and pcDNA3.1-YAP /TAZ group ,and transfection efficiency was detected using qRT -PCR method ;EdU staining ,flow cytometry ,and Transwell cells were applied to detect cell proliferation ,apoptosis ,and in-vasion ,respectively ;Western blot method was applied to detect the expression of proliferating cell nuclear an-tigen (PCNA ),Bcl -2associated X protein (Bax ),N -cadherin ,E -cadherin ,and YAP ,TAZ proteins ;OS nude mouse model was constructed ,and grouped into a control group and an MDZ group ,immunohistochemi-㊃1㊃ʌ基金项目ɔ湖北省卫生健康委科研项目,(编号:WJ2023F090)ʌ通讯作者ɔ涂梦佳cal methods were applied to detect the expression of Ki-67,YAP,and TAZ proteins in transplanted tumor tis-sue,and TUNEL staining was applied to detect apoptosis.Results:The expression levels of YAP and TAZ mRNA and protein in OS cell lines were obviously increased(P<0.05);MDZ obviously reduced the expres-sion of PCNA,N-cadherin,YAP,and TAZ,inhibitd the proliferation and invasion of MG63and U20S cells, increased the expression of Bax and E-cadherin,and promoted cell apoptosis.Overexpression of YAP/TAZ was able to reverse the inhibitory effects of MDZ on the proliferation and invasion of MG63and U20S cells, and promote the apoptosis of MG63and U20S cells(P<0.05);in vivo experiments showed that MDZ obvi-ously reduced the mass and volume of transplanted tumors,and the expression levels of Ki-67,YAP,and TAZ,promoted apoptosis of transplanted tumor cells(P<0.05).Conclusion:MDZ may inhibit the YAP/ TAZ signaling pathway,inhibit the proliferation and invasion of OS cells,and promote apoptosis.ʌKey wordsɔ㊀Midazolam;㊀YAP/TAZ;㊀Osteosarcoma;㊀Proliferation;㊀Apoptosis;㊀Invasion㊀㊀骨肉瘤(osteosarcoma,OS)是一种的原发性骨恶性肿瘤,常见于儿童和青少年㊂尽管手术与化疗相结合极大地改善了OS患者的预后,但转移性或复发性OS 的预后仍然差[1]㊂因此研究新的治疗OS药物,改善患者预后具有重要意义㊂大量研究表明,麻醉药物可抑制OS发生发展,如丙泊酚通过腺苷单磷酸激活蛋白激酶/转录因子叉头框蛋白O1介导的自噬抑制OS 细胞的恶性进展[2]㊂右美托咪定通过与miR-520a-3p-去泛素水解酶1基因相互作用,抑制OS细胞增殖和粘附,同时促进细胞凋亡[3]㊂咪达唑仑(midazolam, MDZ)广泛用于术前镇静㊂研究表明,MDZ可抑制肝细胞癌细胞的增殖㊁侵袭和迁移[4]㊂在OS中,MDZ通过上调circNOL10表达,诱导OS细胞凋亡,并抑制其增殖和迁移[5]㊂但MDZ抑制OS细胞恶性进展涉及其他分子机制仍有待进一步的研究㊂研究表明,Yes 相关蛋白(yes-associated protein,YAP)/PDZ结合基序转录共激活因子(transcriptional