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韩美娟1，2 1. 河北北方学院药学院，2. 河北北方学院附属第一医院药学部，【摘要】铜绿假单胞菌或获得性耐药。
究PA分泌系统毒力因子的致病机制对预防和治疗其引起的感染具有重要意义。
【关键词】铜绿假单胞菌；【中图分类号】Research Progress on the Secretion System and Virulence Factors of Pseudomonas Aeruginosa外膜中的促胰液素复合物组成。
其中促胰液素所包含的结构已知的膜嵌入C 端结构域和模块化的周质N 端结构域，又与聚集底物和连接内膜配置有关。
其独特的结构组成有利于PA 将结构化的效应蛋白经细胞周表1 I 型分泌系统分泌的毒力因子毒力因子分类活性功能AprA 碱性蛋白酶［3］胞外酶锌依赖性金属蛋白酶破坏已形成的嗜中性粒细胞加重宿主感染；切割宿主免疫补体C2和C3；降解细胞因子干扰素（INF ）-λ和肿瘤坏死因子（TNF ）- αHasAp ［4］ 铁卟啉化合物血红素获得蛋白获取建立感染的必需营养素-铁；血红素的获取和代谢对慢性和急性感染至关重要AprX 碱性胞外酶锌依赖性金属尚不明确蛋白酶［5］蛋白酶表2 Ⅱ型分泌系统分泌的毒力因子T2SS 亚型毒力因子分类活性功能Xcp 系统LasA 弹性蛋白酶（金属蛋白酶）［9］胞外酶β-裂解金属肽酶/丝氨酸蛋白酶切割甘氨酸-甘氨酸肽键；分解宿主细胞表面蛋白多糖，影响信号转导；增强LasB 弹性蛋白酶的裂解活性LasB 弹性蛋白酶［10］胞外酶M4热熔素肽酶家族/锌依赖性金属蛋白酶对宿主防御系统具有强降解性（例如表面活性蛋白a 和D 、补体和粘蛋白）；降解宿主细胞组织蛋白（例如弹性蛋白和胶原蛋白）；阻碍体内离子转运并损伤天然免疫系统，还可能释放利于细菌存活的营养素，在初始发病阶段发挥重要作用PlcH ［11］胞外酶溶血性磷脂酶C 降解磷脂酰胆碱或鞘磷脂，破坏真核细胞结构，为细菌生长提供营养PlcN ［12］胞外酶非溶血性磷脂酶C 降解磷脂酰丝氨酸和磷脂酰胆碱产生磷酸酯PlcB ［13］胞外酶磷脂酰乙醇胺-磷脂酶C水解磷脂酰胆碱和磷脂酰乙醇胺；破坏宿主细胞外膜和遗传物质完整性CbpD 几丁质结合蛋白［14］胞内酶裂解性多糖单加氧酶为细菌在血液中存活提供条件，其催化活性对毒力有实质贡献；影响先天免疫和后天免疫外毒素A （ToxA ）［15］胞外酶ADP -核糖基转移酶参与宿主蛋白合成，突出细胞毒性作用；破坏宿主固有免疫和获得性免疫；损伤细胞组织PrpL［16］胞外酶赖氨酸特异性内肽酶（蛋白酶Ⅳ）降解纤溶酶原免疫球蛋白和补体成分；抑制成纤细胞、角质形成细胞和内皮细胞的生成；切割白细胞介素（IL -22），破坏先天防御屏障LipA ［17］ 胞外酶三酰基甘油酰水解酶通过静电作用与细菌外膜基质中的多糖海藻酸盐相互作用，促进生物膜的形成LipC［18］胞外酶脂肪酶促进细胞聚集，影响细菌生物膜的遗传特性，直接或间接的影响黏液性生物膜的形成；可能还与细菌游动以及鼠李糖脂的形成有关PhoA ［19］ 胞外酶碱性磷酸酶去除核苷酸、蛋白质以及生物碱中的磷酸基团；在碱性条件下发挥出更强的作用PaAP ［20］胞外酶氨肽酶满足细菌利用蛋白质作为营养供应的需求；与细菌外膜囊泡形成有关；影响细菌生物膜的形成绿脓素（PCN ）［21］次级代谢产物，三环吩嗪氧化-还原活性两性离子具有肠道吸收、血浆蛋白结合潜力和跨生物膜通透性；重要的氧化还原性毒力因子；诱导中性粒细胞凋亡，破坏免疫调节功能，增加细胞毒性Hxc 系统LapA 磷酸结合蛋白［22］胞外酶碱性磷酸酶在细菌生物膜的形成中至关重要Txc 系统CbpE ［23］膜蛋白甲壳素结合蛋白与CbpD 具有部分同源性；与细菌的发育、黏附和信号转导有关；可当作碳源质分泌到宿主细胞的外环境，进而导致一系列的细胞损伤［7］。
S ECURITY E NGINEERING WITH P ATTERNSMarkus Schumacher1and Utz Roedig2Darmstadt University of TechnologyDepartment of Computer Science1IT Transfer Office(ITO)markus.schumacher@ito.tu-darmstadt.de2Industrial Process and System Communications(KOM)utz.roedig@kom.tu-darmstadt.de6th March2002AbstractConducting digital business requires secure network and application architectures.The recently increasing occurrence of severe attacks has shown,however,that we will still need quite some time and effort to reachsecurity standards of IT systems alike the standard already usual in otherfields.At present,there is a huge gapbetween theory and the code of practice.Whereas scientists work on formal approaches for the specification andverification of security requirements,practitioners have to meet the users’requirements.The Pattern Communityrecognized this problem,too.Patterns literally capture the experience from experts in a structured way.Thusnovices can benefit from know-how and skills of experts.Hence,we propose to apply the pattern approach to thesecurity problem.We show that recent security approaches are not sufficient and describe how Security Patternscontribute to the overall process of security engineering.A Security Pattern System provides linkage betweenSecurity Patterns.Thus dependencies between specific security problems can be considered in a comprehensiveway.1IntroductionConducting digital business requires secure network and application architectures.The result of a well-engineered security system must be an architecture that ensures specific security aspects such as privacy,confidentiality, integrity and availability.The occurrence of the Love Letter virus or the Distributed Denial-of-Service attacks against famous web sites in the beginning of2000has shown,however,that we will still need quite some time and effort to reach security standards of IT systems alike the standard already usual in otherfields.The following examples show that we obviously do not learn from past security incidents and that we make the same mistakes over and over again:1.Virus Attacks.In March1999,the Melissa macro virus within a Microsoft Word document having beendistributed via the email program Microsoft Outlook,caused severe damage(about80million dollars).The reason was that Microsoft Word was able to(mis)use the e-mail interface of Microsoft Outlook.In March2000,the Love Letter virus caused a damage of10billion dollars.This virus used almost the same methods as thefirst one,i.e.the e-mail functions of Microsoft Outlook could be used to spread all around the world.2.Virtual Private Networks(VPN).The specification of the Point to Point tunneling Protocol(PPTP)isconsidered to be secure.During the implementation for the Windows NT platform some basic mistakes have been made though[SM98].The establishment of an encrypted link requires a shared secret between both VPN endpoints.Unfortunately,the chosen shared secret based on the Windows password scheme, which is known to be breakable and insecure.As a consequence,the overall implementation of PPTP was insecure.A very important issue is a more comprehensive view of security since security obeys the rule that a chain is as weak as its weakest link.At present,there is a huge gap between theory and the code of practice.Scientists develop formal approaches for the specification and verification of security