下载文档原格式
AMERICAN BRANDPermethrin Granules sFor Lawns and around Homes, Commercial & Industrial BuildingsContains Permethrin to provide contact and residual control of troublesome outdoorinsects and pests as listed, for up to 4 weeks. 10 lbs. treats up to 5000 sq. ft.AMERICAN BRANDPermethrin Granules sactive inGredient:Permethrin* .............................................................................0.25%Other Ingredients ..................................................................99.75%total: ..............................................................................100.00%•cis-trans ratio: min . 55% (+/-) cis, max 45% (+/-) trans.KEEP OUT OF REACH OF CHILDRENCAUTIONPrecaucion al uSuario: Si usted no entende la etiqueta, busque a alguien para que se la explique austed en detalle. (If you do not unterstand the label, find someone to explain it to you in detail.)FIRST AIDiF SWalloWed: Call a poison control center or doctor immediately for treatment advice. Have person sip a glass of water if able to swallow. Do not induce vomiting unless told so by the poison control center or doctor. Do not give anything by mouth to an unconscious person.iF in eyeS: Hold eye open and rinse slowly and gently with water for 15 to 20 minutes. Remove contact lenses, if present after 5 minutes, then continue rinsing. Call a poison control center or doctor for treatment advice.iF on SKin or clotHinG: Take off contaminated clothing. Rinse skin immediately with plenty of water for 15 to 20 minutes. Call a poison control center or doctor for treatment advice.iF inHaled: Move person to fresh air. If person is not breathing, call 911 or an ambulance, then give artificial respiration, preferably mouth-to-mouth if possible. Call a poison control center or doctor for further treatment.You may also call 1-800-858-7378 for emergency medical treatment advice.See back of bag for additional precautionary statements.distributed By:voluntary PurcHaSinG GrouPS, inc.230 Fm 87BonHam, teXaS 75418ePa reg. no. 55431-4-7401 ePa est. no. 7401-tX-1net Wt. 25 lB (11.34 kg)Precautionary StatementSHazardS to HumanS and domeStic animalSCAUTION: Harmful if swallowed, inhaled or absorbed through skin. Causes moderate eye irritation. Avoid inhalation of dust. Avoid contact with skin, eyes or clothing. Wash thoroughly after han-dling. Avoid contamination of food and feed.environmental HazardSThis product is extremely toxic to fish. Do not apply directly to water. Drifts and runoff from treated areas may be hazardous to aquatic organisms in neighboring areas. Do not contaminate water when disposing of equipment wash waters.directionS For uSeIt is a violation of Federal law to use this product in a manner inconsistent with its labeling. This product is permitted for outdoor use around the home. May be used on ornamental and recreational turf grass areas. Not for use on Sod or Turf Farms. For outdoor lawn or perimeter insect control around homes, commercial and industrial buildings. Wear protective household gloves while using. Food utensils such as teaspoons and measuring cups should not be used for food purposes after use with pesticides.ready to uSeKills on contact, protects your home from invading ants. Also stops Spiders, Roaches, Fleas, Brown dog ticks.FOr LAwNs ANd TUrF grAss: Kills Grubs, Fleas, Ants, Crickets, Brown dog ticks, Fire ants, Chinch bugs, Armyworms, Billbugs, Cutworms, Earwigs, Mole crickets, Sod webworms, and other insects listed on this label.Barrier/band Treatment around Commercial buildings and homes.The following pests are commonly found around or near foundations of commercial buildings and houses as well in lawns. Some pests attempt to enter the structure. Apply a 3 to 5 foot barrier band of AMERICAN BRAND PERMETHRIN GRANULES next to the buildings foundation for control of insects such as Ants, Armyworms, Brown dog ticks, Chinch bugs, Clover mites, Crickets, Cutworms, Earwigs, Fleas, Lawn moths-Sod webworms, Leafhoppers, Millipedes, Mole crickets, and Sowbugs. APPLICATION RATE: Use 3.2 ounces for each 100 sq. ft. area 5 ft. x 20 ft. or 2 lbs. per 1000 sq. ft. of area.HOw TO UsE: Distribute product evenly over the area to be treated. Lightly water in the granules after application. Do not mow treated area until granules have been watered in.sPOT TrEATMENT FOr ANTs: Apply 1 1/2 teaspoonful over and around each ant hill. Water lightly after application. Reapply if insects reappear and after hard rains.SPreader SettinGSspreader settings for pounds of this product to use per 1000 sq. ft.spreader Make and Model grass or dichondra Lawns2 pounds Heavy growth st. Augustine Lawns3 poundssoil Insects as listed2 1/2 poundsCentral Imperial MakeNo. 1662-G6 1/27 1/47Central Suburbia Lawn SpreaderNo.1622-06 1/27 1/47Cyclone ModelB-1 Rotary4 1/25 4 3/4Greenfield Model#1056 1/27 1/4 6 3/4Ortho Drop Spreader Model30004 1/25 1/25Ortho Broadcast Spreader#40004 1/276J.C. Penney Model#60057 1/487 1/2J.C. Penney RotarySpreader576ScottsModel 3545 4 1/2ScottsModel PF 3F H H/ISears CraftsmanModel 671.1919867 6 1/2VW&R MasterlineR75FBF H H/IdirectionS For aPPlyinG american Brand PermetHrin GranuleSTreatment Areas Target Pests Remarks Use RateBand treatment around outside of house foundation Ants, Brown dog ticks, Clover mites,Crickets, Earwigs, Fleas, Millipedes,Roaches, Silverfish, Sowbugs(Pillbugs),SpidersThese pests are commonly found around or near foun-dations of houses as well as in lawns. Certain of thesepests may enter houses. Apply a 3 to 5 ft band of gran-ules around the dwelling. You may water lightly after ap-plication. Repeat if new insects appear.3.2 oz. for each 100 sq. ft. area(5 x 20 ft.) or 2 lbs. per 1000 sq. ft.equal to 87 lbs. per acre.HOW TO USEFor overall lawn applications: Turfgrass and Dichondra #1 Determine and measure the sq. ft. of the area to be treated. #2 Refer to spreader settings for your application rate. Check and calibrate your spreader as per your spreader manual. #3 Apply as directed in this chart.SURFACE INSECTSAnts, Armyworms, Brown dog ticks,Chinch bugs, Clover mites, Crickets,Cutworms, Earwigs, Fleas, Lawnmoths, (Sod webworms), Leafhoppers,Millipedes, SowbugsApply with a fertilizer spreader. See spreader settingschart on this label. Apply when grass is dry. It is notnecessary to water in the insecticide for the control ofsurface insects. Repeat if new insects appear.For application with other type ofequipment apply at the rate of 2 lbs.per 1000 sq. ft. or 3.2 oz. per 100sq. ft. For heavy lawns such asSt. Augustine grass increase rate to3 lbs. per 1000 sq. ft. equal to4.8 oz. per 100 sq. ft.SOIL INSECTSAtaenius grubs, Billbug grubs, Japa-nese beetle grubs, June beetle grubs,Mole crickets, White grubs.Water grass thoroughly after application with 1/4 to 1/2inch water to release the insecticide to the soil level. Forbillbug control apply when activity is first observed orwhen chewed or brown grass indicates damage fromthese insects. For white grub control except dung beetle,treat any time between late July and early October. Treatagain in late summer or early fall if a 2nd generationoccurs.For these soil insects use the rateof 2 1/2 lbs. per 1000 sq. ft. equalto 109 lbs. per acre.Spot Treatments Ants Water lightly after applying 1 1/2 teaspoonfuls/ant hill.Repeat if ants reappear Spread granules over and around each ant hill.Imported Fire Ants Thoroughly and gently sprinkle a minimum of 1 gallon ofwater over each mound after application. High pressurewatering may cause ants to migrate reducing product ef-fectiveness. Apply in cool weather, 65° to 80° in earlymorning or late evening hours. Treat new ant mounds ifthey appear.Gently apply 1 cup of granules over and 2 ft around each ant mound. This product works within 24 hours.StoraGe and diSPoSalsTOrAgE: Store product in its original container (closed), in a locked, cool, dry, area out of reach of children and pets.dIsPOsAL: Completely empty bag into application equipment and use as directed. IF EMPTY: Do not reuse this bag. Place in trash or offer for recycling if suitable.IF PArTLY FILLEd: Call your local solid waste agency for disposal instructions. Never place unused product down any indoor or outdoor drain.sPILL: In case of spill, sweep up and use as directed or dispose of material according to product disposal directions above.IMPOrTANT INFOrMATION: rEAd BEFOrE UsINg PrOdUCT.conditionS oF Sale and limitation oF Warranty and liaBility noticeRead the entire Directions for Use and Conditions of Sale and Limitation of Warranty and Liability before buying or using this product. If the terms are not acceptable, return the product at once, unopened, and the purchase price will be refunded. The Directions for Use of this product should be followed carefully. It is impossible to eliminate all risks inherently associated with the use of this product. Crop injury, ineffectiveness or other unintended consequences may result because of such factors as manner of use or application, weather, or crop conditions, presence of other materials or other influencing factors in the use of the product, which are beyond the control of Voluntary Purchasing Groups, Inc. or Seller. Buyer and User shall assume all such risks, and Buyer and User agree to hold Voluntary Purchasing Groups, Inc. and Seller harmless for claims relating to such factors. Voluntary Purchasing Groups, Inc. warrants that this product conforms to the chemical description on the label and is reasonably fit for the purposes stated in the Directions for Use, subject to the inherent risks referred to above, when used in accordance with directions under normal use conditions. This warranty does not extend to the use of this product contrary to label instructions, or under abnormal conditions or under conditions not reasonably foreseeable to or beyond the control of Seller or Voluntary Purchasing Groups, Inc., and Buyer and User assume the risk of any such use. VOLUNTARy PURCHASING GROUPS, INC. MAKES NO WARRANTIES OF MERCHANT-ABILITy OR OF FITNESS FOR A PARTICULAR PURPOSE NOR ANy OTHER EXPRESS OR IMPLIED WARRANTy EXCEPT AS STATED ABOVE. In no event shall Voluntary Purchasing Groups, Inc. or Seller be liable for any incidental, consequential or special damages resulting from the use or handling of this product. THE EXCLUSIVE REMEDy OF THE USER AND BUyER AND THE EXCLUSIVE LIABILITy OF VOLUNTARy PURCHASING GROUPS, INC., AND SELLER FOR ANy AND ALL CLAIMS, LOSSES, INJURIES OR DAMAGES (INCLUDING CLAIMS BASED ON BREACH OF WARRANTy, CONTRACT, NEGLIGENCE, TORT, STRICT LIABILITy OR OTHERWISE RESULTING FROM THE USE OR HANDLING OF THIS PRODUCT SHALL BE, AT THE ELECTION OF VOLUNTARy PURCHAS-ING GROUPS, INC. OR SELLER THE REPLACEMENT OF THE PRODUCT OR COMPENSATION LIMITED TO DAMAGES NOT EXCEEDING THE FAIR MARKET PURCHASE PRICE, AND SHALL NOT INCLUDE INCIDENTIAL OR CONSEQUENTAL DAMAGES. Voluntary Purchasing Groups, Inc. and Seller offer this product and Buyer and User accept it, subject to the foregoing conditions of sale and limitations of warranty and of liability, which may not be modified except by written agreement signed by the duly authorized representative of Voluntary Purchasing Groups, Inc.。