co-activator with PDZ-binding motif,TAZ)信号通路与OS细胞增殖和细胞凋亡过程,华蟾素注射液和维替泊芬通过下调YAP/TAZ通路,具有抗OS的作用[6-7]㊂基于此,本研究以MG63㊁U20S为研究对象,通过体内外实验探讨MDZ对MG63㊁U20S细胞增殖㊁凋亡和侵袭的影响及潜在作用机制,以期为OS的治疗提供新的参考㊂1㊀材料与方法1.1㊀细胞来源:人OS细胞系MG63㊁U20S㊁HOS㊁Saos2及人正常成骨细胞系hFOB1.19购于购于美国ATCC㊂SPF级BALB/c裸鼠购自武汉贝赛模式生物科技有限公司(许可证号:SCXK(鄂)2022-0029),6-8周龄,体重20g左右㊂本研究经本院动物伦理委员会批准同意(批号:2022042615-04)㊂1.2㊀主要材料㊁仪器:咪达唑仑(国药准字H20153019)江苏九旭药业有限公司;兔源一抗PCNA (ab15498)㊁YAP㊁(ab81183)㊁TAZ(ab307148)㊁Ki67㊁(ab16667)㊁N-cadherin(ab18203)㊁E-cadherin (ab18203))㊁GAPDH(货号:ab9485)以及HRP标记的羊抗兔二抗(货号:ab6721)美国Abcam公司;Annexin VFITC/PI检测试剂盒(4101-100T)上海晶风生物科技有限公司;细胞蛋白提取试剂盒(01126B)上海圻明生物科技有限公司;TUNEL凋亡检测试剂盒(D21014)上海雅吉生物科技有限公司;实时荧光定量PCR试剂盒(KMR027230)温州科淼生物科技有限公司;MTT试剂盒(M1020)北京雅安达生物技术有限公司;细胞转染试剂盒(AMEKO403)上海联硕生物科技有限公司;EdU染色(C10310)广州市锐博生物科技有限公司;KEEBIO-VE180蛋白电泳仪购于上海嘉鹏科技有限公司;680型全自动酶标仪购于上海君翼仪器设备有限公司;7500实时荧光定量PCR仪购于广州维基科技有限公司;A16K-R离心机购于湖南安君研仪器有限公司;DM3000-DM3000光学显微镜购于德国Leica㊂1.3㊀qRT-PCR法检测YAP㊁TAZ的表达:用Trizol试剂提取细胞的总RNA㊂反转录试剂盒以RNA为模板合成cDNA后,按照实时荧光定量PCR试剂盒配置反应体系㊂用实时荧光定量PCR仪检测YAP㊁TAZ的表达㊂YAP㊁TAZ的相对表达量用2-ΔΔCT法计算㊂1.4㊀MTT法检测MG63㊁U20S细胞增殖活性:将MG63㊁U20S接种到96孔板中常规培养(1ˑ104/孔) 24h,设6个复孔㊂加入含0㊁12.5㊁25㊁50㊁100㊁200μmoL/L MDZ[5]的培养液24h后加入MTT溶液(20μL/孔),检测490nm波长处检测各孔的吸光度,计算细胞增殖抑制率(%)和半数抑制浓度(IC50)㊂㊃2㊃表1㊀引物序列基因上游引物(5'-3')下游引物(5'-3') YAP ACCACAGCTCAGCATCTTCG TGGCTTGTTCCCATCCATCAG TAZ GCTGCTTCTGGACCAAGTACA AGATGTGGCGGAGTTTCAGG GAPDH CTTTGGTATCGTGGAAGGACTC GTAGAGGCAGGGATGATGTTC1.5㊀细胞转染:将MG63㊁U20S细胞接种到6孔板中(1.2ˑ106个),将细胞分为control组㊁MDZ组㊁pcD-NA3.1组㊁pcDNA3.1-YAP/TAZ组,利用转染试剂盒进行转染48h㊂qRT-PCR法检测转染效率㊂除control 组外,其余各组用含有100μmoL/L MDZ的培养基培养干预24h㊂1.6㊀细胞增殖检测:将转染成功的各组MG63㊁U20S 细胞接种于96孔板中(1ˑ104个/mL),细胞贴壁后加入5-乙炔基-2'-脱氧尿苷(EdU)稀释溶液(100μL/孔),孵育2h,磷酸盐缓冲液(PBS)洗涤,经固定㊁通透后,先后加入1ˑApollo㊁4',6-二脒基-2-苯基吲哚(DAPI)染色液,荧光显微镜下观察并拍照,计算Edu 阳性细胞率㊂1.7㊀细胞凋亡检验:将转染成功的各组MG63㊁U20S 细胞接种于6孔板中培养24h㊂加入5μL的膜联蛋白V-异硫氰酸荧光素(Annexin V-FITC)和碘化丙啶(PI)染液,遮光染色15min,上流式细胞仪检测细胞凋亡㊂1.8㊀细胞侵袭实验:Transwell小室用Matrigel覆盖后,上室加入MG63㊁U20S细胞悬液(200μL),下室加RPMI1640培养基(600μL),常规培养24h㊂下室细胞经固定,结晶紫染色(20min)㊁PBS冲洗后,镜下观察侵袭数㊂1.9㊀蛋白表达检测:Western