requirements of IT-systems.The prac-titioners follow specific methodologies in order to meet the users’requirements such as performance,usability, and reliability.Unfortunately,security is merely treated as add-on.Furthermore,today’s software engineering practices don’t consider security sufficiently.The Pattern Community is aware of this problem,too.Patterns are a concept to solve recurring design prob-lems in a literary form.As written in[Cop97]”each pattern is a relationship between a certain context,a certain system of forces which occurs repeatedly in that context,and a certain spatial configuration which allows these forces to resolve themselves.”Patterns capture the experience from experts in a structured way.Thus novices can benefit from know-how and skills of people who have put more effort into understanding contexts,forces,and solutions than novices have done or want to do.As there is a visible deficit in security engineering,we propose to apply the pattern approach to the security problem.A Security Pattern System provides linkage between various Security Patterns.Thus dependencies between specific security problems can be considered in a comprehensive way.As shown in this paper Security Patterns have several advantages:•Novices can act as security experts.•Security experts can identify,name and discuss both problems and solutions more efficiently.•Problems are solved in a structured way.•Dependencies of components can be identified and considered appropriately.The remainder of this paper is organized as follows:in Section2we describe basic reasons for the problems in thefield of IT-security.In Section3we compare some well-known methodological approaches for establishing security.We have a look at the benefits and drawbacks of each concept.In Section4the concept of security patterns is introduced.We redefine a template for security patterns,present some basic definitions and the related work.In Section5we envision the possibilities of security engineering with patterns.In order to underline this we show the benefits of this approach with a real world scenario.Finally,a summary of the contribution of this document and an outlook into the future work is given in Section6.Appendix A contains the pattern system we used in this document.2Security and the Human FactorThe main reason for the existing problems is that security engineering is no technical problem in thefirst place. Security is always implemented by humans on request of humans.Thus the human factor influences IT-security in significant ways:•Security engineering by non security experts.With the exception of cryptography,the science of IT security is quite new and covers multiple disciplines such as operating systems,computer networks and software engineering.It is very difficult to make IT-systems secure as there are many different components and mechanisms involved.In addition,trust relationships change frequently,which makes an analysis of all security requirements very hard.One cannot assume that the average system developer or architect isa skilled security expert,too.We should notice that in most cases security engineering is done by non-security experts.For example,the developer of an IT-system has to implement the necessary security features to some extent as an add-on.In most cases the developer is an expert regarding the system’s functionality,but not regarding its security.•Structured problem solution.When people try tofind a solution for a problem,they often follow an ad-hoc approach.The impact on elements aside the adopted solution are overseen in most cases[Dor97].A good example is the handling of system passwords:a system administrator wants to prevent that theusers chose weak passwords.Thus he configures the system to enforce strong passwords,i.e.a user cannot enter passwords that don’t meet certain criteria(password must not be contained in a dictionary).This solution is straight forward.Unfortunately,the administrator doesn’t realize that nobody can remember strong passwords any longer.As a consequence,users might start to write their passwords on a slip of paper that could be found under their keyboard,or even worse,pinned on their monitor.•Scope dependencies and completeness.IT-security is a very complex area with manifold dependencies.Even if all dependencies are identified,it might be difficult to consider all of them appropriately.A Web server serves as a good example:the administrator canfigure out whether the Web server software is secure or not.But probably he cannot decide whether the combination of Web server,operating system and hardware components is secure.The overall system is too complex to be considered as a whole.Often, the developer or administrator of a specific component doesn’t even want to worry about security outside his area of responsibility,e.g.the Webmaster only cares about his Web server.Security engineering making sense should be possible without knowledge of the whole security scope and all its dependencies.