高三宇宙奥秘英语阅读理解30题1<背景文章>Black holes are one of the most fascinating and mysterious phenomena in the universe. A black hole is formed when a massive star collapses at the end of its life. The gravitational pull of a black hole is so strong that nothing, not even light, can escape from it.The formation of a black hole begins with the collapse of a massive star. As the star runs out of fuel, it can no longer support its own weight and begins to collapse. The collapse continues until the star reaches a critical density, at which point it becomes a black hole.Black holes have several unique characteristics. One of the most notable is their event horizon, which is the boundary beyond which nothing can escape. Another characteristic is their intense gravitational field, which can distort the space and time around them.Black holes can have a significant impact on the surrounding celestial bodies. They can attract and swallow nearby stars and planets, and their gravitational pull can also affect the orbits of other celestial bodies.Scientists are still working to understand black holes better. They use a variety of tools and techniques, such as telescopes and computer simulations, to study these mysterious objects. Despite significant progressin recent years, there is still much that we don't know about black holes.1. What is a black hole formed by?A. A small star collapsing.B. A massive star collapsing.C. A planet collapsing.D. A moon collapsing.答案：B。
二叠纪-三叠纪灭绝事件二叠纪-三叠纪灭绝事件（Permian–Triassic extinction event）是一个大规模物种灭绝事件，发生于古生代二叠纪与中生代三叠纪之间，距今大约2亿5140万年[1][2]。
若以消失的物种来计算，当时地球上70%的陆生脊椎动物，以及高达96%的海中生物消失[3]；这次灭绝事件也造成昆虫的唯一一次大量灭绝。
计有57%的科与83%的属消失[4][5]。
在灭绝事件之后，陆地与海洋的生态圈花了数百万年才完全恢复，比其他大型灭绝事件的恢复时间更长久[3]。
此次灭绝事件是地质年代的五次大型灭绝事件中，规模最庞大的一次，因此又非正式称为大灭绝（Great Dying）[6]，或是大规模灭绝之母（Mother of all mass extinctions）[7]。
二叠纪-三叠纪灭绝事件的过程与成因仍在争议中[8]。
根据不同的研究，这次灭绝事件可分为一[1]到三[9]个阶段。
第一个小型高峰可能因为环境的逐渐改变，原因可能是海平面改变、海洋缺氧、盘古大陆形成引起的干旱气候；而后来的高峰则是迅速、剧烈的，原因可能是撞击事件、火山爆发[10]、或是海平面骤变，引起甲烷水合物的大量释放[11]。
目录? 1 年代测定? 2 灭绝模式o 2.1 海中生物o 2.2 陆地无脊椎动物o 2.3 陆地植物? 2.3.1 植物生态系统? 2.3.2 煤层缺口o 2.4 陆地脊椎动物o 2.5 灭绝模式的可能解释? 3 生态系统的复原o 3.1 海洋生态系统的改变o 3.2 陆地脊椎动物? 4 灭绝原因o 4.1 撞击事件o 4.2 火山爆发o 4.3 甲烷水合物的气化o 4.4 海平面改变o 4.5 海洋缺氧o 4.6 硫化氢o 4.7 盘古大陆的形成o 4.8 多重原因? 5 注释? 6 延伸阅读? 7 外部链接年代测定在西元二十世纪之前，二叠纪与三叠纪交界的地层很少被发现，因此科学家们很难准确地估算灭绝事件的年代与经历时间，以及影响的地理范围[12]。
收稿日期：2013-12-24基金项目：国家自然科学基金项目(30925030；81172885)*通信作者：E-mail ：nsxia@夏宁邵，男，1964年7月生，教授，厦门大学国家传染病诊断试剂与疫苗工程技术研究中心主任，公共卫生学院院长，全国体外诊断产业技术创新战略联盟理事长。
长期致力于病毒性疾病的应用基础及转化研究。
主持研制出全球第一个商品化重组戊型肝炎疫苗及30余种传染病诊断试剂盒并均实现产业化，研制的重组人乳头瘤病毒16/18型二价疫苗正在进行三期临床试验。
所主持项目获国家技术发明二等奖、国家科技进步二等奖、中国专利金奖各1项，获授权专利22项，在Lancet 、PNAS 等SCI 刊物发表论文90余篇。
大肠杆菌表达的病毒样颗粒疫苗李少伟，夏宁邵*(厦门大学国家传染病诊断试剂与疫苗工程技术研究中心，厦门 361102)摘要：重组病毒样颗粒是病毒衣壳蛋白外源表达的重要形式，形态结构与天然病毒高度相似，位于纳米尺度的大小易于被免疫系统识别，可激发机体产生保护性免疫反应，且不含有病毒基因，因此，是一种理想的疫苗形式，也是基于结构进行疫苗设计的重要结构载体。
目前已上市的乙型肝炎疫苗、人乳头瘤病毒疫苗和戊型肝炎疫苗等基因工程疫苗均采用病毒样颗粒形式。
大肠杆菌表达系统被广泛用于基因工程药物的生产，具有安全性好、生产周期短、易于放大生产等优点，在病毒样颗粒疫苗应用上具有良好前景。
本文综述了利用大肠杆菌研制戊型肝炎疫苗和人乳头瘤病毒疫苗的进展，特别是这些病毒样颗粒疫苗的表达及组装、表位结构特征和临床试验结果。
关键词：疫苗；病毒样颗粒；大肠杆菌；戊型肝炎病毒；人乳头瘤病毒；重组蛋白Virus-like particle-based vaccine development usingEscherichia coli expression systemLI Shaowei, XIA Ningshao *(National Institute of Diagnostics and Vaccine Development in infectious disease, Xiamen University, Xiamen 361102, China )Abstract: Virus-like particles (VLPs) are generated by the self-assembly of viral structural protein usingvarious expression systems. VLPs ensemble native virus particles in morphology and maintain key immune epitopes as authentic virus. In light of nanometer-sized particles with diameters of 20- 60 nm, VLPs were shown to be a passport to immune recognition, thus being capable of eliciting strong protective immune responses. Recombinant VLP-based vaccines have superior safety profiles due to lack of any viral genome. There are several licensed and highly successful VLP-based vaccines produced using recombinant DNA《生命的化学》2014年34卷1期· 14 ·专题：新兴疫苗technology, such as recombinant hepatitis B, human papillomavirus and hepatitis E vaccine. E.coli expression system was well-established in production of biotherapeutics. This production platform for recombinant VLP-based vaccines has many advantages, such as rapid replication cycle and amenability for scale-up for commercial scale production. This review outlines the success of hepatitis E and human papillomavirus vaccines derived from E.coli. We highlight the protein expression, particle assembly, key epitope structure and clinical trials of these VLP-based vaccines.Key words: prophylactic vaccine; virus-like particles; Escherichia coli; hepatitis E virus; human papillomavirus; recombinant protein疫苗是预防病毒性疾病最为成功的干预手段。
Giant magnetoresistanceFrom Wikipedia, the free encyclopediaGiant magnetoresistance (GMR) is a quantummechanical magnetoresistance effect observed in thinfilm structures composed of alternating ferromagneticand nonmagnetic layers.The effect manifests itself as a significant decrease(typically 10-80%) in electrical resistance in thepresence of a magnetic field. In the absence of anexternal magnetic field, the direction of magnetizationof adjacent ferromagnetic layers is antiparallel due to aweak anti-ferromagnetic coupling between layers. Theresult is high-resistance magnetic scattering as a resultof electron spin.When an external magnetic field is applied, themagnetization of the adjacent ferromagnetic layers isparallel. The result is lower magnetic scattering, andlower resistance.[1] The effect is exploited commercially by manufacturers of hard disk drives. The 2007 Nobel Prize in physics was awarded to Albert Fert and Peter Grünberg for the discovery of GMR.DiscoveryGMR was discovered in 1988 in Fe/Cr/Fe trilayers by a research team led by Peter Grünberg of the Jülich Research Centre (DE), who owns the patent. It was also simultaneously but independently discovered in Fe/Cr multilayers by the group of Albert Fert of the University of Paris-Sud (FR). The Fert group first saw the large effect in multilayers that led to its naming, and first correctly explained the underlying physics. The discovery of GMR is considered the birth of spintronics. Grünberg and Fert have received a number of prestigious prizes and awards for their discovery and contributions to the field of spintronics including the 2007 Nobel Prize in Physics.Founding results of Fert et al.Contents1 Discovery2 Types of GMR2.1 Multilayer GMR2.2 Spin valve GMR2.3 Pseudo Spin Valve2.4 Granular GMR3 Applications4 References5 See also6 External linksTypes of GMRMultilayer GMRIn multilayer GMR two or more ferromagnetic layers are separated by a very thin (about 1 nm) non-ferromagnetic spacer (e.g. Fe/Cr/Fe). At certain thicknesses the RKKY coupling between adjacent ferromagnetic layers becomes antiferromagnetic, making it energetically preferable for themagnetizations of adjacent layers to align in anti-parallel. The electrical resistance of the device is normally higher in the anti-parallel case and the difference can reach more than 10% at room temperature. The interlayer spacing in these devices typically corresponds to the secondantiferromagnetic peak in the AFM-FM oscillation in the RKKY coupling.The GMR effect was first observed in the multilayer configuration, with much early research into GMR focusing on multilayer stacks of 10 or more layers.Spin valve GMRIn spin valve GMR two ferromagnetic layers are separatedby a thin non-ferromagnetic spacer (~3 nm), but withoutRKKY coupling. If the coercive fields of the twoferromagnetic electrodes are different it is possible toswitch them independently. Therefore, parallel and anti-parallel alignment can be achieved, and normally theresistance is again higher in the anti-parallel case. Thisdevice is sometimes also called a spin valve.Research to improve spin valves is intensely focused onincreasing the MR ratio by practical methods such asincreasing the resistance between individual layersinterfacial resistance, or by inserting half metallic layersinto the spin valve stack. These work by increasing the distances over which an electron will retain its spin (thespin relaxation length), and by enhancing the polarizationeffect on electrons by the ferromagnetic layers and the interface. The magnetic properties ofnanostructures (and all properties) are dominated by surface and interface effects due to the high local ratio of atoms as compared to the bulk.At the National University of Singapore, Z.Y. Leong and collaborators experimented with the interfacial resistance principle to show the magnetoresistance is suppressed to zero in NiFe/Cu/NiFe spin-valve at high amounts of interfacial resistance.The crucial trick for maximizing GMR ratio is to find the optimal resistance and polarization of the interface between layers to