【精品文档】封印者资料-实用word文档 (9页)

ISTQB CertificationSample Question PapersCopyright © Abhishek Kumar TiwariAll rights reserved. No part of this material may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or by any information storage and retrieval system, without prior permission from the owner. Contact the owner for the information on foreign rights.Question Paper 1:1.An input field takes the year of birth between 1900 and 2004The boundary values for testing this field area.0,1900,2004,2005b.1900, 2004c.1899,1900,2004,2005d.1899, 1900, 1901,2003,2004,20052.Which one of the following are non-functional testing methods?a.System testingability testingc.Performance testingd.Both b & c3.Which of the following tools would be involved in the automation of regressiontest?a.Data testerb.Boundary testerc.Capture/Playbackd.Output comparator.4.Incorrect form of Logic coverage is:a.Statement Coverageb.Pole Coveragec.Condition Coveraged.Path Coverage5.Which of the following is not a quality characteristic listed in ISO 9126 Standard?a.Functionalityabilityc.Supportabilityd.Maintainability6.To test a function, the programmer has to write a _________, which calls thefunction to be tested and passes it test data.a.Stubb.Driverc.Proxyd.None of the above7.Boundary value testinga.Is the same as equivalence partitioning testsb.Test boundary conditions on, below and above the edges of input andoutput equivalence classesc.Tests combinations of input circumstancesd.Is used in white box testing strategy8.Pick the best definition of qualitya.Quality is job oneb.Zero defectsc.Conformance to requirementsd.Work as designed9.Fault Masking isa.Error condition hiding another error conditionb.Creating a test case which does not reveal a faultc.Masking a fault by developerd.Masking a fault by a tester10.One Key reason why developers have difficulty testing their own work is :ck of technical documentationck of test tools on the market for developersck of trainingck of Objectivity11.During the software development process, at what point can the test process start?a.When the code is complete.b.When the design is complete.c.When the software requirements have been approved.d.When the first code module is ready for unit testing12.In a review meeting a moderator is a person whoa.Takes minutes of the meetingb.Mediates between peoplec.Takes telephone callsd.Writes the documents to be reviewed13.Given the Following programIF X < YTHEN Statement 1;ELSE IF Y >= ZTHEN Statement 2;ENDMcCabe’s Cyclomatic Complexity is :a. 2b. 3c. 4d. 514.How many test cases are necessary to cover all the possible sequences ofstatements (paths) for the following program fragment? Assume that the twoconditions are independent of each other : -…………if (Condition 1)then statement 1else statement 2fiif (Condition 2)then statement 3fi…………a.Test Casesb. 3 Test Casesc. 4 Test Casesd.Not achievable15. Acceptance test cases are based on what?a.Requirementsb.Designc.Coded.Decision table16. “How much testing is enough?”a.This question is impossible to answerb.This question is easy to answerc.The answer depends on the risk for your industry, contract and specialrequirementsd.This answer depends on the maturity of your developers17. A common test technique during component test isa.Statement and branch testingability testingc.Security testingd.Performance testing18.Statement Coverage will not check for the following.a.Missing Statementsb.Unused Branchesc.Dead Coded.Unused Statement19.Independent Verification & Validation isa.Done by the Developerb.Done by the Test Engineersc.Done By Managementd.Done by an Entity Outside the Project’s sphere of influence20.Code Coverage is used as a measure of what ?a.Defectsb.Trends analysisc.Test Effectivenessd.Time Spent TestingQuestion Paper 2:Q2Regression testing should be performed:v) every week w) after the software has changed x) as often as possible y) when the environment has changed z) when the project manager says NOTE: Only one answer per question Q1A deviation from the specified or expected behaviour that is visible to end-users is called:a) an error b) a faulta) v & w are true, x, y & z are false b) w, x & y are true, v & z are false c) w & y are true, v, x & z are false d) w is true, v, x, y & z are falseQ3IEEE 829 test plan documentation standard contains all of the following except c) a failure d) a defecta) test items b) test deliverables c) test tasks d) test specificationsQ4When should testing be stopped?a) when all the planned tests have been run b) when time has run outc) when all faults have been fixed correctlyd) it depends on the risks for the system being testedQ5Order numbers on a stock control system can range between 10000 and 99999 inclusive. Which of the following inputs might be a result of designing tests for only valid equivalence classes and valid boundaries?a) 1000, 50000, 99999 b) 9999, 50000, 100000 c) 10000, 50000, 99999 d) 10000, 99999, 100000Q6 Consider the following statements about early test design:i. early test design can prevent fault multiplicationii. faults found during early test design are more expensive to fixiii. early test design can find faultsiv. early test design can cause changes to the requirementsv. early test design normally takes more efforta) i, iii & iv are true; ii & v are falseb) iii & iv are true; i, ii & v are falsec) i, iii, iv & v are true; ii is falsed) i & ii are true; iii, iv & v are falseQ7 Non-functional system testing includes:a) testing to see where the system does not function correctlyb) testing quality attributes of the system including performance and usabilityc) testing a system function using only the software required for that functiond) testing for functions that should not existQ8 Which of the following is NOT part of configuration management?a) auditing conformance to ISO 9000b) status accounting of configuration itemsc) identification of test versionsd) controlled library accessQ9Which of the following is the main purpose of the integration strategy for integration testing in the small?a) to ensure that all of the small modules are tested adequatelyb) to ensure that the system interfaces to other systems and networksc) to specify which modules to combine when, and how many at onced) to specify how the software should be divided into modulesQ10 What is the purpose of a test completion criterion?a) to know when a specific test has finished its executionb) to ensure that the test case specification is completec) to set the criteria used in generating test inputsd) to determine when to stop testingQ11 Consider the following statements:i. an incident may be closed without being fixed.ii. incidents may not be raised against documentation.iii. the final stage of incident tracking is fixing.iv. the incident record does not include information on test environments.a) ii is true, i, iii and iv are falseb) i is true, ii, iii and iv are falsec) i and iv are true, ii and iii are falsed) i and ii are true, iii and iv are falseQ12 Given the following code, which statement is true about the minimum number of test cases required for full statement and branch coverage?Read pRead qIF p+q > 100 THENPrint"Large"ENDIFIF p > 50 THENPrint "p Large"ENDIFa) 1 test for statement coverage, 3 for branch coverageb) 1 test for statement coverage, 2 for branch coveragec) 1 test for statement coverage, 1 for branch coveraged) 2 tests for statement coverage, 2 for branch coverageQ13 Consider the following statements:i. 100% statement coverage guarantees 100% branch coverage.ii. 100% branch coverage guarantees 100% statement coverage.iii. 100% branch coverage guarantees 100% decision coverage.iv. 100% decision coverage guarantees 100% branch coverage.v. 100% statement coverage guarantees 100% decision coverage.a) ii is True; i, iii, iv & v are Falseb) i & v are True; ii, iii & iv are Falsec) ii & iii are True; i, iv & v are Falsed) ii, iii & iv are True; i & v are FalseQ14 Functional system testing is:a) testing that the system functions with other systemsb) testing that the components that comprise the system function togetherc) testing the end to end functionality of the system as a wholed) testing the system performs functions within specified response timesQ15 Incidents would not be raised against:a) requirementsb) documentationc) test casesd) improvements suggested by usersQ16 Which of the following items would not come under Configuration Management?a) operating systemsb) test documentationc) live datad) user requirement documentsQ17 Maintenance testing is:a) updating tests when the software has changedb) testing a released system that has been changedc) testing by users to ensure that the system meets a business needd) testing to maintain business advantageQ18 What can static analysis NOT find?a) the use of a variable before it has been definedb) unreachable (“dead”) codec) memory leaksd) array bound violationsQ19 Which of the following techniques is NOT a black box technique?a) state transition testingb) LCSAJc) syntax testingd) boundary value analysisQ20 Beta testing is:a) performed by customers at their own siteb) performed by customers at the software developer's sitec) performed by an Independent Test Teamd) performed as early as possible in the lifecycleQ21 Given the following types of tool, which tools would typically be used by developers, and which by an independent system test team?analysisi. statictestingii. performancemanagementiii. testanalysisiv. dynamica) developers would typically use i and iv; test team ii and iiib) developers would typically use i and iii; test team ii and ivc) developers would typically use ii and iv; test team i and iiid) developers would typically use i, iii and iv; test team iiQ22 The main focus of acceptance testing is:a) finding faults in the systemb) ensuring that the system is acceptable to all usersc) testing the system with other systemsd) testing from a business perspectiveQ23 Which of the following statements about component testing is FALSE?a) black box test design techniques all have an associated test measurement techniqueb) white box test design techniques all have an associated test measurement techniquec) cyclomatic complexity is not a test measurement techniqued) black box test measurement techniques all have an associated test design techniqueQ24 Which of the following statements is NOT true?a) inspection is the most formal review processb) inspections should be led by a trained leaderc) managers can perform inspections on management documentsd) inspection is appropriate even when there are no written documentsQ25 A typical commercial test execution tool would be able to perform all of the following, EXCEPT:a) calculating expected outputsb) comparison of expected outcomes with actual outcomesc) recording test inputsd) reading test values from a data fileQ26 The difference between re-testing and regression testing is:a) re-testing ensures the original fault has been removed; regression testing looks forunexpected side-effectsb) re-testing looks for unexpected side-effects; regression testing ensures the original fault hasbeen removedc) re-testing is done after faults are fixed; regression testing is done earlierd) re-testing is done by developers; regression testing is done by independent testersQ27 Expected results are:a) only important in system testingb) only used in component testingc) most useful when specified in advanced) derived from the codeQ28 What type of review requires formal entry and exit criteria, including metrics:a) walkthroughb) inspectionc) management reviewd) post project reviewQ29 Which of the following uses Impact Analysis most?a) component testingb) non-functional system testingc) user acceptance testingd) maintenance testingQ30 What is NOT included in typical costs for an inspection process?a) setting up forms and databasesb) analysing metrics and improving processesc) writing the documents to be inspectedd) time spent on the document outside the meetingQ31 Which of the following is NOT a reasonable test objective:a) to find faults in the softwareb) to prove that the software has no faultsc) to give confidence in the softwared) to find performance problemsQ32 Which expression best matches the following characteristics of the review processes:1. led by the author2. undocumented3. no management participation4. led by a moderator or leader5. uses entry and exit criterias) inspectiont) peer reviewu) informal reviewv) walkthrougha) s = 4 and 5, t = 3, u = 2, v = 1b) s = 4, t = 3, u = 2 and 5, v = 1c) s = 1 and 5, t = 3, u = 2, v = 4d) s = 4 and 5, t = 1, u= 2, v = 3Q33 Which of the following is NOT part of system testing?a) business process-based testingb) performance, load and stress testingc) usability testingd) top-down integration testingQ34 Which statement about expected outcomes is FALSE?a) expected outcomes are defined by the software's behaviourb) expected outcomes are derived from a specification, not from the codec) expected outcomes should be predicted before a test is rund) expected outcomes may include timing constraints such as response timesQ35 The standard that gives definitions of testing terms is:a) ISO/IEC 12207b) BS 7925-1c) ANSI/IEEE 829d) ANSI/IEEE 729Q36 The cost of fixing a fault:a) is not importantb) increases the later a fault is foundc) decreases the later a fault is foundd) can never be determinedQ37 Which of the following is NOT included in the Test Plan document of the Test Documentation Standard?a) what is not to be testedb) test environment propertiesc) quality plansd) schedules and deadlinesQ38 Could reviews or inspections be considered part of testing?a) no, because they apply to development documentationb) no, because they are normally applied before testingc) yes, because both help detect faults and improve qualityd) yes, because testing includes all non-constructive activitiesQ39 Which of the following is not part of performance testing?a) measuring response timesb) recovery testingc) simulating many usersd) generating many transactionsQ40 Error guessing is best used:a) after more formal techniques have been appliedb) as the first approach to deriving test casesc) by inexperienced testersd) after the system has gone liveQuestion Paper 3:1. What is failure?A. Deviation from expected result to actual resultB. Defect in the software.C. Error in the program code.D. Fault in the system.2. People who don’t participate in technical reviewsA. AnalystsB. ManagementC. DevelopersD. Testers3. What type of testing is done to supplement the rigorous testing?A. Regression testing.B. Integration testing.C. Error GuessingD. System testing.4. Capture and replay facilities are least likely to be used to ….A. Performance testingB. Recovery testingC. GUI testingD. User requirements.5. What is the smallest number of test cases required toProvide 100% branch coverage?If(x>y) x=x+1; else y=y+1;while(x>y){y=x*y; x=x+1;}A. 1B. 2C. 3D. 46. Cyclomatic complexity is used to calculateA. number of independent paths in the basis set of a programB. number of binary decisions + 1C. upper bound for the number of tests that must be conducted to ensure that all statements have been executed at least onceD. number of branches and decisions7. If a candidate is given an exam of 40 questions, should get 25 marks to pass (61%) and should get 80% for distinction, what is equivalence class.A. 23, 24, 25B. 0, 12, 25C. 30, 36, 39D. 32,37,408. Match the following:1. Test estimation2. Test control3. Test monitoringa. measures of tracking processb. effort required to perform activitiesc. reallocation of resourcesA. 1-b, 2-c, 3-aB. 1-b, 2-a, 3-cC. 1-c, 2-a, 3-bD. 1-a, 2-b, 3-c9. One of the following is not a part of white box testing asper BS7925-II standards.A. Random testingB. Data Flow testing.C. Statement testing.D. Syntax testing.10. Exclusive use of white box testing in a test-phase will:A. Ensure the test item is adequately tested.B. Make the need for black-box testing redundant.C. Run the risk that the requirements are not satisfied.D. Suffices for the unit testing phase.11. Match the following.1. Configuration identification2. Configuration control3. Status reporting4. Configuration auditinga. Maintains of CI’s in a libraryb. Checks on the contents of the libraryc. Function recording and tracking problems.d. Requires the all CI’s and their versions in the systemare knownA. 1-d, 2-c, 3-d, 4-a.B. 1-d, 2-a, 3-c, 4-b.C. 1-a, 2-b, 3-d, 4-c.D. 1-c, 2-b, 3-a, 4-d.12. Cost of the reviews will not include.A. Review process itselfB. Metrics analysisC. Tool support.D. Process improvement.13. What type of testing will you perform on internet banking solution?A. System integrationB. Functional testingC. Non-functional testing.D. Requirements testing14. Which tool will be used to test the flag memory leaks and unassigned pointersA. Dynamic analysis toolB. Static Analysis tool.C. Maintenance tool.D. Configuration tool.15. Which of the following is not included in Test Plan.A. Features to be tested.B. Environmental needs.C. Suspension criteria.D. Expected results.16. A piece of software has been given….what tests in the Following will you perform?1) Test the areas most critical to business processes2) Test the areas where faults will be maximum3) Test the easiest functionalitiesA. 1&2 are true and 3 is false.B. 1,2&3 are true.C. 1 is true, 2&3 are false.D. 1&2 are false, 3 is true.17. Amount of testing performed will not depend onA. Risks involvedB. Contractual requirementsC. Legal requirementsD. Test data.18. Which of the following provides the biggest potential cost saving from use of CAST?A. Test managementB. Test designC. Test planningD. Test execution19. Testing is not done to ….A. Find faultsB. Improve qualityC. Check user friendliness.D. Improve software accuracy20. Software quality is not relevant to …A. CorrectnessB. UsabilityC. ViabilityD. Reusability.21. Which of the following are false?A. Incidents should always be investigated and resolved.B. Incidents occur when expected and actual results differ.C. Incidents can be analyzed to assist in test process improvement.D. An incident can be raised against documentation.22. Which of the following is a type of non-functional testing?A. Usability testing.B. Statement Coverage.C. Dataflow testing.D. Cause-effect graphing.23. To make a test effective it is most important that:A. It is easy to execute.B. It is designed to detect faults if present.C. The expected outcome is specified before execution.D. It is unlikely to delay progress.24. Error guessing is:A. An appropriate way of deriving system tests.B. Only used if good requirements are not available.C. Only used when good requirements are available.D. The most appropriate way of deriving system tests.25. A standard for software testing terminology is:A. IEEE 802.11B. ISO 9001C. BS 7925-1D. BS 7925-226. Which of the following is true of V-model?A. It includes the verification of designs.B. It states that modules are tested against user requirements.C. It specifies the test techniques to be used.D. It only models the testing phase.27. Which of the following is NOT part of a high level test plan?A. Functions not to be tested.B. Environmental requirements.C. Analysis of Specifications.D. Entry and Exit criteria.28. When do you stop testing?A. When the specified number of faults are found.B. When the test completion criteria are met.C. When all high and medium priority tests are complete.D. When all statements have been executed.29. Which of the following is least important in test management?A. Estimating test duration.B. Incident Management.C. Configuration Management.D. De-bugging.30. How would you estimate the amount of re-testing likely to be required?A. Metrics from previous similar projects.B. Discussions with the development team.C. Time allocated for regression testing.D. Both A & B.31. Which of the following statements is true of static analysis:A. Compiling code is not a form of static analysis.B. Static analysis need not be performed before imperative code is executed.C. Static analysis can find faults that are hard to find withdynamic testing.D. Extensive statistic analysis will not be needed if white-Box testing is to be performed.32. Regression testing always involvesA. Testing whether a known software fault been fixed.B. Executing a large number of different tests.C. Testing whether modifications have introduced adverseside effects.D. Using a test automation tool.33. A field failure occurs when multiple users access a system.Which of the following is true?A. This is an acceptable risk of a multi-user system.B. Insufficient functional testing has been performed.C. This indicates an important non-functional requirement was not specified and tested.D. It is not possible to test against such events prior to release.34. Integration testing in the large involves:A. Testing the system when combined with other systems.B. Testing a sub-system using stubs and drivers.C. Testing a system with a large number of users.D. Combing software components and testing them in onego.35. Data flow analysis studies:A. How rapidly data is transferred through a program.B. The rate of change of data values as a program executes.C. The use of data on paths through the code.D. The intrinsic complexity of the code.36. The oracle assumption is that:A. There is some existing system against which test outputmay be checked.B. The tester knows everything about the software undertest.C. The tester can routinely identify the correct outcome ofa test.D. Tools are used to check the results of testing.36 The following text will be used in Q.37 and Q.38. In a system designed to work out the tax to be paid:An employee has $4000 of salary tax freeThe next $1500 is taxed at 10%The next $28000 is taxed at 22%Any further amount is taxed at 40%37. To the nearest $ which of these is a valid Boundary Value Analysis test case?A. $1500B. $32001C. $28000D. $3350138. Which of these groups of numbers would fall into the same equivalence class?A. $5800; $28000; $32000B. $0; $200; $4200C. $5200; $5500; $28000D. $28001; $32000; $3500039. Which of the following is NOT a characteristic of User Acceptance Testing?A. Use of automated test execution tools.B. Testing performed by users.C. Testing against acceptance test criteria.D. Integration of system with user documentation.40. For software to be reliable it must:A. Be easy to maintain.B. Be unlikely to cause a failure.C. Never fail under any circumstances.D. Be written according to coding standardsQuestion Paper 4:1 We split testing into distinct stages primarily because:a) Each test stage has a different purpose.b) It is easier to manage testing in stages.c) We can run different tests in different environments.d) The more stages we have, the better the testing.2 Which of the following is likely to benefit most from the use of test tools providing test capture and replay facilities?a) Regression testingb) Integration testingc) System testingd) User acceptance testing3 Which of the following statements is NOT correct?a) A minimal test set that achieves 100% LCSAJ coverage will also achieve 100% branch coverage.b) A minimal test set that achieves 100% path coverage will also achieve 100% statement coverage.c) A minimal test set that achieves 100% path coverage will generally detect more faults than one that achieves 100% statement coverage.d) A minimal test set that achieves 100% statement coverage will generally detect more faults than one that achieves 100% branch coverage.4 Which of the following requirements is testable?a) The system shall be user friendly.b) The safety-critical parts of the system shall contain 0 faults.c) The response time shall be less than one second for the specified design load.d) The system shall be built to be portable.5 Analyse the following highly simplified procedure:Ask: “What type of ticket do you require, single or return?”IF the customer wants ‘return’Ask: “What rate, Standard or Cheap-day?”IF the customer replies ‘Cheap-day’Say: “That will be £11:20”ELSESay: “That will be £19:50”ENDIFELSESay: “That will be £9:75”ENDIFNow decide the minimum number of tests that are needed to ensure that allthe questions have been asked, all combinations have occurred and allreplies given.a) 3b) 4c) 5d) 66 Error guessing:a) supplements formal test design techniques.b) can only be used in component, integration and system testing.c) is only performed in user acceptance testing.d) is not repeatable and should not be used.7 Which of the following is NOT true of test coverage criteria?a) Test coverage criteria can be measured in terms of items exercised by a test suite.b) A measure of test coverage criteria is the percentage of user requirements covered.c) A measure of test coverage criteria is the percentage of faults found.d) Test coverage criteria are often used when specifying test completion criteria.8 In prioritising what to test, the most important objective is to:a) find as many faults as possible.b) test high risk areas.c) obtain good test coverage.d) test whatever is easiest to test.9 Given the following sets of test management terms (v-z), and activity descriptions (1-5), which one of the following best pairs the two sets?v – test controlw – test monitoringx - test estimationy - incident managementz - configuration control1 - calculation of required test resources2 - maintenance of record of test results3 - re-allocation of resources when tests overrun4 - report on deviation from test plan5 - tracking of anomalous test resultsa) v-3,w-2,x-1,y-5,z-4b) v-2,w-5,x-1,y-4,z-3c) v-3,w-4,x-1,y-5,z-2d) v-2,w-1,x-4,y-3,z-510 Which one of the following statements about system testing is NOT true?a) System tests are often performed by independent teams.b) Functional testing is used more than structural testing.c) Faults found during system tests can be very expensive to fix.d) End-users should be involved in system tests.11 Which of the following is false?。