•Time dependencies.IT-Security is also time-dependent.A system that is considered to be secure today may be insecure tomorrow.Statements about the system’s security are only valid with time references.For instance,the strength of cryptographic algorithms typically is founded on difficult mathematical problems.If a geniusfinds a solution for such a problem,security suddenly disappears.Security engineering could be done more efficiently if people get support with handling the aspects described above.This support should comprise appropriate methods and tools.3Methodologies for Security EngineeringIn recent years a number of security methodologies have been developed to assist organizations in establishing and maintaining security.These methods differ regarding their structure,granularity,flexibility,usability,costs, or whether they are formal or informal.In this section we outline the characteristics of some well known methods. Then we describe how the aspects stated in the previous Section are addressed.3.1Security PolicySecurity activities usually begin with the development of a security policy.In general policies describe the use of an institution’s information.At a system level,the security policy enumerates objects and assets as well as threats targeted on them.Furthermore,the security-level envisaged is described[CZ95].Subsequently this security policy is applied to IT-systems.In practice,the security policy is a simple informal text document.Regarding the problems stated in Section2,an informal security policy has several serious drawbacks:it does not help to recognize dependencies between various aspects,it contains a monolithic and linear description of problems and solutions,interferences of different sections are not obvious.Furthermore,real-world security policies never reflect the actual security situation and are always out-dated,as many parameters(e.g.requirements or attacks)change frequently.Besides,the person writing the document has to know a lot about security,as no aspect of security should be missed.3.2Evaluation CriteriaThe evaluation of the security of IT-systems is very important for military,intelligence and more and more public organizations.Thus a variety of evaluation criteria and security guideline documents have been developed by various governments in cooperation with some large organizations.Prominent examples are the Trusted Computer System Evaluation Criteria(also known as“Orange Book”)and the Common Criteria.All criteria define different levels at which IT-systems can be evaluated and compared.The levels represent different sets of functionality and an increasing level of assurance.The primary goal is to prove that the system fulfills certain requirements regarding its protection mechanisms and that the correctness of the implementation meets a certain assurance level.However,evaluation criteria have the following drawbacks:they focus on individual targets of evaluation (TOE).Therefore dependencies to other IT-systems and components could be missed easily.Besides,the evalu-ation of IT-systems according to any criteria is a costly and time-consuming task.Thus it is difficult to keep the evaluation results of a TOE up-to-date.Furthermore,it is difficult for the layman to perform such evaluations as the process very complex and requires a lot of background knowledge.3.3Tree RepresentationsSeveral approaches with symbolic tree representations have been developed.Typically they are often based on AND/OR trees that allow some basic calculations if values are assigned to the nodes such as the cost of an attack. The values can be propagated up the tree to the root.Another result could be which attack would be the most likely one,if the values possible and impossible are assigned to the tree nodes.There are Fault Trees(determine system failures that could stop the functionality of a system,see[KM94]),Threat Trees(identify threats to a system,see[Amo94])and Attack Trees(identify possible attacks to a system,see[Sch99]).As the tree approaches cover only single aspects,they can only be a part of an overall security methodology. Tree approaches are a good way to identify threats,faults,or attacks in a systematic way.On the other hand the process of creating any of these trees still requires a lot of security know-how.If you are a novice in thefield of security,it is very likely that you will forget some aspects but as written in[Sch00]“creating(attack)trees requires a certain mindset and takes practice”and“you’ll get better with time”.As a sufficient coverage of all considered aspects(e.g.attacks or threats)is desired,something like a(e.g.public)tree repository is needed. Furthermore,periodical reviews have to defined in order to keep the trees up-to-date.3.4Formal MethodsSecurity is a current hot topic in the formal methods community.Typically formal methods are applied to proto-cols for authentication,fair exchange,electronic commerce,and electronic auctions.Policies in a formal way and especially authorization policies are used in the area of management of dis-tributed