Reconstitution of prion infectivity
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新东方谢真真:2014年11月08日托福阅读真题智课网整理第一套题第一篇:版本一:生命组成元素elements有很多,主要有氢和氦,还说了一些惰性元素,虽然不同星球元素比例不同但是所须具备的元素都有。
生命必须存在于有atmosphere和liquid的星球,两个条件同时满足,因为atmosphere可以防止辐射,liquid可以让元素之间反应从而形成organic的分子版本二:生命组成元素,element有九十多种,life element包括25种;然后讲了一下heavy element和氢、氦占元素的百分比,古老星球的heavy element百分比很低;地球生物最重要的元素是碳,其他星球具有生命所需的元素,所以可能有生命存在,并且可以based on 碳或者硅或者其他元素;atmosphere和liquid必须存在,有利于元素相互反应,形成生命。
词汇题不太记得了,貌似有一个widespread among第二篇:版本一:美国十九世纪lumber开始是农民卖,后来产业化,还说了technology促进了发展,有方法可以让开采全面进行而不是在冬天停滞,另外有方法解决冬天结冰导致木材搬运不同的问题,还说有铁路建设,尽管有些地方很偏远,但是由于对木材的持续需求建了版本二:讲美国五大湖地区的lumber,在十九世纪开始产业化,技术的发展使木材可以搬运到全国各地,stream水运木材,有一个啥技术可以控制水运的方向,使运输更快速;还有一个是在dry冬天,freeze减少摩擦力,解决冬天木材很难运的问题,可以运很重的木材了;另外就是铁路运输。
其他的细节就不太记得了第三篇:版本一:megafauna为什么灭绝说了一个主要的原因是人类活动,有两人还制作了模型检验,另外一个人支持这个观点,也有什么证据,说有remains在人类活动的地方,后来反驳,说few remains在那个地方,然后前面制作数学模型的人就说这反而能支持他们的观点,但是又有一个人说新西兰也有一个地方的什么动物,灭绝也是因为人类活动,但却留下很多evidence版本二:magafauna灭绝的问题,科学家提出是人类kill的,解释了一下为什么climate不会是灭绝的原因:很久之前有一次climate变化但没导致m灭绝,这次climate变化也不会导致灭绝。
小学上册英语第3单元期末试卷考试时间:100分钟(总分:100)B卷一、综合题(共计100题共100分)1. 填空题:The ________ was a famous historical figure known for his peace efforts.2. 选择题:What is the opposite of 'tight'?A. LooseB. FirmC. SecureD. Strong答案:A3. 听力题:I put my _____ (衣服) in the closet.4. 填空题:The _______ (The Great Famine) devastated Ireland in the 19th century.5. 选择题:What is the capital city of New Zealand?A. WellingtonB. AucklandC. ChristchurchD. Dunedin答案: A6. 选择题:What do we call a place where you can buy books?A. LibraryB. BookstoreC. SchoolD. Grocery storeI like to draw pictures of my ________ (玩具名) and imagine their adventures.8. 听力题:Some planets have _____ made of gas and clouds.9. 填空题:The _______ (The Civil Rights Act) aimed to dismantle segregation laws.10. 填空题:Certain plants are known for their ability to improve ______ health in the environment. (某些植物因其改善环境健康的能力而闻名。
小学上册英语第3单元测验试卷英语试题一、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.The chemical symbol for tellurium is _____.2.What do you call a baby cow?A. CalfB. FoalC. KidD. LambA3.What do you call the person who takes care of animals?A. VeterinarianB. FarmerC. ZookeeperD. All of the above4. A reaction that occurs in a closed system is called a ______ reaction.5.My favorite thing about school is ______.6. A ______ is a large area of flat land at a high elevation.7.What is the largest ocean?A. AtlanticB. IndianC. ArcticD. PacificD8.What do you call a baby horse?A. CalfB. FoalC. PupD. KidB9.The Amazon __________ is the longest river in South America.10.I love to _____ with my friends. (play)11.The dog is a loyal ________________ (宠物).12.The ______ is essential for many ecosystems.13. A chemical reaction can occur in _____ solutions.14. A physical change does not produce a new ______.15.What do you call a person who studies insects?A. EntomologistB. BiologistC. ZoologistD. BotanistA16. A compound that contains both carbon and hydrogen is called a ______.17.We eat ______ together as a family. (dinner)18.The __________ (历史的名人) inspire future generations.19.The ______ is a talented actress.20.The process of combining elements to form a compound is called ______.21.Which of these is an insect?A. SpiderB. ButterflyC. WormD. Snail22.What do we call the day of the week after Wednesday?A. MondayB. TuesdayC. ThursdayD. FridayC23.What is the name of the famous mouse created by Walt Disney?A. Mickey MouseB. Jerry MouseC. Stuart LittleD. Tom CatA24.urban forestry) enhances city landscapes. The ____25.The process of adding more solute to a saturated solution does not increase the _______.26.The process of changing from a solid to a gas without becoming liquid is called _______.27.Which planet is known for its rings?A. MarsB. SaturnC. JupiterD. Neptune28.The signing of the Treaty of __________ ended World War I. (凡尔赛)29.This ________ (玩具) is perfect for all ages.30.What is the term for a group of wolves?A. PackB. FlockC. SchoolD. GaggleA31. A chemical property describes how a substance _____ with others.32.The _____ (盒子) is full of toys.33.My dad is _______ (cooking) dinner.34.The _____ (花坛) in front of my house is colorful and vibrant.35.What do you call a large body of freshwater?A. LakeB. OceanC. RiverD. Sea36.The Earth's inner structure includes the ______ and outer core.37.We eat lunch at ___. (noon)38.I have a _____ (question/answer) for you.39.The _____ (铃铛) rings at noon.40.What do you call the act of saving money?A. SpendingB. InvestingC. BudgetingD. All of the above41. A lion is known as the ________________ (森林之王).42.The hummingbird can hover in one ________________ (位置).43.We have a test in ___. (math)44.What is the main ingredient in sushi?A. RiceB. PastaC. BreadD. Corn45.My favorite subject is ________.46.The __________ (湿地) is home to many birds.47.The _____ (海豹) is often found in cold waters.48.What do we call a person who studies the past?A. HistorianB. ArchaeologistC. AnthropologistD. SociologistA49.What is the name of the famous mountain range in South America?A. RockiesB. AndesC. AlpsD. HimalayasB50.The __________ is a famous historical building.51. A ____ has a shiny shell and moves slowly.52.The ________ was a significant moment in the history of public health.53.We develop ________ (strategies) for success.54. A ______ (鸽子) is often seen in city parks.55.The Amazon rainforest is located in ________ America.56.She has a new ________.57.I want to be a ___ (scientist/artist).58.The frog is jumping ___. (high)59. A ____ is often kept as a pet and enjoys playing fetch.60.The ice cream is _______ (melting) in the sun.61.Which planet is furthest from the sun?A. MarsB. JupiterC. SaturnD. NeptuneD62. (Great Depression) started in 1929. The ____63.What kind of animal is a parrot?A. MammalB. ReptileC. BirdD. Fish64.His favorite color is ________.65. A ______ is a geological feature that can provide insights into the past.66.What is the opposite of "near"?A. CloseB. FarC. NextD. TogetherB67.What is the capital of the Philippines?A. ManilaB. CebuC. DavaoD. Zamboanga68.小果子) grows on trees. The ___69. A dolphin is known for its playful ________________ (性格).70.The __________ is a famous area known for its artistic contributions.71. A solution that does not conduct electricity is called a ______ solution.72.What is the main ingredient in bread?A. RiceB. FlourC. SugarD. Milk73.Electrons are found outside the _____ (nucleus) of an atom.74. A ______ (蝙蝠) is a nocturnal animal.75.This _____ (球) is very bouncy.76. A chemical reaction can absorb or release ______.77.The first settlers in America were looking for _______. (新机会)78.Planting trees can help combat ______ change. (种树可以帮助抵御气候变化。
a rXiv:n ucl-t h /96v15Sep2Signature of a Pairing Transition in the Heat Capacity of Finite Nuclei S.Liu and Y.Alhassid Center for Theoretical Physics,Sloane Physics Laboratory,Yale University,New Haven,Connecticut 06520,U.S.A.