The presence of these two patterns in both humans and mouse suggests their importance in the evolution of mammalian X chromo-somes.Our sample of functional retroposed genes in the mammalian genomes is likely at least an order of magnitude smaller than the actual number(10,11).Notably,our analyses exclude retrocopies maintaining introns,such as partially processed retrogenes(35)or chi-meric genes(36),which would implicate even more genes.Finally,other mechanisms of interchromosomal gene movement are also likely influenced by the aforementioned se-lective forces.Thus,we expect many more genes to be subject to the gene traffic de-scribed herein.To elucidate the age of retrogene move-ments,we dated the human duplications involv-ing X-linked parents or retrogenes both by comparison to the mouse genome sequence and by sequence divergence analysis(16).Most copies that escape X linkage(12/15)as well as most copies that obtain X linkage(10/13)orig-inated before the human-mouse split(Fig.2, tables S7and S8).Duplicates in the mouse genome show the same pattern,consistent with this notion.Thus,both patterns result from an-cient evolutionary forces common to eutherian mammals.However,this process appears to be an ongoing characteristic of eutherian X evolu-tion,because6/28events have occurred subse-quent to the human-mouse split in the human lineage,6/33retropositions have occurred with-in the pastϳ80million years in the mouse lineage,and some of these retroduplicate pairs have high sequence similarity(Ͼ95%)at syn-onymous sites.This chromosome-biased gene origination appears to be an important process actively driving the differentiation of the X chromosome in mammals and suggests that this differentiation is still in progress.References and Noteshn,N.M.Pearson,K.Jegalian,Nature Rev.Genet.2,207(2001).2.H.Skaletsky et al.,Nature423,825(2003).3.J.A.Marshall Graves et al.,Cytogenet.Genome Res.96,161(2002).4.J.R.McCarrey,Bioscience44,20(1994).5.M.J.Lercher,A.O.Urrutia,L.D.Hurst,Mol.Biol.Evol.20,1113(2003).6.P.J.Wang,J.R.McCarrey,F.Yang,D.C.Page,NatureGenet.27,422(2001).7.J.C.Venter et al.,Science291,1304(2001).8.B.Lewin,Genes VII(Oxford University Press,NewYork,2000).9.E.Betran,K.Thornton,M.Long,Genome Res.12,1854(2002).10.P.M.Harrison et al.,Genome Res.12,272(2002).11.L.Z.Strichman-Almashanu,M.Bustin,ndsman,Genome Res.13,800(2003).12.E.Betra´n,W.Wang,L.Jin,M.Long,Mol.Biol.Evol.19,654(2002).13.J.Brosius,Science251,753(1991).nder et al.,Nature409,860(2001).15.T.Hubbard et al.,Nucleic.Acids.Res.30,38(2002).16.Materials and methods are available as supportingmaterial on Science Online.17.R.H.Waterston et al.,Nature420,520(2002).18.Z.Zhang,P.Harrison,M.Gerstein,Genome Res.12,1466(2002).19.C.-I.Wu,E.Y.Xu,Trends Genet.19,243(2003).20.C.Richler,H.Soreq,J.Wahrman,Nature Genet.2,192(1992).21.H.H.Dahl et al.,Genomics8,225(1990).22.D.J.Elliott et al.,Hum.Mol.Genet.9,2117(2000).23.M.Taira et al.,J.Biol.Chem.265,16491(1990).24.B.Dass et al.,J.Biol.Chem.276,8044(2001).25.M.Parisi et al.,Science299,697(2003).26.J.M.Ranz,C.I.Castillo-Davis,C.D.Meiklejohn,D.L.Hartl,Science300,1742(2003).27.V.Reinke et al.,Mol.Cell6,605(2000).28.T.Fujii et al.,EMBO Rep.3,367(2002).29.A.Kong et al.,Nature Genet.31,241(2002).30.H.A.Wichman,Genetica86,287(1992).ngley et al.,Genet.Res.52,223(1988).32.J.A.Bailey,L.Carrel,A.Chakravarti,E.E.Eichler,Proc.Natl.Acad.Sci.U.S.A.97,6634(2000).33.Z.Gu,H.Wang,A.Nekrutenko,W.H.Li,Gene259,81(2000).34.B.Charlesworth,J.A.Coyne,N.H.Barton,Am.Nat.130,113(1987).35.M.B.Soares et al.,Mol.Cell.Biol.5,2090(1985).36.A.Courseaux,J.-L.Nahon,Science291,1293(2001).37.We thank K.Thornton,Y.Chen,T.Nagylaki,C.-I.Wu,J.Spofford,B.L.Sidlauskas,T.M.Martin,and twoanonymous reviewers for helpful comments.Sup-ported by grants NSF Career MCB-0238168and NIHGM-065429-01A1;a Packard Fellowship(to M.L.),anNSF Predoctoral Fellowship(to J.J.E.),an EmmyNoether fellowship from the Deutsche Forschungs-gemeinschaft(to H.K.);and the startup funds(to E.B.)from University of Texas at Arlington.Supporting Online Material/cgi/content/full/303/5657/537/DC1Materials and MethodsTables S1to S9Fig.S1References4August2003;accepted26November2003A Map of the Interactome Networkof the Metazoan C.elegansSiming Li,1*Christopher M.Armstrong,1*Nicolas Bertin,1*Hui Ge,1*Stuart Milstein,1*Mike Boxem,1*Pierre-Olivier Vidalain,1*Jing-Dong J.Han,1*Alban Chesneau,1,2*Tong Hao,1Debra S.Goldberg,3Ning Li,1Monica Martinez,1Jean-Franc¸ois Rual,1,4Philippe Lamesch,1,4Lai Xu,5†Muneesh Tewari,1Sharyl L.Wong,3Lan V.Zhang,3Gabriel F.Berriz,3Laurent Jacotot,1‡Philippe Vaglio,1‡Je´roˆme Reboul,1§Tomoko Hirozane-Kishikawa,1Qianru Li,1Harrison W.Gabel,1Ahmed Elewa,1ሻBridget Baumgartner,5Debra J.Rose,6Haiyuan Yu,7Stephanie Bosak,8Reynaldo Sequerra,8Andrew Fraser,9Susan E.Mango,10 William M.Saxton,6Susan Strome,6Sander van den Heuvel,11Fabio Piano,12Jean Vandenhaute,4Claude Sardet,2Mark Gerstein,7Lynn Doucette-Stamm,8Kristin C.Gunsalus,12 J.Wade Harper,5†Michael E.Cusick,1Frederick P.Roth,3David E.Hill,1¶Marc Vidal1¶#To initiate studies on how protein-protein interaction(or“interactome”)networksrelate to multicellular functions,we have mapped a large fraction of the Caeno-rhabditis elegans interactome network.Starting with a subset of metazoan-specificproteins,more than4000interactions were identified from high-throughput,yeasttwo-hybrid(HTϭY2H)screens.Independent coaffinity purification assays exper-imentally validated the overall quality of this Y2H data set.Together with alreadydescribed Y2H interactions and interologs predicted in silico,the current version ofthe Worm Interactome(WI5)map containsϳ5500interactions.Topological andbiological features of this interactome network,as well as its integration withphenome and transcriptome data sets,lead to numerous biological hypotheses.To further understand biological processes,it isimportant to consider protein functions in thecontext of complex molecular networks.Thestudy of such networks requires the availabilityof proteome-wide protein-protein interaction,or“interactome,”maps.The yeast Saccharomy-ces cerevisiae has been used to develop a eu-karyotic unicellular interactome map(1–6).Caenorhabditis elegans is an ideal model forstudying how protein networks relate to multi-cellularity.Here we investigate its interactomenetwork with HT-Y2H.As Y2H baits,we selected a set of3024worm predicted proteins that relate directly orindirectly to multicellular functions(7).Gateway-cloned open reading frames(ORFs)were available in the C.elegans ORFeome1.1(8)for1978of these selected proteins.Ofthese,81autoactivated the Y2H GAL1::HIS3reporter gene as Gal4DNA binding domainfusions(DB-X),and24others conferred tox-icity to yeast cells.The remaining1873baitswere screened against two different Gal4ac-tivation domain libraries(AD-wrmcDNA andAD-ORFeome1.0),each with distinct,yet complementary,advantages(7).We maximized the specificity of the Y2H system by applying stringent experimental and bioinformatics criteria(fig.S1).To eliminate interactions that originated from nonspecific promoter activation,we only considered DB-X–AD-Y pairs if they activated at least two out of three different Gal4-responsive promoters. Positives were subsequently retested in fresh yeast cells,and their AD-Y identities were de-termined with interaction sequence tags(ISTs) obtained by sequencing the corresponding polymerase chain reaction(PCR)products(9). The AD-Y reading frame was verified for each IST to avoid the recovery of out-of-frame pep-tides.In total,ϳ16,000ISTs were obtained.Having applied those criteria,we subdivided the interactions into three confidence classes (fig.S1):those that were found at least three times independently and for which the AD-Y junction is in frame(“Core-1,”858interactions); those in frame found fewer than three times and that passed the retest(“Core-2,”1299interac-tions);and all other Y2H interactions found inour screens(“Non-Core,”1892interactions).The Core data set(Core-1and Core-2)contains2157high-confidence interactions between502DB-X baits and1039AD-Y preys.After col-lapsing22interactions that occur in both DB-X–AD-Y and DB-Y–AD-X configurations,a totalof2135unique interactions are obtained(tableS1).The Non-Core data set contains1892inter-actions between531DB-X baits and1395AD-Y preys.Altogether,Core and Non-Coreconstitute the“First-Pass”data set,with a total of4027distinct interactions.Out of2783and1505interactions found with AD-wrmcDNA and AD-ORFeome1.0,respectively,239interactionswere identified with both libraries.To estimate the coverage of the HT-Y2Hdata sets,we manually searched the baitsscreened here for known interactors inWormPD(10).This search gave rise to108interactions,referred to as the“literature”data set(table S1).The Core and Non-Coredata sets recapitulated eight and two interac-tions in this benchmark data set,respectively.Thus,our overall rate of coverage for theFirst-Pass data set isϳ10%[(8ϩ2)/108)].To evaluate the accuracy of the HT-Y2H datasets,we reasoned that interactions detected in twodifferent binding assays are unlikely to be exper-imental false-positives.A representative sample ofY2H interaction pairs from each of these threesubsets(33for Core-1,62for Core-2,and48forNon-Core)was randomly selected,and tested in acoaffinity purification(co-AP)glutathioneS-transferase(GST)pull-down assay(Fig.1).Baitand prey ORFs were transiently transfected into293T cells as GST-bait and Myc-prey fusions,respectively.For potential interaction pairs whereboth proteins were expressed at detectable levels,the co-AP success rates were14out of17(82%)for Core-1,17out of29(59%)for Core-2,and8out of23(35%)for Non-Core(table S2).Thesedata demonstrate that our three data sets contain alarge proportion of highly reliable interactions andcorroborate their expected relative qualities.In addition to experimental screens,we alsoperformed in silico searches for potentially con-served interactions,or“interologs,”whose or-thologous pairs are known to interact in one ormore other species(9,11).Starting from ahigh-confidence yeast interaction data set(7),reciprocal best-hit BLAST searches(E-valueՅ10Ϫ6)were performed against the worm pre-dicted proteome.In all,949potential worminterologs were identified,constituting the in-terologs data set(7).In addition,the Y2Hinteractome maps that have been previouslygenerated for individual biological processes(including vulval development,protein degra-dation,DNA damage response,and germlineformation)(9,12–14)were pooled to define the“scaffold”data set.The HT-Y2H,literature,interologs,and scaffold data sets were com-bined into Worm Interactome version5(WI5),containing5534interactions and connecting15%of the C.elegans proteome(table S1).WI5gives rise to a giant network component of2898nodes connected by5460edges(Fig.2A).Similar to other biological networks(15),theworm interactome network exhibits small-world and scale-free properties(Fig.2B)(7).This data set also allowed us to analyze whetheror not evolutionary recent proteins tend topreferentially interact with each other ratherthan with ancient proteins.We subdivided thenodes of the network into three classes:748proteins with a clear ortholog in yeast(“an-cient”),1314proteins with a clear ortholog inDrosophila,Arabidopsis,or humans but not inyeast(“multicellular”),and836proteins withno detectable ortholog outside of C.elegans(“worm”)(7).These three groups seem to con-nect equally well with each other(Fig.2C),whichsuggests that new cellular functions rely on acombination of evolutionarily new and ancientelements,consonant with the classic proposal ofevolution as a tinkerer that modifies and adds topre-existing structures to create new ones(16).Previous studies have related interactomedata with genome-wide expression(transcrip-1Dana-Farber Cancer Institute and Department of Genetics,Harvard Medical School,44Binney Street, Boston,MA02115,USA.2Institut de Ge´ne´tique Mo-le´culaire,Centre National de la Recherche Scienti-fique UMR5535,1919Route de Mende,34293Mont-pellier Cedex5,France.3Department of Biological Chemistry and Molecular Pharmacology,Harvard Medical School,250Longwood Avenue,Boston,MA 02115,USA.4Unite´de Recherche en Biologie Mole´cu-laire,Faculte´s Notre-Dame de la Paix,61Rue de Bruxelles,5000Namur,Belgium.5Verna and Marrs Department of Biochemistry and Molecular Biology, Program in Cell and Molecular Biology,Biophysics, and Biology,Baylor College of Medicine,One Baylor Plaza,Houston,TX77030,USA.6Department of Bi-ology,Indiana University,Jordan Hall142,1001East Third Street,Bloomington,IN47405,USA.7Depart-ment of Molecular Biophysics and Biochemistry and Department of Computer Science,Yale University, 266Whitney Avenue,New Haven,CT06520,USA. 8Agencourt Bioscience Corporation,100Cummings Center,Suite107G,Beverly,MA01915,USA.9The Wellcome Trust Sanger Institute,Wellcome Trust Ge-nome Campus,Hinxton,Cambridge,CB101SA,UK. 10Huntsman Cancer Institute,University of Utah, 2000Circle of Hope,Salt Lake City,UT84112,USA.11Massachusetts General Hospital Cancer Center, Building149,13th Street,Charlestown,MA02129, USA.12Department of Biology,New York University, 1009Silver Building,100Washington Square East, New York,NY10003,USA.*These authors contributed equally to this work.†Present address:Department of Pathology,Harvard Medical School,200Longwood Avenue,Boston,MA 02115,USA.‡Present address:Modul-Bio,232Boulevard Sainte-Marguerite,13009Marseille,France.§Present address:INSERM,Unite´119,Institut Paoli Calmettes,13009Marseille,France.࿣Present address:Program in Gene Function and Ex-pression,University of Massachusetts,55Lake Ave-nue,North Worcester,MA01605,USA.¶Present address:Center for Cancer Systems Biology and Department of Cancer Biology,Dana-Farber Cancer Institute and Department of Genetics,Harvard Medical School,44Binney Street,Boston,MA02115,USA.#To whom correspondence should be addressed.E-mail:marc_vidal@.Fig.1.Coaffinity purification assays.Shown are10examples from the Core-1,Core-2,and Non-Core data sets.The top panels show Myc-tagged prey expression after affinity purification on glutathione-Sepharose,demonstrating binding to GST-bait.The middle and bottom panels show expression of Myc-prey and GST-bait,respectively.The lanes alternate between extracts expressing GST-bait proteins(ϩ)and GST alone(–).ORF pairs are identified in table S1with the lane number corresponding to the order in which they appear in the table.tome)and phenotypic profiling (phenome)data in S.cerevisiae (17).To investigate to what extent different functional genomic assays should correlate in the context of a multicellular organism,we overlapped WI5with C.elegans transcriptome and phenome data sets.Based on a C.elegans transcriptome com-pendium data set (18),we calculated Pearson correlation coefficients (PCCs)for gene pairs involved in Y2H interactions and compared them with randomized data sets (Fig.2D).About 150Core interactions (9.5%)corre-sponded to gene pairs with significantly high-er PCCs than expected from random (P Ͻ0.05)(table S3).Thus,those pairs can be considered “more biologically likely ”be-cause two completely independent approach-es point to a functional relationship between the corresponding genes.The remaining pairs are labeled “without additional evidence.”Indeed,it is important to note that lack of coexpression does not suggest that the corre-sponding interactions are irrelevant.Indeed,75%of literature pairs,defined as biological-ly relevant,do not correlate with transcrip-tome data (Fig.2D).We also systematically examined Y2H interactions where both proteins belong to common C.elegans expression clusters,or “Topomap mountains ”(18).As an example,a highly connected subnetwork derived from mountain 29(Fig.2E)contains seven pro-teins (ABU-1,ABU-8,ABU-11,PQN-5,PQN-54,PQN-57,and PQN-71)that share common domains (DUF139domain and cysteine-rich repeat).Furthermore,these pro-teins are all expressed in the pharynx (19–21),which suggests that they may act togeth-er in pharynx function or development.For relatively small-scale S.cerevisiae and C.elegans interactome data sets,physical interactions pointed to genes that share sim-ilar phenotypes when knocked out or knocked down (17).To evaluate this idea for the C.elegans interactome,we assembled a collection of phenotypic data based on RNA interference (RNAi)knockdown experiments from WormBase (7,22),and we calculated the percentage of protein interaction pairs that share embryonic lethal phenotypes for the interaction data sets and theirrandomizedFig.2.Analysis of the WI5network.(A )Nodes (representing proteins)are colored according to their phylogenic class:ancient (red),multicellular (yellow),and worm (blue).Edges represent protein-protein interactions.The inset highlights a small part of the network.(B )The proportion of proteins,P(k ),with different numbers of interacting partners,k ,is shown for C.elegans proteins used as baits or preys and for S.cerevisiae proteins.(C )The pie charts show the proportion of interacting preys found in Y2H screens that fall into each phylogenic class.Also shown is the distribution of all preys found and all preys searched in the AD-ORFeome1.0library.(D )Overlap with transcriptome (see text)(18),Pearson correlation coefficients (PCCs)were calculated and graphed for each pair of proteins in the interaction data sets and their corresponding randomized data sets.The red area to the right corresponds to interactions that show a significant relationship to expression profiling data (P Ͻ0.05).(E )Interac-tions between proteins in Topomap mountain 29(18).The dash-circled proteins belong to the same paralogous family (sharing more than 80%homology)and are thus collapsed into one set of interactions.(F )Proportion of interaction pairs where both genes are embryonic lethal (P Ͻ10–7).controls and found a twofold enrichment for the Core and First-Pass data sets (Fig.2F).Similar correlations were also observed for the maternal sterile phenotype and four groups of postembryonic phenotypes (23).Because protein-protein interactions for which both genes are coexpressed across many conditions and show similar pheno-type(s)when knocked down should be considered particularly likely,the global cor-relations described above illustrate how biological hypotheses can be derived from overlapping interactome,transcriptome,and phenome data sets (table S3).In S.cerevisiae ,two proteins that have many interaction partners in common are more likely to be related biologically (24).We exam-ined the C.elegans interactome network for the presence of highly connected neighborhoods by determining the mutual clustering coefficient between proteins in the network (table S4)(24).As an example,we examined the properties of one of the clusters containing such a high-scoring protein pair:VAB-3/C49A1.4(Fig.3).VAB-3and C49A1.4have strong similarity to the products of the Drosophila genes eyeless (ey )and eyes absent (eya ),respectively,but not to each other.EY and EYA are components of a conserved network of transcription factors that regulate eye development (25).VAB-3and C49A1.4are part of a highly interconnected subnetwork in WI5(Fig.3)with proteins that are known or suspected to be functionally linked to VAB-3and C49A1.4,or to their respective orthologs in other organisms.These include (i)EGL-27,which negatively regulates MAB-5in her-maphrodites (26)and is linked to MAB-5through C49A1.4;(ii)WRT-2,an interactor of C49A1.4with similarity to Drosophila Hedgehog,which alleviates repression of eya expression by Cubitus interruptus (27);and (iii)CEH-33and CEH-35,two of four mem-bers of the sine oculis homeobox gene fami-ly,which is involved in the same Drosophila regulatory network of transcription factors as ey and eya (28).Finally,eight proteins in this cluster are annotated in WormPD as involved in membrane function,which suggests a functional relationship between the eyeless transcription network and membrane activity.Together with interologs and previously described interactions,the Y2H data set pro-vides functional hypotheses for thousands of uncharacterized proteins in the C.elegans proteome.Integration with other functional genomic data indicates that the correlation between transcriptome and interactome data,although significant,is lower than what would be expected from observations made in yeast (17).This observation applies to both the Y2H data set described here and well-characterized worm interactions from the literature-derived data set (Fig.2D).This may occur because,unlike unicellular organ-isms,metazoans are complicated by the fact that biological processes may occur different-ly in the organism,across various organs,tissues,or single cells.Our current interactome map also illustrates how a human interactome project would benefit from an ORFeome cloning project using re-combinational cloning systems,such as Gate-way (8).Indeed,recombinationally cloned ORFs can be shuffled at will into various ex-pression vectors needed for different types of protein interaction assays,as exemplified by our ability to transfer bait-and prey-encoding ORFs into Myc-and GST-tagged vectors to validate Y2H interactions.References and Notes1.E.M.Marcotte et al .,Science 285,751(1999).2.M.Pellegrini,E.M.Marcotte,M.J.Thompson,D.Eisenberg,T.O.Yeates,Proc.Natl.Acad.Sci.U.S.A.96,4285(1999).3.P.Uetz et al .,Nature 403,623(2000).4.T.Ito et al .,Proc.Natl.Acad.Sci.U.S.A.98,4569(2001).5.Y.Ho et al .,Nature 415,180(2002).6.A.C.Gavin et al .,Nature 415,141(2002).7.See supporting material on Science Online.8.J.Reboul et al .,Nat.Genet.34,35(2003).9.A.J.M.Walhout,R.Sordella,X.Lu,J.L.Hartley,Science 287,116(2000).10.M.C.Costanzo et al .,Nucleic Acids Res.29,75(2001).11.L.R.Matthews et al .,Genome Res.11,2120(2001).12.A.Davy et al .,EMBO Rep.2,821(2001).13.S.J.Boulton et al .,Science 295,127(2002).14.A.J.M.Walhout et al .,Curr.Biol.12,1952(2002).15.S.H.Strogatz,Nature 410,268(2001).16.F.Jacob,Science 196,1161(1977).17.H.Ge,A.J.M.Walhout,M.Vidal,Trends Genet.19,551(2003).18.S.K.Kim et al .,Science 293,2087(2001).19.J.Gaudet,S.E.Mango,Science 295,821(2002).20.M.Hanazawa,M.Mochii,N.Ueno,Y.Kohara,Y.Iino,Proc.Natl.Acad.Sci.U.S.A.98,8686(2001).21.F.Urano et al .,J.Cell Biol.158,639(2002).22.L.Stein,P.Sternberg,R.Durbin,J.Thierry-Mieg,J.Spieth,Nucleic Acids Res.29,82(2001).23.H.Ge,unpublished observations.24.D.S.Goldberg,F.P.Roth,Proc.Natl.Acad.Sci.U.S.A.100,4372(2003).25.S.Wawersik,R.L.Maas,Hum.Mol.Genet.9,917(2000).26.Q.Ch’ng,C.Kenyon,Development 126,3303(1999).27.K.S.Pappu et al .,Development 130,3053(2003).28.C.Dozier,H.Kagoshima,G.Niklaus,G.Cassata,T.R.Burglin,Dev.Biol.236,289(2001).29.We thank members of M.V.’s laboratory for theirinput and help;C.Boone,G.Achaz and D.Allinger for discussions;the sequencing staff at Agencourt Bio-sciences for technical assistance;the ORFeome meet-ing participants for their input;C.McCowan,T.Cling-ingsmith,and C.You for administrative assistance;and C.Fraughton for laboratory support.This work was supported by a grant from NHGRI and NIGMS awarded to M.V.Other support includes an NSF award (K.C.G.);NIGMS grants (S.v.d.H.,S.E.M.,J.W.H.);a Department of Defense Predoctoral Fellow-ship (B.B.);an award from the Ligue Nationale Contre Le Cancer (e ´quipe labelise ´e)(C.S.,A.C.);an institu-tional HHMI grant (F.P.R.,G.F.B);and Fellowships from EMBO (P.-O.V.),NSF (D.S.G),Ryan,Milton (S.L.W.),Fu (L.V.Z.),and Leukemia Research Founda-tion (M.E.).Supporting Online Material/cgi/content/full/1091403/DC1Material and Methods Fig.S1Table S1to S5References11September 2003;accepted 1December 2003Published online 2January 2004;10.1126/science.1091403Include this information when citing thispaper.Fig.3.Graphical representation of a highly interconnected subnetwork around VAB-3and C49A1.4.Biological functional classes were obtained from WormPD (10).。