systems.There exists different languages[DDLS01]to describe policies in a formal way and also mechanisms to check consistency and conflicts[SX01].Typically formal methods are applied only to specific problem areas such as smart-cards or cryptographic rger systems cannot be handled with formal methods due to increased complexity and dependencies. They deliver important contributions to thefield of security.But the layman cannot be expected to have the required mathematical background.The verification of a system can be only as good as the assumptions that have been postulated.Thus the biggest challenges of the formal method community will always be to improve the specification capabilities.This could be a difficult task as systems become more and more complex.Other exercises will be to push the integration of formal methods into the overall process of engineering,to obtain tool support,and to cope with partial specifications.3.5Semi-formal ApproachesData-,function-and object-oriented modeling methods are counted among the semi-formal approaches.They are characterized through different graphical elements but also linguistic elements.An example of data modeling techniques is Entity Relationship Modeling(ERM),for object-oriented modeling the Unified Modeling Language (UML).The usage of UML class diagrams offer the possibility to describe a role,his characteristics and relations between roles.Statecharts can be used to describe the behavior of elements or roles.Activity graphs,sequence diagrams,and collaboration diagrams are used to describe the cooperation of the different elements of the whole system.Condensed,regarding to the aspects from Section2,semi-formal approaches,especially UML,offer a struc-tured way to describe systems and possibilities to show dependencies between elements.The disadvantage of these methods is that an automated validation isn’t possible.However,UML can be made more formal by writing constraints in Object Constraint Language(OCL)as part of the model.The advantages of these methods espe-cially of UML over the formal methods are that they are human-readable,depict and useful for non-modeling experts also.4Security PatternsPatterns are a hot topic in the software community.They describe recurring solutions to common problems in a given context and system of forces[Ale79].Meanwhile patterns can be found in many problem domains,e.g. Human Computer Interaction(HCI)or Teaching and Learning.In this section we define some basic terms and concepts of security patterns.Furthermore,we present the related work.4.1Template for Security PatternsPatterns are a literary format for capturing insights and experience of expert designers and communicating it to novices.Both the definitions of a security pattern and a security pattern system that are presented below are based on the descriptions that are given in[BMR+96].We show what additional aspects are necessary to make a pattern to a security pattern.Definition1(Security Pattern)A security pattern describes a particular recurring security problem that arises in specific contexts and presents a well-proven generic scheme for its solution.The more information a pattern has,the more important structure becomes[Vli96].Structure leads to uni-formity of patterns.Thus people can compare them easily and search for information in a systematic way.Less structure means more informal text,which might befine for casual reading but unacceptable for comparison and reference purposes.Introducing the key elements of security patterns we follow the Mandatory Elements Present pattern[MD96].We make use of the terminology given in the Common Criteria[CC99].•Name:Certainly security patterns aren’t different to“normal”patterns with regard to their name.The name of the pattern becomes a part of the vocabulary of the community.It should be easy to remember and refer to.A good name should be evocative and give an image what the pattern might be about.•Context(and Related Patterns):Based on a scenario the context of the security pattern is illustrated.The general conditions under which the problem does occur and which forces do emerge are described.It is useful to list context setting security patterns,too.As some countermeasures may introduce other vulnerabilities,additional security patterns should be considered in the related patterns section,too.The same is valid for problems that are solved partly or couldn’t be considered within the given security pattern.That way a pattern hierarchy will be formed.•Problem:The Problem statement defines the problem that will be solved by the security pattern.The major aspects of the problem are elaborated by the viewpoint of the Forces to be solved.In thefield of security a problem occurs whenever a system component is protected in an insufficient way against abuse.Generally spoken we have to deal with generic Threats i.e.a potential for the violation of security.A threat is a possible danger that exploit Vulnerabilities.A typical threat action is an Attack that lead to security violations such as disclosure,deception,disruption and usurpation.Thus we propose to use Attack Trees (see section3.3)in order to identify attacks in a systematic way.Other Forces could be trade-offs between security and other aspects such as usability and performance,too.The perception of threats is determined by certain Security Objectives that may be written down as Security Policy Statements.