(February 8,2008)Abstract The heat capacity of iron isotopes is calculated within the interacting shell model using the complete (pf +0g 9/2)-shell.We identify a signature of the pairing transition in the heat capacity that is correlated with the suppression of the number of spin-zero neutron pairs as the temperature increases.Our results are obtained by a novel method that significantly reduces the statis-tical errors in the heat capacity calculated by the shell model Monte Carlo approach.The Monte Carlo results are compared with finite-temperature Fermi gas and BCS calculations.Typeset using REVT E XPairing effects infinite nuclei are well known;examples include the energy gap in the spectra of even-even nuclei and an odd-even effect observed in nuclear masses.However,less is known about the thermal signatures of the pairing interaction in nuclei.In a macroscopic conductor,pairing leads to a phase transition from a normal metal to a superconductor below a certain critical temperature,and in the BCS theory[1]the heat capacity is characterized by afinite discontinuity at the transition temperature.As the linear dimension of the system decreases below the pair coherence length,fluctuations in the order parameter become important and lead to a smooth transition.The effects of both staticfluctuations[2,3] and small quantalfluctuations[4]have been explored in studies of small metallic grains.A pronounced peak in the heat capacity is observed for a large number of electrons,but for less than∼100electrons the peak in the heat capacity all but disappears.In the nucleus,the pair coherence length is always much larger than the nuclear radius,and large fluctuations are expected to suppress any singularity in the heat capacity.An interesting question is whether any signature of the pairing transition still exists in the heat capacity of the nucleus despite the largefluctuations.When only static and small-amplitude quantal fluctuations are taken into account,a shallow‘kink’could still be seen in the heat capacity of an even-even nucleus[5].This calculation,however,was limited to a schematic pairing model.Canonical heat capacities were recently extracted from level density measurements in rare-earth nuclei[6]and were found to have an S-shape that is interpreted to represent the suppression of pairing correlations with increasing temperature.The calculation of the heat capacity of thefinite interacting nuclear system beyond the mean-field and static-path approximations is a difficult problem.Correlation effects due to residual interactions can be accounted for in the framework of the interacting nuclear shell model.However,atfinite temperature a large number of excited states contribute to the heat capacity and very large model spaces are necessary to obtain reliable results.The shell model Monte Carlo(SMMC)method[7,8]enables zero-andfinite-temperature calculations in large spaces.In particular,the thermal energy E(T)can be computed versus temperature T and the heat capacity can be obtained by taking a numerical derivative C=dE/dT.However,thefinite statistical errors in E(T)lead to large statistical errors in the heat capacity at low temperatures(even for good-sign interactions).Such large errors occur already around the pairing transition temperature and thus no definite signatures of the pairing transition could be identified.Furthermore,the large errors often lead to spurious structure in the calculated heat capacity.Presumably,a more accurate heat capacity can be obtained by a direct calculation of the variance of the Hamiltonian,but in SMMC such a calculation is impractical since it involves a four-body operator.The variance of the Hamiltonian has been calculated using a different Monte Carlo algorithm[9],but that method is presently limited to a schematic pairing interaction.Here we report a novel method for calculating the heat capacity within SMMC that takes into account correlated errors and leads to much smaller statistical ing this method we are able to identify a signature of the pairing transition in realistic calculations of the heat capacity offinite nuclei.The signature is well correlated with the suppression in the number of spin-zero pairs across the transition temperature.The Monte Carlo approach is based on the Hubbard-Stratonovich(HS)representation of the many-body imaginary-time propagator,e−βH= D[σ]GσUσ,whereβis the in-verse temperature,Gσis a Gaussian weight and Uσis a one-body propagator that de-scribes non-interacting nucleons moving influctuating time-dependent auxiliaryfieldsσ. The canonical thermal expectation value of an observable O can be written as O = D[σ]GσTr(OUσ)/ D[σ]GσTr Uσ,where Tr denotes a canonical trace for N neutrons and Z protons.We can rewriteO = [Tr(OUσ)/Tr Uσ]ΦσWsamples.In particular the thermal energy can be calculated as a thermal average of the Hamilto-nian H .The heat capacity C =−β2∂E/∂βis then calculated by estimating the derivative as a finite differenceC =−β2E (β+δβ)−E (β−δβ)D [σ±]G σ±(β±δβ)Tr U σ±(β±δβ),(3)where the corresponding σfields are denoted by σ±.To have the same number of time slices N t in the discretized version of (3)as in the original HS representation of E (β),we define modified time slices ∆β±by N t ∆β±=β±δβ.We next change integration variables in (3)from σ±to σaccording to σ±=(∆β/∆β±)1/2σ,so that the Gaussian weight is left unchanged G σ±(β±δβ)≡exp −αn12|v α|(σα(τn ))2∆β =G σ(β)(v αare the interaction ‘eigenvalues’,obtained by writing the interaction in a quadratic form αv αˆρ2α/2,where ˆραare one-body densities).Rewriting (3)using the measure D [σ](the Jacobian resulting from the change in integration variables is constant and canceled between the numerator and denominator),we findE (β±δβ)= Tr HU σ±(β±δβ)Tr U σ(β)Φσ W Tr U σ(β)Φσ W ≡H ±among the quantities H±and Z±,which would lead to a smaller error for C.The covariances among H±and Z±as well as their variances can be calculated in the Monte Carlo and used to estimate the correlated error of the heat capacity.We have calculated the heat capacity for the iron isotopes52−62Fe using the complete (pf+0g9/2)-shell and the good-sign interaction of Ref.[10].Fig.1demonstrates the sig-nificant improvement in the statistical Monte Carlo errors.On the left panel of thisfigure we show the heat capacity of54Fe calculated in the conventional method,while the right panel shows the results from the new method.The statistical errors for T∼0.5−1MeV are reduced by almost an order of magnitude.The results obtained in the conventional calculation seem to indicate a shallow peak in the heat capacity around T∼1.25MeV,but the calculation using the improved method shows no such structure.The heat capacities of four iron isotopes55−58Fe,calculated with the new method,are shown in the top panel of Fig. 2.The heat capacities of the two even-mass iron isotopes (56Fe and58Fe)show a different behavior around T∼0.7−0.8MeV as compared with the two odd-mass isotopes(55Fe and57Fe).While the heat capacity of the odd-mass isotopes increases smoothly as a function of temperature,the heat capacity of the even-mass isotopes is enhanced for T∼0.6−1MeV,increasing sharply and then leveling off,displaying a ‘shoulder.’This‘shoulder’is more pronounced for the isotope with more neutrons(58Fe). To correlate this behavior of the heat capacity with a pairing transition,we calculated the number of J=0nucleon pairs in these nuclei.A J=0pair operator is defined as usual by∆†= a,m a>0(−1)j a−m aj a+1/2a†ja m aa†ja−m a,(5)where j a is the spin and m a is the spin projection of a single-particle orbit a.Pair-creation operators of the form(5)can be defined for protons(∆†pp),neutrons(∆†nn),and proton-neutrons(∆†pn).The average number ∆†∆ of J=0pairs(of each type)can be calculated exactly in SMMC as a function of temperature.The bottom panel of Fig.2shows the number of neutron pairs ∆†nn∆nn for55−58Fe.At low temperature the number of neutron pairs for isotopes with an even number of neutrons is significantly larger than that forisotopes with an odd number of neutrons.Furthermore,for the even-mass isotopes we observe a rapid suppression of the number of neutron pairs that correlates with the‘shoulder’observed in the heat capacity.The different qualitative behavior in the number of neutron pairs versus temperature between odd-and even-mass iron isotopes provides a clue to the difference in their heat capacities.A transition from a pair-correlated ground state to a normal state at higher temperatures requires additional energy for breaking of neutron pairs, hence the steeper increase observed in the heat capacity of the even-mass iron isotopes. Once the pairs are broken,less energy is required to increase the temperature,and the heat capacity shows only a moderate increase.It is instructive to compare the SMMC heat capacity with a Fermi gas and BCS calcula-tions.The heat capacity can be calculated from the entropy using the relation C=T∂S/∂T. The entropy S of uncorrelated fermions is given byS(T)=− a[f a ln f a+(1−f a)ln(1−f a)],(6) with f a being thefinite-temperature occupation numbers of the single-particle orbits a.Above the pairing transition-temperature T c,f a are just the Fermi-Dirac occupancies f