blot检测蛋白表达,利用蛋白提取试剂盒得到MG63㊁U20S细胞的总蛋白,经定量㊁电泳分离㊁转聚偏氟乙烯(PVDF)膜后加入按照1ʒ1500稀释的一抗PCNA㊁Bax㊁N-cadherin㊁E-cad-herin㊁YAP㊁TAZ㊁GAPDH在4ħ孵育过夜,按照1ʒ1000稀释HRP标记的二抗室温孵育90min,以GAP-DH为内参,分析各蛋白表达㊂1.10㊀体内实验:于BALB/c裸鼠右侧背部皮下注射MG63细胞悬液,1周后,随机将裸鼠分成对照组㊁MDZ 组,每组6只㊂MDZ组腹腔注射5mg/kg MDZ,对照组腹腔注射等量生理盐水,2d/1次,干预4周㊂处死小鼠并分离肿瘤,测量肿瘤质量与体积㊂1.11㊀免疫组化法检测Ki-67㊁YAP㊁TAZ蛋白表达:将裸鼠瘤组织,固定于4%多聚甲醛中,石蜡包埋,切片㊁封闭和透化后,使用按照1ʒ1000稀释的抗Ki-67㊁YAP㊁TAZ抗体孵育过夜,在室温下与HRP标记的二抗(1ʒ1000)孵育1h,DAB溶液孵育3~15min,使用光学显微镜拍摄图像,黄色细胞被视为阳性细胞㊂使用Imagepro Plus6.0分析阳性细胞率㊂1.12㊀TUNEL染色检测凋亡:将1.3.9中肿瘤切片用3%双氧水处理10min,PBS洗涤3次㊂严格按照TUNEL试剂盒进行加反应液㊂二氨基联苯胺染色后,切片用梯度醇脱水,用二甲苯透明,用中性胶密封㊂在显微镜下观察细胞凋亡的变化㊂并计算细胞凋亡率㊂1.13㊀统计与分析:采用SPSS25.0软件对数据进行统计分析,计量资料以( xʃs)表示㊂多组间比较采用单因素方差分析,组间两两比较采用SNK-q检验㊂检验水准为α=0.05,P<0.05表示差异有统计学意义㊂2㊀结㊀果2.1㊀YAP㊁TAZ在不同细胞中的表达:与hFOB1.19细胞比较,MG63㊁U20S㊁HOS㊁Saos2中YAP㊁TAZ mR-NA及蛋白表达水平升高(P<0.05),YAP㊁TAZ mRNA 及蛋白表达水平在HOS㊁Saos2较高,MG63㊁U20S中较低,后续实验选择MG63㊁U20S细胞㊂见图1㊁表2㊂图1㊀不同细胞中YAP、TAZ蛋白表达㊃3㊃表2㊀YAP TAZ在不同细胞中的表达( xʃs,n=6)分组YAP mRNA TAZ mRNA YAP/GAPDH TAZ/GAPDH hFOB1.19 1.00ʃ0.01 1.00ʃ0.010.36ʃ0.070.23ʃ0.04 MG63 1.85ʃ0.19∗ 1.90ʃ0.17∗ 1.45ʃ0.11∗ 1.53ʃ0.13∗U20S 1.78ʃ0.18∗ 1.73ʃ0.15∗ 1.32ʃ0.12∗ 1.35ʃ0.11∗HOS 2.25ʃ0.23∗ 2.19ʃ0.22∗ 1.67ʃ0.15∗ 1.78ʃ0.16∗Saos2 1.94ʃ0.21∗ 2.18ʃ0.19∗ 1.58ʃ0.13∗ 1.64ʃ0.14∗㊀㊀注:与hFOB1.19细胞比较,∗P<0.05表3㊀不同浓度的MDZ对MG63U20S细胞的增殖抑制率( xʃs,n=6)分组增殖抑制率(%) MG63㊀㊀㊀㊀㊀㊀U20S0μmoL/L0.00ʃ0.000.00ʃ0.00 12.5μmoL/L7.15ʃ2.27∗ 6.81ʃ2.15∗25μmoL/L18.57ʃ2.49∗15.42ʃ2.36∗50μmoL/L33.26ʃ3.68∗34.57ʃ3.42∗100μmoL/L47.91ʃ4.31∗49.28ʃ3.90∗200μmoL/L65.24ʃ4.97∗65.37ʃ4.68∗IC50104.71μmoL/L102.09μmoL/L ㊀㊀注:与0μmoL/L比较,∗P<0.052.2㊀MDZ对MG63㊁U20S细胞增殖活性的影响:与㊀0μmoL/L比较,MG63㊁U20S细胞增殖抑制率在0㊁12. 5㊁25㊁50㊁100㊁200μmoL/L MDZ干预下以剂量依赖性的方式显著增加(P<0.05),MG63㊁U20S的IC50分别为104.71㊁102.09μmoL/L,本研究选取100μmoL/L MDZ进行后续实验㊂见表3㊂2.3㊀MDZ对MG63㊁U20S细胞中YAP㊁TAZ表达的影响:与control组比较,MDZ组MG63㊁U20S细胞中YAP㊁TAZ mRNA水平显著降低(P<0.05);与pcD-NA3.1组比较,pcDNA3.1-YAP/TAZ组MG63㊁U20S 细胞中YAP㊁TAZ mRNA水平显著升高(P<0.05), pcDNA3.1-YAP/TAZ转染成功㊂见表4㊂2.4㊀MDZ对MG63㊁U20S增殖能力的影响:与control 组比较,MDZ组MG63㊁U20S细胞Edu阳性率显著降低(P<0.05);与pcDNA3.1组比较,pcDNA3.1-YAP/ TAZ组MG63㊁U20S细胞Edu阳性率显著升高(P<0.05)㊂见图2㊁图3和表5㊂表4㊀各组MG63U20S细胞中YAP TAZ表达比较( xʃs,n=6)分组MG63YAP mRNA㊀㊀㊀㊀TAZ mRNA U20SYAP mRNA㊀㊀㊀㊀㊀TAZ mRNA control组 1.00ʃ0.07 1.00ʃ0.06 