yield high performance from the spin valve.Spacer materials include Cu (copper), and ferromagnetic layers use NiFe (permalloy), which are both widely studied and meet industrial requirements.Current perpendicular to plane (CPP) Spin valve GMR is the configuration that currently yields the highest GMR and thus is the configuration used in hard drives. Research is ongoing in the older current-in-plane configuration, and in the tunneling magnetoresistance (TMR) spin valves which enable diskSpin-valve GMRdrive densities exceeding 1 Terabyte per square inch.Pseudo Spin ValvePseudo Spin Valve devices are very similar to the spin valve structures. The significant difference is the coercivities of the ferromagnetic layers. In a pseudo spin valve structure a soft magnet will be used for one layer; where as a hard ferromagnet will be used for the other. This allows the applied field to flip the magnetization of one layers before the other, thus providing the same anti-ferromagnetic affect that is required for GMR devices. For Pseudo Spin valve devices to work they generally require the thickness of the non-magnetic layer to be thick enough so that exchange coupling is kept to a minimum. It is imperative to prevent the interaction between the two ferromagnetic layers in order to exercise complete control over the device.Granular GMRGranular GMR is an effect that occurs in solid precipitates of a magnetic material in a non-magnetic matrix. To date, granular GMR has only been observed in matrices of copper containing cobalt granules. The reason for this is that copper and cobalt are immiscible, and so it is possible to create the solid precipitate by rapidly cooling a molten mixture of copper and cobalt. Granule sizes vary depending on the cooling rate and amount of subsequent annealing. Granular GMR materials have not been able to produce the high GMR ratios found in the multilayer counterparts.ApplicationsGMR has been used extensively in the read heads in modern hard drives and magnetic sensors. Another application of the GMR effect is in magnetoresistive random access memory (MRAM), a type of non-volatile semiconductor memory. GMR has triggered the rise of a new field of electronics called spintronics.References1.^ Stoner-Leeds (/research/gmr.htm)L. L. Hinchey and D. L. Mills (1986). "Magnetic properties of superlattices formed from ferromagnetic and antiferromagnetic materials (/abstract/PRB/v33/p3329)". Physical Review B33(5): 3329–3343. doi:10.1103/PhysRevB.33.3329 (/10.1103/PhysRevB.33.3329)./abstract/PRB/v33/p3329.P. Grünberg, R. Schreiber, Y. Pang, M. B. Brodsky, and H. Sowers (1986). "Layered Magnetic Structures: Evidence for Antiferromagnetic Coupling of Fe Layers across Cr Interlayers(/abstract/PRL/v57/p2442)". Physical Review Letters57 (19): 2442–2445.doi:10.1103/PhysRevLett.57.2442 (/10.1103/PhysRevLett.57.2442)./abstract/PRL/v57/p2442.C. Carbone and S. F. Alvarado (1987). "Antiparallel coupling between Fe layers separated by a Crinterlayer: Dependence of the magnetization on the film thickness(/abstract/PRB/v36/p2433)". Physical Review B36 (4): 2433.doi:10.1103/PhysRevB.36.2433 (/10.1103/PhysRevB.36.2433)./abstract/PRB/v36/p2433.M. N. Baibich , J. M. Broto, A. Fert, F. Nguyen Van Dau, F. Petroff, P. Eitenne, G. Creuzet, A.Friederich, and J. Chazelas (1988). "Giant Magnetoresistance of (001)Fe/(001)Cr Magnetic Superlattices (/abstract/PRL/v61/p2472)". Physical Review Letters61 (21): 2472–2475.doi:10.1103/PhysRevLett.61.2472 (/10.1103/PhysRevLett.61.2472)./abstract/PRL/v61/p2472.G. Binasch, P. Grünberg, F. Saurenbach, and W. Zinn (1989). "Enhanced magnetoresistance in layeredmagnetic structures with antiferromagnetic interlayer exchange(/abstract/PRB/v39/p4828)". Physical Review B39 (7): 4828–4830.doi:10.1103/PhysRevB.39.4828 (/10.1103/PhysRevB.39.4828)./abstract/PRB/v39/p4828.A. E. Berkowitz, J. R. Mitchell, M. J. Carey, A. P. Young, S. Zhang, F. E. Spada, F. T. Parker, A. Hutten,Hutten, and G. Thomas (1992). "Giant magnetoresistance in heterogeneous Cu-Co alloys(/abstract/PRL/v68/p3745)". Physical Review Letters68 (25): 3745–3748.doi:10.1103/PhysRevLett.68.3745 (/10.1103/PhysRevLett.68.3745)./abstract/PRL/v68/p3745.John Q. Xiao, J. Samuel Jiang, and C. L. Chien (1992). "Giant magnetoresistance in nonmultilayer magnetic systems (/abstract/PRL/v68/p3749)". Physical Review Letters68 (25): 3749–3752. doi:10.1103/PhysRevLett.68.3749 (/10.1103/PhysRevLett.68.3749)./abstract/PRL/v68/p3749.Z.Y. Leong, S.G. Tan, M.B.A. Jalil, S. Bala Kumar, and G.C. Han (Journal of Magnetism and Magnetic Materials). "Magnetoresistance modulation due to interfacial conductance of current perpendicular-to-plane spin valves". Journal of Magnetism and Magnetic Materials310 (2): e635–637.doi:10.1016/j.jmmm.2006.10.679 (/10.1016/j.jmmm.2006.10.679).See alsoMagnetoresistanceColossal magnetoresistanceTunnel magnetoresistanceExternal linksGiant Magnetoresistance: The Really Big Idea Behind a Very Tiny Tool(/education/tutorials/magnetacademy/gmr/) National High Magnetic Field LaboratoryPresentation of GMR-technique (IBM Research)(/research/gmr.html)Nobel prize in physics 2007(/nobel_prizes/physics/laureates/2007/index.html) - Nobel Foundation (also Scientific background (/nobel_prizes/physics/laureates/2007/phyadv07.pdf) PDF (472 KB)Retrieved from "/wiki/Giant_magnetoresistance"Categories: Condensed matter physics | Electric and magnetic fields in matter | Quantum electronics | Spintronics | Computer storage technologiesHidden categories: Cleanup from November 2007 | All pages needing cleanupThis page was last modified on 13 May 2009 at 15:43.Text is available under the Creative Commons Attribution/Share-Alike License; additional terms may apply. See Terms of Use for details.Wikipedia® is a registered trademark of the Wikimedia Foundation, Inc., a non-profitorganization.。
小学上册英语第6单元综合卷英语试题一、综合题(本题有100小题，每小题1分，共100分.每小题不选、错误，均不给分)1.In school, we have a library where we can borrow ______ (书籍). I love discovering new stories.2.What is the main ingredient in jelly?A. SugarB. GelatinC. FruitD. WaterC3.The fish in the aquarium are very _______ (水族馆里的鱼非常_______).4.The primary structure of proteins is made up of ______.5.What is 15 + 15?A. 25B. 30C. 35D. 40B6.The parrot can ______ (说话) and mimic sounds.7.I have two _____ at home. (cats)8.What is 6 x 7?A. 42B. 36C. 48D. 549.The Earth's surface is constantly changing due to erosion and ______.10.What is the main purpose of a compass?A. To tell timeB. To find directionC. To measure distanceD. To calculate speedB11.My _________ (玩具箱) is filled with memories from my childhood.12.I found a ________ under the leaf.13.The frog jumps from ________ to ________.14.The __________ (历史的循环) shows patterns in human behavior.15.The _____ (狮子) is a powerful predator.16.In a combustion reaction, the products are usually carbon dioxide and _____.17., the wind can be really __________. (强劲的) Sparrows18.What do you call a person who helps sick people?A. TeacherB. DoctorC. EngineerD. ChefB19.My uncle is a ____ (doctor) who helps sick people.20.How many eyes does a typical human have?A. OneB. TwoC. ThreeD. FourB21.What do you call a large, slow-moving animal with a shell?A. TurtleB. TortoiseC. SnailD. ArmadilloB22.What do you call the time when the sun is highest in the sky?A. MorningB. NoonC. EveningD. Night23.The flowers are _____ in the sunshine. (smiling)24.Many _______ are found in tropical regions.25.The first person to summit Mount Everest was _______ Hillary.26. A tapir has a short ______ (鼻子).27.The _______ (小刺猬) has sharp spines for protection.28.The _____ is a phenomenon where the moon blocks the sun.29. A __________ is a mixture where one substance is dissolved in another.30.The flowers are _______ (opening) in the spring.31.They are watching a _____ (movie/show) together.32.My dog likes to bark at passing ______ (汽车).33.Certain plants can ______ (帮助) mitigate climate change.34.Every summer, I go to _______ (地方) with my family. We create many _______ (记忆).35.The Earth's crust is rich in ______ elements.36.What do we wear on our heads?A. ShoesB. HatC. GlovesD. ScarfB37.My aunt loves to volunteer at the ____ (animal shelter).38.The _____ (大象) uses its trunk to drink water.39.The ________ was a significant turning point in the fight for freedom.40.My mom is my caring _______ who loves me very much.41.What do we call the act of giving someone a gift?A. PresentingB. BestowingC. OfferingD. All of the AboveD42.The Pacific Ocean is to the ________ of the Americas.43.The land beneath our feet is called the ______.44.The capital of Singapore is _______.45.What do you call the process of removing trees from a forest?A. AfforestationB. DeforestationC. ReforestationD. UrbanizationB46.Did you see a _______ (小野猪) in the woods?47.I have a ______ (玩具枪) that makes sounds. I pretend to be a ______ (超级英雄).48.