a= [1+eβ(ǫa−µ)]−1,whereµis the chemical potential determined from the total number of particles andǫa are the single-particle energies.Below T c,it is necessary to take into account the BCS solution which has lower free energy.Since condensed pairs do not contribute to the entropy,the latter is still given by(6)but f a are now the quasi-particle occupancies[1],1f a=(ǫa−µ)2+∆2are the quasi-particle energies,where the gap∆(T)and the chemical potentialµ(T)are determined from thefinite-temperature BCS equations.In practice,we treat protons and neutrons separately.We applied the Fermi gas and BCS approximations to estimate the heat capacities of the iron isotopes.To take into account effects of a quadrupole-quadrupole interaction,we used an axially deformed Woods-Saxon potential to extract the single-particle spectrumǫa[11].A deformation parameterδfor the even iron isotopes can be extracted from experimental B(E2)values.However,since B(E2)values are not available for all of these isotopes,we used an alternate procedure.The excitation energy E x(2+1)of thefirst excited2+state in even-even nuclei can be extracted in SMMC by calculating J2 βat low temperatures and using a two-state model(the0+ground state and thefirst excited2+state)where J2 β≈6/(1+eβE x(2+1)/5)[12].The excitation energy of the2+1state is then used in the empirical formula of Bohr and Mottelson[13]τγ=(5.94±2.43)×1014E−4x(2+1)Z−2A1/3to estimate the meanγ-ray lifetimeτγand the corresponding B(E2).The deformation pa-rameterδis then estimated from B(E2)=[(3/4π)Zer20A2/3δ]2/5.Wefind(using r0=1.27 fm)δ=0.225,0.215,0.244,0.222,0.230,and0.220for the even iron isotopes52Fe–62Fe, respectively.For the odd-mass iron isotopes we adapt the deformations in Ref.[14].The zero-temperature pairing gap∆is extracted from experimental odd-even mass differences and used to determine the pairing strength G(needed for thefinite temperature BCS solu-tion).The top panels of Fig.3show the Fermi-gas heat capacity(dotted-dashed lines)for 59Fe(right)and60Fe(left)in comparison with the SMMC results(symbols).The SMMC heat capacity in the even-mass60Fe is below the Fermi-gas estimate for T<∼0.5MeV, but is enhanced above the Fermi gas heat capacity in the region0.5<∼T<∼0.9MeV. The line shape of the heat capacity is similar to the S-shape found experimentally in the heat capacity of rare-earth nuclei[6].We remark that the saturation of the SMMC heat capacity above∼1.5MeV(and eventually its decrease with T)is an artifact of thefinite model space.The solid line shown for60Fe is the result of the BCS calculation.There are two‘peaks’in the heat capacity corresponding to separate pairing transitions for neutrons (T n c≈0.9MeV)and protons(T p c≈1.2MeV).Thefinite discontinuities in the BCS heat capacity are shown by the dotted lines.The pairing solution describes well the SMMC results for T<∼0.6MeV.However,the BCS peak in the heat capacity is strongly suppressed around the transition temperature.This is expected in thefinite nuclear system because of the strongfluctuations in the vicinity of the pairing transition(not accounted for in themean-field approach).Despite the largefluctuations,a‘shoulder’still remains around the neutron-pairing transition temperature.The bottom panels of Fig.3show the number of spin-zero pairs versus temperature in SMMC.The number of p-p and n-p pairs are similar in the even and odd-mass iron isotopes. However,the number of n-n pairs at low T differs significantly between the two isotopes. The n-n pair number of60Fe decreases rapidly as a function of T,while that of59Fe decreases slowly.The S-shape or shoulder seen in the SMMC heat capacity of60Fe correlates well with the suppression of neutron pairs.Fig.4shows the complete systematics of the heat capacity for the iron isotopes in the mass range A=52−62for both even-mass(left panel)and odd-mass(right panel). At low temperatures the heat capacity approaches zero,as expected.When T is high,the heat capacity for all isotopes converges to approximately the same value.In the intermediate temperature region(T∼0.7MeV),the heat capacity increases with mass due to the increase of the density of states with mass.Pairing leads to an odd-even staggering effect in the mass dependence(see also in Fig.2)where the heat capacity of an odd-mass nucleus is significantly lower than that of the adjacent even-mass nuclei.For example,the heat capacity of57Fe is below that of both56Fe and58Fe.The heat capacities of the even-mass58Fe,60Fe,and62Fe all display a peak around T∼0.7MeV,which becomes more pronounced with an increasing number of neutrons.In conclusion,we have introduced a new method for calculating the heat capacity in which the statistical errors are strongly reduced.A systematic study in several iron isotopes reveals signatures of the pairing transition in the heat capacity offinite nuclei despite the largefluctuations.This work was supported in part by the Department of Energy grants putational cycles were provided by the San Diego Supercomputer Center (using NPACI resources),and by the NERSC high performance computing facility at LBL.REFERENCES[1]J.Bardeen,L.N.Cooper and J.R.Schrieffer,Phys.Rev.108,1175(1957).[2]B.Muhlschlegel,D.J.Scalapino and R.Denton,Phys.Rev.B6,1767(1972).[3]uritzen,P.Arve and G.F.Bertsch,Phys.Rev.Lett.61,2835(1988).[4]uritzen,A.Anselmino,P.F.Bortignon and R.A.Broglia,Ann.Phys.(N.Y.)223,216(1993).[5]R.Rossignoli,N.Canosa and P.Ring,Phys.Rev.Lett.80,1853(1998).[6]A.Schiller,A.Bjerve,M.Guttormsen,M.Hjorth-Jensen,F.Ingebretsen,E.Melby,S.Messelt,J.Rekstad,S.Siem,S.W.Odegard,arXive:nucl-ex/9909011.[7]ng,C.W.Johnson,S.E.Koonin and W.E.Ormand,Phys.Rev.C48,1518(1993).[8]Y.Alhassid,D.J.Dean,S.E.Koonin,ng,and W.E.Ormand,Phys.Rev.Lett.72,613(1994).[9]S.Rombouts S,K.Heyde and N.Jachowicz,Phys.Rev.C58,3295(1998).[10]H.Nakada,Y.Alhassid,Phys.Rev.Lett.79,2939(1997).[11]Y.Alhassid,G.F.Bertsch,S.Liu and H.Nakada,Phys.Rev.Lett.84,4313(2000).[12]H.Nakada,Y.Alhassid,Phys.Lett.B436,231(1998).[13]S.Raman et al.,Atomic Data and Nuclear Data Tables42,1(1989).[14]P.M¨o ller et al,Atomic Data Nucl.Data Tables59,185(1995),P.M¨o ller,J.R.Nix,and K.-L.Kratz,Atomic Data Nucl.Data Tables66,131(1997);G.Audi et al,Nucl.Phys.A624,1(1997).FIGURES00.51 1.52T (MeV)051015C 00.51 1.52T (MeV)FIG.1.The SMMC heat capacity of 54Fe.The left panel is the result of conventional SMMCcalculations.The right panel is calculated using the improved method (based on the representation (4)where a correlated error can be accounted for).0.40.81.21.6T (MeV)357911<∆+n n∆n n >051015CFIG.2.Top panel:the SMMC heat capacity vs.temperature T for55Fe(open circles),56Fe(solid diamonds),57Fe(open squares),and58Fe(solid triangles).Bottom panel:the number ofJ =0neutron pairs versus temperature for the same nuclei.0.511.5T (MeV)0246810<∆+∆>05101520C60Fe0.51 1.52T (MeV)59FeFIG.3.Top:Heat capacity versus T for 60Fe(left)and59Fe(right).The Monte Carlo resultsare shown by symbols.The dotted-dashed lines are the Fermi gas calculations,and the solid line (left panel only)is the BCS result.The discontinuities (dashed lines)correspond to a neutron (T c ∼0.9MeV)and proton (T c ∼1.2MeV)pairing transition.Above the pairing-transition temperature,the BCS results coincide with the Fermi gas results.Bottom panels:The number of J =0n -n (circles),p -p (squares),and n -p (diamonds)pairs vs.T for60Fe(left)and59Fe(right).0.40.8 1.2 1.6T (MeV)051015C00.40.8 1.2 1.6T (MeV)53Fe 55Fe 57Fe 59Fe 61FeFIG.4.The heat capacity of even-even (left panel)and odd-even (right panel)iron isotopes.。