•Solution:This section describes the Solution of the Problem.Appropriate solutions are determined by the Context of the pattern.Countermeasures have to be applied in order to reduce the Risk.For each Threat/Attack there should be at least one countermeasure.It is useful to warn from pitfalls(how does this pattern become an Anti-Pattern)and refer to variants of the pattern.There are also some optional elements that can be applied if they improve the comprehension of a security pattern.The Aliases section lists other names by which this security pattern might be known.Certain diagrams can be used to illustrate Structure of and Interactions between the participants of a security pattern.Security has impacts on many other requirements such as performance and usability.Thus it could be helpful to enlist the Consequences of the application of an security pattern.The benefits and drawbacks of a security pattern can be discussed.In order to illustrate the application of the pattern,concrete Examples could be provided[MD96]. Useful are code or configuration samples as well as some sketches.Analogies to real-world scenarios are also suitable such as the Running Example of a military base that were used in[YB97].As we have already denoted, security is always hard to proof.In fact it’s much more easier to show that something went wrong.We can use a Counterexamples section in order to show how the Security pattern can be applied in the wrong way,i.e.it becomes an Anti-Security pattern.Analogous to[BMR+96]we prefer the notion of a security pattern system.A security pattern doesn’t exist in isolation,there are many interactions with other security patterns.The security pattern system describes the relationships and the linkage between individual security patterns.Thus dependencies between specific security problems can be considered in a comprehensive way.As thefield of security is very broad,we cannot speak of a pattern language,that implies the complete coverage of every aspect of the problem rmally a security pattern system can be defined as follows:Definition2(Security Pattern System)A security pattern system is a collection of security patterns,together with guidelines for their implementation,combination and practical use in security engineering.A formal model of a pattern system,that is independent of a problem domain,has been introduced in[Bor00]. In[Sch01a]we applied a slightly modified version of this model.It turned out that it is sufficient to refer only to mandatory elements of patterns[MD96].These elements allows to represent pattern systems as directed acyclic graphs and to perform basic reasoning,e.g.if a cycle occurs there must be something wrong with the process of abstraction.Many pattern practitioners think that formal approaches should not be applied to patterns [BMR+96].Formal models are,however,especially required in thefield of security to proof that certain security requirements are met.In5.2we describe what we expect from the usage of a formal model in the context of security patterns.As a security pattern system covers only certain aspects of security,it should meet at least the following requirements[BMR+96]:it should comprise a sufficient base of security patterns;all its security patterns should be described uniformly;the relationships between security patterns have to be exposed;it must support the security engineering process;its own evolution must be supported.4.2Related WorkWe are aware of the following contributions which focus on security related patterns.An up-to-date list of patterns related to security can be found at[Sch01b].In order to reflect the evolution of security patterns they are presented in chronological order.•Application Security.In[YB97]a framework to build secure applications is introduced.In order to take advantage of the security of underlying systems,an interface for a Secure Access Layer is described.The Single Access Point restricts the entries into the application.The Check Point allows to make appropriate decisions when dealing with security breaches.In order to achieve access control,Roles that grant or deny rights are assigned to groups of users.A Session allows to distribute global user information such as the user’s identity.Eventually two patterns that deal with human computer interface(HCI)aspects are provided.According to their Role the user’s possibilities are restricted with a Limited View of legal options or they are given a Full View With Errors,when their privileges are not sufficient to perform certain actions.Although the secure access layer is introduced the linkage to low-level security services such as cryptog-raphy isn’t stated explicitly.