Exam:190-622Titl e :Notes Domino 6 Managing Servers and Users��Ver : 01.09.06QUESTION 1Which one of the following explains why Hans cannot retrieve an HTML file whichhe knows is on the Domain server?A. The CGI script are not enabled.B. The default URL is set to ?Open.C. Hans has Depositor access to the Domino server.D. The HTML files are not located in the HTML directory listed in the server or Web site configuration documents.Answer: AQUESTION 2Jim's Notes workstation's Smart Upgrade did not work. Which one of the following could be the cause of Smart Upgrade failing?A. Jim did not access his mail database.B. Jim's Personal Address Book has the filename JIM.NSF.C. Jim's Location document does not correctly specify his home server.D. Jim does not have administrator access to his Windows 2000 workstation. Answer: AQUESTION 3When a policy selected for a particular user?A. During registrationB. During workstation setupC. When the user's certifier ID is createdD. When the user accesses their mail serverAnswer: BQUESTION 4Jessica wants to assign an explicit policy to a user. Which one of the following documents should be modified to assign the policy to the user?A. The policy documentB. The user's Person documentC. The office Location documentD. The mail server's configuration documentAnswer: BQUESTION 5Which one of the following statements about the Smart Upgrade database isTRUE?A. The database must have ODS version 41.B. The database must have the filesystem SMUPKIT.NSFC. The database must contain the relevant Notes RS information.D. The database must exist on at least one server in the domain.Answer: DQUESTION 6Amy wants to decommission an existing source server. Before transferring the applications, she runs a Decommission Server Analysis report. Which one of the following types of information does this tool provide?A. Source server information onlyB. Target server information onlyC. A compression between source server and target serverD. A detailed report of everything transferred from the souce server to te target server once the transfer takes placeAnswer: CQUESTION 7In addition to Notes database, which one of the following files on a Domino server should be backed up in order that new users can created in case of the server?A. The CACHE.DSK fileB. Certifier ID filesC. The user's DESKTOP.DSK fileD. The server's BOOKMARK.NSF file.Answer: BQUESTION 8Brie left the Create New Replicas field blank in a Server document. Who can create new replica databases on that server?A. No oneB. AdministratorsC. Local Domain ServerD. Everyone in the domainAnswer: AQUESTION 9Which one of the following statements about the compact server task is TRUE?A. The compact process re-indexes views.B. By default, compact runs every day at 3 P.M.C. Compact may be scheduled via a program document.D. Compact is listed in the TASKS line on all Notes 6/6.5 workstations.Answer: CQUESTION 10Josh has cross certified the /BOX organization to his /ACME organization. An administrator from the BOX organization has cross certified the /ACME to the/BOX organization. When a /BOX server attempts to assess a /ACME server, it receives a " You are not authorized to access this server" error. Which one of the following could be the cause of this error?A. The Box server's ID does not contain a copy of the Acme certifier.B. The Box server's ID file needs to be updated with the cross certificate.C. The Box administrator needs to add the Box server to the LocalDomainServer group.D. The Box server has not been granted access to the Acme server in the Acme server document.Answer: DQUESTION 11Suzanne, a Notes administrator sets the "Allow Anonymous Notes connections" setting in the Server document to yes. Which one of the following levels of accesswill Bob, Notes user, have when he accesses a database with the following settings:-Default- Editor*/ACME - ReaderAnonymous - AuthorOtherDomainServer -NoAccessA. ReaderB. AuthorC. DefaultD. No AccessAnswer: BQUESTION 12Amy is setting up session-based name-and-password authentication. Which one ofthe following is required for this to work?A. JavaB. COBRAC. CookiesD. JavaScriptAnswer: CQUESTION 13Jeff cross certified the Organizational Unit (OU) /NYC/ACME with his organization's /BOX Organizational (O) certifier. Which one of the following statements are correct for this scenario?A. Users or server with /NYC/BOX certificate can access /ACME servers.B. Users with an ACME ID can access any of Box's severs that have a /NYC/BOX certificate.C. Any user or server ID with the /NYC/ACME certificate can access any server in the /BOX organization if they have the appropriate cress certificate.D. Server with the /NYC/ACME certificate can access any server in the /BOX organization if they have the appropriate cress certificate but no .NYC/AME user can access /BOX servers.Answer: AQUESTION 14Robert's windows 200 Domain server has frozen with no error output on theserver's screen or other indication that anything is wrong. Which on of the followig can cause this problem?A. The server was replicating with another serverB. The server's transaction log was being updated.C. Robert has forced mail routing to another server.D. Microsoft windows Quick Edit mode was enabled for the server console window. Answer: DQUESTION 15Bob left the Create New Databases field empty in a Server document. Who is authorized to create new databases on the server?A. No oneB. Administrators onlyC. LocalDomainServer onlyD. Anyone with access to the serverAnswer: DQUESTION 16Which one of the following statements about transaction logging is TRUE?A. The transaction log is always named TRANSLG.NSFB. Transaction logging must be enabled on all Domain 6/6.5 servers.C. All database changes are written to a transaction log in a batch.D. Transaction logging is enabled by default on all Domain 6/6.5 servers. Answer: CQUESTION 17Raul wants to review reads and writes to databases from various Notes user. Where would he locate this information?A. LOG.NSFB. DOMLOG.NSFC. ACTINFO.NSFD. ACTIVIOTY.NSFAnswer: AQUESTION 18Which one of the following statements about Domino SSL server security is TRUE?A. SSL must be enabled for all server ports.B. SSL can only be enabled for POP3, IMAP and SMTP.C. If a server is using SSL, users must access the server using a browser.D. You can require SSL connections for all databases on a server or for an individual database.Answer: DQUESTION 19Dawn used the Administration Process (AdminP) to convert a non-roaming Note user to a roaming Notes user. In which one of the following does the Administration Process update the user's status from non-roaming to roaming?A. Person documentB. Server documentC. Location documentD. Configuration documentAnswer: AQUESTION 20Orin changes the name of a group using the Domino Administrator client. How often does the Administration process (AdminP) update group names in Author and Reader Names fields?A. DailyB. HourlyC. WeeklyD. ImmediatelyAnswer: CQUESTION 21Jonathan has been moved out of one group and into another. How long before this change will be effective throughout the seventeen servers in his domain?A. One hour.B. ImmediatelyC. It is dependent on the replication schedule for NAMES.NSF.D. The Administrator Process (AsminP) will change his group membership throughout the domain within 24 hours.Answer: CQUESTION 22Maria got married ad changed her last name. She needs to have her name changedin the Domino Directory. Which one of the Following actions did the administrator use to change Maria's name?A. Edit PersonB. Move to folderC. Rename Selected PeopleD. Recertify Selected PeopleAnswer: CQUESTION 23Karen uses the Administration Process (AdminP) to move a user's main file to a different mail server. Which one of the following must Karen do in order for thenew mail file to be created and the old mail file to be deleted?A. Update the user's Location documents.B. Modify the mailserver field in the user's person documentC. Approve the deletion of the old mail file in the ADMIN4.NSF database.D. Give the user rights to create new databases on the new mail server.Answer: CQUESTION 24When a user is deleted from the Domino Directory, which one of the following tasks must be approved in the ADMIN4.NSF database by the administrator before theAdministration process (AdminP) performs the task?A. Remove the user from groups.B. Delete the user's mail database.C. Add the user to a Terminations groupD. Remove the Person document from the domain's Domino Directly.Answer: BQUESTION 25Jennifer is moving user from /Madrid/ACME to /Boston/ACME. Which one of the following will she need to perform this task?A. The Web Administrator clientB. Just the destination certifierC. Author access to the domain's Domino DirectoryD. Both the /Madrid/ACME and the /Boston/ACME certifier ID filesAnswer: DQUESTION 26Which one of the following processes ensures that group names in ACLs are updated when an administrator changes the name of the group in the domain's Domino Directory?A. UpdateB. UpdallC. ConvertD. Administration Process (AdminP)Answer: DQUESTION 27Which one of the following may cause the error "You are not authorized to use the server on remote consol"?A. The user is not authorized to use the server.B. The server does not allow remote administration.C. The user's ID file is not cress certified with the server.D. The person in not listed in the Administrators field of the Server document. Answer: DQUESTION 28Jose wants to see the replication topology map of his Notes domain. To see this topology, what must he do?A. Load the MAPS EXTRACTOR server task.B. Load the TOPMAPS EXTRACTOR server task.C. Load the MAPTOP EXTRACTOR server task.D. Load the TOPOLOGY EXTRACTOR server task.Answer: AQUESTION 29Which one of the following would be helped by using the Mail Trace feature?A. A user cannot read encrypted e-mail.B. All mail messages to a particular user are not routing properly.C. Mail is not routing from outside your domain to your domain's mail servers.D. Mail messages from one user in your domain are not routing properly to external users.Answer: BQUESTION 30Geoff wants to force replication of NAMES.NSF between ServerA/Acme and ServerB/Acme. Which one of the following console commands will accomplish this?A. LOAD REPLICATIONB. REP SERVERA SERVERBC. REP SERVERB/ACME NAMESD. TELL SERVERA REP SERVERB NAMESAnswer: CQUESTION 31Both Server A and Server B are in the domain and in the same Domain Network Name. What is the minimum number of Connection Documents needed for replication to occur in both directions?A. TwoB. OneC. ThreeD. NoneAnswer: BQUESTION 32Which one of the following may prevent the Administration Process (AdimnP)from updating a user's name in their ID file?A. The user accepted the name change.B. The CERTLOG.NSF database does not include a copy of the user's certificate.C. The user's name is not listed in the Domino Directory's Access Control List (ACL).D. The user did not accept the name change in the first twenty-four hours after the administrator initiated it.Answer: BQUESTION 33An agent that is designed to run "On schedule more than once daily" does not complete. Which one of the following could cause this problem?A. The agent must be initiated by user action.B. The agent can only run hourly, weekly or monthly.C. The developer must start the agent manually the first time.D. The agent has exceeded the value in the Max Execution time in the Agent Manager settings of the Server document.Answer: DQUESTION 34Koki, a Domino administrator, is trying to move a server within the certification hierarchy by running the administration Process task. He recertifies the server witha different certifier ID but the change never tasks place. Which one of the following will cause the recertifying process to fail?A. The server has a hierarchical certificate.B. The Administration Process was enabled.C. The Certification Log was placed on the Domino Administrator workstation.D. A replica copy of the Administration Requests database was placed was placed on every server in the domain.Answer: CQUESTION 35Which one of the following may cause the error "You are not autjorized to accessthe server"?A. The user's ID file has expired.B. The server's ID file has expired.C. The cross certificate exists on both directions.D. The Access List in the Server document denies the user access.Answer: DWhich one of the following types of information is stored in an .NSD file?A. Current server statusB. Notes storage directoriesC. Information regarding a system crashD. HTTP access information for the serverAnswer: CQUESTION 37Both Server A and Server B are in the Acme domain, are correctly listed in DNS, are in the same Domino Network Name. How many Connection Document are required mail to route for Server B and from Server A to Server B and form Server B to Server A?A. OneB. TwoC. ThreeD. NoneAnswer: DQUESTION 38Which one of the following may cause the error "Unable to find path to server" on a Notes workstation?A. The user's ID file has expired.B. Server's hostname is not resolving.C. The Personal Address Book has been deleted.D. Server does to have a Connection document to the server.Answer: BQUESTION 39Jeff is trying to create a replica of STATUS.NSF but is unable to create the local copy. Which one of the following could cause this problem?A. Jeff only has Reader access in the database's Access Control List (ACL).B. Jeff only has Author access in the database's Access Control List (ACL).C. Jeff only has Editor access in the database's Access Control List (ACL).D. Jeff has not been given the right to replicate or copy documents in the database's Access Control List (ACL).Answer: DWhat is the minimum level of access a Notes 6/6 5 user must have to run the Out of Office agent?A. AuthorB. EditorC. ManagerD. DesignerAnswer: BQUESTION 41Jose is creating a new password for himself. Which one of the following will password quality checking indicate as the least secure password?A. Mixed case passphrasesB. All uppercase passwordsC. Passphrases containing numbers and punctuationD. Would found in Notes dictionaries during spell checkAnswer: DQUESTION 42Kurt created an event generator. Which one of the following will this do?A. Restart the event task.B. Initiate a task a server.C. Create entries in the EVLOG.NSF databases.D. Monitor a task a task or statistic by probing a server.Answer: AQUESTION 43Which one of the following controls when an agent runs on a server?A. The Agent Manager taskB. Server document, Agent Settings field.C. Configuration document, Agent Setting filed.D. The AGENTSCHED parameter in the server's NOTES.INI file.Answer: BQUESTION 44Why did Sophia create an event handler?A. To define a threshold for a statistic.B. So that server sets quotas on database size.C. In order to have the server log events to the EVENTS4.NSF database.D. To be notified via page if the server's available disk space fails below 25%. Answer: DQUESTION 45What is the DOMLOG.NSF database used for?A. To track all users who access the Domino serverB. To track users who access the Domino server via HTTPC. To track users who access the Domino server via NNTPD. To track user who authenticate with the Domino server.Answer: BQUESTION 46Which one of the following best describes the available directory architecture in a Domino 6 domain?A. A centralized directory architectureB. Both distributed and centralized directory architecturesC. None of the answers applyD. A distributed directory architectureAnswer: BNotes & Domino 6 supports both a distributed directory architecture and a central directory architecture.In a Distributed Directory architecture in a Domino domain, in which all servers use the standard Domino Directory in a central directory architecture,some servers store Configuration Directories (contains configuration settings only) and then use the standard Domino Directories on remote servers for lookups. QUESTION 47What is the significance of Kit Type=1 and where is it located?A. Server, in the Notes.iniB. Workstation, in the server documentC. Workstation, in the Notes.iniD. Server, in the configuration documentAnswer: CSpecifies which program you are running:1 - Workstation2 - ServerQUESTION 48Which on of the following is the proper syntax for forcing replication via a Domino server console command?A. Replicate [databasename] servernameB. Replicate servername [databasename]C. Replicate sourceservname/sourcedatabasenamedestinatationservername/destinationdatabasenameD. Replicate servername/databasenameAnswer: BSyntax: Replicate servername [databasename]Description: Forces replication between two servers (the server where you enter this command and the server you specify). Use the server's full hierarchical name. If the server name is more than one word, enclose the entire name in quotes. To force replication of a particular database that the servers have in common, specify the database name after the server name. The initiating server (where you're currently working) first pulls changes from the other server, and then gives the other server the opportunity to pull changes from it. You can use this command to distribute changes quickly or to troubleshoot a replication or communication problem.QUESTION 49Mary needs to rebuild all used views in her database. Which one of the following commands could she issue on the Domino server console?A. UPDALL -ALLB. UPDALL -*C. UPDALL -RD. UPDALL -XAnswer: CRebuild: All used viewsUPDALL -RRebuilds all used views. Using this option is resource-intensive, so use it as a last resort to solve corruption problems with a specific database.QUESTION 50What database is created when you run the Decommission Server Analysis tool?A. Log AnalysisB. DecommissionC. Server analysisD. ResultsAnswer: DWhen you run the Decommission Server Analysis tool, you create a Results databasecontaining detailed information comparing the source server and the target server. The source server is the server being removed from service, and the target server is the server taking the place of the source server. The source and the target servers must be Domino servers that have hierarchical names and that are in the same domain.QUESTION 51Which of the following uniquely identifies a document across all replicas of a database?A. UNIDB. NotelIDC. ReplicalDD. OFIDAnswer: AThe UNID (universal ID) is a 32-character combination of hexadecimal digits (0-9, A-F) that uniquely identifies a document across all replicas of a database.QUESTION 52A Program document can not be used to run which of the following types of commands or programs?A. API programsB. UNIX shell scriptsC. OS/2 command filesD. Java appletsAnswer: DA Program document is used to automatically run a server task at a specific time. You can also use a Program document to run an OS/2 command file, a UNIX shell script or program, or an API program. You can use the Program document to schedule tasksand/or programs.QUESTION 53Sandra has migrated all of her users and applications from Nagano/CertFX toMoscow/CertFX. Which one of the following can she use to ensure she has transferred all database and settings?A. Tivoli Server Analyzer for Lotus DominoB. The administration client action "Show Server Difference"C. Cluster comparison toolD. Server decommission Analysis toolAnswer: DThe Decommission Server Analysis tool generates a categorized list of items that were analyzed. Each category represents a different aspect of a server's configuration thatneeds attention. Within each category, items are listed alphabetically. Each item lists any differences between the source and the target server's settings or values. In the Results database, you can view the categorized list of the items that were analyzed. QUESTION 54A Security Policy Settings document controls which of the following?A. The Notes and Internet passwordsB. None of the aboveC. The administration ECL as well as Notes and Internet passwordsD. The administration ECL as well as Internet passwordsAnswer: CA Security policy settings document controls the Administration ECL as well as Notes and Internet passwords.QUESTION 55Which one of the following can you specify in the setup profile when performing a seamless upgrade?A. To which groups the client must be addedB. Which mail file template to apply according to the Lotus Notes client versionC. All of the aboveD. Which address book template to apply according to the Lotus Notes client version Answer: BWhen the Domino server receives the call to upgrade a mail file template, the server checks for a Desktop policy settings document or Setup Profile assigned to the user. The Desktop policy settings document and the Setup Profile contain a "Mail Template Information" section. This section is new to Setup Profiles in Notes 6. In this section, you can specify which mail file template to apply according to the Lotus Notes client version. QUESTION 56Which one of the following is not an Event Generator document?A. TCP server even generatorB. NRPC server event generatorC. Task status event generatorD. Statistic event generatorAnswer: BMonitoring Configuration documents define and configure what constitutes an event and how the event is handled. You can also use these documents to customize the message that appears on screen when an event occurs. The Monitoring Configuration documents are stored in EVENTS4.NSF.Event Generator documents are also stored in the Monitoring Configuration database, EVENTS4.NSF.QUESTION 57Which one fo the following best describes the information that can be found in the DOMLOG.nsf database?A. Both Lotus Notes and web activity on the Domino serverB. Lotus Notes activity on the Domino serverC. Web activity on the Domino serverD. Activity trends and health reporting of the Domino serverAnswer: CYou can log your server activity and Web server requests to the Domino Web server log (DOMLOG.NSF) database. This option may be preferable if you want to create views and view data in different ways. Logging to a database is somewhat slower than logging to text files, especially at very busy sites, and the size of the database can become large so that maintenance becomes an issue. However, if you use the Domino Web server log, you can treat this information as you would other Notes databases, and you can use built-in features to analyze the results.QUESTION 58Michael needs to force replicate between the following two servers:Notes Hub/Tools/ CertkillerSametime Hub/Tools/ CertkillerMichael is using a remote console on SametimeMichael is using a remote console on Sametime Hub/Tools/ Certkiller . Which oneof the following Domino server console commands did he issue?A. REPLICATE NotesHubB. REPLICATE Notes HubC. REPLICATE Notes Hub/Tools/ CertkillerD. REPLICATE Notes Hub/Tools/ Certkiller "Answer: DSyntax: Replicate servername [databasename]Description: Forces replication between two servers (the server where you enter this command and the server you specify). Use the server's full hierarchical name. If the server name is more than one word, enclose the entire name in quotes. To force replication of a particular database that the servers have in common, specify the database name after the server name. The initiating server (where you're currently working) first pulls changes from the other server, and then gives the other server the opportunity to pull changes from it. You can use this command to distribute changes quickly or to troubleshoot a replication or communication problem.Which one of the following is not an available policy settings document?A. Ecplicit policiesB. Exception policiesC. Implied policiesD. Organizational policiesAnswer: CA policy is a collection of individual policy settings documents.There are three types of policies: Organizational policies, Explicit policies and Exception policies.Organizational policies are applied automatically to users or groups based on the organizational structure.Explicit policies, like they sound, are assigned explicitly. They apply settings directly to a user, instead of through the organizational hierarchy.Exception policies allow users to control their own user settings.QUESTION 60Which one of the following statements about transaction logging is TRUE?A. Transaction logging is used to support Domino clusteringB. Logged transactions are stored in memoryC. Logged transactions are written to disk in a batchD. Transaction logging is enabled by default on all Domino 6 serversAnswer: CTransaction logging captures all the changes that are made to databases and writes them to a transaction log. The logged transactions are written to disk in a batch when resources are available or at specified intervals.QUESTION 61Hoke needs to make design changes to forms and views of a production database.He plans to make the changes locally in a template and then have the server updatethe production database with the changes. Which one of the following server tasksdoes the administrator run in order for the changes to be propagated?A. The REPLICA server taskB. The FIXUP -D server taskC. The DESIGN server taskD. The UPDALL server taskAnswer: CThe DESIGN or designer task updates all database design elements from their master template(s). The DESIGN task runs daily by default at 1 AM.A user has been added to a group with author access in the salesdatabase. When she enters the database she finds that she is able to change her co-workers documents as well as her own. Why?A. She is listed individually in the ACL with reader accessB. The ACL has become corrupt and needs fixed upC. None of the aboveD. She is listed in another group in the ACL with editor accessAnswer: DA name is included in two or more groups. The name receives the access of the group with the highest access.QUESTION 63Many needs to rebuild all used views in her database. Which one of the following commands could she issue on the Domino server console?A. UPDALL -RB. UPDALL -XC. UPDALL -ALLD. UPDALL -*Answer: ARebuild: All used viewsUPDALL -RRebuilds all used views. Using this option is resource-intensive, so use it as a last resort to solve corruption problems with a specific database.QUESTION 64Which one of the following should not be included in a NNN?A. Domino servers on a WANB. Domino servers on Dial-upC. None of the aboveD. Domino servers on a LANAnswer: BThe Domino Server Setup program automatically places all servers that are in a Domino domain and that run the same network protocol in the same Notes named network (NNN). In the Server document, the setup program assigns each NNN a default name in the format portname network.After you complete the Server Setup program, rename the NNN for each network port in the Server document. It is useful if the name reflects both the location of the network and its protocol. For example, if your company has a TCP/IP network and has LANs in Boston and San Francisco, change the name of the NNN in Boston to "TCPIP Boston。