1 Surface reconstruction of the surgicalfield from stereoscopic microscopeviews in neurosurgeryO.J.Fleig,F.Devernay,J.-M.Scarabin and P.JanninLaboratoire SIM,Facultéde Médecine,Universitéde Rennes1,2Avenue du Pr.Léon Bernard,35043Rennes Cedex(France)Carl Zeiss S.A.,60Route de Sartrouville,78230Le Pecq(France)Team ChIR,INRIA,Sophia-Antipolis(France)Department of Neurosurgery,University Hospital of Rennes,(France)We present a technique to reconstruct the surface of the surgicalfield during neurosurgery.The images are taken with a stereo camera mounted on a surgical microscope,which is part of a neuronavigation system.We use an intensity-based method to calculate a dense disparity map over the whole image extend.Correspondences are established by exploiting the epipolar ge-ometry and comparing image intensities along corresponding epipolar lines.The method does not require a strong calibration.Only the colineation denoted by the fundamental matrix,which maps corresponding epipolar lines from one image to the other,has to be known.The results show that our method is able to reconstruct the surface of the surgicalfield upto a few areas.It is limited at points of specular reflections of the wet brain surface and points of uniform texture. Keywords:Stereoscopic reconstruction;neuronavigation;stereo microscope;augmented re-ality;augmented virtuality1.IntroductionIn neurosurgery,guidance systems,which correlate patient imaging data to the surgicalfield using tracked probes or surgical microscopes,have become standard equipment.The most ad-vanced systems dispose of an additional head-up display showing relevant information on a two-dimensional graphical overlay directly within one of the oculars of the microscope.Our experience has shown that to take full advantage of augmented reality,more complex colour and stereoscopic displays are needed for a better understanding and interpretation of the overlaid in-formation[1].Therefore,this basic overlay will be replaced in future by more sophisticated three-dimensional displays being able to render stereoscopic views of a virtual scene.Today’s augmented reality,where the real scene’s information is enhanced with additional information, might be backed by augmented virtuality[2,3].Augmented virtuality fuses multimodal scene information acquired during planning with real-time images from the surgicalfield.To ade-2 quately merge real and virtual information for both augmented reality and virtuality we need to know surface information on the real scene.E.g.to determine which part of a virtual object would be occluded by the real scene,we need to know the surface of the surgicalfield.With the surgical microscope we dispose of the possibility to record corresponding stereo images from the right and left ocular,without expensive additional hardware.A UDETTE proposes in his work on brain deformation[4]the use of a commercial range-sensor and structured laser light to reconstruct the brain surface.S KRINJAR uses a strongly calibrated stereo rig and an integration technique to compute the surface normals from the partial derivatives of intensities in both images[5].Integration techniques accumulate errors and constrain the surface to be continuous and globally follow the Lambertian reflectance model. We also use an intensity-based method,where point correspondences are established by com-paring intensities within a defined window in the left and right image[6].This method works on surfaces which are only locally Lambertian and may contain discontinuities in the disparity. Intensity-based methods calculate dense disparity maps in opposition to feature based methods, which recover only sparse disparity maps.2.Material and Methods2.1.AcquisitionThe Department of Neurosurgery at the University Hospital of Rennes disposes of an SMN zoom lens microscope from C ARL Z EISS(Oberkochen,Germany).A localiser tracks patient, probe,and microscope position.A pair of stereoscopic single CCD cameras mounted on the microscope is used to record still colour images with an off-the-shelf video grabber card on a desktop PC.A video channel switch driven by the parallel port of the PC selects between the left and right ocular.Focus and zoom parameters as well as the tracked microscope position are transferred from the navigation system to the PC via serial port connection.A simple user interface under MS-Windows initiates grabbing of an image pair and stores it along with zoom and focus settings and the microscope position.2.2.Epipolar geometry and fundamental matrixTo reconstruct the surface from two images,point correspondences have to be established. We take advantage of the epipolar geometry to simplify the search for corresponding points [6,7].The epipolar geometry constraints points lying on an epipolar line in the left image to be projected onto the corresponding epipolar line in the right image.The fundamental matrix is a colineation that gives the correspondence between epipolar lines,and allows to establish the equation of the epipolar line in the right image corresponding to a point given in the left image.Tofind a match to a point from the left image,the search is limited to the corresponding epipolar line in the right image.The fundamental matrix is calculated from one or multiple pairs of images with the same settings for zoom and focus.To minimise numerical instability we used multiple views at the same settings by generating additional sets of images of a calibration grid. The colineation between lines in the right and left images described by the fundamental matrix has seven degrees of freedom and can be solved using least square method based on at least eight pairs of corresponding points[8].Points are extracted automatically using curvature operators and matches are automatically established using a relaxation based algorithm.3 2.3.RectificationTo simplify point comparisons the fundamental matrix is used to rectify the image pairs in the way that corresponding epipolar lines are parallel to the abscissa at the same value for the ordinate in the image coordinate system[7,9].Figure1shows the rectified image of the left ocular.2.4.Disparity mapAfter rectification for each point of the left images,a corresponding point has to be found on the epipolar with the same ordinate value.The luminance within a window of predefined size is compared using a zero-mean normalised sum of squared differences(ZNSSD)criterion:,with for the intensity at image point and4Figure1.Rectified image of the left micro-scope view.Figure2.Disparity map from ZNSSD crite-rion.Figure3.Disparity map after medianfilteringand local planefitting.Figure4.Reconstructed surface,textured-mapped with the original image.The holescorrespond to the areas where the disparitymap contains no information.5 the image pairs.Overall calculation of the disparity map performs very good.However,the matching fails on various regions and points in the images and leaves multiple holes in the disparity map.Examination of the regions which were not reconstructed shows that most of them are satu-rated in one or both of the original images due to specular reflection.Other regions which were not reconstructed are areas on the border of the surgicalfield with gauze or cloth where there is no texture to exploit for the luminance comparison.Although the brain surface is speckled with tiny vessels which give it enough texture for reconstruction there are some areas on larger gyri of uniform colour.Those areas are not recovered by the reconstruction either.4.Discussion and ConclusionWe have demonstrated that the stereoscopic reconstruction of the brain is feasible from only the two images of a stereoscopic microscope view.The main problem is the particularity of the intra-operative images of the brain surface.The microscope’s light source is parallel to the optical axes which,in conjunction with the wet brain surface,causes a large amount of specular reflection.We cannot calculate the surface by luminance comparison for pixels in these reflection areas.But the results are still satisfactory as we do not need to know the exact surface of every point.For visual application like the already mentioned occlusion problem it seems sufficient tofill the holes in the disparity map with an interpolation technique by exploiting an a priori knowledge about the surface.Such an a priori knowledge is the fact that the reconstructed surface is a single surface and relatively smooth.For other applications like distance measures for brain-shift evaluation it is preferable not to interpolate,since