1.00ʃ0.08 1.00ʃ0.10 MDZ组0.16ʃ0.04∗0.19ʃ0.03∗0.10ʃ0.01∗0.13ʃ0.02∗pcDNA3.1组0.13ʃ0.030.15ʃ0.020.12ʃ0.020.15ʃ0.01 pcDNA3.1-YAP/TAZ组0.76ʃ0.09#0.54ʃ0.07#0.48ʃ0.05#0.69ʃ0.08#㊀㊀注:与control组比较,∗P<0.05;与pcDNA3.1组比较,#P<0.052.5㊀MDZ对MG63㊁U20S细胞凋亡的影响:与control 组比较,MDZ组MG63㊁U20S细胞凋亡率显著升高(P<0.05);与pcDNA3.1组比较,pcDNA3.1-YAP/TAZ组MG63㊁U20S细胞凋亡率显著降低(P<0.05)㊂见图4和表6㊂㊃4㊃图2㊀Edu 实验检测MG63细胞增殖(ˑ100)图3㊀Edu 实验检测U20S 细胞增殖(ˑ100)表5㊀各组MG63U20S 细胞Edu 阳性率比较( xʃs,n =6)分组Edu 阳性率(%)MG63㊀㊀㊀㊀㊀㊀U20S control 组78.30ʃ6.5472.48ʃ5.39MDZ 组21.46ʃ3.18∗22.67ʃ3.35∗pcDNA3.1组22.51ʃ3.2720.93ʃ3.11pcDNA3.1-YAP /TAZ 组56.78ʃ5.21#58.62ʃ4.87#㊀㊀注:与control 组比较,∗P<0.05;与pcDNA3.1组比较,#P<0.05图4㊀流式细胞仪检测细胞凋亡表6㊀各组MG63U20S 细胞凋亡率比较( xʃs ,n =6)分组凋亡率(%)MG63㊀㊀㊀㊀㊀㊀㊀U20S control 组9.24ʃ1.288.37ʃ1.19MDZ 组57.82ʃ3.91∗54.62ʃ3.78∗pcDNA3.1组58.63ʃ4.3752.94ʃ3.46pcDNA3.1-YAP /TAZ 组32.01ʃ3.12#28.91ʃ2.58#㊀㊀注:与control 组比较,∗P<0.05;与pcDNA3.1组比较,#P<0.052.6㊀MDZ 对MG63㊁U20S 细胞侵袭的影响:与control 组比较,MDZ 组MG63㊁U20S 细胞侵袭数显著降低(P <0.05);与pcDNA3.1组比较,pcDNA3.1-YAP /TAZ 组MG63㊁U20S 细胞侵袭数显著升高(P<0.05)㊂见图5和表7㊂图5㊀各组细胞侵袭情况比较(ˑ100)图6㊀各组细胞中PCNA ㊁Bax ㊁N -cadherin ㊁E -cadherin ㊁YAP ㊁TAZ 蛋白表达比较注:A :control 组;B :MDZ 组;C :pcDNA3.1组;D :pcDNA3.1-YAP /TAZ 组㊃5㊃表7㊀各组MG63U20S 细胞侵袭数比较( xʃs ,n =6)分组侵袭细胞数(个)MG63㊀㊀㊀㊀㊀㊀U20S control 组163.58ʃ12.46145.94ʃ10.35MDZ 组74.98ʃ8.15∗57.31ʃ6.46∗pcDNA3.1组69.32ʃ7.8354.96ʃ6.28pcDNA3.1-YAP /TAZ 组128.19ʃ10.92#113.62ʃ9.67#㊀㊀注:与control 组比较,∗P<0.05;与pcDNA3.1组比较,#P<0.052.7㊀MDZ 对PCNA ㊁Bax ㊁N -cadherin ㊁E -cadherin ㊁YAP ㊁TAZ 蛋白表达的影响:与control 组比较,MDZ 组MG63㊁U20S 细胞PCNA ㊁N -cadherin ㊁YAP ㊁TAZ 表达显著降低,Bax ㊁E -cadherin 表达显著升高(P <0.05);与pcDNA3.1组比较,pcDNA3.1-YAP /TAZ 组MG63㊁U20S 细胞PCNA ㊁N -cadherin ㊁YAP ㊁TAZ 表达显著升高,Bax ㊁E -cadherin 表达显著降低(P<0.05)㊂见图6㊁表8㊁表9㊂表8㊀各组MG63细胞蛋白表达比较( xʃs,n =6)分组PCNA Bax N -cadherin E -cadherin YAP TAZ control 组 1.37ʃ0.120.31ʃ0.06 1.60ʃ0.150.36ʃ0.08 1.45ʃ0.11∗ 1.53ʃ0.13∗MDZ 组0.36ʃ0.07∗ 1.54ʃ0.14∗0.43ʃ0.07 1.65ʃ0.13∗0.35ʃ0.08∗0.61ʃ0.10∗pcDNA3.1组0.34ʃ0.05 1.57ʃ0.150.51ʃ0.10 1.68ʃ0.140.42ʃ0.070.57ʃ0.08pcDNA3.1-YAP /TAZ 组1.12ʃ0.11#0.62ʃ0.10#1.28ʃ0.13#0.75ʃ0.11#1.17ʃ0.13#1.26ʃ0.12#㊀㊀注:与control 组比较,∗P<0.05;与pcDNA3.1组比较,#P<0.05表9㊀各组U20S 细胞蛋白表达比较( xʃs,n =6)分组PCNA Bax N -cadherin E -cadherin YAP TA control 组 1.56ʃ0.140.21ʃ0.04 1.47ʃ0.130.28ʃ0.05 1.32ʃ0.12 1.35ʃ0.11MDZ 组0.43ʃ0.07∗ 1.28ʃ0.11∗0.39ʃ0.05∗ 1.37ʃ0.12∗0.29ʃ0.05∗0.32ʃ0.05∗pcDNA3.1组0.41ʃ0.05 1.34ʃ0.130.37ʃ0.04 1.42ʃ0.130.33ʃ0.060.35ʃ0.07pcDNA3.1-YAP /TAZ 组1.27ʃ0.13#0.73ʃ0.10#1.12ʃ0.11#0.58ʃ0.07#1.09ʃ0.10#1.18ʃ0.11#㊀㊀注:与control 组比较,∗P<0.05;与pcDNA3.1组比较,#P<0.052.8㊀MDZ 对裸鼠移植瘤生长的影响:体内实验表明,与对照组比较,MDZ 组小鼠移植瘤质量与体积明显下降,移植瘤细胞凋亡率显著升高(P <0.05);MDZ 组移植瘤中,Ki -67㊁YAP ㊁TAZ 蛋白表达水平显著降低(P <0.05),见图7~9和表10㊂表10㊀各组裸鼠移植瘤生长和Ki -67YAP TAZ 蛋白表达比较( xʃs,n =6)分组移植瘤体积(mm 3)移植瘤质量(g )Ki -67阳性率(%)YAP 阳性率(%)TAZ 阳性率(%)细胞凋亡率(%)对照组1318.46ʃ124.57 1.58ʃ0.1237.69ʃ2.5341.95ʃ3.5843.57ʃ4.10 6.94ʃ1.37MDZ 组647.89ʃ98.34∗0.49ʃ0.08∗15.43ʃ1.19∗16.31ʃ1.47∗18.61ʃ2.06∗35.28ʃ2.45∗㊀㊀注:与对照组比较,∗P<0.05㊃6㊃图7㊀各组移植瘤组织生长情况(n =6)图8㊀免疫组化检测移植瘤组织中Ki -67㊁YAP ㊁TAZ 蛋白表达(ˑ100)图9㊀TUNEL 染色检测各组移植瘤细胞凋亡(ˑ200)3㊀讨㊀论OS 是一种起源于间充质组织的恶性肿瘤,具有易转移㊁预后差和短期死亡率高的特点㊂由于其发展迅速和早期转移,患者的5年生存率不理想㊂化疗联合手术治疗仍然是骨肉瘤的主要治疗方法,然而,药物引起的副作用和手术后肿瘤复发会降低患者的生活质量并降低患者的生存率㊂因此,寻找安全有效的治疗药物是当前研究的重点㊂麻醉剂在肿瘤复发和转移中起至关重要的作用㊂研究发现,七氟醚可抑制OS 细胞的增殖和侵袭转移能力,同时降低MG63细胞对顺铂的敏感性[8]㊂布比卡因治疗提高了OS 细胞的凋亡率,减弱了侵袭和迁移能力,肿瘤异种移植实验中,布比卡因显著抑制裸鼠OS 细胞生长[9]㊂盐酸罗哌卡因通过下调Livin 基因,降低了OS 细胞对多柔比星的耐药性,具有抗OS 的作用[10]㊂MDZ 是一种苯二氮卓类药物,具有起效快,作用短的特点,此外,还具有显著的催眠㊁抗焦虑和镇静特性㊂除了用作麻醉剂外,MDZ 还可能具有预防或抑制肿瘤发展的能力㊂Kang 等[4]研究结果显示,于裸鼠腹腔内注射MDZ 可减少肝细胞癌肿瘤的发展,并提高肝细胞癌抗程序性死亡配体1(PD -1)免疫治疗的效率㊂MDZ 通过调节miR -194-5p /hook 微管栓系蛋白3(HOOK3)轴来增强非小细胞肺癌耐药细胞中的顺铂敏感性,MDZ 抑制非小细胞肺癌细胞增殖和迁移,促进细胞凋亡,MDZ 可作为临床实践中非小细胞肺癌治疗的辅助药物[11]㊂MDZ 在体内抑制胰腺导管腺癌小鼠肿瘤大小,通过下调细胞周期蛋白和细胞周期蛋白依赖性激酶抑制胰腺导管腺癌增殖,并在体外诱导细胞凋亡[12]㊂本研究发现,MDZ 显著降低MG63㊁U20S 细胞的Edu 阳性细胞率㊁侵袭数㊁移植瘤质量和体积㊁同时降低增殖㊁侵袭相关蛋白(Ki -67㊁PCNA ㊁N -cad-herin )表达水平,升高细胞凋亡率和凋亡蛋白Bax 表达㊂提示,MDZ 可抑制MG63㊁U20S 细胞增殖和侵袭,促进细胞凋亡,具有抗OS 的作用㊂Hippo 通路调节细胞增殖㊁凋亡㊁侵袭和转移,对多数肿瘤发生中起关键作用㊂YAP 和TAZ 是Hippo 通路是Hippo 途径的下游效应因子,在肝癌㊁乳腺癌㊁肺癌等许多类型的肿瘤中经常观察到YAP /TAZ 的表达升高,YAP /TAZ 激活不仅使化疗㊁放疗或免疫治疗产生耐药性,也可与TEAD 家族共激活剂结合,促进癌细胞增殖㊁侵袭性迁移和转移[13]㊂研究发现,YAP /TAZ 在OS 组织和细胞中高表达,YAP /TAZ 被认为是骨肉瘤的新型预后标志物和治疗靶点[14]㊂本研究发现,MDZ 可显著降低体内体外YAP ㊁TAZ mRNA 及蛋白表达,过表达YAP /TAZ 可逆转MDZ 对MG63㊁U20S 细胞恶性行为的抑制作用,提示,MDZ 可能通过抑制YAP /TAZ 信号通路,抑制MG63㊁U20S 增殖和侵袭,促进凋亡㊂综上所述,MDZ 可能通过抑制YAP /TAZ 信号通路,抑制OS 细胞增殖和侵袭,促进凋亡㊂MDZ 可作为治疗OS 的潜在药物㊂后续将继续研究MDZ 对OS 的作用及其他作用分子机制㊂㊃7㊃ʌ参考文献ɔ[1]㊀Chen C ,Xie L ,Ren T ,et al.Immunotherapy for osteosarco-ma :fundamental mechanism ,rationale ,and recent break-throughs [J ].Cancer Lett ,2021,500(1):1-10.