__________ are used in skincare products for their moisturizing properties.49.What do you call a story told by a narrator?A. PlayB. DramaC. NarrativeD. Poem50. A _______ (小刺猬) comes out at night to forage.51.The ______ can live a long time.52.What do we call a person who designs clothes?A. TailorB. Fashion designerC. ModelD. StylistB53. A _______ change is when the appearance changes, but the substance remains the same. (物理)54. A ________ (植物遗传多样性) is crucial for resilience.55.I enjoy listening to audiobooks while __________.56.What is the opposite of heavy?A. LightB. DenseC. SolidD. ThickA57.My favorite type of ________ (饮料) is soda.58.The ancient Greeks established the concept of ________ (市民权).59.I find ________ (天文学) very interesting.60.My __________ (玩具名) is very __________ (形容词) and colorful.61.I can ______ (认真) consider different perspectives.62.I have a _____ (question/answer) for you.63.Which planet is known for its storm called the Great Red Spot?A. SaturnB. JupiterC. MarsD. NeptuneB64.The law of conservation of mass states that mass cannot be _______ or destroyed.65. A reaction that absorbs heat is called an ______ reaction.66. A ______ is a structured approach to inquiry.67.What is the name of the famous Italian dish made with dough and toppings?A. PastaB. PizzaC. RisottoD. LasagnaB Pizza68.My dad encourages me to be __________ (有创造力的).69.The _____ (小猴子) swings from tree to tree.70.Gravity pulls objects ______ to the ground.71.I want to _______ (学习) about animals.72.The capital of Argentina is _______.73.My favorite hobby is ______ (摄影). I love capturing beautiful ______ (瞬间).74.What is the main purpose of a compass?A. Measure temperatureB. Show directionC. Tell timeD. Measure distanceB75.The _____ (teacher/student) is reading.76.The dog is ___ (friendly/scary).77.The ancient city of ________ was known for its advanced architecture.78.We have a ______ (丰富的) schedule for school events.79.Plants need ______ (水) to survive and grow.80.n be very _________. (长) Snakes d81.What do we call a person who studies mathematics?A. MathematicianB. StatisticianC. EngineerD. All of the above82.How many continents are there?A. FiveB. SixC. SevenD. Eight83. A _______ can be a great project for kids.84.The __________ (文化传统) are passed down through generations.85.My brother likes to watch ____ (cartoons) on weekends.86.What do we call the study of animals?A. BotanyB. ZoologyC. GeologyD. EcologyB87.Carbon atoms can form long _____ of molecules.88.The bumblebee helps pollinate ________________ (花).89.The chemical formula for calcium nitrate is _____.90.My ________ (玩具) is a great conversation starter.91.What do we call the action of putting something in water?A. SoakingB. DryingC. FloatingD. Sinking92.The ______ has a long tongue.93. A meerkat is social and lives in ______ (群体).94.I think kindness is essential in our daily lives. Simple acts of kindness can brighten someone’s day. I try to spread kindness by __________ whenever I can.95.I enjoy going to the ______ (森林) for hikes and picnics. Nature is a wonderful escape.96.What is your favorite __________?97.The coach, ______ (教练), motivates us to improve.98.The chef, ______ (厨师), creates delicious desserts.99.In math, we learn how to ________ (加法) and ________ (减法). It’s really________ (有趣).100. A _______ is a reaction that occurs in the presence of water.。
托福阅读TPO33(试题+答案+翻译)第3篇:灭绝事件托福阅读TPO33阅读原文E某tinction Episodes of the Past【1】It was not until the Cambrian period, beginning about 600 million years ago, that a great proliferation of macroscopic species occurred on Earth and produced a fossil record that allows us to track the rise and fall of biodiversity. Since the Cambrian period, biodiversity has generally risen, but there have been some notable e 某ceptions. Biodiversity collapsed dramatically during at least five periods because of mass e某tinctions around the globe. The five major mass e某tinctions receive most of the attention, but they are only one end of a spectrum of e某tinction events. Collectively, more species went e某tinct during smaller events that were less dramatic but more frequent. The best known of the five major e某tinction events, the one that saw the demise of the dinosaurs, is the Cretaceous-Tertiary e某tinction.【2】Starting about 280 million years ago, reptiles were the dominant large animals in terrestrial environments. In popular language this was the era “when dinosaurs ruled Earth,” when a wide variety of reptile species occupying many ecological niches. However, no group or species can maintain its dominance indefinitely, and when, after over 200 million years, the age of dinosaurs came to a dramatic end about 65 million years ago, mammals began to flourish, evolving from relatively few types of small terrestrial animals into the myriad of diverse species, including bats and whales, that we know today. Paleontologists label this point in Earth’s history as the end of the Cretaceous period and the beginning of the Tertiary period, often abbreviated as the K-T boundary. This time was alsomarked by changes in many other types of organisms. Overall, about 38 percent of the families of marine animals were lost, with percentages much higher in some groups Ammonoid mollusks went from being very diverse and abundant to being e某tinct. An e某tremely abundant set of planktonic marine animals called foraminifera largely disappeared, although they rebounded later. Among plants, the K-T boundary saw a sharp but brief rise in the abundance of primitive vascular plants such as ferns, club mosses, horsetails, and conifers and other gymnosperms. The number of flowering plants (angiosperms) was reduced at this time, but they then began to increase dramatically.【3】What caused these changes? For many years scientists assumed that a cooling of the climate was responsible, with dinosaurs being particularly vulnerable because, like modern reptiles, they were ectothermic (dependent on environmental heat, or cold-blooded). It is now widely believed that at least some species of dinosaurs had a metabolic rate high enough for them to be endotherms (animals that maintain a relatively consistent body temperature by generating heat internally). Nevertheless, climatic e某planations for the K-T e某tinction are not really challenged by the ideas that dinosaurs may have been endothermic, because even endotherms can be affected by a significant change in the climate.【4】E某planations for the K-T e某tinction were revolutionized in 1980 when a group of physical scientists led by Luis Alvarez proposed that 65 million years ago Earth was stuck by a 10-kilometer-wide meteorite traveling at 90,000 kilometers per hour. They believed that this impact generated a thick cloud of dust that enveloped Earth, shutting out much of the incoming solar radiation and reducing plant photosynthesis to very low levels. Short-term effects mighthave included huge tidal waves and e某tensive fires. In other words, a series of events arising from a single cataclysmic event caused the massive e某tinctions. Initially, the meteorite theory was based on a single line of evidence. At locations around the globe, geologists had found an unusually high concentration of iridium in the layer of sedimentary rocks that was formed about 65 million years ago. Iridium is an element that is usually uncommon near Earth’s surface, but it is abundant in some meteorites. Therefore, Alvarez and his colleagues concludedthat it was likely that the iridium in sedimentary rocks deposited at the K-T boundary had originated in a giant meteorite or asteroid. Most scientist came to accept the meteorite theory after evidence came to light that a circular formation, 180 kilometers in diameter in diameter and centered on the north coast of the Yucatan Peninsula, was created by a meteorite impact about 65 million years ago.托福阅读TPO33阅读试题1.The word "proliferation" in the passage (paragraph 1) is closest in meaning toA.decline.B.e某tinction.C.increase.D.migration.2.Paragraph 1 supports which of the following statements about life on Earth before the Cambrian period?A.Biodiversity levels were steady, as indicated by the fossil record.B.Levels of biodiversity could not be tracked.C.The most dramatic e某tinction episode occurred.D.Few microscopic species e某isted.3.Which of the sentences below best e某presses the essential information in the highlighted sentence in the passage? Incorrect choices change the meaning in important ways or leave out essential information.A.The dominance of dinosaurs came to an end 65 million years ago, at which time mammals began to flourish and diversify.B.Because no group of species can remain dominant forever, mammals became the dominant group when dinosaurs became e某tinct.C.After being the dominant group for more than 200 million years, the age of dinosaurs came to a dramatic end 65 million years ago.D.The diverse group of mammals that we know today, including bats and whales, evolved from small terrestrial forms that had been dominated by dinosaurs.4.According to paragraph 2, why are dinosaurs popularly said to have "ruled Earth" during the Cretaceous period?A.Dinosaurs were the only species of reptile that e某isted during the whole of the Cretaceous period.B.Dinosaurs won the battle for food resources over mammals during the Cretaceous period.C.Dinosaurs survived e某tinction during the Cretaceous period, whereas many other animal species did not.D.Dinosaurs were the physically and ecologically dominant animals during the Cretaceous period.5.According to paragraph 2, which of the following speciesinitially increased in number at the K-T boundary?A.inifera.C.onoid mollusks.6.Why does the author note that "even endotherms can be affected by a significant change in the climate"(paragraph 3)?A.To argue that there was a significant climate at the time that endothermic dinosaurs became e某tinct.B.To argue that climate change caused some dinosaurs to evolve as endotherms.C.To support the view that at least some of the dinosaurs that became e某tinct were endotherms.D.To defend climate change as possible e某planation for the e某tinction of dinosaurs.7.The word "generated"(paragraph 4) in the passage is closest in meaning toA.collected.B.produced.C.spread.D.added.8.The word "e某tensive"(paragraph 4) in the passage is closestin meaning toA.widespread.B.erous.D.subsequent.9.According to paragraph 4, all of the following contributed to the massive e某tinctions of the K-T period E某CEPT:A.tidal waves.B.fires.C.insufficient solar .10.According to paragraph 4, which of the following statements e 某plains the importance of the discovery of high levels of iridiumrocks?A.It provided evidence that overe某posure to solar radiation led to the K-T e某tinction.B.It showed that more than one cataclysmic event was responsible for the K-T e某tinction.C.It suggested that the cause of the K-T e某tinction may have been a meteorite striking Earth.D.It provided evidence that the K-T e某tinction occurred 65 million years ago.11.According to paragraph 4, which of the following is true about the Yucatan Peninsula?A.The circular