•Cryptographic Software.Cryptography is the classic area of IT-security.In[BRD98]a set of nine pat-terns for building cryptographic software components is introduced.The focus is on the traditional aspects of security,i.e.confidentiality,integrity,authentication and non-repudiation.The Information Secrecy pattern describes how to keep messages secret from an attacker(confidentiality).The Message Integrity pattern shows how to prevent that an attacker modifies or replaces messages without the sharing of cryp-tographic keys.The Sender Authentication pattern illustrates how messages can be authenticated with the usage of cryptographic keys.The Signature pattern describes how it can be prevented that communicating parties cannot repudiate a message(non-repudiation).Based on this four generic cryptographic patterns, the remaining ones are derived,such as Secrecy with Integrity and Secrecy with Sender Authentication.Together they form a Generic Object-Oriented Cryptographic Architecture(GOOCA).•The Authenticator Pattern.The work presented in[BDdVF99]describes a single Pattern that“performs authentication of a requesting process before deciding access to distributed objects.”With respect to secu-rity the authors identified relationships to patterns for authorization as introduced in[FH97]and[NG98]•Authorization Patterns.Several contributions deal with authorization.In[HLF00]the security func-tions of authentication,access control and datafiltering in a distributed environment are combined in a framework.This framework consists of the following patterns:Data Filter[FF99],Bodyguard[NG98], RPC Client[HF99]and Authenticator[BDdVF99].These patterns and a security model structure build the Object Filter and Access Control Framework.In[Fer00]some authorization patterns are discussed,namely Authorization Rules and Role-Based Access Control.The clue is that metadata constraints are used to define authorization.Furthermore,architectural levels(based on the Layer pattern[BMR+96])are defined whereas each level has its own security mech-anism and security enforcement.The combination of a layered architecture that includes a metalevel and patterns are a promising approach for a security pattern system.Picking up this work,a pattern language for security models is given in[FP01].There are patterns for established security models:the Authorization pattern(access matrix),Role-Based Access Control pattern and Multilevel Security Pattern(Bell-LaPadula).It is suggested how these abstract patterns can be applied to all levels of the layered architecture.The work on security patterns has evolved over the past years.Today a mixture of single patterns,frameworks and pattern languages is available.The following topics are,however,left for future work:•As written in[MM97],a sense of scale is missing from most software design patterns.Especially in thefield of security it is very important to have several levels of abstraction and different views on IT-systems.It is left for future work to identify a suitable classification scheme for security patterns.Good starting points are the pattern categories that are introduced in[BMR+96]:architectural patterns,design patterns and idioms.Other promising work is the Layer model described in[Fer00]and the OM-AM framework introduced in[San00].Such approaches help to“put things in perspective and to emphasize the encompassing nature of security[FP01].•The application of security patterns at other layers has to be considered.For example the Check Point pattern[YB97]does also apply at the system and distribution layer:firewalls are in fact check points.•Besides,the pattern approach can be applied to many other security concepts such as cryptographic keys, protocols(e.g.SSL)and algorithms(such as hashes,signatures,and encryption)that could be included ina security pattern system(mining and refactoring of security patterns).•All security patterns have certain commonalities,or“junction points”where they could be grouped to-gether,to form“the”pattern language(or as we prefer to say pattern system)for the security domain.In future we should think about suitable junction points and concepts for merging security patterns should be developed.The layer approach given in[Fer00]and[FP01]provides a good starting point in this direction.5Security Engineering with PatternsIn this Section we explain why security patterns are a suitable approach for security engineering.Then we envision the possibilities of tool supported security engineering with patterns.We introduce our latest activities in this area,too.Then we discuss the benefits of this approach with the aid of a real world scenario in the area of secure networks.The patterns used in this Section can be found in Appendix A.Finally we discuss research topics that aren’t covered in a satisfactory way.5.1Patterns and IT SecuritySecurity patterns are a suitable approach for security engineering.As shown in the following all important aspects that were given in the problem statement in Section2can be covered:•Security engineering by non security experts.Security patterns capture the know-how and skills of security experts.Thus Security Patterns enable novices to act as security experts.Experts use the security patterns as a common and powerful vocab-ulary in order to deal with security problems and solutions.The members of a security pattern community can identify,name and discuss both problems and solutions more efficiently.The systematic analysis of known security breaches will reveal bad practices(anti-patterns).Based on this,refactored solutions can be developed and published as best practice security patterns.