[2]㊀Dai L ,Li S ,Li X ,Jiang B.Propofol inhibits the malignant de-velopment of osteosarcoma U2OS cells via AMPK /FΟΧO1-mediated autophagy [J ].Oncol Lett ,2022,24(3):310-317.[3]㊀Yan R ,Jin S ,Liu H ,et al.Dexmedetomidine inhibits cell ma-lignancy in osteosarcoma cells via miR -520a -3p -YOD1in-teractome [J ].Biochem Biophys Res Commun ,2021,543(1):56-64.[4]㊀Kang J ,Zheng Z ,Li X ,et al.Midazolam exhibits antitumourand enhances the efficiency of Anti -PD -1immunotherapy inhepatocellular carcinoma [J ].Cancer Cell Int ,2022,22(1):312-325.[5]㊀季英龙,周全军,冀国力.咪达唑仑抑制骨肉瘤细胞增殖㊁迁移并诱导其凋亡[J ].局解手术学杂志,2023,32(7):586-591.[6]㊀Chen Y ,Wang Y ,Zhai Y ,et al.Cinobufacini injection sup-presses the proliferation of human osteosarcoma cells by in-hibiting PIN1-YAP /TAZ signaling pathway [J ].Front Phar-macol ,2023,14(1):1081363-1081374.[7]㊀Li Y ,Yang S ,Yang S.Verteporfin inhibits the progression ofspontaneous osteosarcoma caused by trp53and Rb1deficien-cy in ctsk -expressing cells via impeding hippo pathway [J ].Cells ,2022,11(8):1361-1372.[8]㊀鲁飞,王强,谷淼.气体麻醉剂影响骨肉瘤细胞侵袭转移能力及化疗敏感性的分子机制[J ].临床和实验医学杂志,2020,19(24):2614-2618.[9]㊀Wang Z ,Song Y ,Zhang H ,et al.Local anesthetic levobupiva-caine inhibits stemness of osteosarcoma cells by epigenetical-ly repressing MAFB though reducing KAT5expression [J ].Aging (Albany NY ),2022,14(6):2793-2804.[10]㊀于鹏,尹天翔,罗彩云,等.盐酸罗哌卡因对骨肉瘤细胞增殖㊁侵袭㊁凋亡的影响及其机制[J ].中国应用生理学杂志,2021,37(6):654-659,664.[11]㊀Zhang X ,Han Z ,Li Z ,et al.Midazolam impedes lung carci-noma cell proliferation and migration via EGFR /MEK /ERK signaling pathway [J ].Open Med (Wars ),2023,18(1):20230730-20230740.[12]㊀Oshima Y ,Sano M ,Kajiwara I ,et al.Midazolam exhibits an-titumour and anti -inflammatory effects in a mouse model ofpancreatic ductal adenocarcinoma [J ].Br Anaesth ,2022,128(4):679-690.