the accuracy of the reconstructed points is more important than the number of these points.The authors are aware of the fact that validation is of immediate concern for the further use of this method.To validate the accuracy of the reconstruction,we plan to apply the algorithm on known synthetic scene with real intra-operative images as object texture.The microscope calibration has to be recovered in order to pass from the for the moment only projective recon-struction to an euclidian reconstruction.We also have to evaluate the reconstruction of images at a higher magnification.At a higher magnification we might probably loose some texture from the vessels and have larger areas of unrecoverable uniform intensity.We consider intra-operative stereoscopic images from the surgical microscope as a sofar un-exploited additional modality in multimodal neurosurgery.We already mentioned brain-shift estimation and occlusion as possible applications.A surface reconstruction gives us also the possibility to update a scene depending on the progress of the intervention or to verify or estab-lish registration to the patient.There is also the possibility to calculate the volume of resection. Our method does not require expensive additional equipment as long as it is possible to capture intra-operative images from a surgical microscope.REFERENCES1.Pierre Jannin,Oliver J.Fleig,Eric Seigneuret,Christophe Grova,Xavier Morandi,andJean-Marie Scarabin.A data fusion environment for multimodal and multi-informational puter Aided Surgery,5:1–10,2000.6 2.Damini Dey,Piotr J.Slomka,David G.Gobbi,and Terry M.Peters.Mixed Reality Mergingof Endoscopic Images and3-D Surfaces.In Medical Image Computing and Computer-Assisted Intervention(MICCAI),volume1935of Lecture Notes in Computer Science,pages 796–803.Springer Verlag,2000.3.P.Jannin,A.Bouliou,E.Journet,and J.M.Scarabin.Ray-traced texture mapping for en-hanced virtuality in image-guided neurosurgery.In Stud Health Technol Inform,volume29, pages553–563,1996.4.M.A.Audette,K.Siddiqi,and T.M.Peter.Level-set surface segmentation and fast corticalrange image tracking for computing intrasurgical deformations.In Medical Image Com-puting and Computer-Assisted Intervention(MICCAI),volume1679of Lecture Notes in Computer Science,pages788–797.Springer Verlag,1999.5.Oskar Skrinjar,Hemant Tagare,and James Duncan.Surface growing from stereo images.In IEEE Computer Society Conference on Computer Vision and Pattern Recognition(CVPR 2000),volume II,pages571–576,Hilton Head Island,SC,USA,june2000.6.R.Klette,K Schüns,and A puter Vision:Three-Dimensional Data fromImages.Springer,1998.7.Olivier Faugeras.Three-Dimensional Computer Vision:A Geometric Viewpoint.MIT Press,Cambridge,Massachusetts,1993.8.Zhengyou Zhang,Rachid Deriche,Olivier Faugeras,and Q.T.Luong.A robust techniquefor matching two uncalibrated images through the recovery of the unknown epipolar geom-etry.Technical Report RR-2273,Inria,Institut National de Recherche en Informatique et en Automatique,1994.9.N.Ayache.Artificial Vision for Mobile Robots:Stereo Vision and Multi-sensory Percep-tion.MIT Press,Cambridge,MA,1991.10.Frédéric Devernay and puting differential properties of3-D shapesfrom stereoscopic images without3-D models.In Proceedings of the International Confer-ence on Computer Vision and Pattern Recognition,pages208–213,1994.11.Frederic Devernay and Olivier Faugeras.From projective to euclidean reconstruction.InProceedings of the International Conference on Computer Vision and Pattern Recognition, pages264–269,1996.。
小学下册英语第二单元期中试卷考试时间:90分钟(总分:110)B卷考试人:_________题号一二三四五总分得分一、综合题(共计100题共100分)1. 填空题:The flower grows from a ______.2. 选择题:Which holiday is celebrated on December 25?a. Thanksgivingb. Halloweenc. Christmasd. Easter答案:C3. 选择题:What do we call a person who catches fish?A. FishermanB. HunterC. FarmerD. Forager4. 填空题:My dad is __________ (强壮的) and tall.5. 听力题:The ice is _______ (slippery).6. 选择题:What do we call the science of numbers?A. ChemistryB. PhysicsC. MathematicsD. Biology答案:CThe ________ (兰花) is a beautiful flower that comes in many colors.8. 填空题:Martin Luther King Jr. fought for _____ rights.9. 选择题:What do we call the process of taking in oxygen and releasing carbon dioxide?A. RespirationB. PhotosynthesisC. DigestionD. Fermentation答案:A10. 选择题:What do you call the process of making something?A. ProductionB. ManufacturingC. CreationD. All of the above答案: D11. 选择题:Which animal is known for its ability to swim well?A. CatB. DogC. FishD. Horse答案:C12. community governance structure) facilitates decision-making. 填空题:The ____13. 选择题:What is the capital of the Netherlands?A. AmsterdamB. RotterdamC. The HagueD. Utrecht14. 听力题:I _____ (like/hate) broccoli.15. 填空题:We need to _______ (鼓励)彼此。
《业务营销化》1 问街上一般的人什么是营销时,他们会告诉你那大概就是“卖东西的”。
这从根本上说是正确的,但营销不是简单的销售行为,而是怎样做成的销售。
我们都被全天候不间断营销所围绕,而我们每一个人都已经以我们自己的方式成了一名营销人。
2 专家是怎么定义营销的呢?根据美国市场营销协会,市场营销是一种组织职能,是为组织自身及利益相关者(stakeho ldersn. 利益相关者;股东)而创造、传播、传递客户价值,管理客户关系的一系列过程。
3 根据世界市场营销协会对营销的定义,“核心的经营理念是指导通过交换来识别和满足个人和组织需要的过程,从而为各方创造出众的价值。
”4最后,英国特许营销学会说,“营销是有利地识别,预测,和满足顾客需求的管理过程”。
5 如果我们只是看这三个定义的共性,我们可以看出,营销本质上(in essence)是:a)发现和给顾客他们所想要的和需要的东西,b)通过做这些来获利。
4Ps或5Ps营销策略6 密歇根州立大学(Michiga n State Univers ity)的杰罗姆·麦卡锡(JeromeMcCarth y)教授在20世纪50年代写了一本书并且定义了4Ps营销策略,包括产品、渠道、价格和促销。
这本书为这个星球上最古老的专业提供了一个清晰的结构,而这个结构成为市场营销的定义。
7 为了更好地理解营销,你应该有你自己对术语的定义。
例如,我认为营销是对产品的价格、分配、促销以及人员进行控制,满足顾客以获得利益。
控制是个充满感情的词语,尤其在我们谈及控制人的时候。
无论怎样,控制是很重要的,因为作为一名营销人员,我要控制市场营销的每一个工具并且操纵它们来使市场的影响力达到最大化。
8 作为一名营销经理,我控制一个产品的形象、味道和触感。
Reconstitution of Prion Infectivity from SolubilizedProtease-resistant PrP and Nonprotein Components of Prion Rods*Received for publication,August 28,2000,and in revised form,January 2,2001Published,JBC Papers in Press,January 4,2001,DOI 10.1074/jbc.M007815200Gideon M.Shaked‡,Zeev Meiner‡,Inbal Avraham‡,Albert Taraboulos§¶,and Ruth Gabizon‡ʈFrom the ‡Department of Neurology,The Agnes Ginges Center for Human Neurogenetics,Hadassah University Hospital,and §Department of Molecular Biology,Hebrew University Medical School,Jerusalem 91120,IsraelThe scrapie isoform of the prion protein,PrP Sc ,is the only identified component of the infectious prion,an agent causing neurodegenerative diseases such as Creutzfeldt-Jakob disease and bovine spongiform en-cephalopathy.Following proteolysis,PrP Sc is trimmed to a fragment designated PrP 27–30.Both PrP Sc and PrP 27–30molecules tend to aggregate and precipitate as amyloid rods when membranes from prion-infected brain are extracted with detergents.Although prion rods were also shown to contain lipids and sugar po-lymers,no physiological role has yet been attributed to these molecules.In this work,we show that prion infec-tivity can be reconstituted by combining Me 2SO-solubi-lized PrP 27–30,which at best contained low prion in-fectivity,with nonprotein components of prion rods (heavy fraction after deproteination,originating from a scrapie-infected hamster brain),which did not present any infectivity.Whereas heparanase digestion of the heavy fraction after deproteination (originating from a scrapie-infected hamster brain),before its combination with solubilized PrP 27–30,considerably reduced the reconstitution of infectivity,preliminary results suggest that infectivity can be greatly increased by combining nonaggregated protease-resistant PrP with heparan sulfate,a known component of amyloid plaques in the brain.We submit that whereas PrP 27–30is probably the obligatory template for the conversion of PrP C to PrP Sc ,sulfated sugar polymers may play an important role in the pathogenesis of prion diseases.PrP Sc ,the abnormal isoform of PrP C ,is the only known compo-nent of the prion,an agent causing fatal neurodegenerative disor-ders such as bovine spongiform encephalopathy and Creutzfeldt-Jakob disease (1).It has been postulated that prion diseases propagate by the conversion of PrP C molecules into protease-resistant and insoluble PrP Sc molecules by a mechanism in which PrP Sc serves as a template (2).Whereas some PrP Sc may be insol-uble in vivo (3),it is well documented that