C族4级战斗任务&故事线任务V1.1奥运最新版攻略 (2)前言 (2)任务地点介绍 (2)任务基本社会技能 (2)任务基本配置 (2)任务介绍 (3)狂暴 (3)阻止小偷 (3)货柜递送 (3)侦察1/3（古斯塔） (4)侦察1/3（血袭者） (4)侦察2/3（古斯塔/血袭者） (4)侦察3/3（古斯塔/血袭者） (4)袭击 (5)海盗屠杀 (5)海盗侵略 (5)双匪劫（古斯塔） (6)双匪劫（血袭者） (6)无赖的奴隶贩子1/2 (6)无赖的奴隶贩子2/2 (6)大举进犯 (7)自由无人机的骚扰 (7)无人机的袭击 (7)抢救矿冶设施 (8)扎XXX的右使者 (8)危难中的少女 (8)古斯塔斯间谍 (9)血袭者间谍 (9)未授权的军队 (9)拦截走私船 (10)疤痕 (10)世界碰撞（古斯塔） (10)世界碰撞（血袭者） (11)复仇之火（古斯塔） (11)复仇之火（血袭者） (11)古斯塔斯(天使的妄想)的妄想 (12)截击破坏者 (13)死人不会说话 (13)封锁线 (13)腹背受敌（1/5） (14)腹背受敌（3/5） (14)腹背受敌（4/5） (15)腹背受敌（5/5） (15)故事线任务可以增加势力声望 (15)绑架（A Case Of Kidnapping） (15)船厂盗窃犯（古斯塔）（Shipyard Theft） (16)船厂盗窃犯（血袭者）（Shipyard Theft） (16)隐藏行踪（Covering Your Tracks） (16)C族4级战斗任务&故事线任务V1.1奥运最新版攻略前言相信大家对于我写的/thread-126838-1-1.html这个帖子有点印象，当时写这个帖子的时候比较仓促，而且正好处于LP比较值钱的时候，所以我当时这个帖子是以如何快速得到LP为主要目的，因此可能不适合很多新来做任务的朋友，加上时间已经过去4个月了，所以特地在奥运期间，重新将该任务帖子整理编辑，以方便新人老人共勉，另外该帖完成后地址会在C任务党频道“CE龙门客栈”中置顶，还望各位多多支持，谢谢。