formation there was caused by a meteorite impact65 million years entary rocks from that area have the lowest iridium concentration of any rocks on Earth.C.There is evidence that a huge tidal wave occurred there 65 million years ago.D.Evidence found there challenged the meteorite impact theory.12.Which of the following can be inferred from paragraph 4 about the meteorite theory?A.The data originally presented as evidence for the theory were eventually rejected.B.Many scientists did not accept it when it was first proposed.C.It has not been widely accepted as an e某planation for the K-T e某tinction.D.Alvarez subsequently revised it after a circular formation was found in the Yucatan Peninsula.13. Look at the four squares [■] that indicate where the following sentence could be added to the passage. Where would thesentence best fit? This focused on the chemical composition of ancient rocks.E某planations for the K-T e某tinction were revolutionized in 1980 when a group of physical scientists led by Luis Alvarez proposed that 65 million years ago Earth was stuck by a 10-kilometer-wide meteorite traveling at 90,000 kilometers per hour. They believed that this impact generated a thick cloud of dust that enveloped Earth, shutting out much of the incoming solar radiation and reducing plant photosynthesis to very low levels. Short-term effects might have included huge tidal waves and e某tensive fires. In other words, a series of events arising from a single cataclysmic event caused the massive e某tinctions. ■【A】Initially, the meteorite theory was based on a single line of evidence. ■【B】At locations around the globe, geologists had found an unusually high concentration ofiridium in the layer of sedimentary rocks that was formed about 65 million years ago. ■【C】Iridium is an element that is usually uncommon near Earth's surface, but it is abundant in some meteorites. ■【D】Therefore, Alvarez and his colleagues concluded that it was likely that the iridium in sedimentary rocks deposited at the K-T boundary had originated in a giant meteorite or asteroid. Most scientist came to accept the meteorite theory after evidence came to light that a circular formation, 180 kilometers in diameter in diameter and centered on the north coast of the Yucatan Peninsula, was created by a meteorite impact about 65 million years ago.14. Prose SummaryThe K-T e某tinction 65 million years ago is the best known of the five major e某tinction episodes since the Cambrian period.A.Collectively, the five major e某tinction episodes resulted inthe elimination of a larger number of species than did all the minor e某tinction events.B.The K-T e某tinction eliminated the dinosaurs and ammonoid mollusks but was followed by the diversification of mammals and gymnospermous plants.C.An e某treme cooling of the climate could not have caused theK-T e某tinction of dinosaurs, because, while most dinosaurs depended on environmental heat, some did not.D.The K-T e某tinction of the dinosaurs is the only mass e某tinction that has been e某plained by the impact of a meteorite.E.In 1980 Luis Alvarez proposed that the K-T e某tinction was caused by ecological disasters brought about by the impact of a meteorite striking Earth.F.A high concentration of iridium in sedimentary rocks at the K-T boundary and a large impact crater in the Yucatan Peninsula from 65 million years ago strongly support Alvarez' hypothesis.托福阅读TPO33阅读答案1.proliferation，繁殖，激增，对应C。
中国粉体技术CHINA POWDER SCIENCE AND TECHNOLOGY 第27卷第2期2021年3月Vol. 27 No. 2Mar. 2021文章编号:1008-5548 (2021) 02-0017-05 doi ：10.13732/j.issn.l008-5548.2021.02.003碳气凝胶超细粉体的可控制备技术于照亮，彭文联，刘清海，张彤，代晓东(军事科学院防化研究院，北京102205)摘要：基于球磨技术建立碳气凝胶超细粉体制备方法，系统地研究制备工艺中球磨时间、球料比、助磨剂等制备条件对 制备产物的影响，揭示制备参数对碳气凝胶粉体特性的影响规律。
结果表明：延长球磨时间有利于降低大粒子在产物中 比例，但大于4 h 的球磨时间对小粒子影响不大，存在“粉碎极限”效应；随着球料比的增加，中位径2(50)先降低再升 高,球料质量比为50：1时，得到的2(50)最小值为2.58叩；添加助磨剂会增大产物的粒径,且不同的助磨剂增幅不同。
关键词：碳气凝胶；超细粉体；球磨工艺；可控制备中图分类号:TB383.3 文献标志码:AControllable preparation techniche of carbon aerogel ultrafine powderYU Zhaoliang , PENG Wenlian , LIU Qinghai , ZHANG Tong , DAI Xiaodong(Chemical Defense Institute , Academy of Military Science , Beijing 102205 , China)Abstract : Carbon aerogel ultrafine powders were prepared by ball-milling method. The effects of preparation parameters such as ball-milling time, quantity ratio of ball-sample and addition of grinding aid, on the properties of products were systematically investigated. The results show that the proportion of large particles in particle size distribution of products decrease with increas ing in ball-milling time, but the ball grinding time above 4 h has little influence on small particles , and that Z)v (50) in particle size distribution of products first decrease and then increase with increasing in mass ratio of ball-sample , the lowest Z)v (50) value is 2. 58 jxm when the mass ratio is 50-1, and that the particle size of products increase with the addition of the solutions of grinding aids and the influence of different grinding aids is different.Keywords : carbon aerogels ； ultrafine powder ； ball-milling technology ； controllable preparation碳气凝胶是三维空间网状结构中填充气体介质的碳基凝胶材料,具有轻质、孔隙率大、可设计性强、 比表面积大、导热系数小等优点⑴，在许多领域应用价值较高，如储能材料：2-4\吸附材料⑸、隔热材 料⑹、防护材料⑺、光电干扰材料Z9］等。
J OURNAL OF V IROLOGY,Dec.2006,p.11678–11685Vol.80,No.23 0022-538X/06/$08.00ϩ0doi:10.1128/JVI.00940-06Copyright©2006,American Society for Microbiology.All Rights Reserved.Mimivirus Giant Particles Incorporate a Large Fraction of Anonymousand Unique Gene Productsᰔ†Patricia Renesto,1*Chantal Abergel,2Philippe Decloquement,1Danielle Moinier,3Saı¨d Azza,1 Hiroyuki Ogata,2Patrick Fourquet,4Jean-Pierre Gorvel,4and Jean-Michel Claverie2Unite´des Rickettsies,CNRS UMR6020,IFR-48,Faculte´de Me´decine,27Boulevard Jean Moulin,1Information Ge´nomique& Structurale,CNRS UPR2589,IBSM,Parc Scientifique et Technologique de Luminy,163Avenue de Luminy,Case934,2Institut de Biologie Structurale et Microbiologie,31Chemin Joseph Aiguier,3and Centre d’Immunologie INSERM-CNRS-Universite´de la Me´diterrane´e de Marseille-Luminy,Parc Scientifique et Technologique de Luminy,413288Marseille,FranceReceived9May2006/Accepted5September2006Acanthamoeba polyphaga mimivirus is the largest known virus in both particle size and genome complexity.Its1.2-Mb genome encodes911proteins,among which only298have predicted functions.The composition ofpurified isolated virions was analyzed by using a combined electrophoresis/mass spectrometry approachallowing the identification of114proteins.Besides the expected major structural components,the viral particlepackages12proteins unambiguously associated with transcriptional machinery,3proteins associated withDNA repair,and2topoisomerases.Other main functional categories represented in the virion includeoxidative pathways and protein modification.More than half of the identified virion-associated proteinscorrespond to anonymous genes of unknown function,including45“ORFans.”As demonstrated by bothWestern blotting and immunogold staining,some of these“ORFans,”which lack any convincing similarity inthe sequence databases,are endowed with antigenic properties.Thus,anonymous and unique genes consti-tuting the majority of the mimivirus gene complement encode bonafide proteins that are likely to participatein well-integrated processes.Acanthamoeba polyphaga mimivirus(mimivirus)is the larg-est virus isolated so far(23).Based on its highly specific char-acteristics,this double-stranded-DNA icosahedral virus(47)is thefirst member of the new Mimiviridae family(33,43).Com-putational annotation of its 1.2-Mb genome(33)revealed many atypical features,including the presence of key transla-tion enzymes,a full complement of DNA repair pathway com-ponents,and the unique presence of three different topoisom-erases(of types IA,IB,and II)(2,33).Another unique characteristic of mimivirus is the presence of nearly identical promoter sequence motifs upstream of half of its911protein-encoding genes(42),which are presumably associated with proteins expressed during the early or late-early phase.Only 23%of the predicted coding genes exhibit convincing homol-ogy to proteins of known function,and39%of them do not exhibit a clear(E values,Ͻ10Ϫ5)sequence database match (33).Such coding regions without sequence similarity to other genes in databases are considered orphan open reading frames (ORFs)and termed“ORFans”(12).The origin and function of ORFan genes are still a matter of controversy,with opinions ranging from considering them pieces of junk DNA(1,8,40, 44)to seeing them as quickly evolving sequences encoding normally expressed functional proteins(38,39).Recent clinical evidence raised the possibility that mimivirus might be a hu-man pathogen causing pneumonia(4,24,34),as suspected when it wasfirst isolated from a cooling tower following an outbreak of pneumonia(23).Mass spectrometry-based analysis has recently emerged as a technique of choice to identify more comprehensively the set of viral proteins associated with viral particles(19,29,49).We now present the application of this technique to the largest known,and presumably most complex,viral particle,Acanth-amoeba polyphaga mimivirus.MATERIALS AND METHODSSample preparation for bidimensional(2D)gel electrophoresis.Acanth-amoeba polyphaga mimivirus(23)was purified through a sucrose gradient(25%) and washed twice with phosphate-buffered saline(PBS)in the presence of protease inhibitors(Complete;Roche,Mannheim,Germany).The resulting pellet was solubilized in40mM Tris-HCl,pH7.5,supplemented with2%(wt/vol) sodium dodecyl sulfate(SDS;Sigma-Aldrich)and60mM dithiothreitol(DTT), followed by5min of heating at95°C.The insoluble fraction was removed by centrifugation(12,000ϫg,4°C,10min),and soluble proteins were precipitated using a PlusOne2-D cleanup kit(Amersham Biosciences)to remove SDS.The final pellet was resuspended in solubilization buffer{7M urea,2M thiourea,4% (wt/vol)3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate(CHAPS)} and stored atϪ80°C until isoelectric focusing(IEF)was performed.2D gel electrophoresis and silver staining.Immobiline DryStrips(18cm,pH 4to7or6to11;Amersham)were rehydrated overnight using350l rehydration buffer(8M urea,2%[wt/vol]CHAPS,60mM DTT,2%[vol/vol]Immobiline pH gradient(IPG)buffer[Amersham])containing200g of solubilized mimivirus proteins,and IEF was carried out according to the manufacturer’s protocol (Multiphor II;Amersham).Before the second-dimension electrophoresis was performed,strips were equilibrated twice in10ml equilibration buffer(30% [vol/vol]glycerol,2%[wt/vol]SDS,6M urea,50mM Tris-HCl,bromophenol blue,pH8.8)for15min.This buffer was supplemented with65mM DTT for the first equilibration and with100mM iodoacetamide for the second one.The strips were then embedded in0.5%agarose,and the proteins were resolved by10% SDS-polyacrylamide gel electrophoresis(SDS-PAGE)(Ettan DALT;Amer-sham)at5W/gel for30min,followed by4to5h at17W/gel.Following migration,gels were stained by a method compatible with mass spectrometry (36).Spots excised from the gel were stored atϪ20°C until identification.For*Corresponding author.Mailing address:Unite´des Rickettsies, CNRS UMR6020,IFR-48,Faculte´de Me´decine,27Boulevard Jean Moulin,13385Marseille,France.Phone:33491324630.Fax:33491 387772.E-mail:patricia.renesto@medecine.univ-mrs.fr.