•Structured problem solution.Security patterns prevent ad-hoc problem solutions,as they help tofind proven solutions in a systematic and structured way.As security patterns are linked to related security patterns,the impact of certain decisions becomes evident.Side-effects and alternative solutions can be identified easily.•Scope dependencies and scope completeness.。
The Four Stages of Bee GrowthThe life cycle of a bee is a fascinating journey that unfolds through four distinct stages: egg, larva, pupa, and adult. Each phase contributes to the development of a fully functional bee, essential for the survival and prosperity of the hive.The journey begins with the egg stage, the first and most fundamental phase of bee growth. The queen bee, the reproductive heart of the colony, is responsible for laying these eggs. She selects a cell within the honeycomb, a hexagonal structure built by the worker bees, as the perfect incubator for the next generation. The eggs are tiny, yet they carry the genetic blueprint for the future bees. Once laid, the eggs are incubated by the surrounding worker bees, who maintain a constant temperature within the hive to ensure optimal conditions for growth.After about three days, the eggs hatch into larvae, the second stage of bee development. These larvae are tiny, white, and worm-like, completely dependent on the worker bees for nourishment. The larvae are fed a diet of royal jelly, a nutrient-rich secretion produced by the glands of worker bees. This diet is crucial for the larvae's growth and development, particularly for those destined to become queens, as theyrequire a higher concentration of royal jelly to reach their full potential.As the larvae grow and molt several times, they enter the pupal stage, a transitional phase between larva and adult. During this time, the larva undergoes profound physical changes, transforming from a legless grub into a recognizable bee. The pupal stage lasts for approximately eight to ten days, during which the bee's eyes, antennae, legs, and wings develop. This is a critical period, as any disruption to the pupal chamber or changes in environmental conditions can fatally impact the developing bee.Finally, the bee emerges from the pupal case as a fully formed adult. This emergence, known as eclosion, marks the beginning of the bee's adult life. Depending on its caste, the bee will assume different roles within the hive. Worker bees, the most numerous caste, are responsible for a wide range of tasks, including foraging for food, caring for the young, and maintaining the hive. Queen bees are the reproductive queens of the colony, while drone bees, the male members of the hive, are primarily involved in mating with queens.Each bee, regardless of its role, contributes to the overall health and success of the hive. The intricate dance theyperform to communicate the location of food sources, their tireless efforts to gather nectar and pollen, and their dedication to the care of the young are all essential components of the hive's survival.The four stages of bee growth are not only fascinating in their complexity but also serve as a testament to the resilience and adaptability of these remarkable creatures. From the delicate egg to the fully functional adult bee, each phase represents a crucial step in the continuation of the bee species. The hive, a bustling metropolis of activity and cooperation, is a testament to the power of collective effort and the beauty of nature's design.Moreover, the bee's lifecycle offers valuable insights into the natural world and the interconnectedness of all life. Bees play a crucial role in pollination, a process that is essential for the reproduction of many plant species and, ultimately, the maintenance of our ecosystems. Their decline, due to factors such as habitat loss, pesticide use, and climate change, has sparked global concern over the impact on biodiversity and agricultural productivity.Understanding the stages of bee growth, therefore, not only enhances our appreciation for these insects but alsohighlights the importance of protecting them and their habitats. As we learn more about the intricate lives of bees, we gain a deeper understanding of the natural world and our role in preserving it for future generations.In conclusion, the four stages of bee growth represent a remarkable journey from the minuteness of the egg to the vibrancy of the adult bee. Each phase is essential for the development of a healthy and functional bee, crucial for the survival and prosperity of the hive. By understanding and respecting the lifecycle of bees, we can better appreciate their role in nature and work towards safeguarding their future.。