贪婪洞窟看了个游戏贪婪洞窟，评价很高，下载后感觉很好玩。

现在玩到噩梦61层，谈谈个人玩的经验首先，贪婪洞窟是个比较耗费时间的游戏（对于平民玩家）。

贪婪洞窟符文前期地图会刷符文，加攻击、加防御、加蓝、加魔力。

贪婪洞窟任务不充值又想拿钻石的方法只有做任务了，每十层会有35个任务，运气好的可以拿到400+加钻石。

贪婪洞窟老奶奶商店转动转盘前上传存档，，如果没有转到好东西可以，再下载可以让再次转动转盘。

贪婪洞窟科恩帽子科恩帽子，可以说是要玩穿整个游戏必备的装备，同时去老奶奶哪里买个手套，多刷刷买双幸运高的手套。

加点方面我选择的是吸血流：属性点主加攻击，防御，血量。

核心技能：虹吸斩，三连击。

主要装备属性选择：攻击，防御，生命，魔法恢复。

加点方面，幸运必点10，技能下方辅助技能点左边叁个陷阱隔空顺移点陷阱抗性是因为陷阱真的很痛，隔空可以远远拿装，顺移则是方便跑路。

1-20层，没什么可说的，随便凑凑一身金装，然后把武器强化+3继续闯到20打boss。

前期任务好做，尽量多做做任务赚钻石。

钻石前期留着不要用。

21-40层，初期比较费劲，慢慢做任务，等40层之前的任务做的差不多的时候，一身的装备也应该可以打boss了，boss比较简单，从21-40层开始刷药水，带够药水就可以过boss了，很简单。

40层boss开始掉至关重要的装备了贪婪洞窟科恩帽子，这时候做任务、成就等得的钻石应该也有1500+了，这时候先去老奶奶那里买科恩手套，同时尽可能的强化。

41-之后的所有楼层打法，首先科恩双件套备齐，尽可能穿一身强化+3的幸运装，每层开始前先穿幸运装进入，各种摸箱子，41层之后就开始出现橙装了，当身上装备大多都是+3的金装时候就可以打boss了，当然装备的优先级是攻击，防御，生命，魔法恢复。