†Supplemental material for this article may be found at http://jvi/.ᰔPublished ahead of print on13September2006.11678 at Penn State Univ on February 12, 2008 Downloaded fromaccuracy,the spectra of at least two separate samples of each protein were analyzed and compared.In-gel digestion and MALDI-TOF mass spectrometry(MS).Spots excised from silver-stained gels were destained and subjected to in-gel digestion with trypsin(sequencing-grade modified porcine trypsin;Promega,Madison,WI) (36).Tryptic peptides were then extracted from the gel by successive treatments with5%formic acid and50%acetonitrile–5%formic acid.Extracts were pooled and dried in a Speedvac evaporator.Peptides resuspended in an␣-cyano-4-hydroxycinnamic acid matrix solution(prepared by diluting3times a saturated solution in50%acetonitrile–0.3%trifluoroacetic acid)were then spotted on the matrix-assisted laser desorption ionization(MALDI)target.Mass analyses were performed on a MALDI-time offlight(MALDI-TOF)Bru¨ker Ultraflex spec-trometer(Bru¨ker Daltonique,Wissembourg,France).Mass spectra were inter-nally calibrated using autolytic peptides from trypsin.Tryptic peptide mass lists were used to identify the proteins,using Mascot software.Searches were per-formed against all available sequences in public databases,including those for eukaryotes().Immunization and Western blotting.Acanthamoeba polyphaga mimivirus pro-teins resolved by2D gel electrophoresis were transferred onto nitrocellulose membranes(Semi-Phor unit;Hoefer Scientific,San Francisco,CA).Membranes were then blocked in PBS supplemented with0.2%Tween20and5%nonfat dry milk(PBS-Tween-milk)for1.5h before incubation with anti-Acanthamoeba polyphaga mimivirus sera.The sera were obtained from BALB/c mice immunized by three intraperitoneal injections(with15-day intervals between injections)of5g of purified viral particles resuspended in CpG as an adjuvant.After1h of incubation(1:6,400dilution in PBS-Tween-milk),membranes were washed three times with PBS-Tween and probed with horseradish peroxidase-conjugated goat anti-mouse secondary antibodies(1:1,000;Amersham).Detection was achieved by chemiluminescence(ECL;Amersham).SDS-PAGE coupled with electrospray ionization–ion-trap MS/MS analysis. An Acanthamoeba polyphaga mimivirus sample(175g)prepared as described above was separated by SDS-PAGE on an11%acrylamide gel(22),and the proteins were revealed afterwards by Coomassie blue R-250staining.Bands of 1-mm thickness were systematically cut from the gel,resulting in49pieces to be analyzed.These bands were reduced and alkylated before trypsin digestion,and the resulting peptides were analyzed through liquid chromatography(LC) (Finnigan Surveyor HPLC system;Thermo Electron,San Jose,CA)coupled to an electrospray ionization–ion-trap mass spectrometer(LCQ-Deca XP;Ther-mofinnigan).Tryptic peptides were resuspended in20l formic acid at5%in water,and1/20of the whole extract obtained from each piece of gel was desalted on a C18trapping column(Zorbax300SB-C18[5-m inner diameter,5by0.3 mm];Agilent)by a20-min washing step with0.1%formic acid.Peptide separa-tion was achieved by a60-min linear gradient of acetonitrile(0to65%)in0.2% formic acid on a C18reverse-phase column(PicoFrit column[5-m BioBasic C18, 300-Åpore size,75-m inner diameter,100-mm long,15-m tip]).The peptides were then ionized at a capillary temperature of160°C with a2-kV spray voltage.A collision energy of35%was applied for the MS/MS scan events.The MS/MS spectrum of the three most intense peaks was obtained after each full MS scan. The dynamic exclusion features were set at a repeat count of2within0.5min, with an exclusion duration of3min.Protein identification was performed using the TurboSequest algorithm(11)in the Bioworks3.2software package(Thermo Electron)and the Acanthamoeba polyphaga mimivirus database(33).TurboSequest automatically identifies pro-teins by matching a peptide’s experimental MS/MS spectrum to a predicted mass spectrum for amino acid sequences within databases.Identified peptides were evaluated using the cross-correlation number(X corr)versus the charge state. Criteria for positive identification of peptides were X corr values ofϾ1.8for singly charged ions,Ͼ2.5for doubly charged ions,andϾ3.5for triply charged ions. Only the best match was considered,with two or more unique peptides identi-fying a protein.Four proteins with masses below19kDa were assigned by a single peptide after visual inspection of the MS/MS spectrum.Protein glycosylation analysis.A purified pellet of Acanthamoeba polyphaga mimivirus was resuspended in-mercaptoethanol(Sigma-Aldrich)and incu-bated overnight at4°C.The insoluble fraction was removed by centrifugation (12,000ϫg,4°C,10min).Proteins present in the supernatant were separated by SDS-PAGE on a15%acrylamide gel(22)and visualized by Coomassie R-250 staining.Glycoproteins were revealed using a GlycoProfile III kit(Sigma-Aldrich),which allows specific and sensitive in-gelfluorescence detection of glycoproteins.Sequence annotation and ORF functional predictions.ORF sequences were annotated using homology searches against all available databases,as previously described(33).In addition,anonymous(homologous to proteins of unknown function)and orphan sequences were reanalyzed using the3D structure-based homology program FUGUE(37),generating more tentative structural and/or functional assignments(indicated within parentheses in Table1).Only assign-ments scored as“likely”or“certain”were reported.Immunogold labeling.Immunolabeling of ultrathin sections for electron mi-croscopy was performed as previously described(24).Briefly,ultrathin sections (70nm)were cut using an ultramicrotome(Ultracut E;Leica),collected on 300-mesh nickel grids without Formvar(Electron Microscopy Sciences),and then incubated in PBS supplemented with0.2%bovine serum albumin(Roche Diagnostics).The sections were then treated with0.05M lysine(Sigma-Aldrich) in order to neutralize putative residual aldehyde groups.After repeated washings with PBS,the sections were then incubated for3h at37°C under a humidified atmosphere with a monoclonal anti-Mimi_L725antibody(Mab6E2;1:100)(23) diluted in PBS supplemented with3%nonfat dry milk.The secondary reaction used goat anti-mouse antibodies conjugated to25-nm colloidal gold particles (Aurion EM Reagents,The Netherlands),followed by washings with PBS and H2O.Gold particles were visualized using an R-GENT SE-EM silver enhance-ment kit(Aurion)according to the instructions of the manufacturer.After washings and aldehydefixation,a treatment with5%uranyl acetate(ICN Bio-medicals)was done to produce intense electron-opaque staining,and samples were viewed with a Philips electron microscope(MORGAGNI268D)at80kV.A negative control experiment was performed with preimmune serum.RESULTSGlobal proteomic analysis of mimivirus particle by LC-MS/ MS.A critical parameter for every proteome project is the purity of the sample to be analyzed.This is particularly true when considering intracellular pathogens.For this study,the absence of major eukaryotic debris was assessed by negative-staining electron microscopy(Fig.1A).Mimivirus proteins separated by one-dimensional SDS-PAGE(Fig.1B)were frac-tionated in49pieces of gel,and corresponding tryptic digests were analyzed by capillary LC-MS/MS.Alternatively,mimivi-rus samples were resolved by2D gel electrophoresis(Fig.2), and protein identification was achieved by MALDI-TOF–MS. These complementary approaches allowed114proteins to be identified.These are listed in Table1,together with their predicted functions(where applicable),and are classified into broad functional categories.The diversity of functions repre-sented in the viral particle is quite large,with bonafide“struc-tural”proteins amounting for a small fraction of the number of proteins.We considered onlyfive candidates,whose sole role is to constitute the virion particle,as belonging to such a class. They are an A16L-like virion-associated membrane protein, the major core protein,the capsid protein D13L,a putative spore coat assembly factor,and a lipocalin-like outer mem-brane lipoprotein.Transcriptional enzymes and factors(12 gene products)constitute the largest functional category asso-ciated with the viral particle.This set includes allfive DNA-directed RNA polymerase subunits as well as two helicases, one mRNA guanylyltransferase,and four transcription factors. Incidentally,one of the best identification scores was obtained with the-subunit of the RNA polymerase(MIMI_R501), where the intron computationally identified within the gene was found to be excised as predicted(33).The next