most PrP Sc ,as well as its protease-resistant core denominated PrP 27–30,precipitate into insoluble aggregates (also known as prion rods)when membranes from scrapie-infected brains are extracted with detergents such as sarkosyl (4).In addition to PrP Sc ,prion aggregates were shown tocontain nonprotein components,which include sphingolipids as well as polysaccharides (5–7).The traces of nucleic acids present in prion rods are believed to be too small to function as coding tools (8).No physiological role has ever been attributed to any nonprotein components of prion rods.Disruption of prion rods into detergent protein lipid com-plexes resulted in the retention of their protease resistance property concomitantly with an increase in their prion infec-tivity,suggesting that solubilized PrP Sc is more infectious than the aggregated prion protein (9).Contrarily,disruption of prion rods by sonication and SDS resulted in a protease-sensitive PrP with complete loss of infectivity (10).As opposed to methods to disrupt prion aggregates,we have recently introduced a new experimental procedure that results in the production of nonaggregated PrP Sc or PrP 27–30mole-cules by inhibition of the primary detergent-induced aggrega-tion used for rod formation (11).When membranes from brains of hamsters terminally ill with scrapie were incubated in the presence of Me 2SO in addition to sarkosyl and subsequently applied to a sucrose density gradient,the protease-resistant PrP molecules (PrP 27–30)were divided between the light fractions,containing soluble or poorly aggregated PrP 27–30molecules,and the heaviest fractions,containing insoluble and heavily aggregated PrP 27–30molecules (11).Interestingly,when light and heavy fractions of such gradients,containing similar concentrations of protease-resistant PrP,were inocu-lated into hamsters,the infectivity of the light fractions was lower by more than 2logs than the infectivity of the heavy fractions.Light fractions produced in parallel in the absence of Me 2SO,which did not contain any detectable PrP,presented the same low infectivity as the Me 2SO light fractions,suggest-ing that this residual infectivity was not due to the presence of Me 2SO-solubilized PrP 27–30molecules.We attribute the low infectivity present in both light fractions to the fact that the brain extracts were applied to the sucrose gradient from the top,and therefore some small prion aggregates,containing undetectable PrP 27–30,may not have sedimented.In this work,we investigated whether molecules other than protease-resistant PrP might have a physiological role in prion infectivity.To this effect,we combined the low infectious pro-tease-resistant Me 2SO-solubilized PrP described above with nonprotein components that remain in prion aggregates sub-sequent to denaturation and harsh protease digestion (NPH Sc ).1In some experiments we substituted the NPH Sc frac-*The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked “advertisement ”in accordance with 18U.S.C.Section 1734solely to indicate this fact.¶Supported by a grant from the Israel Center for the Study of Emerging Diseases.ʈSupported by grants from the European Community and by the Agnes Ginges Center for Human Neurogenetics.To whom correspond-ence should be addressed:Dept.of Neurology,Hadassah University Hospital,Jerusalem,Israel 91120.Fax:972-2-6429441;E-mail:gabizonr@.il.1The abbreviations used are:NPH,nonprotein heavy;NPH Sc ,NPH fraction originating from a scrapie-infected hamster brain;PK,protein-ase K;L,light;H,heavy;GndSCN,guanidium thiocyanate;H Sc ,heavy fraction originating from a scrapie-infected hamster brain;NPH N ,heavy fraction after deproteination,originating from a normal hamsterbrain;L Sc ,light fraction from a scrapie-infected hamster brain;L Sc Me 2SO,light fraction from a scrapie-infected hamster brain solubilized with Me 2SO.T HE J OURNAL OF B IOLOGICAL C HEMISTRYVol.276,No.17,Issue of April 27,pp.14324–14328,2001©2001by The American Society for Biochemistry and Molecular Biology,Inc.Printed in U.S.A.This paper is available on line at 14324at SHANGHAI JIAOTONG UNIVERSITY on March 21, 2016/Downloaded fromtion for heparan sulfate,a known component of prion rods.Our results show that the addition of the deproteinized sedimented fraction (NPH Sc )to low infectious solubilized protease-resis-tant PrP restores the prion infectivity to its original values.We therefore propose that in addition to PrP Sc ,prion infectivity may depend upon,or at least be largely facilitated by,the presence of other components of prion rods.EXPERIMENTAL PROCEDURESSucrose Gradients—Three hundred l of 10,15,20,25,30,and 60%sucrose in phosphate-buffered saline were loaded into centrifuge tubes adapted for the TLS-55rotor of the TL 100ultracentrifuge (Beckman Instruments)to form a zonal gradient.Normal or scrapie brain mem-branes (25l containing 15g/ml protein)were diluted with STE buffer (100m M NaCl,10m M Tris,pH 7.4,1m M EDTA)containing 2%sarkosyl to a final volume of 240l.When appropriate,Me 2SO (10%)was added to the brain extract and incubated for 16h at 4°C before the extract was loaded on top of the gradient and centrifuged in a TLS-55tube ultra-centrifuge (Beckman)at 55,000rpm (g av ϭ100,000)for 1h at 20°C.Before immunoblotting with anti-PrP monoclonal antibody 3F4(12),gradient samples of equal volume were collected and digested with proteinase K (PK,40g/ml)for 60min at 37°C.Sample Preparation—The three top fractions of each gradient were pooled and denominated fraction L (light).The three pooled bottom fractions (H,heavy)were tested either directly or after a deproteination treatment that included the following:1)denaturation with 4.5M gua-nidium thiocyanate (GndSCN)(final concentration)for 15min,2)pre-cipitation with methanol to wash out the GndSCN,3)resuspension in 2%sarkosyl STE buffer before digestion with PK (100g/ml for 60min at 37°C)to form NPH (non protein heavy)fractions.To form the combined fractions,original samples (L,NPH,or HS (heparan sulfate))were mixed at equal volumes and incubated for 16h at 4°C.Original samples as well as combinations (detailed in Fig.2)were assayed for the presence of PrP and infectivity.All volumes of original samples were adjusted before inoculation to contain the same concentration of L or NPH samples present in the mixtures.Heparan Sulfate—400l of the L Sc Me 2SOsample were incubated with bovine kidney heparan sulfate (Sigma)for 16h (4mg/ml)at 25°C.Theresulting fraction was denominated HS/L Sc Me 2SO.Heparanase Digestion—400l of the NPH Sc sample were precipi-tated in methanol and resuspended in STE buffer before the addition of 25units/ml of heparinase III (Sigma)for 16h at 37°C.The resulting sample was denominated NPH*.In Vivo Infectivity Experiments—Five male Syrian hamsters,4weeks old,were inoculated intracerebrally with 50l of each of the samples to be tested for prion infectivity.Animals were tested daily.Prion titers were measured by monitoring the incubation period until the appear-ance of symptoms (13).Before inoculation into hamsters,samples con-taining only L or H fractions were supplemented with 2%sarkosyl to retain similar concentrations of the components in the infectivity assay.Immunoblotting of Brain PrP Sc —10%(w/v)of brain tissuefromF IG .1.Solubilization of PrP 27–30by Me 2SO.a ,membranes from scrapie-infected brains extracted with 2%sarkosyl in the presence or absence of 10%Me 2SO or from normal brain extracted with sarkosyl only were subjected to a sucrose density gradient (10–60%)as described (11).Fractions collected from the gradients were digested in the pres-ence and absence of PK (40g/ml for 60min at 37°C)and immuno-blotted with monoclonal antibody 3F4.N ,normal;Sc,scrapie.b ,light fractions produced in the presence of Me 2SO (lane 1)were diluted several times (10-fold)with 2%ne 2,10-fold dilution;lane 3,100-fold dilution;lane 4,1000-fold dilution;lane 5,light fraction produced in the absence of Me 2SO.F IG .2.Outline of prion reconstitution experiment.The three top fractions of each gradient