这里说说魔法恢复，这个是为你攥药水过boss最核心的属性了。

保证普通关卡回魔在20以上就行，做好这些就可以轻轻松松的过boss了，普通100层很简单，个人幸运属性到200+，摸箱子基本一趟10+金装，3+橙装。

ORIGINAL ARTICLEAdenosine A 1receptor regulates osteoclast formation by altering TRAF6/TAK1signalingW.He &B.N.CronsteinReceived:31October 2011/Accepted:19January 2012/Published online:5February 2012#Springer Science+Business Media B.V .2012Abstract Adenosine is an endogenous nucleoside that mod-ulates many physiological processes through four receptor subtypes (A 1,A 2a ,A 2b ,A 3).Previous work from our labora-tory has uncovered a critical role for adenosine A 1receptor (A 1R)in osteoclastogenesis both in vivo and in vitro.Our current work focuses on understanding the details of how A 1R modulates the receptor activator of NF-κB ligand (RANKL)-induced signaling in osteoclastogenesis.Osteo-clasts were generated from mouse bone marrow precursors in the presence of RANKL and macrophage-colony stimulat-ing factor.A pharmacological antagonist of A 1R (DPCPX)inhibited RANKL-induced osteoclast differentiation,including osteoclast-specific genes (Acp5,MMP9,β3Integrin ,αv Integ-rin ,and CTSK )and osteoclast-specific transcription factors such as c-fos and nuclear factor of activated Tcells cytoplasmic 1(NF ATc1)expression in a dose-dependent manner.DPCPX also inhibited RANKL-induced activation of NF-κB and JNK/c-Jun but had little effect on other mitogen-activated protein kinases (p38and Erk).Finally,immunoprecipitation analysis showed that blockade of A 1R resulted in disruption of the association of tumor necrosis factor receptor-associated factor 6(TRAF6)and transforming growth factor-β-activated kinase 1(TAK1),a signaling event that is important for activation of NF-κB and JNK,suggesting the participation of adenosine/A 1R in early signaling of RANKL.Collectively,these data demonstrated an important role of adenosine,through A 1R in RANKL-induced osteoclastogenesis.Keywords Adenosine A 1receptor .Osteoclastogenesis .TAK1.TRAF6.NF-κBIntroductionOsteoclasts,which are derived from monocytic/macrophagic hematopoietic cells in response to macrophage colony-stimulating factor (M-CSF)and receptor activator of NF-κB ligand (RANKL),a member of the tumor necrosis factor (TNF)cytokine superfamily,play a critical role in bone remod-eling.Increased osteoclast activity is seen in many osteopenic disorders,including postmenopausal osteoporosis [1,2],Paget's disease [3,4],bone metastases [5,6],periodontitis [7,8],and rheumatoid arthritis [9,10].Mature osteoclasts are giant,multinucleated cells that synthesize and directionally secrete bone matrix-degrading enzymes,including cathepsin K,matrix metalloproteinase-9(MMP9),and tartrate-resistant acid phosphatase (TRAP).The bone-resorbing activity of osteoclasts requires their adherence to the bone surface and subsequent development of ruffled borders and sealing zones.A substantial body of evidence suggests that adhesion mole-cules,including the integrin αv β3,play an important role in regulating migration,adhesion,polarization,and activation of osteoclasts [11–14].Activation of the NF-κB complex is a key early event in RANKL-induced osteoclast formation.Mammalian cells have five NF-κB family members (RelA/p65,RelB,c-Rel,NF-κB1/p105,and NF-κB2/p100)which contain an N-terminal Rel homology domain (RHD)with sequences for dimerization,DNA binding,and nuclear localization.Mice that lack both the p50and p52subunits of NF-κB developed severe osteo-petrosis due to a defect in osteoclast differentiation.Mice lacking c-Fos,a component of the transcription factor activator protein-1(AP-1),also develop osteopetrosis due to a completeElectronic supplementary material The online version of this article (doi:10.1007/s11302-012-9292-9)contains supplementary material,which is available to authorized users.W.He :B.N.Cronstein (*)New York University School of Medicine,550First Avenue,New York,NY 10016,USA e-mail:cronsb01@Purinergic Signalling (2012)8:327–337DOI 10.1007/s11302-012-9292-9block in osteoclast differentiation[15].AP-1binding sites have been identified in the promoters of several osteoclast genes including Acp5,β3integrin,carbonic anhydrase II,and NF ATc1 [16–19].Moreover,AP-1cooperates with other transcription factors(e.g.,NF-κB and NFA Tc1)to regulate RANKL-induced transcription osteoclast-specific genes[20].Binding of RANKL to RANK activates other signals that are critical for osteoclast formation as well including activation of mitogen-activated protein kinases(MAPKs),namely the extracellular signal-regulated kinase(Erk),c-Jun N-terminal kinase(JNK),and p38kinase.Genetic and biochemical studies indicate that the activation of JNK/c-Jun is indispensible for RANKL-induced osteoclast formation and mice from JNK null mice or c-Jun-deficient mice fail to form osteoclasts and suffer from osteopetrosis[21,22].Another signaling protein,transformation growth factor-β(TGF-β)activated kinase-1(TAK1)has been implicated in RANKL-induced osteoclastogenesis[23–25].Upon RANK receptor engagement,the cytoplasmic domain of RANK inter-acts with an adaptor protein,tumor necrosis factor-receptor-associated factor6(TRAF6),and endogenous TAK1is recruited to the TRAF6complex.The phosphorylation and activation of TAK1subsequently leads to MAPKs and inhib-itoryκB kinase(IKK)activation,the prerequisite event nec-essary to induce NF-κB.In this process,TAK1-associated binding protein-2(TAB2)acts as a bridge linking TRAF6to TAK1[26].Although the mechanism by which TAK1is activated is not fully understood,many studies have revealed the critical role of the lysine-63-linked polyubiquitination by TRAF6in the activation of TAK1[27,28].Adenosine is an endogenous nucleoside that modulates many physiological processes through four receptor sub-types(A1,A2a,A2b,A3).Recent studies in our laboratory have revealed a novel role for adenosine/A1receptor(A1R) in osteoclastogenesis:A1R activation is required for both osteoclast formation and function in vitro and only function in vivo,as demonstrated using pharmacologic inhibitors and mice lacking adenosine A1receptors[29,30].The disparity between in vitro and in vivo osteoclast formation is reminis-cent of a similar disparity in osteoclast formation in vitro and in vivo in mice lacking either TRAF6or Atp6v0d2in which osteoclasts are present in vivo,although functionally defec-tive[31]and do not form from precursors in vitro[32,33]). One possible explanation for these discrepancies is the pres-ence of other factors in the in vivo microenvironment which can partially compensate the A1R,TRAF6or Atp6v0d2 deficiency,such as TGF-β[32].In this work,we further probed the signaling pathways by which adenosine/A1R activation mediates its effect on osteoclastogenesis.We report here that adenosine A1R activation is required for appropriate formation of TRAF6/TAK1complexes and the resulting activation of NF-κB,the critical signaling step in osteoclastogenesis.MethodsAntibodies and reagentsCommercially available antibodies were purchased from the following resources:IκB,p-c-Jun,c-Jun,p-Erk,p65,TAK1, TRAF6,NFATc1(Santa Cruz Biotechnology Inc),p-p-38, p38,Erk,p-JNK,JNK(Cell Signaling Technology),p84, andβ-actin(abcam).Recombinant murine M-CSF and murine RANKL were from R&D System Inc.Sodium thiosul-fate and silver nitrate were purchased from Sigma.Osteoclast cultureFor generation of bone marrow-derived osteoclasts,primary bone marrow cells from6to8-week-old mice were cultured as described previously[30].Briefly,bone marrow was extracted from femora and tibia of mice.The cells were grown in completeα-MEM(Invitrogen)containing10% fetal bovine serum for24h.Then the non-adherent BMMs were collected and replated in culture dishes at1×105cells/cm2 density with murine M-CSF(30ng/ml)for2days.Cells at this stage were considered M-CSF-dependent bone marrow macro-phages(BMMs)and used as osteoclast precursors.Induction of differentiation to osteoclasts was achieved by culturing the BMM cells with the osteoclastogenic medium contain-ing M-CSF(30ng/ml)and recombinant murine RANKL (30ng/ml).The day cells were treated with differentiation medium(RANKL+M-CSF)was counted as day0.Typically, cells were TRAP-positive multinuclear at day5after the initiation of culture with RANKL.To test the effect of A1R antagonist,DPCPX,these osteoclast precursors(BMMs)were cultured in the osteoclastogenic medium with or without different doses of DPCPX for5days.The culture medium was replaced with fresh medium containing these reagents every3days.TRAP staining and bone resorption assayOsteoclast differentiation was evaluated by staining for TRAP using a leukocyte acid phosphatase kit(Sigma-Aldrich). TRAP-positive multinucleated cells(≥3nuclei)were counted as osteoclasts.For bone resorption assay,The BMMs were seeded on BD BioCoat Osteologic MultiTest slides(BD Biosciences)and cultured in the osteoclastogenic medium with or without different doses of DPCPX for8days.At the end of culture,cells were removed from chamber slides with bleach and stained by V on Kossa method.Briefly,slides were stained with5%silver nitrate for30min and then fixed in5%sodium carbonate in25%formalin for5min to remove un-reacted silver nitrate.The surface areas of resorption pits were mea-sured and analyzed using Labworks Image Acquisition and Analysis software4.0(UVP).Using the software,the percentileof pixels of white in a gray scale was measured as a represen-tation of the percentage of resorption area to total area.Real-time PCRTotal RNA was isolated from culture cells using RNeasy Kit (Qiagen).cDNAwas synthesized from1μg of total RNA using the SuperScript First-Strand Synthesis System(Invitrogen)in a volume of20μl.Real-time PCR was performed using Master SYBR Green Kit(Strategene).Primers are listed in Table1. PCR conditions were95°C for5min followed by38cycles of 95°C for30s,58°C for30s,and72°C for30s.Each experiment was done in triplicate,and results were standardized against the message level ofβ-actin.The comparative CT method was used to calculate the expression levels of RNA transcripts.Western blotFor RANKL signaling,bone marrow cells were grown in 30ng/ml M-CSF for2days.Cells were then washed with PBS and treated with30ng/ml M-CSF and50ng/ml RANKL or without different doses of DPCPX for the indicated times. Total cell lysate(30μg)were collected and subjected to western blot analysis with the indicated antibodies.Nuclear extracts (15μg)were prepared using NE-PER kit(Pierce)according to the manufacturer's instruction and loaded in10%SDS-PAGE gels and immunoblotted with different antibodies.NF-κB p65DNA-binding analysisNuclear extracts were obtained from primary osteoclast precursor cells as above and assayed for NF-κB transcription-al activity using an ELISA(Cayman Chemicals)according to the manufacturer's instruction.Nuclear extracts(15μg)were incubated into a96-well plate coated with oligonucleotide containing the consensus NF-κB response element.Absor-bance at450nm was read in a plate reader,and the data were calculated as ratio to control(BMMs treated with M-CSF only).Immunoprecipitation assayCells were washed once with ice-cold PBS and lysed in ice-cold IP lysis buffer(Pierce)according to the manufac-turer's instruction with freshly added proteinase inhibitor cocktail(Sigma)and phosphatase inhibitor(Cayman Chem-icals).Cleared lysates(3–5mg total protein)were preincubated with TRAF6antibody for overnight and complexes separated using protein G agarose(25μl per sample,Pierce)before gel electrophoresis.Statistical analysisData are shown as the means±S.D from at least three independent experiments.Statistical analysis was done by using the Prism4.02(GraphPad Software).All data was evaluated using an analysis of variance(ANOV A)followed by Bonferroni post hoc.P<0.05was considered to be significant (*P<0.05,**P<0.01,***P<0.001).ResultsA1R blockade suppresses osteoclastogenesisand bone-resorbing activity in primary BMMs cultured with RANKL and M-CSF in a dose-dependent manner We have previously demonstrated that adenosine regulates osteoclast formation through A1R[30].We therefore examinedTable1Oligonucleotides used for quantitative real-timePCR Target mouse gene Sequence Gene bank reference β-actin(F)5′-ACTATTGGCAACGAGCGGTT-3′NM_007393.3(R)5′-CAGGATTCCATACCCAAGAAGGA-3′Acp5(F)5′-CGTCTCTGCACAGATTGCAT-3′NM_007388(R)5′-TGAAGCGCAAACGGTAGTA-3′Ctsk(F)5′-GGAGGCGGCTATATGACCA-3′NM_007802(R)5′-ACAACTTTCATCCTGGGCCCA-3′MMP-9(F)5′-CCTGTGTGTTCCCGTTCATCT-3′NM_013599.2(R)5′-GCCATACAGTTTATCCTGGTCA-3′Integrinβ3(F)5′-TTTGCCCAGCCTTCCAGCCCA-3NM_016780.2(R)5′-CGGTAATCCTCCTCAGAGCA-3′Integrinαv(F)5′-AACATCACCTGGGGCATTCA-3′NM_008402.2(R)5′-TGAGGTGGTCGGACACGTTT-3NFATc1(F)5′-CTCGAAAGACAGCACTGGA-3′AF309389.1(R)5′-AGGTGCTGGAAGGTGTACT-3′c-fos(F)5′-GAACAACACACTCCATGCGG-3′NM_010234.2(R)5′-GGAGGACCTTACCTGTTCGTGA-3′the effect of A1R blockade on osteoclast formation and func-tion in vitro using pharmacological tools.After5-day culture in osteoclast differentiation medium(M-CSF and RANKL (30ng/ml each)),murine primary osteoclast precursors were differentiated into large,multinucleated TRAP+cells(Fig.1a). When the A1R-selective antagonist,DPCPX,was added at the beginning of culture(day0),a dose-dependent inhibition of osteoclastogenesis was observed,as we previously reported (Fig.1a).DPCPX at1and10μM inhibited RANKL-induced osteoclast formation by about51%and76%,respectively(P< 0.001compared with control treated with RANKL+M-CSF, for both).Even though DPCPX may also block A2A and A2B receptors,it is unlikely that interaction with these receptors plays any role in inhibiting osteoclastogenesis since stimulation of both A2A and A2B receptors inhibits osteoclast differentiation([34]and Supplemental Figure1).Consistent with the effect on osteoclast formation,resorption pit forma-tion by mature osteoclasts at day8of culture was inhibited by 1μM DPCPX by61%(Fig.1a and b,P<0.05)and nearly eliminated by10μM DPCPX(by95%)(Fig.1a,b,P<0.01). DPCPX treatment is not cytotoxic at the concentrations used (MTT assay,data not shown),indicating that the anti-osteoclastogenic effect of DPCPX was not due to toxic effects on osteoclast precursor cells.A similar pattern of dose-dependent inhibition by adenosine A1R blockade was observed with regard to RANK-induced expression of known osteoclast marker genes,including Acp5,Ctsk,MMP9,and Integrinαvβ3(Fig.1c).DPCPX ataFig.1Suppression of osteoclast formation and function by A1R-selective antagonist.Murine BMMs(1×105cell/cm2)were cultured with M-CSF and RANKL(30ng/ml each),with or without various concentrations of DPCPX for5days in48-well plates for TRAP staining(a upper panel),or8days on calcium phosphate-coated slides (BD biosciences)and for pit formation assay(a lower panel),respec-tively.b Numbers of TRAP-positive multinuclear cells containing more than three nuclei(TRAP+MNC)were counted.For pit formation assay,the slides were stained with von Kossa reagent.Bone resorption percentage was quantified using Labworks Image Acquisition and Analysis software4.0.The percentile of pixels of white in a gray scale was measured as a representation of the percentage of resorption area to total area(bone resorption,percent).c BMMs were cultured with M-CSF and RANKL(30ng/ml each),with or without various concen-trations of DPCPX in6-well plates for5days prior to RNA extraction and real-time PCR for Integrinαv,β3,Acp5,Ctsk,and MMP9.β-actin served as PCR control.Relative expression was calculated relative to M-CSF only cells(fold value1).Values are shown as means±S.D.of at least three independent experiments.