largest functional group is constituted of nine gene products,all of which are associated with oxidative pathways.Some of these enzymes might help the virus to cope with the oxidative stress generated by the host defense.The protein/lipid modification functional category is also well represented,including a phos-phoesterase and a lipase,which are eventually used to digest the cell membrane,two protein kinases,and a protein phos-phatase.Finally,five proteins associated with DNA topology and damage repair were identified,including the topoisomer-V OL.80,2006MIMIVIRUS PROTEOME11679at Penn State Univ on February 12, 2008 Downloaded fromTABLE 1.Mimivirus proteins identified in this studyProtein name and categoryORF a2D spot no.bDNA topology and repairTopoisomerase IB (pox-like)MIMI_R194NP Topoisomerase IA (bacterial type—untwisting)MIMI_L221NP DNA polymerase family XMIMI_L318NPRegulator of chromosome condensation RCC1MIMI_R3451,2,3,4,5DNA UV damage repair endonucleaseMIMI_L687NP TranscriptionDNA-directed RNA polymerase subunit 5MIMI_L23593DNA-directed RNA polymerase subunit 2MIMI_L244NP VV D6R-like helicaseMIMI_R350NP DNA-directed RNA Pol2G subunitMIMI_L37655D6R-like putative early transcription factorMIMI_L377NP African swine fever virus (ASFV)NP868R-like mRNA capping enzyme MIMI_R382NP DNA-directed RNA polymerase subunit L MIMI_R47037DNA-directed RNA polymerase subunit 1MIMI_R501NP VV I8-like helicaseMIMI_L540NP NPH-1-like transcription termination factor MIMI_L538NP TFIIB-like transcription initiation factor MIMI_L544NP NPH-1-like transcription termination factor MIMI_R563NPOxidative pathwaysCholine dehydrogenase MIMI_R13569,70,71,72Amine oxidoreductaseMIMI_R18810Glutaredoxin (ESV128type)MIMI_R195NP Thioredoxine domain MIMI_R36296Thioredoxine domainMIMI_R44367Zn-dependent alcohol dehydrogenase MIMI_L498NP Putative oxidoreductase (C terminus)MIMI_L893NP Putative oxidoreductase (N terminus)MIMI_L894NP Thiol oxidoreductase E10R (C-C bonding)MIMI_R59687Protein/lipid modification Ubiquitin-specific proteaseMIMI_L293NP Serine/threonine protein phosphatase MIMI_R307NP PhosphoesteraseMIMI_R398NP F10L-like S/T protein kinase MIMI_R400NP Putative S/T protein kinase MIMI_L516NP Putative triacylglycerol lipase MIMI_R52645,46Prolyl 4-hydroxylase MIMI_L593NP Particle structureA16L-like virion-associated membrane protein MIMI_L65NP Major core protein MIMI_L41011,12Capsid protein D13LMIMI_L4256,7,8,9Putative spore coat assembly factorMIMI_L454NP Lipocalin-like outer membrane lipoprotein MIMI_R877NP OtherCytoskeletal protein MIMI_L29421,22RNA methyl transferaseMIMI_R383NP tRNA (uracil-5-)methyltransferase MIMI_R407*88,89Putative exoribonuclease MIMI_R528NP Cytochrome P450MIMI_L532*75Putative elongation factor EF-Tu homolog MIMI_R55373,74Proline-rich proteinMIMI_R61059,60Mannose-6-phosphate isomeraseMIMI_L61268Phosphatidyl-ethanolamine-binding protein MIMI_R644NP Chemotaxis MIMI_R706NP Chemotaxis MIMI_R72144ORFan MIMI_L48*65,66ORFanMIMI_L208NP Unknown (WD repeat)MIMI_L264NP ORFanMIMI_L274NP Chilo iridescent virus 380R-like anonymous domain MIMI_R301NP ORFan MIMI_L309NP ORFan MIMI_L33083ORFanMIMI_L352NPContinued on facing page11680RENESTO ET AL.J.V IROL .at Penn State Univ on February 12, 2008 Downloaded fromase IA (MIMI_L221)and a DNA UV damage repair endonu-clease (MIMI_L687),which were never described before as viral proteins.Other identified proteins belonging to this cat-egory are a tRNA methyltransferase and a putative mannose-6-phosphate isomerase (see Table S1in the supplemental ma-terial).Yet the most abundant class of proteins associated with the mimivirus particle corresponds to those with unknown function (65proteins),among which are 45ORFans exhibiting no convincing sequence match in the databases.We noticed that the fraction of ORFan gene products found to be associ-TABLE 1—ContinuedProtein name and categoryORF a2D spot no.bORFan MIMI_L389NP ORFanMIMI_L399NP Unknown (PBCV-1A488R)MIMI_L417NPORFanMIMI_L44223,24,25,26,30,31,32,33,34,35,36ORFanMIMI_L452NP Unknown (ankyrin repeat)MIMI_L484NP ORFan (biotinyl carrier)MIMI_L485*95ORFanMIMI_L488NP ORFan (coenzyme A binding)MIMI_L492NP UnknownMIMI_L515NP ORFan (nuclease)MIMI_L533NP ORFan (leucine zipper)MIMI_L55097Unknown MIMI_L56778,79,80,81,82ORFan MIMI_L585NP ORFanMIMI_L59177ORFan (leucine zipper)MIMI_L64794ORFanMIMI_L68852,53Unknown (WD repeat)MIMI_L69027,28,29Unknown (COG 3979)MIMI_L701NP ORFan (fusion protein ͓Nipah virus ͔)MIMI_L72485,86ORFan MIMI_L72547,48,49,50,51ORFan MIMI_L778NPUnknown MIMI_L82913,14,15,16,17,18,54,62ORFanMIMI_L851NP ORFan (aromatic amino acid hydrolase)MIMI_L87243ORFan MIMI_L899NP ORFan MIMI_R160NP Unknown MIMI_R161NPUnknown MIMI_R25338,39,40,76ORFan MIMI_R326NP UnknownMIMI_R327NP Unknown (ASFV C475L)MIMI_R34141ORFanMIMI_R347NP Unknown (ASFV S273R)MIMI_R355NP ORFan (apolipophorin III)MIMI_R38742UnknownMIMI_R40219ORFan (kinase)MIMI_R403NP ORFan MIMI_R457NP ORFan MIMI_R45991,92ORFan MIMI_R463NP Unknown MIMI_R472NP UnknownMIMI_R486NPUnknown (PBCV-1A352L)MIMI_R48956,57,58ORFanMIMI_R557NPORFan (acetyltransferase)MIMI_R58463,64,84ORFanMIMI_R646NP Unknown (WD repeat)MIMI_R64820ORFan MIMI_R65361ORFan MIMI_R658NP ORFan MIMI_R679NP ORFan MIMI_R691NP ORFan MIMI_R69290ORFan MIMI_R695NP ORFanMIMI_R705NP ORFan (alpha-synuclein)MIMI_R710NP Unknown MIMI_R722NP ORFanMIMI_R727NPa *,ORF products identified solely through 2D MS.bNP,proteins not positioned on 2D gels.V OL .80,2006MIMIVIRUS PROTEOME 11681at Penn State Univ on February 12, 2008 Downloaded fromated with the virion (45/114[39.47%])is equivalent to their proportion within the whole mimivirus genome (39%),a find-ing consistent with the notion that these genes are in no way different from other mimivirus genes and probably all encode bona fide proteins of importance to virus physiology.In a landmark study,Iyer et al.(16)identified a set of nucleocyto-plasmic large DNA virus core genes that they further classified into four classes according to their decreasing levels of con-servation across the main nucleocytoplasmic large DNA virus clades.We found that mimivirus particles incorporated four ofthe nine class I core gene products (including the major capsid protein),none of the class II core gene products,five of the class III gene products,and three of the class IV gene products (see Table S1in the supplemental material).Finally,a small fraction (13/114)of the products originated from genes pre-ceded by the highly conserved predicted mimivirus promoter (found in front of about half of all mimivirus genes)(42),confirming its putative role in governing the transcription of early or late-early genes (see Table S1in the supplemental material).Posttranslational modifications.As shown on 2D gels,most of the mimivirus proteins were not resolved into single spots but rather as a train of spots (Fig.2).This was the case,for instance,for the capsid protein D13L (spots 6to 9),which is glycosylated (Fig.3).We observed that 66%of ORFan-en-coded proteins exhibited molecular weights corresponding to their in silico predictions.Others,such as the MIMI_L442ORFan gene product,were not located at their expected lo-cations on the gel.Extensive analysis of the 11corresponding isoforms showed that tryptic peptides from spot 23matched exclusively with the central part of the protein,while spots 24to 26and 30to 36matched the N-and C-terminal extremities of the full-length 140-kDa precursor,respectively.In other cases,both the mature protein and cleavage products were identified.Other cases of discrepancy might correspond to computational errors in the identification of the true N-termi-nal boundary of the protein (i.e.,the bona fide initiation codon).The same problem was noticed for proteins with func-tional attributes.According to both the areas and intensities of silver-stained spots,we can assume that some of the ORFan-encoded proteins are strongly expressed (spots 47to 51,MIMI_L725;spots 52to 53,MIMI_L688;and spot 61,MIMI_R653).Only four proteins not detected byLC-MS/MSFIG.1.Electron micrograph and SDS-PAGE of mimivirus.(A)Purified mimivirus particles (bar,500nm)separated by 11%SDS-PAGE and stained with Coomassie blue R250.(B)Bands in gels were excised for subsequent LC-MS/MS analysis after in-gel trypsin diges-tion.Identified proteins are listed in Table 1(also see details in Table S1in the supplementalmaterial).FIG.2.2D gel electrophoresis patterns of proteins.Acanthamoeba polyphaga mimivirus was separated using 18-cm pI 4to 7(left)or 6to 11(right)IPG strips for the first dimension (IEF)followed by 10%linear SDS-PAGE for the second dimension.Spots revealed by silver staining were cut out from the gel and subjected to trypsin digestion followed by MALDI-TOF–MS analysis.Molecular sizes and pI ranges are indicated.Identified proteins are listed in Table 1(also see details in Table S1in the supplemental material).11682RENESTO ET AL.J.V IROL .at Penn State Univ on February 12, 2008 Downloaded fromwere identified through 2D electrophoresis coupled with MALDI-TOF–MS.Antigenic properties of ORFan-encoded mimivirus proteins.To investigate the antigenicity of mimivirus proteins,immuno-blots were performed on samples resolved by 2D-PAGE,using sera from mice immunized with the whole particle (Fig.4).Interestingly,two ORFan-encoded proteins,namely,MIMI_L724(218amino acids)and MIMI_L725(224amino acids),were specifically recognized,while no protein was recognized by preimmune sera.Both proteins are rich in cysteine residues and might be involved in a cross-linked structure accessible at the periphery of the particle.Immunogold labeling using an anti-MIMI_L725monoclonal antibody further revealed gold particles surrounding the capsid core structure of mimivirus,confirming the exposure of the corresponding epitope to the surface (Fig.5).DISCUSSIONThe proteomic analysis of mimivirus allowed us to identify 114distinct proteins.Most of these proteins were identified through 1D electrophoresis coupled with LC-MS/MS,as