[13]㊀Thompson BJ.YAP /TAZ :drivers of tumor growth ,metasta-sis and Resistance to Therapy [J ].Bioessays ,2020,42(5):e1900162-e1900177.[14]㊀Chim LK ,Williams IL ,Bashor CJ ,et al.Tumor -associatedmacrophages induce inflammation and drug resistance in amechanically tunable engineered model of osteosarcoma[J ].Biomaterials ,2023,296(1):122076-122086.ʌ文章编号ɔ1006-6233(2024)01-0008-08Si -SETDB1通过SPG20甲基化对肺腺癌细胞迁移侵袭的影响赵宝山1,㊀杨㊀阳2,㊀孙光蕊1,㊀郑竞雄1,㊀梁宗英1(1.承德医学院附属医院胸外科,㊀河北㊀承㊀德㊀0670002.河北省胸科医院临床药理试验部,㊀河北㊀石家庄㊀050000)ʌ摘㊀要ɔ目的:检测肺腺癌中SETDB1和SPG20甲基化水平,探究下调SETDB1对SPG20甲基化及A549细胞迁移和侵袭的影响㊂方法:选取60例肺腺癌组织和正常肺组织,qRT -PCR 检测mRNA 相对表达量;免疫组织化学法和Western blot 检测蛋白表达量;基因甲基化水平应用焦磷酸测序法检测;构建并合成SETDB1的小干扰RNA ,脂质体介导法转染细胞,qRT -PCR 检测mRNA 相对表达量㊁Wesrernblot 检测蛋白表达㊁焦磷酸测序法检测基因甲基化㊁MTT 检测细胞增殖活性㊁划痕愈合检测细胞迁移㊁Transwell 小室实验检测细胞侵袭㊂结果:qRT -PCR 结果显示肺腺癌组织中SPG20表达下调(P <0.05),而SETDB1表达上调(P <0.05);Western blot 结果显示肺腺癌组织中SETDB1蛋白表达量显著高于正常肺组织,而SPG20蛋白在癌组织中呈低表达,低于正常肺组织㊂在癌组织中SPG20基因甲基化率显著高于正常肺组织,差异有统计学意义㊂SPG20甲基化水平与SETDB1表达呈正相关性;肺腺癌细胞中SETDB1呈高表达,而SPG20呈低表达;正常支气管上皮细胞中SETDB1呈低表达,SPG20则呈高表达㊂RNA 干扰SETDB1可使A549细胞内SETDB1mRNA 和蛋白表达量显著降低,SPG20基因甲基化率㊃8㊃ʌ基金项目ɔ河北省自然科学基金资助项目,(编号:H2021406045);承德市科学技术研究与发展计划项目,(编号:201904A029)ʌ通讯作者ɔ梁宗英。
- 175 -①宁夏医科大学 宁夏 银川 750000②宁夏医科大学总医院通信作者：马雅玲SMILE对角膜生物力学影响的研究进展魏嘉① 马雅玲② 【摘要】 近年来，全飞秒激光小切口角膜基质透镜取出术（small incision lenticule extraction，SMILE）逐渐成为矫正屈光不正的主流手术方式，虽然其以安全性、有效性、可预测性等优点受到广泛推崇，但仍有术后角膜扩张病例的报道及术后角膜生物力学稳定性显著降低的研究结果，因此仍然需要大量的研究来探讨导致角膜生物力学稳定性降低的相关因素。
本文对角膜生物力学的概述、角膜生物力学的测量方法及SMILE 对角膜生物力学的影响进行综述。
【关键词】 角膜生物力学 全飞秒激光小切口角膜基质透镜取出术 角膜 doi：10.14033/ki.cfmr.2023.33.044 文献标识码 A 文章编号 1674-6805（2023）33-0175-06 Research Progress of the Effect of SMILE on Corneal Biomechanics/WEI Jia, MA Yaling. //Chinese and Foreign Medical Research, 2023, 21(33): 175-180 [Abstract] In recent years, small incision lenticule extraction (SMILE) has gradually become a mainstream surgical practice for correcting refractive errors. Although it is widely praised for its advantages such as safety, effectiveness, and predictability, there are still reports of postoperative corneal dilation cases and research results showing a significant decrease in corneal