were pooled and denominated fraction L.The three pooled bottom fractions (H)were tested either directly or after a deproteination treatment that included the following:1,dena-turation with 4.5M GndSCN (final concentration)for 15min;2,precip-itation with methanol to wash out the GndSCN;3,resuspension in 2%sarkosyl STE buffer before digestion with PK (100g/ml for 60min at 37°C)to form NPH fractions.When applicable,NPH samples were digested by heparanase (25units/ml of heparinase III (Sigma)for 16h at 37°C).To form the combined fractions,original samples (L,NPH,or H)were mixed at equal volumes and incubated for 16h at 4°C.The HS concentration was 4mg/ml.Original samples as well as combinations (detailed in Fig.1)were assayed for the presence of PrP,infectivity,andstructure by electron microscopy.NPH Sc *,NPH Sc sample digested with heparanase;L N ,light fraction from a normal hamsterbrain.F IG .3.Protease-resistant PrP concentration and pattern in inoculum samples (a )and in infected animal brain samples (b ).a ,PrP immunoblot of 10-l samples from each inoculum.b ,immunoblot of 10-l samples of 10%scrapie hamster brain homogenates infectedwith the samples shown in a .Lanes:1,H Sc ;2,NPH Sc ;3,L Sc Me 2SO;4,L Sc ;5,NPH Sc /L Sc Me 2SO ;6,HS/L Sc Me 2SO ;7,NPH N /L Sc Me 2SO;8,NPH Sc /L Sc ;9,NPH Sc */L Sc Me 2SO.All samples were digested prior to immunoblotting with 40g/ml for 60min at 37°C.Reconstitution of Prion Infectivity 14325at SHANGHAI JIAOTONG UNIVERSITY on March 21, 2016/Downloaded fromscrapie-infected hamsters (frozen at Ϫ80°c following flash freezing in liquid nitrogen)was homogenized in cold sucrose buffer (10m M Tris,0.3M sucrose in phosphate-buffered saline).2%sarkosyl was added to 50-l samples before digestion with 40g/ml PK for 60min at 37°C.Statistical Analysis—To compare the study groups,Anova and the nonparametric Kruskal-Wallis test were applied.In addition,multiple pairwise comparisons were performed using the Dunnett and Scheffe methods.The tests were performed using the SPSS for Windows com-puter program.RESULTSBrain membranes from scrapie-infected and uninfected ham-sters were extracted with sarkosyl in the presence and absence of Me 2SO and,following an overnight incubation,applied to a 10–60%sucrose gradient as described (11).Gradient fractions were digested with PK and immunoblotted with ␣PrP mono-clonal antibody 3F4(Fig.1a ).As can be seen in the figure,the light fraction prepared in the presence of Me 2SO contained a considerable fraction of the total PrP 27–30.To assure that the concentration of PrP 27–30present in the light fractions ob-tained with Me 2SO was significantly higher than that present in the light fraction without Me 2SO,we immunoblotted with an anti-PrP antibody several 10-fold dilutions of the Me 2SO scrapie light fraction.As can be seen in Fig.1b ,the concentra-tion of PrP 27–30in the Me 2SO light fractions was at least 1000times larger than its concentration in the light fraction produced without Me 2SO.Fig.2presents the organization of the reconstitution exper-iments and the samples used for infectivity assays.The three first fractions (of 12),as well as the three last fractions of each sucrose gradient,were pooled and denominated L and H frac-tions,respectively.All gradient fractions were digested with 40g/ml PK at 37°C for 60min.Part of each H fraction was totally denatured with 4.5M GndSCN and,after methanol precipitation,digested again with PK to produce the NPH fraction.Subsequently,L and NPH fractions from different sources (normal and scrapie-infected with and without Me 2SO)were used to create the combined samples specified in Fig.2.All samples to be evaluated for infectivity were precipitated by methanol (to remove traces of Me 2SO)and resuspended into inoculation buffer (1%bovine serum albumin in phosphate-buffered saline)to contain PrP 27–30at comparable concentra-tions (Fig.3a ).No protease-resistant PrP was detected in the NPH Sc samples following the denaturation/protease digestion treatment (Fig.3a ,lane 2).PrP 27–30was also absent from the light samples produced in the absence of Me 2SO,as well as in the NPH Sc /L Sc sample (Fig.3a ,lanes 4and 8).When the bioassays were completed,we also tested the brains of the animals inoculated with each sample for the concentration and electrophoretic pattern of PrP after PK digestion (Fig.3b ).Although incubation times for the different samples varied widely (see Table I),the concentration of PrP 27–30,as well as the banding pattern of the protein,was the same regardless of the inocula administered to the hamsters.Histoblot analysis of all brains were also identical (data not shown).This suggests that the manipulations performed in this work did not produce a new prion strain.Although it was repeatedly shown that different strains of prions can be characterized by these param-eters (14–18),end point titration analyses are required to prove this point conclusively.All samples described in Fig.2(original and combined)were bioassayed for prion infectivity (Fig.4and Tables I and II).T ABLE ISummary of infectivity experimentsIn order to compare the study groups,Anova and the nonparametric Kruskal-Wallis test were applied.In addition,multiple pairwise comparisons were performed using the Dunnett and Scheffe methods.The tests were performed using the SPSS for Windows computer program.The sample abbreviations used are as described for Figs.2and 3.Number of trialsExperiment 1Experiment 2Experiment 3Total animalsIncubation (mean ϮS.E.)TiterdaysH Sc34(87.8Ϯ3.1)5(96.6Ϯ4.2)10(83.9Ϯ2.35)1988.0Ϯ2.1 6.7NPH Sc 35(not sick)5(not sick)5(not sick)15Not sick 0L ScMe 2SO 33(117.7Ϯ2.0)5(120.6Ϯ2.3)9(111.4Ϯ3.6)17115.2Ϯ2.2 4.0NPH Sc /L Sc Me 2SO35(90.6Ϯ3.1)5(94.6Ϯ2.6)5(80.8Ϯ0.9)1588.7Ϯ2.0 6.6NPH Sc /L N15(not sick)5Not sick NPH N /L Sc Me 2SO16(114Ϯ2.5)6114.0Ϯ2.5 4.1L Sc34(113.8Ϯ1.6)4(122.5Ϯ1.2)4(107.5Ϯ1.7)12114.6Ϯ2.0 4.0NPH Sc /L Sc 15(112.0Ϯ3.8)5112.0Ϯ3.8 4.2L Sc Me 2So /HS16(89.8Ϯ2.7)689.8Ϯ2.7 6.4NPH Sc */L ScMe 2SO25(95Ϯ3.4)5(109.2Ϯ3.3)10102.1Ϯ3.44.7T ABLE IISignificance of the variability among treatment groups [p values]Following ANOVA and the nonparametric Kruskal Wallis test showing a significant variability between treatment groups,post hoc multiple pairwise comparisons were performed in order to define the significance (p values)of the variability between treatment groups.Significant differences (asterisks)were calculated with the Dunnett method.Numerical values (in this case representing no difference between the samples)were calculated using the method of Scheffe for multiple comparison.H ScNPH ScL Sc Me 2SO NPH Sc /L ScMe 2SO NPH Sc /L NNPH N /L ScMe 2SOL Sc NPH Sc /L ScL Sc Me 2SO /HS NPH Sc */L ScMe 2SOH Sc**a** 1.000******** 1.000*NPH Sc ****** 1.000**********L ScMe 2SO ******** 1.000 1.0000.999***NPH Sc /L Sc Me 2SO1.000*********** 1.000*NPH Sc /L N** 1.000**************NPH N /L Sc Me 2SO**** 1.000**** 1.000 1.000**L Sc**** 1.000**** 1.000 1.000***NPH Sc /L Sc ****0.999*** 1.000 1.000*0.805L Sc Me 2SO /HS1.000**** 1.000*******NPH Sc */L ScMe 2SO*********0.805*a The mean difference is significant at the 0.001level.bThe mean difference is significant at the 0.05level.Reconstitution of Prion Infectivity14326 at SHANGHAI JIAOTONG UNIVERSITY on March 21, 2016/Downloaded fromTable I presents the individual and accumulative results of three infectivity experiments performed by intracerebellar in-oculation of Syrian hamsters with the samples described in Fig.2.Table II presents the significance of the variability among the treatment groups (p values).Although p Ͻ0.05(*)is considered significant enough in this kind of test,we also noted the extremely significant comparisons where p was smaller than 0.001(**).p values of 1or close to 1suggest similarity between samples.The disease incubation times for animals inoculated with similarly prepared samples in the different experiments were pooled in the general calculations because no statistically significant difference was found between them.We also calculated the titers (log ID 50)from