this approach eliminated the methodological deficiencies encoun-tered in separating low-abundance and hydrophobic proteins that are difficult to resolve by 2D electrophoresis (31).Com-pletion of 2D gels coupled to MALDI-TOF–MS therefore provided additional and complementary information,as con-cluded from two recently published vaccinia virion proteome analyses (6,48).First,for the majority of proteins identified from these gels,a good correlation between observed and theoretical pI and M w values was observed.Analysis of 2D gels also revealed the existence of several isoforms due,in part,to protein glycosylation.Mimivirus contains six ORFs encoding putative glycosyltransferases and is thus,like Paramecium bur-saria Chlorella virus (PBCV-1)(14,46),likely to provide its own glycosylation machinery (33).The 2D gel profiles revealed cleaved proteins,a phenomenon found in many viruses and associated with synthesis and maturation steps (18,27,45).However,possible partial degradation of our samples due to contaminating proteases (although antiproteases were used)cannot be formally excluded,despite the reproducibility of our gel profiles.This study also provided direct evidence that a large number of ORFan gene products contribute to the making of the mimivirus particle,with some of them acting as major compo-nents (based on the areas and intensities of the corresponding spots on 2D gels).Moreover,we demonstrated that some of these ORFan proteins are endowed with antigenic properties.Thus,proteins encoded by MIMI_L724and MIMI_L725wereFIG.3.Glycosylation pattern of Acanthamoeba polyphaga mimivi-rus proteins detected by fluorescence assay.Mimivirus proteins were separated by 10%SDS-PAGE (A)or by 2D electrophoresis using 7-cm IPG strips (pI 3to 10)(B),and glycosylated proteins were revealed with GlycoProfile ne 1,standard glycosylation markers;lane 2,mimivirus.FIG.4.Western blot analysis of mimivirus proteins.Mimivirus pro-teins were separated by 2D electrophoresis using 7-cm IPG strips (pI 3to 10)and transferred to nitrocellulose membranes before incuba-tion with serum from an immunized mouse as described in Materials andMethods.FIG.5.Localization of Mimi_L725-encoded epitope.Ultrathin sections of mimivirus-infected amoebae were immunogold labeled with a monoclonal antibody raised against the MIMI_L725-encoded protein followed by goat anti-mouse antibodies conjugated to 25-nm colloidal gold particles before immunoelectron microscopy analysis.Bar,200nm.V OL .80,2006MIMIVIRUS PROTEOME 11683at Penn State Univ on February 12, 2008 Downloaded fromrecognized by sera of mice experimentally infected with mim-ivirus.The antigenicity of MIMI_L724as well as that of other ORFan proteins,encoded by MIMI_L330,MIMI_L442,and MIMI_L591,was also observed in a case of human infection (34).The surface exposure of the MIMI_L725epitopes was further evidenced by immunogold staining with a monoclonal antibody raised against this protein.In the future,complemen-tary experiments will be planned to localize other identified antigens.It is noteworthy that no eukaryotic proteins were identified in our samples,confirming their purity and demon-strating that eukaryotic proteins are not encapsidated within mimivirus particles.In contrast,proteomic analyses of vaccinia virus carried out by two distinct laboratories identified a sig-nificant proportion(Ͼ20%)of host proteins associated with purified intracellular mature virions(6,17).Virus-associated host proteins include highly conserved proteins such as-actin (6).This observation,associated with the fact that two amoebal genomes are available in the databases(10,25),led us to discard a possible failure of identification.Genome replication and assembly of viruses often take place in specific cytoplasmic compartments,termed the viral factory(28),that may be in-strumental in limiting the packaging of eukaryotic components within the viral particle.This hypothesisfits well with our own observation of mimivirus factories in infected amoebae(un-published data).Pending future studies of mimivirus physiology and future characterization of the virus-host interaction,the functions predicted for the proteins associated with the virion allow us to speculate on a possible molecular scenario for the early stage of the virus cycle.For instance,it is likely that the encapsidated lipolytic and proteolytic enzymes are part of the mechanism allowing the virus to gain access to the amoeba cytoplasm from its initial vacuolar location.The two topoisomerases(IA and IB)might then be involved in the ejection of the viral DNA from the particle and its injection into the cytoplasm,as well as allowing transcription to take place.These enzymes,which regulate the superhelical density of DNA(7),could indeed provide a driving force for DNA ejection into the bacterial cytoplasm by using the energy gained from changes of DNA topology(5).The DNA repair machinery components might then turn to action,allowing the viral DNA to be repaired prior to starting its transcription.Finally,the completeness of the transcription machinery components found in the viral particle is highly reminiscent of what has been described for poxviruses(15,49),strongly suggesting that early transcription could take place in the host cytoplasm immediately upon mimi-virus infection.Altogether,these results contribute to the ongoing debate on the evolutionary origin of the gene content of large DNA viruses epitomized by mimivirus.Part of the community con-siders large viral genomes a“bag of genes”randomly accumu-lating unused laterally transferred genes(26).However,the recent release of two whole amoeba genomes(10,25)does not favor this hypothesis(30).The fact that some of the mimivirus ORFans match unknown DNA sequences obtained from the Sargasso Sea suggests that mimivirus’s uniqueness might well be related to a poor knowledge on viruses from the environ-ment(13),which could partly explain the huge proportions of unmatched sequences in metagenomic studies(9).According to this view,the sequences of these genes might quickly lose any resemblance to their homologues under no functional con-straint.Ourfinding of multiple ORFan products in viral par-ticles rather suggests that ORFan proteins participate in a highly coordinated multimolecular process normally associated with tight evolutionary constraints.The unprecedented conser-vation of mimivirus promoters(42),as well as evidence for gene duplication generating well-conserved paralogues(20, 41),is also in favor of a well-ordered rather than chaotic model of genome evolution.In conclusion,we believe that this work fuels the debate on the nature of viruses.Mimivirus was thought to be as complex and large as microorganisms of comparable genome size,such as rickettsiae or mycoplasmas(21).As a matter of fact,this proteomic analysis confirms that the virus’s protein complexity is comparable to that of Rickettsia conorii(35).This observa-tion,in association with the apparently common origin of the capsid protein of mimivirus and those of viruses infecting dif-ferent hosts in the three domains of life,i.e.,Eukarya,Bacteria, and Archaea(3),makes the question of the origin of viruses more relevant than ever(32).Finally,recent metagenomic studies indicated that up to70%of randomly sampled DNA sequences from various environments have no database match, while10%have similarities with viruses.Our data,with the finding of mimivirus relatives in the Sargasso Sea,suggest that environmental ORFans may indeed correspond to bonafide viral proteins whose functions and origins remain to be discov-ered.ACKNOWLEDGMENTSWe thank Jean-Yves Patrice for his help with Acanthamoeba polyphaga mimivirus culture,Claude Nappez for production of anti-bodies,and Bernard Campagna for immunolabeling and electron mi-croscopy analysis.We gladly acknowledge the use of the bioinformatics platform of Marseille-Nice Ge´nopole.REFERENCES1.Amiri,H.,W.Davids,and S.G.Andersson.2003.Birth and death of orphangenes in Rickettsia.Mol.Biol.Evol.20:1575–1587.2.Benarroch,D.,J.-M.Claverie,D.Raoult,and S.Shuman.2006.Character-ization of mimivirus DNA topoisomerase IB suggests horizontal gene trans-fer between eukaryal viruses and bacteria.J.Virol.80:314–321.3.Benson,S.D.,J.K.H.Bamford,D.H.Bamford,and R.M.Burnett.2004.Does common architecture reveal a viral lineage spanning all three domains of life?Mol.Cell.16:673–685.4.Berger,P.,L.Papazian,M.Drancourt, Scola,J.P.Auffray,and D.Raoult.2006.Ameba-associated microorganisms and diagnosis of noso-comial pneumonia.Emerg.Infect.Dis.12:248–255.5.Bustamante,C.,Z.Bryant,and S.B.Smith.2003.Ten years of tension:single-molecule DNA mechanics.Nature421:423–427.6.Chung,C.S.,C.H.Chen,M.Y.Ho,C.Y.Huang,C.L.Liao,and W.Chang.2006.Vaccinia virus proteome:identification of proteins in vaccinia virus intracellular mature virion particles.J.Virol.80:2127–2140.7.Corbett,K.D.,and J.M.Berger.2004.Structure,molecular mechanisms,and evolutionary relationships in DNA topoisomerases.Annu.Rev.Biophys.Biomol.Struct.33:95–118.8.Dujon,B.1996.The yeast genome project:what did we learn?Trends Genet.12:263–270.9.Edwards,R.A.,and F.Rohwer.2005.Viral metagenomics.Nat.Rev.Mi-crobiol.3:504–510.10.Eichinger,L.,J.A.Pachebat,G.Glockner,M.A.Rajandream,R.Sucgang,M.Berriman,J.Song,R.Olsen,K.Szafranski,Q.Xu,B.Tunggal,S.Kummerfeld,M.Madera,B.A.Konfortov,F.Rivero,A.T.Bankier,R.Lehmann,N.Hamlin,R.Davies,P.Gaudet,P.Fey,K.Pilcher,G.Chen,D.Saunders,E.Sodergren,P.Davis,A.Kerhornou,X.Nie,N.Hall,C.Anjard, L.Hemphill,N.Bason,P.Farbrother,B.Desany,E.Just,T.Morio,R.Rost,C.Churcher,J.Cooper,S.Haydock,N.van Driessche,A.Cronin,I.Good-head,D.Muzny,T.Mourier,A.Pain,M.Lu,D.Harper,R.Lindsay,H.Hauser,K.James,M.Quiles,M.Madan Babu,T.Saito,C.Buchrieser,A.Wardroper,M.Felder,M.Thangavelu, D.Johnson, A.Knights,H.Loulseged,K.Mungall,K.Oliver,C.Price,M.A.Quail,H.Urushihara,J.11684RENESTO ET AL.J.V IROL.at Penn State Univ on February 12, 2008 Downloaded from。