*P<0.05,**P<0.01,and ***P<0.001compared to RANKL+M-CSF cellsconcentration of 1μM or more significantly reduced the transcription of these genes at day 5of culture.Similarly,rolofylline (KW3902,a more selective and potent A 1R antag-onist)inhibited RANKL-induced gene expression of Ctsk by KW3902(1μM,supplemental Figure 2,P ≤0.001compared with control treated with RANKL±M-CSF),further confirm-ing the receptor specificity of the inhibitory effect of DPCPX we observed in RANKL-induced osteoclastogenesis.Because the selective A 1receptor agonist N 6-cyclopentyladenosine neither directly affected Ctsk expression nor reversed the DPCPX-mediated inhibition of Ctsk expression (Supplemen-tal Figure 3),our results are consistent with the hypothesis that the A 1receptor is constitutively active and DPCPX acts as an inverse agonist to inhibit osteoclastogenesis.A 1R blockade diminishes induction of the transcription factors c-Fos and NFATc1in primary BMMsThe essential role of transcription factors c-Fos and NFATc1in RANKL signaling is now well established.Hence,we determined whether the inhibitory effect of A 1R blockade also leads to regulation of these factors.NFATc1is a master switch for terminal differentiation of osteoclasts;we there-fore examined the expression of NFATc1at day 4of culture.As shown in Fig.2a,b ,whereas NFATc1mRNA levels were augmented about 13.88-fold and protein levels were in-creased about 49.1-fold by RANKL treatment (relative to M-CSF only),these increases were completely abolished by DPCPX at concentrations of 1and 10μM.Previous studies of signaling pathways in osteoclast for-mation indicate that NFATc1is downstream of c-Fos in osteo-clastogenesis regulated by RANKL [17,20].We next determined whether adenosine A 1R blockade inhibits c-fos expression at day 3of culture and found that DPCPX abro-gated RANKL-induced c-fos expression was observed (Fig.2c ).The increase in the c-Fos message induced by RANKL (about twofold)was significantly decreased by DPCPX in a dose-dependent fashion.These results indicate that A 1R blockade directly diminishes the transcription of c-Fos and NFATc1byRANKL.Fig.2Suppression of the RANKL-induced expression of transcrip-tion factors NFATc1and c-fos by A 1R-selective antagonist.BMMs were cultured with M-CSF and RANKL (30ng/ml each),with or without various concentrations of DPCPX for 4days (a ,b )or 3days (c ).Total RNA was isolated and NFATc1(a )and c-fos (c )mRNA levels were quantified by real-time PCR.Relative expression in mRNA levels was calculated relative to M-CSF only cells (fold value 1).Values are shown as means±S.D.of four independent experiments.b The NFATc1protein expression was determined by immunoblotting.Pro-tein band intensities were quantified by densitometry and corrected with β-actin.Protein level (fold of change)was expressed as fold change compared with M-CSF only.Values are shown as means±S.D.of three independent experiments.*P <0.05,**P <0.01compared to RANKL+M-CSF cellsA 1R blockade inhibits RANKL-induced JNK/c-Junactivation,but not that of p38and Erk in primary BMMs An increasing body of information indicates that RANKL stimulates NF-κB and MAPK activation and subsequently elicits the activation of essential transcription factors,such as AP-1and NFATc1,required for osteoclast differentiation.Since we found that DPCPX profoundly blocks the induc-tion of c-fos and NFATc1expression by RANKL,we next examined the two major RANKL-induced signaling path-ways,MAPKs and NF-κB.Our findings confirm prior reports that RANKL induces rapid phosphorylation of the JNK,c-Jun,p38,and Erk in primary bone marrow culture (Fig.3a ).With the addition of DPCPX to the cultures,we observed a substantial suppression of the RANKL-induced phosphorylation of JNK and c-Jun at 15min (P <0.05for both,Fig.3b ),whereas the activation of p38and Erk were not affected.The activation of c-Jun by JNK is an important mechanism involved in osteoclastogenesis [21].Since the expression of c-fos was down-regulated by A 1R blockade (Fig.2c )and it is known that AP-1comprises Fos/Jun dimers,our findings indicate that blockade of adenosine A 1R impaired the RANKL-induced activation of AP-1,the critical factor in osteoclastdifferentiation.Fig.3Inhibition of RANKL-induced activation of JNK/c-Jun,but not that of Erk and p38,by A 1R-selective antagonist.BMMs were stimu-lated with RANKL/M-CSF,with or without 1μM DPCPX for various times.a Cells were lysed and equal amounts of proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies.b Protein band intensities were quantified by densitometry,and the levels of phosphorylated MAPKs were normalized to the total levels of corresponding MAPKs.The ratio of phosphorylated MAPKs/total MAPKs in the RANKL-stimulated cells was relative to that of cells treated with M-CSF only (fold value 1).The densitometry values are shown as means±S.D.of three independent experiments.The figure shows representative data from one of three replicate experiments.*P <0.05using an analysis of variance (ANOV A)followed by Bonferroni post hocA1R blockade inhibits RANKL-induced NF-κB activation in primary BMMsNF-κB signaling in osteoclasts has been extensively studied. Recent study suggests that NF-κB activation is the upstream signaling molecule of c-Fos in osteoclastogenesis[35].Dele-tion of the NF-κB subunits p50and p52causes severe osteo-petrosis through the absence of osteoclasts[36,37].We therefore examined the effects of DPCPX on the activation of NF-κB by RANKL.To this end,we stimulated BMMs with RANKL in the presence or absence of different doses of DPCPX and assessed the degradation of IκB in whole cell lysates at5min and the presence of p65in nuclei at10min by western blot analysis.As previously reported,there is cyto-solic degradation of IκB and nuclear translocation of p65in response to RANKL(Fig.4a).DPCPX markedly reduced IκB degradation in the cytosol and nuclear translocation of p65in a dose-dependent manner(Fig.4b).Consistent with these findings,we observed that RANKL induced significant p65 DNA binding activity starting from2min and continuing as long as30min(Fig.4c).With the addition of1μM of DPCPX to the cultures,we observed a significant inhibition of RANKL-induced p65transcriptional(DNA binding)activity at5min and15min(P<0.05for both).These results indicate that the A1R-mediated inhibition of RANKL-induced osteo-clastogenesis is mediated through two major pathways,inhibi-tion of NF-κB and JNK pathways,which regulate transcription of c-Fos and NFA Tc1in osteoclast precursors.A1R blockade disrupts RANKL-induced formationof TRAF6-TAK1complex in primary BMMsTAK1,a MAPK kinase kinase can form complexes with RANK and TRAF6,and was up-regulated in RANKL-induced osteoclastogenesis([23–25].RANKL-induced oste-oclast differentiation requires TAK1for NF-κB activation[25]. Herefore,we determined whether DPCPX affects the recruit-ment,and interaction of TAK1with TRAF6by RANKL.In line with previous reports,we found a striking induction of TAK1association with TRAF6following stimulationwithFig.4Inhibition of RANKL-induced activation NF-κB by A1R-se-lective antagonist.BMMs were stimulated with RANKL/M-CSF,with or without various concentrations of DPCPX.IκB degradation in whole lysates was determined at5min and nuclear localization of p65was determined at10min with western blot analysis(a).Protein band intensities were quantified by densitometry(b).For IκB degra-dation,the levels of IκB were normalized toβ-actin,and the data are expressed as percentages of control(M-CSF only).For p65nuclear translocation,the levels of p65were normalized to p84and the data are expressed as the fold changes to control(M-CSF only).The densitom-etry values are shown as means±S.D.of three independent experiments.The figure shows representative data from one of three replicate experi-ments.p65DNA binding activity was measured with an ELISA-based assay(c).BMMs were stimulated with RANKL/M-CSF,with or without 1μM DPCPX for various times.Equal amounts of nuclear proteins were collected and incubated into a96-well plate coated with oligonucleotide containing the consensus NF-κB response element.Absorbance at 450nm was read in a plate reader and the data were calculated as ratio to control(M-CSF only).Values are shown as means±S.D.of four independent experiments.*P<0.05,**P<0.01,and***P<0.001 compared to RANKL+M-CSF cellsRANKL.The addition of1μM DPCPX in culture medium completely abolished the RANKL-induced TAK1/TRAF6as-sociation without affecting the total levels of TAK1at5min (P<0.01,Fig.5a,b).Taken together,our results suggest that adenosine A1R activation is required for association of TAK1 with RANK and that blockade of A1R prevents this activation step thereby diminishing TAK1-mediated NF-κB activation. DiscussionIn the present study,we have confirmed,using the selective A1R antagonist DPCPX,that endogenous adenosine,acting via A1receptor is an important regulator of RANKL-induced osteoclastogenesis.Moreover,we found that A1R blockade impairs the activation of NF-κB by RANKL, blocks phosphorylation of JNK and inhibits the transcription of c-fos and NFATc1.These findings indicate that adenosine A1R activation is required for signaling at RANK,and A1R blockade leads to disruption in the signaling pathway for osteoclast formation.These findings also provide a solid explanation for our prior observation that A1R-selective antagonists are most effective in preventing osteoclast for-mation when included at early stages of osteoclastogenesis (before day3of culture)[30].Activation of NF-κB by RANKL is a crucial event in the early stages of osteoclastogenesis.A recent study using p50/p52double knockout splenocytes and ectopic expression of c-Fos shows that NF-κB is upstream from c-Fos and required for c-Fos activation in RANKL-stimulated osteoclast precursors[35].Similarly,in the present study,we found that induction of c-fos by RANKL was prevented by A1R blockade-mediated abrogation of NF-κB activation. Although the consensus NF-κB binding site has not been recognized in the c-Fos promoter,and there is no direct evidence of activation of c-Fos by NF-κB,our findings and others support the hypothesis that NF-κB is upstream of c-Fos in osteoclastogenesis.Adenosine has been reported to activate the JNK pathway through A1R in many other systems[38,39].Our study is the first report to demonstrate that adenosine/A1R is also required for RANKL-induced JNK/c-Jun activation in BMMs.Although the precise role of JNK/c-Jun in osteoclastogenesis has yet to be determined,there is increasing evidence that JNK/c-Jun activa-tion is essential for efficient osteoclastogenesis[22].It has been reported that osteopetrosis develops in transgenic mice with dominant-negative c-Jun in their osteoclast lineage cells[21].A more recent study showed that the JNK/c-Jun pathway is required for an anti-apoptotic effect of RANKL in multinucle-ated osteoclasts[40].However,unlike mature osteoclasts,c-Jun activation by RANKL is not involved in the anti-apoptotic effect of JNK in bone marrow macrophages[22].In line with these observations,blockade of A1R had little effect on cell viabilityinFig.5Disruption of RANKL-induced formation of TRAF6/TAK1 complex by A1R-selective antagonist.a BMMs were stimulated with RANKL/M-CSF,with or without1μM DPCPX for various times.Cell extracts were immunoprecipitated(IP)with anti-TRAF6antibody.The immunoprecipitates were then analyzed by immunoblotting with anti-TAK1antibody and anti-TRAF6antibody(upper two panels).Whole-cell extracts were immunoblotted with anti-TAK1antibody(bottom panel).Protein band intensities were quantified by densitometry(b). The level of TAK1were normalized to TRAF6and the data are expressed as fold change to control(M-CSF only).Values are shown as means±S.D.of four independent experiments.The figure shows representative data from one of four replicate experiments.**P<0.01using an analysis of variance(ANOV A)followed by Bonferroni post hocmouse BMMs(data not shown).These findings collectively argue for an essential role of JNK/c-Jun in osteoclastogenesis but not prevention of osteoclast apoptosis.Our observation that inhibition of activation of JNK and NF-κB by blockade of A1R suggests that these molecules may share a common pathway to induce osteoclast precursor dif-ferentiation.Recent studies have provided more insights into the molecular mechanism underlying the activation of these pathways.In particular,new findings reveal that TRAF6 associates with TAK1through an adaptor protein TAB2. The formation of the complex of TRAF6,TAB2,and TAK1 leads to the activation of TAK1which subsequently phosphor-ylates NF-κB-inducing kinase(NIK)and eventually leads to the activation of the NF-κB pathway[23,41,42].In addition, activated TAK1also activates the JNK pathway down-stream of RANK[24,43].In support of this hypothesis, we found that the association of RANKL/RANK in oste-oclast precursors derived from bone marrow cells evokes a rapid and dramatic accumulation of TRAF6/TAK1com-plex,which is clearly diminished by A1R blockade.This observation is consistent with the previous report that adenosine A1R blockade results in loss of cellular TRAF6 in RAW264.7cells[30].These results suggest that aden-osine A1R blockade-mediated blockade of TRAF6-TAK1 complexes triggers TRAF6degradation.We therefore pos-tulate that the pivotal effect of adenosine A1R in promo-tion of osteoclast formation is the proper formation of TRAF6-TAB2-TAK1complex(Fig.6).Further studies will be required to clarify the role of each component of this complex in mediating adenosine/A1R regulation ofosteoclastogenesis as well as the mechanism by which A1R regulates formation of this complex.Recent studies suggest that the usual classification of GPCR-active agents as agonists,antagonists,or inverse ago-nists(agents that diminish activity of a constitutively active receptor)needs refinement.Activation of GPCRs regulates cellular function by activating G proteins and,as more recently described,activatingβ-arrestin with distinct downstream sig-naling and functional consequences([44]).It has recently been recognized that some receptor active agents preferentially stimulate G protein signaling whereas others stimulate the alternative pathway and this phenomenon has recently been shown to be important for adenosine receptor physiology[44]. Because we found that stimulation of the A1receptor does not affect osteoclastogenesis whereas antagonism of A1receptors inhibits osteoclastogenesis,our results are most consistent with the hypothesis that the A1receptor is constitutively active and that blockade of the A1receptor inhibits osteoclastogen-esis by acting as an inverse agonist.Thus,it is possible that DPCPX is acting as an inverse agonist in the setting of A1R-mediated regulation of osteoclast function.Alternatively, many cell types release or generate adenosine at the cell surface and these low levels of adenosine are sufficient to activate A1R,a phenomenon blocked by DPCPX and other receptor antagonists.Previous studies have demonstrated a role for adenosine and adenosine A1R in regulating formation of multinucleated giant cells from peripheral blood monocytes[45–47],a find-ing consistent with the observations here.Similarly,in prelim-inary studies,we have observed that both DPCPX and rolofylline block osteoclast formation from human bone marrow-derived precursors(He,Mazumder and Cronstein, unpublished observations).Thus,it is likely that the observa-tions on murine osteoclast formation reported here are rele-vant to human osteoclasts as well.Adenosine may either be released from cells via bidirectional adenosine transporters on the cell membrane or adenosine may be generated by hydrolysis of extracellular adenosine nucleo-tides by such extracellular phosphohydrolases as nucleoside triphosphate phosphohydrolase(CD39),ecto-5′nucleotidase (CD73),tissue non-specific alkaline phosphatase(TNAP), among others.CD39and CD73are expressed on the surface of murine osteoclast precursors(He,W.and Cronstein,BN, unpublished),although their roles in producing extracellular adenosine have not been established.Because adenosine A1R can be fully activated by adenosine levels which are inthe Fig.6A proposed scheme of adenosine/A1R-mediated regulation of RANKL-induced osteoclast formation.During osteoclastogenesis,the critical transcription factors c-fos and NFATc1are induced by RANKL. NFATc1is known to be master switch of osteoclastogenesis.Blockade of A1R with DPCPX inhibits osteoclast formation through interfering the RANKL-induced formation of TRAF6/TAK1signaling complex followed by de-activation of NF-κB and JNK and subsequently leading to the reduction of osteoclast formation by RANKL。