the median of disease incubation time as described (13).However,because the accu-racy of titers calculated from disease incubation times (in days)is a debatable issue,we based all the statistical analyses di-rectly on the disease incubation times.A graphic representa-tion of the results can be seen in Fig.4.Whereas very high infectivity was present in the H Sc frac-tion,no infectivity whatsoever was observed when this fraction was first denatured with 4.5M GndSCN and then digested with PK,resulting in the NPH Sc fraction.These results indicate that the NPH Sc fraction cannot convert in vivo PrP C into PrP Sc ,because even after a long incubation time (more than 300days),no animals inoculated with these samples present any disease symptoms.Moreover,no traces of PrP 27–30were observed in their brains even after 300days (Fig.3a ),suggest-ing that no subclinical infection was established in these ani-mals.Preliminary experiments (data not shown)also suggest that NPH Sc cannot convert PrP C to PrP Sc in vitro ,because the brain inoculation of combined fractions containing NPH Sc and light fractions from normal hamsters without PK digestion (containing large quantities of PrP C )did not result in any disease symptoms or PrP Sc accumulation after more than 300days.As shown here and in our previous work (11),whereas sam-ples H Sc and L Sc Me 2SOcontained similar concentrations of PrP 27–30(Fig.3a ),only the aggregated fraction,H Sc ,presented high prion infectivity.This can be seen by comparing the titer,incubation time,and p values for both samples (Tables I and II).However,when the noninfectious NPH Sc was incubatedwith the L Sc Me 2SO fraction to form the NPH Sc /L Sc Me 2SO,infectivity was restored to the levels observed in the H Sc samples.This was not the case for the NPH Sc /L Sc combination,suggestingthat the presence of nonaggregated PrP 27–30in the L Sc Me 2SOwas essential for the restoration of prion infectivity.No increase in infectivity was detected when L Sc Me 2SOwas combined with NPH N .This may imply that a putative second prion component is not present in normal brain.Although it may be so,it is more probable that such a second component needs to combine with protease-resistant PrP in a specific fashion to sediment into the heavy fractions of the sucrose gradient in significant quantities.One of the candidate molecules for a prion second component is HS.This sugar polymer was found in brain amyloid deposits of Alzheimer’s disease as well as of prion diseases (19).In addition,sulfated sugars seem to have an important role in the metabolism of PrP Sc (20).Molecules such as pentosan sulfate and low molecular weight heparin have been shown repeatedly to inhibit the production of PrP Sc in scrapie-infected neuroblas-toma cells (ScN2a cells).HS itself has been shown to either increase or reduce PrP Sc accumulation in these cells,depending on the experimental setup (21–23).These molecules have also been shown to inhibit prion disease pathogenesis in vivo (24–26).This suggests that sulfated sugars of specific size and properties may either help form prions or disrupt prion forma-tion,probably depending on some kind of competition mechanism.To test whether one of the NPH Sc components is an HS-like molecule,we digested the NPH Sc fraction with heparanase in two of the reconstitution experiments,prior to its combinationwith the L Sc Me 2SOfraction.In the third experiment,we substi-tuted HS for NPH Sc .As can be seen in Fig.4,the combined results of these three experiments suggest a role for HS in prion infectivity.Whereas the infectivity of the reconstitutedsample containing heparanase-digested NPH Sc (NPH Sc */L Sc Me 2SO )was higher than that of the light fractions presentinglow infectivity,such as L Sc or L Sc Me 2SO,it was significantlylowerF IG .4.Bioassay of inoculated samples.Male Syrian hamsters,4weeks old,were inoculated intracerebrally with 50l of each of the samples to be tested for prion infectivity.Animals were tested daily.Prion titers were measured by monitoring the incubation period until the appearance of symptoms (13).a ,graphic representation of disease incubation time as described in Table I;b ,infectivity titers as de-scribed in Table I.Titers were calculated from incubation times as described (13).Reconstitution of Prion Infectivity14327at SHANGHAI JIAOTONG UNIVERSITY on March 21, 2016/Downloaded fromthan the infectivity of both the HSc and the NPHSc/LScMe2SOfractions that present high infectivity(see Table II for p val-ues).In addition,the HS/LScMe2SO combined sample showed aconsiderably higher infectivity than the LScMe2SO sample alone. In addition,the clinical features of the disease as well as theneuropathology of the animals infected with HS/LScMe2SO were identical to those of the classical263strain in Syrian hamsters(data not shown and Ref.27),suggesting that the HS/LScMe2SO sample did not contain a new prion strain.Extensive experiments are required to establish whether the effect of HS is unique or whether other sugar polymers,sul-fated or not,may serve as the backbone of prion rods.DISCUSSIONThe results presented here indicate that production of prion infectivity requires the presence both of protease-resistant PrP and of nonprotein components of prion rods and suggest that these components may well be sulfated sugar polymers.It remains to be established whether sulfated sugar polymers are indeed a fundamental component of prions or whether their function,although not essential,greatly facilitates prion prop-agation and the establishment of prion infection. Unsuccessful attempts to dissociate and reconstitute prion infectivity were performed years ago,even before PrP Sc was identified as a necessary components for infectivity(28).Prion infectivity could not yet be associated with protease-resistant PrP molecules produced by an array of in vitro conversion protocols(29–31)and has even been suggested to exist in the absence of detectable protease-resistant PrP in the inocula(32). In view of our results presented here,which suggest both prion components are required,we suggest that prion infectivity can be transmitted by a few(and therefore undetected)molecules of PrP Sc,if associated with the appropriate nonprotein compo-nents.Our results also open the way to a new line of in vitro conversion experiments,which may hopefully result in full in vitro production of prion infectivity.Whereas the function of PrP Sc as a template in the PrP C to PrP Sc conversion stands on solid grounds(33),the pathological role of a putative second component is unclear.A possible rolefor any functional molecule present in the NPHScfractions may be to anchor the PrP Sc molecules associated with it to the appropriate target in the host.Without a polymer such as HS, most PrP Sc molecules may be cleared from the brain before the PrP C to PrP Sc conversion reaction has been established in enough cells to establish a process of infection(34).The kind of sugar polymer used as rod backbone and anchor may play a role in modifying parameters of prion infectivity.The fact that sulfated sugar polymers such as HS may have a crucial function in prion structure and propagation suggests several plausible explanations of the fact that small sulfated sugar polymers such as pentosan sulfate were shown to inhibit the production of PrP Sc in cells(21,22).These molecules may compete with the sugar polymer functioning as prion compo-nent for the right cell targets to which the template PrP Sc molecules should be docked.Otherwise,these molecules may compete with the prion sugar component for the binding of newly formed PrP Sc molecules.Interestingly,polyamines were shown recently to inhibit PrP Sc accumulation from ScN2a cells (35).We suggest that these highly positively charged polymers may bind to the highly negatively charged sulfated sugars and thereby facilitate the clearance of newly formed and still non-aggregated protease-resistant PrP.The results presented here therefore 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