biomechanical stability. Therefore, a large amount of research is still needed to explore the relevant factors leading to a decrease in corneal biomechanical stability. This paper reviews the overview of corneal biomechanics, the measurement methods of corneal biomechanics and effect of SMILE on corneal biomechanics. [Key words] Corneal biomechanics Small incision lenticule extraction Corneal First-author's address: Ningxia Medical University, Yinchuan 750000, China 随着时间的推移，近视已经成为严重的全球公共卫生问题，已经影响了超过30%的人口，而且这一数字还在持续上涨，根据最新的研究，到2050年，全球罹患近视的人口数将超过总人口的50%[1]。
中国组织工程研究 第20卷 第18期 2016–04–29出版Chinese Journal of Tissue Engineering Research April 29, 2016 Vol.20, No.18·研究原著·www.CRTER .org刘慧影，女，1982年生，辽宁省沈阳市人，汉族，辽宁中医药大学在读硕士，主治医师，主要从事脑血管病诊疗方法的研究。
通讯作者：王鹏琴，博士，主任医师，辽宁中医药大学附属第一医院康复科，辽宁省沈阳市 110032中图分类号:R318 文献标识码:B 文章编号:2095-4344 (2016)18-02634-08 稿件接受：2016-03-16 眼针干预脑缺血再灌注模型大鼠神经功能及相关神经营养因子的表达刘慧影1，王鹏琴2，边 颖1，王金春1，魏颖鸿1(1沈阳市第五人民医院神经内一科，辽宁省沈阳市 110023；2辽宁中医药大学附属第一医院康复科，辽宁省沈阳市 110032)引用本文：刘慧影，王鹏琴，边颖，王金春，魏颖鸿. 眼针干预脑缺血再灌注模型大鼠神经功能及相关神经营养因子的表达[J].中国组织工程研究，2016，20(18): 2634-2641.DOI: 10.3969/j.issn.2095-4344.2016.18.008 ORCID: 0000-0003-3166-3344(刘慧影)文章快速阅读：文题释义：神经生长因子：是神经营养因子中最早被发现，目前研究最为透彻的，具有神经元营养和促突起生长双重生物学功能的一种神经细胞生长调节因子，它对中枢及周围神经元的发育、分化、生长、再生和功能特性的表达均具有重要的调控作用。
神经生长因子包含α，β，γ共3个亚单位，活性区是β亚单位，由2个118个氨基酸组成的单链通过非共价键结合而成的二聚体，与人体神经生长因子的结构具有高度的同源性，生物效应也无明显的种间特异性。
脑源性神经营养因子：是体内含量最多的神经营养因子，它通过与酪氨酸激酶B 的结合而发挥作用。
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). In CFE the average separation<x>fluctuates while in CDE one measures the average force to keep the separation constant given by the expression<g>=∂F N(x,T)FIG.7:To see this,we associate anpossible in model C)andshift in the transitionbecomes stiffer.However,itdistribution curves for the‘MID’case.The exact results on short chains of a new semi-microscopic dsDNA that incorporates the directional nature of hydrogen bond show unequivocal signature of an“eye phase”,without going to the long chain limit.This happens for the case with a force acting in the middle of the dsDNA. 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