Size variation of conodont elements of the Hindeodus –Isarcicella clade during the Permian –Triassic transition in South China and its implication for mass extinctionGenming Luo a ,Xulong Lai a ,⁎,G.R.Shi b ,Haishui Jiang a ,Hongfu Yin a ,Shucheng Xie c ,Jinnan Tong c ,Kexin Zhang a ,Weihong He c ,Paul B.Wignall daFaculty of Earth Science,China University of Geosciences,Wuhan 430074,PR ChinabSchool of Life and Environmental Sciences,Deakin University,221Burwood Hwy,Burwood VIC 3125,Australia cKey Laboratory of Geobiology and Environmental Geology,China University of Geosciences,Wuhan 430074,PR China dSchool of Earth and Environment,University of Leeds,Leeds.LS29JT,United KingdomA B S T R A C TA R T I C L E I N F O Article history:Received 17August 2007Received in revised form 1April 2008Accepted 3April 2008Keywords:Multi-episode mass extinction ConodontHindeodus -Isarcicella Size reductionPermian –Triassic transition South ChinaBased on the analysis of thousands of conodont specimens from the Permian –Triassic (P –T)transition at Meishan (the GSSP of P –T Boundary)and Shangsi sections in South China,this study investigates the size variation of Hindeodus and Isarcicella P1elements during the mass extinction interval.The results demonstrate that Hin-deodus –Isarcicella underwent 4episodes of distinct size reduction during the P –T transition at the Meishan Section and 2episodes of size reduction in the earliest Triassic at Shangsi.The size reductions at Meishan took place at the junctions of beds 24d/24e,25/26,27b/27c and 28/29,and at the junctions of beds 28/29c and 30d/31a at Shangsi.The two earliest Triassic size reduction episodes were correlative between the two sections.These changes coincide with some important geological events such as eustatic sea-level changes,anoxic events,carbon isotope oscillations,miniaturization of brachiopods and microbial changes.Through detailed investigation of the palaeoenvironment and the palaeoecology of Hindeodus –Isarcicella ,the authors propose that the main causes of the size reduction was a sharp decline of food availability because of the mass extinction and the anoxic event during the P/T transition.The pattern of size reduction supports suggestions that the end-Permian mass extinction was multi-episodal,consisting of 3extinction events rather than a single catastrophic event.©2008Elsevier B.V.All rights reserved.1.IntroductionThe end-Permian biotic crisis was the largest mass extinction in the fossil record.It eliminated over 90%of species in the oceans (Stanley and Yang,1994;Bambach et al.,2004)and about 70%of vertebrate families on land (Benton,1988;King,1991;Maxwell,1992).The cause or causes and duration as well as the nature of the extinction remain uncertain and actively debated (Wu and Liu,1991;Wignall and Hallam,1993;Isozaki,1997;Kozur,1998;Yin and Tong,1998;Jin et al.,2000;Yin et al.,2001;Wang and Cao,2004;Fang,2004a,b;Grice et al.,2005;Racki and Wignall,2005;Yin et al.,2007a ).The Global Stratotype Section and Point (GSSP)of the Permian –Triassic Boundary at Meishan in Zhejiang Province,China has served as a focal point in this global debate,as it has provided much critical stratigraphical and palaeontological data.As a result,the section has received intensive multidisciplinary studies by various research groups,including lithostratigraphy,biostratigraphy,sedimentology,sequence stratigraphy,isotope geochemistry,eventostratigraphy,and magnetostratigraphy (Yin et al.,2001and references therein).There are several opinions about the causes and patterns of the P –T mass extinction (Isozaki,1997;Kozur,1998;Wang and Cao,2004;Fang,2004a,b;Grice et al.,2005;Racki and Wignall,2005).Some authors have proposed a single-episode catastrophic mass extinction (Jin et al.,2000;Kaiho et al.,2006),while others have argued for a multi-episode mass extinction (Wu and Liu,1991;Wignall and Hallam,1993;Yin and Tong,1998;Fang,2004a,b;Xie et al.,2005;Shen et al.,2006;Yin et al.,2007a ).In this paper,we attempt to test these various scenarios by using the size variation data of a group of conodont species from a single clade from several continuous Permian –Triassic boundary sections in South China.The fundamental question addressed in the study is to see if the sizes of conodont species varied across the PTB and,if so,whether or not the timing of the signi ficant size changes actually corresponded to any of the proposed PTB extinction intervals.A related question,also investi-gated as an integral part of this study,is to elucidate the possible cause (s)for the size change of the conodont species across the PTB.There is now a considerable literature relating size variation in lineages through time to biotic crises caused by environmental changes in earth history.Initially,Urbanek (1993)coined the term “Lilliput effect ”for an observed size reduction of Silurian graptolites during a biotic crisis.Subsequently,other researchers have reported similar size decreases during times of extinction;for example,the sizePalaeogeography,Palaeoclimatology,Palaeoecology 264(2008)176–187⁎Corresponding author.Faculty of Earth Sciences,China University of Geosciences,Wuhan,Hubei 430074,PR China.Tel.:+862767883139;fax:+862787515956.E-mail address:xllai@ (X.Lai).0031-0182/$–see front matter ©2008Elsevier B.V.All rights reserved.doi:10.1016/j.palaeo.2008.04.015Contents lists available at ScienceDirectPalaeogeography,Palaeoclimatology,Palaeoecologyj o u r n a l h o me p a g e :w w w.e l sev i e r.c o m /l oc a t e /pa l a e oof late Devonian conodonts (Girard and Renaud,1996),heart urchins across the Cretaceous/Tertiary boundary (Jeffery,2001),and various organisms around the Permian/Triassic boundary (Twitchett,2001;He et al.,2005;Twitchett,2005a,b;Luo et al.,2006;Twitchett,2006;He et al.,2007;Twitchett,2007).It is widely held that there was no obvious change in conodont fortunes during the end-Permian mass extinction,because many conodont lineages clearly survived through the PTB (e.g.Clark et al.,1986;Jiang et al.,2007).However,the survival of lineages tends to overlook the potential ecological information that can come from the study of large conodont samples.Furthermore,unlike most other organisms,most Late Permian conodont species or their lineages persisted through into the Lower Triassic.Therefore,these conodonts can serve as the best material for the study of the size variation during the P –T transitional period.Luo et al.(2006)reported a size reduction in P1elements of the conodont genus Neogondolella at the bed 24d/24e junction (Upper Permian)at Meishan.However,the limited Neogondolella specimens from the Lower Triassic did not allow us to perform a full-scale size variation study throughout the Permian –Triassic transition.To overcome this shortfall,in this paper wehaveFig.1.A:Location map of study area in South China;Meishan Section (B)and Shangsi Section (C).(after Wingnall et al.,1995and Jiang et al.,2007).Table 1The total number,mean size,standard deviation,percentage of specimens larger than 0.5mm and 95%con fidence interval of the mean for the P1element of Hindeodus –Isarcicella from the P/T transition at the Meishan Section,Changxing,Zhejiang Province Bed24a 24b 24c 24d 24e 252627a 27b 27c 27d 2829Number 793957717289413314127017147Mean (mm)0.4580.4590.4430.5340.4360.5330.3920.4550.5030.4210.4670.5800.459Standard deviation 0.1320.1430.0790.0960.1000.0060.0430.0980.1080.1030.1580.1500.108Percentage (N 0.5mm)29.1133.3320.0076.6225.5310027.6647.3718.4434.4467.8438.3095%con fidence interval of the mean0.49130.49890.70020.56100.49510.58380.40480.47250.51820.43720.48710.60080.49260.42440.40260.18310.51440.35950.48220.36190.42980.48110.40170.44720.55360.4264177G.Luo et al./Palaeogeography,Palaeoclimatology,Palaeoecology 264(2008)176–187chosen hindeodid conodonts because as a lineage they survived across the P–T boundary.The recovery and size measurement of abundant conodont specimens is time-consuming,and this kind of study has only previously been undertaken for the P–T transition(Luo et al., 2006).The present paper examines the size variations,in large samples,of conodonts from the Hindeodus–Isarcicella clade duringtheFig.2.Size distribution histogram of P1elements of Hindeodus–Isarcicella from each bed in ascending order(24a to29except for bed25)through the P/T transition at the Meishan Section A.Thefigures above each histogram are the total number of specimens in each size range.178G.Luo et al./Palaeogeography,Palaeoclimatology,Palaeoecology264(2008)176–187end Permian to earliest Triassic interval,with the aim of improving our understanding of the mass extinction patterns and of biotic recovery.2.Material and methodThirteen successive bulk samples ranging from bed24to bed29 (the PTB is sited at the bottom of bed27c)were collected from Section A at Meishan,Changxing,Zhejiang province(Fig.1).The sample from bed24was divided into5sub-beds(beds24a–e).Bed27was also evenly separated into4sub-beds(beds27a–d).Each of these13 samples weighed20kg.The clay samples collected from beds25and 28were processed directly by water.The other samples were crushed into1cm3size fragments and treated with dilute acetic acid(10%)to dissolve the samples.A 2.80–2.81g/ml gravity liquid made of bromoform(2.89g/ml)and acetone(0.79g/ml)was used in the conodont separation for all the samples.The conodonts were picked one by one under the binocular stereoscope.All the Meishan samples have been entirely processed over20months.The samples from the Shangsi Section have not been completely processed but in the earliest Triassic there are Hindeodus–Isarcicella elements for this work.Over20,000specimens were obtained from this processing.Among these,more than14,000were P1elements of Neogondolella,Hindeodus and Isarcicella,which are important elements during the P/T transition.A binocular stereoscope and micrometer were used to measure the length between the anterior and posterior ends of each well-preserved Hindeodus–Isarcicella P1element.First,the complete elements with both anterior and posterior parts were measured under the binocular stereoscope with the micrometer in the ocular.Secondly,each measured element was arrayed according to its size recorded in the notebook. Thirdly,the size of some randomly-chosen elements was measured and compared to the measurements obtained from thefirst step and found to be nearly the same.Finally,different personnel were employed to measure some of the randomly chosen elements(about30–40elements of each bed)and compared their measurements with those of thefirst person,and again both results were found to be nearly the same.The mean size,distribution histogram,standard deviation and95% confidence interval of the mean size were used to analyse the size distribution and variation of the conodonts.The mean size(X),which reflects the condition of a community,is based on the following equation:X¼1=N4X Ni¼1liwhere X is the mean size,N is the number measured in each bed and li is the length of each element.The95%confidence interval,reflecting whether the size reduction is credible on95%confidence,is based on the following equation:Y¼t a=2SffiffiffiffiNpwhere Y is thefluctuating range,t a=2is the t-test value of1−αconfidence,S is the standard deviation and N is the number measured from each bed.So,the confidence interval is(X−Y,X+Y),where X is the mean size,X−Y is the lower line,while X+Y is the upper line.3.Taxonomy and evolution of Hindeodus–IsarcicellaBoth hindeodid and isarcicellid P1elements are scaphate,with a robust cusp at the anterior and numbers of smaller denticles following the cusp.The hindeodid P1elements are symmetrical or slightly asymmetric,whilst the isarcicellid P1elements are extremely asymmetric.The cavity of hindeodid shows slight swelling but no thickening,whilst that of isarcicellids is both swollen and thickened. Some of the isarcicellid P1elements have a denticle or series of denticles on one side or both sides of the cavity surface.Interpreta-tions of the evolution of the hindeodid–isarcicellid lineage are controversial(Kozur,1996;Ding et al.,1997;Lai,1997;Wang and Wang,1997).Ding et al.(1997)thought that Hindeodus latidentatus–H. parvus–Isarcicella turgida–I.isarcica formed an anagenetic lineage. However,Wang and Wang(1997)replaced I.turgida with I.staeschei in the evolutionary sequence of(Ding et al.,1997).Nevertheless, almost all authors have accepted the idea of an evolutionary sequence from hindeodid to isarcicellid.Nicoll et al.(2002)proposed that the growth pattern of hindeodids occurred by the addition of denticles at the posterior.The length between the anterior and posterior is therefore an important size parameter for Hindeodus–Isarcicella.During the past two decades,the P–T conodonts at the Meishan section have received intensive studies(Lai et al.,1995;Zhang et al., 1995;Ding et al.,1997;Wang and Wang,1997;Mei et al.,1998).Based on large conodont samples,our group has established parallel gondolellid and hindeodid zones at this section(Jiang et al.,2007), and the hindeodid zones in ascending order are tidentatus zone, H.praeparvus zone,H.changxingensis zone,H.parvus zone,Isarcicella staeschei zone and I.isarcica zone from beds24a to29.According to the latest radiometric dates,the absolute age of bed28at the Meishan Section(GSSP)is250.7±0.3Ma,and that of bed25(white clay)is 251.4Ma(Bowring et al.,1998).Mundil et al.(2004)rectified their earlier data(Mundil et al.,2001),and suggested that the duration of the mass extinction was shorter.In any case,the duration from bed25 to28is about0.7million years.This0.7myr interval corresponds to4 hindeodid conodont zones and hence indicates that the evolutionary rates were high for the conodonts.4.Size variation in the Hindeodus–Isarcicella clade4.1.Data from the Meishan sectionThe number(all the complete elements from each bed),mean size and standard deviation of P1elements of Hindeodus–Isarcicella for each bed are shown in Table1,together with the95%confidence interval of the mean and the percentage of Hindeodus–Isarcicella P1 elements larger than0.5mm.The size distribution within each bed is shown in Fig.2.The mean size of all the individuals within a community which can more precisely reflect the living environment of this community is shown in Fig.3,of which the shadow interval shows the95%confidence interval of the mean.The dominant peak for bed24a is0.4–0.5mm,with element length showing a normal distribution(Fig.2).The dominant peak for bed24b is0.3–0.4mm,but with a marked right-skewed distribution (skeweness=0.3112).The dominant peak for bed24c is also0.4–0.5mm,with a normal distribution,although with fewerelements.Fig.3.Variation of the mean size of P1elements of Hindeodus–Isarcicella from the end Permian(bed24a)to early Triassic(bed29)at the Meishan Section.The black area is the 95%confidence interval of the mean size,and the central white circle and line show variation of the mean size.179G.Luo et al./Palaeogeography,Palaeoclimatology,Palaeoecology264(2008)176–187The dominant peak for bed 24d is 0.5–0.6mm,and it also appears to have a normal distribution.The dominant peak for bed 24e is 0.4–0.5mm,with a marked left-skewed trend.Both the two elements of bed 25are larger than 0.5mm,while the elements of bed 26are 0.3–0.5mm,with no element larger than 0.5mm.The dominant peaks for bed 27a and 27b are within 0.4–0.5mm,and their distribution approaches a normal distribution.The dominant peaks for bed 27c and 27d are 0.3–0.4mm,with slight right skew.The dominant peak for bed 28is 0.5–0.7mm,while that for bed 29is 0.4–0.5mm.Fig.3shows the size variation of conodont Hindeodus-Isarcicella P1element from the end Permian (bed 24a)to earliest Triassic (bed 29).The black area stands for the 95%con fidence interval of the mean,and the central white points and line of the area show the variation of mean size of specimens in each bed.As the graph (Fig.3)shows,during the transitions between beds 24e/24d,26/25,27c/27b and 29/28,the characters (mean size,dominant peak,percentage)of size variation exhibit distinct changes.The dominant peak and the mean size for bed 24d are 0.5–0.6mm and 0.534mm respectively,and the percentage with a size larger than 0.5mm is 76.62%,while these characters for bed 24e are 0.4–0.5mm,0.436mm and 25.53%,respectively.The 95%con fidence interval of the mean size shows that the upper line of bed 24e (0.4951)is smaller than the lower line of bed 24d (0.5144),so the size reduction is credible at a 95%con fidence level.The t -test for means shows that the size reduction was distinct (p =0.000).There are only two Hindeodus –Isarcicella P1elements in bed 25,both of which are larger than 0.5mm,with a mean size of 0.533mm,while in bed 26all the elements are less than 0.5mm,and the mean size is only 0.392mm.The t -test for means (p =0.002)indicates the size variation was signi ficant.Also,the size reduction in bed 26is credible as shown by the 95%con fidence interval of the mean shown in Table 1.In beds 27and 28,Hindeodus –Isarcicella replaced Neogondolella as the dominant genera.There are abundant Hindeo-dus –Isarcicella P1elements in these beds.Jiang et al.(2007)identi fied three assemblages from the end Permian (bed 24a)to the earliest Triassic (bed 29)by comparing the ratio of Hindeodus –Isarcicella P1elements to the Neogondolella P1elements,and beds 27and 28belong to their second assemblage.In bed 27b,the dominant peak and themean size are 0.4–0.5mm and 0.503mm,and the percentage with a size larger than 0.5mm is 47.37%,while in bed 27c,the dominate peak and the mean size are 0.3–0.4mm and 0.421mm respectively,and the percentage of size larger than 0.5mm is 18.44%.The upper line (0.4372)of the 95%con fidence interval of the mean at bed 27c is smaller than that of the lower line (0.4811)at bed 27b.The t -test for means (p =0.000)indicated that this reduction of size was undoubted.In bed 27d,the dominant peak is 0.3–0.4mm,the same as bed 27c,but the mean size is 0.467mm,and the percentage with a size larger than 0.5mm is 34.44%.The mean size in bed 28,where the largest specimens in these beds are found,is 0.580mm,the dominant peak is 0.5–0.6mm,and the percentage with a size larger than 0.5mm is 67.84%.However,in bed 29,the dominant peak and mean size are 0.4–0.5mm and 0.459mm,respectively,and the percentage with a size larger than 0.5mm is 38.30%.At the transition between these two beds,all the parameters vary distinctly,and the upper line (0.4926)of the 95%con fidence interval for bed 29is much smaller than the lower line (0.5536)for bed 28.t -test for means shows no reason to believe that the size reduction was not signi ficant (p =0.000).It is interesting to note that the size of P1elements of Hindeodus –Isarcicella in beds 25and 28is comparatively very large,especially in bed 28(see above).Both of these are clay beds,and previous workers have thought that the extinction took place at these levels (Fang,2004a,b;Xie et al.,2005).In order to determine whether or not the size variation of Hindeo-dus –Isarcicella populations was affected by the presence of different species,we investigated the size variation of a single species,Hindeodus typicalis ,from beds 24a to 24e.This reveals fluctuations similar to those shown by the overall population of conodonts,with a peak size in Bed 24d and a marked reduction in Bed 24e (Fig.4).t -test for means shows that the size reduction during this transition was signi ficant (p =0.007).4.2.Data from the Shangsi sectionTable 2,Figs.5and 6show the size variation of P1elements of Hindeodus –Isarcicella and Hindeodus from the Shangsi Section,Sichuan Province.The first change was recognized in thetransitionFig.4.Variation of the mean size of P1elements of Hindeodus typicalis from the end Permian (bed 24a to bed 24e)at the Meishan Section.The black area is the 95%con fidence interval of the mean size,and the central white circle and line show variation of the mean size.Table 2The total number,mean size,standard deviation and 95%con fidence interval of the mean of specimens of the P1element of Hindeodus-Isarcicella of earliest Triassic age from the Shangsi Section,Guangyuan,Sichuan Province Bed28c 28d 29a 29b 29c 30(b +c)30d 31a 31b 3233Number 1111247732104411915Mean(mm)0.3550.4310.1940.3390.3130.3900.4020.3480.3640.3430.455Standard deviation0.05100.0660.0440.1050.0660.0780.1140.0620.13195%Con fidence interval of the mean0.39490.36300.35380.46810.42560.36310.40040.37350.52770.32720.30430.27310.31230.37830.33290.32770.31340.3829Fig.5.Variation of the mean size of P1elements of Hindeodus –Isarcicella from the earliest Triassic of the Shangsi Section,Sichuan Province.180G.Luo et al./Palaeogeography,Palaeoclimatology,Palaeoecology 264(2008)176–187between beds 29c and 28(combined beds 28c and 28d)in the Hin-deodus parvus zone (Lai et al.,1996).The upper line (0.3538)of the 95%con fidence interval of the mean size of bed 29c is larger than the lower line (0.3272)of bed 28,while the upper line of the 75%con fidence interval of the mean size of bed 29c (0.3321)is smaller than that of the lower line of bed 28(0.3381),and the t -test for means (p =0.021)also shows there is no reason to doubt this size reduction..The second size change was during the transition between bed 30d and 31a,in the I.isarcica zone.The upper line (0.3631)of the 95%con fidence interval of the mean size of specimens in bed 31a is smaller than the lower line (0.3783)for bed 30d.So,the size reduction of this episode is credible at a 95%con fidence level,and the t -test for means (p =0.001)also supports this phenomenon.As in the Meishan section,these data show that conodont size underwent two-episodes of reduction at the Shangsi section during the earliest Triassic.A comparison of the mean sizes of elements in these two sections shows that the mean size of hindeodid conodonts from the Shangsi Section is smaller than that of the Meishan Section.There is no reason to doubt that the difference in mean size is creditable as shown by a t -test of the means (p =0.000).This difference in means size between the two sections will be discussed below.5.DiscussionThe genera Hindeodus and Isarcicella survived the P –T mass extinction and then flourished in the earliest Triassic.Taxonomically,both genera present no obvious change after the major extinction events during the P –T transition.However,the Hindeodus –Isarcicella populations show several episodes of size reduction of P1elements in this interval.As shown in Fig.3–6,from the latest Permian to earliest Triassic the mean size of P1elements of Hindeodus –Isarcicella remained nearly the same except for the several times when they experienced obvious size reductions.If the size changes are evidence of biotic crisis (Girard and Renaud,1996;Schmidt et al.,2006;Twitchett,2007),it would indicate that there were several crises that affected the growth of the conodonts in the P/T transition period.Normally,it is dif ficult to distinguish juvenile hindeodids from small adult elements (Dr.Robert Nicoll,personal communication 2006).However,the texture and the thickness of the cavity are different in the juvenile and adults.The cavity of the juvenile is thin and translucent,whilst that of the adult is thick and ing this criterion,we interpret the size reductions of Hindeodus –Isarcicella elements in our study to be indicative of populations with a higher proportion of juveniles.The evolutionary rate of the hindeodid –isarcicellid clade through the PTB interval is very high,re flecting a rapidly changing environment.In this sense,these conodonts did respond to the P –T mass extinction.The temporal pattern of the conodont size variation is also coincident with someother important geological events such as an anoxic event,sea-level changes and carbon isotope oscillations (Fig.7).5.1.Palaeoecology of conodont Hindeodus –IsarcicellaThe palaeoecology of Hindeodus and Isarcicella is still controversial.There are two main views:one viewpoint considered Hindeodus –Isarcicella to be a near-shore,shallow water taxon (Clark,1974;Wardlaw and Collinson,1984;Tian,1993;Hirsch,1994;Baud,1996;Krystyn and Orchard,1996;Orchard,1996);the other supposed that Hindeodus –Isarcicella had a wide facies range and occurred both in shallow and deeper water environments (Behnkn,1975;Clark,1981;Zhang,1990;Kozur,1995,1996;Kozur,1998).Kozur (1995)also pointed out that Hindeodus shows no provincialism and straddles the P/T boundary and is therefore the most suitable genus for de fining the P/T boundary within a phylomorphogenetic line.Lai et al.(2001)and Lai and Zhang (1999),based on the evolution of Hindeodus –Isarcicella and Clarkina (Neogondolella ),and on palaeoen-vironmental interpretations of the Meishan D Section,concluded that Hindeodus was pelagic,found in sediments deposited at different depths,but living in the top layer of the ocean water.They also suggested that the replacement of Clarkina by Hindeodus was caused by the development of anoxic bottom waters during the Permian –Triassic transition and into the Early Triassic.If the upper water became anoxic (Grice et al.,2005;Huang et al.,2007),even Hindeo-dus –-Isarcicella would be affected.Nicoll et al.(2002)also noted that Hindeodus was present in a wide range of marine depositional environments,but they thought the replacement of Neogondolella by Hindeodus –Isarcicella was due to an increase of turbidity levels.If the turbidity of the water was one of the main factors affecting the size reduction of Neogondolella as proposed by Luo et al.(2006),it cannot have been the reason for the size reduction of the conodont Hindeo-dus –Isarcicella .On the basis of previous literature regarding the ecology of Hin-deodus –Isarcicella and the characters of these conodonts at the Meishan A section,the authors consider that Hindeodus –Isarcicella were pelagic conodonts,maybe dwelling in the photic-zone or somewhat deeper,thereby appearing as fossils in both shallow and deep water facies.The anoxic event may have impacted on the evolution of Hindeodus –Isarcicella,which suggested that perhaps implying photic zone anoxia (such as bed 27at the Meishan Section,Grice et al.,2005).There are also different opinions regarding the feeding mechan-isms of conodonts.Nicoll (1985,1987)thought that the conodont apparatus is the filtering array of a microphagous active suspension feeder,while others have interpreted it to be the grasping and food-processing structure of a macrophagous predator or scavenger (Briggs et al.,1983;Aldridge et al.,1987;Purnell,1993).In either case,the abrupt decline of food supplies could have driven the size reduction of the conodont populations,by creating high juvenile mortality.Based on the detailed study of the polygonal microsculpture of Neogondo-lella elements from bed 24e at Meishan (Jiang et al.,2008)indicates an high juvenile mortality at this bed.5.2.Palaeoenvironmental changes during the P/T transition5.2.1.Sea-level changesZhang et al.(1996)studied the sequence stratigraphy of the Meishan D section in detail and proposed that the top contact of bed 24d is a type II sequence boundary,that bed 24e was an upward shallowing parasequence,and that bed 27b to 29belongs to a transgressive systems i et al.(2001)and Lai and Zhang (1999)analyzed the sea-level change bed by bed from bed 24d to 29of the Meishan D section.They thought that the depth of deposition of bed 24d was about 20–60m,while that of bed 24e was about 30–40m.Bed 25,the “white clay bed ”,is composed of montmorillonite –illiteFig.6.Variation of the mean size of P1elements of Hindeodus from the earliest Triassic of the Shangsi Section,Sichuan Province.The black area is the 95%con fidence interval of the mean size,and the central white circle and line show variation of the mean size.181G.Luo et al./Palaeogeography,Palaeoclimatology,Palaeoecology 264(2008)176–187。