Unit 11. constituent (components): component part 成分2. conceive (conceptualize): To form or hold an idea: 构想出：构想出或持有一种想法3. synthesis (making a complex whole by combining ideas): The combining of separate elements or substances to form a coherent whole. 综合：把分别的元素或物质连接成连贯的一个整体4.evolve (develop): To develop or achieve gradually: 使发展：逐渐发展或完成5. venom (poison): A poisonous secretion 毒液：一种诸如蛇、蜘蛛等动物的有毒的分泌物6.matriculation (admission): to be formally admitted to study at a university or college 入学7.proprietary (with the exclusive legal right)：专有的8.highlight (emphasize)：underline 使…显得重要；强调Unit 21.ill-bred (badly behaved): rude or behaving badly,没教养的2.break down (destroy): 损坏3.conceited (self-important): Holding or characterized by an unduly high opinion of oneself; vain. 自负的4.malicious (hateful): very unkind and cruel 充满憎恨;有恶意的pliment (praise): An expression of praise, admiration, or congratulation. 恭维：赞美6.opponent (rival): One that competes 竞争对手7.be entitled to (be worthy of配得上): have the right to do [entitle 有资格]8.alight (get off): To come down and settle, as after flight: 下来9.seniority (high standing through long service)：资历深的人, 年资高的人.Unit 31.scramble (hasty movement)：move quickly 快速移动hasty 急速的2.expertise (knowledge)：Skill or knowledge in a particular area. 专门知识3.disruption (disturbance)：a situation in which something is prevented from continuing in its usual way 扰乱4.renaissance (revival)：A rebirth or revival. 复苏;复兴5.assumption (supposition)：Something taken for granted or accepted as true without proof; 假定，假设6.dexterity (skill of the hand)：Skill and grace in physical movement, especially in the use of the hands; adroitness. 灵巧，敏捷：体育运动熟练或动作优雅，尤指手的使用；灵活7.manipulate (handle with skill)：To operate or control by skilled use of the hands; 通过用手的技巧来操纵或控制8.sleight (skill)：Skillfulness in the use of the hands or body 技巧9.autism (self-absorption)：Abnormal introversion and egocentricity; acceptance of fantasy rather than reality. 孤独症,自我中心主义.10.g et to grip with (cope with): to begin to understand and deal with sth difficult 开始理解并着手处理难题Unit 41.hypothetical (theoretical): based on a situation that is not real, but that might happen 假定的2.put a premium on (encourage): 奖励，鼓励，重视3.demography (scientists in the field of vital [生命的] and social statistics): 人口统计学4.primal (fundamental): Of first importance; 最重要的；主要的5.nascent (burgeoning): Coming into existence; emerging: 开始形成的；出现的/出芽6.prognosis (prediction): A prediction of the probable course and outcome of a disease. 预测：判病7.scalded (burnt): 烫伤的8.convulse (shook violently)：To shake or agitate violently 使…强烈地震动或狂躁不安9.undeterred (not discouraged)：not discouraged 未受挫折的[un 否定+deter 阻止v. + [r] ed =undeterred 未被阻止的] 10.p erforate (penetrate)：To pierce, punch, or bore a hole or holes in; 刺穿11.u nflappable [flap恐慌] (composed)：having the ability to stay calm and not become upset, even in difficult situations 镇定的12.i ncur (invite)：To become liable or subject to as a result of one's actions; bring upon oneself: 招惹Unit 51. phenomenal (extraordinary)：outstanding: 非凡的；杰出的2. unveil (uncovered)：To remove a veil or covering from. 把面纱或覆盖物从…上拿下3. wane (decline)：To decrease gradually in size, amount, intensity, or degree. 衰退4. confide (tell confidentially)：To tell (something) in confidence. 吐露：告知秘密的（事物）5. repel (defeat)：To ward off or keep away; drive back: 击退：击退或赶回；驱除6. give away (reveal)：disclose 揭露泄露7. macabre (frightful)：Suggesting the horror of death and decay; gruesome: 恐怖的，阴森的8. chart (map)：To make a map of 绘制…的图表9. mesmerized (fascinated)：To spellbind; enthrall: 迷人的10. succumb (die from)：由于……死亡11. autopsy (examination of a dead body)：尸体解剖12.piece together (assemble)：To bring together；拼凑Unit 61.awry (wrong)：Away from the correct course; amiss偏差地，错误地2.overblow (exaggerated)：Done to excess; overdone:做过头的；过分的3.churn out (produce in large amounts)：大量生产4.dearth (shortage)：A scarce supply; a lack. 不足；缺乏5.reprimand (reproach)：To reprove severely, especially in a formal or official way；严厉训斥，尤指以正式的或官方的形式6.be attributed to (be due to) :归因于7.fatal (deadly)：Causing or capable of causing death；致命的：引起死亡的8.vendor (seller)：someone who sells things, especially on the street 街边摊贩9.voluntary (unforced)：Arising from or acting on one's own free will；自愿的10.s ubmit (send)：give sth (to sb/sth) 递交11.o versight (supervision)：Watchful care or management; 照管，监督12.c ounsel (advise)：to advise someone. 建议Unit 71.avarice (greed)：Immoderate desire for wealth; cupidity. 贪婪2.memoir (records of one`s life and experiences)：An account of the personal experiences of an author. 自传3.anecdote (short narratives concerning interesting events) : 轶事，趣闻：4.register (records of names or events): A formal or official recording of items, names, actions or events 正式记录，官方登记5.whisk away (going lightly and quickly)：轻,迅速地走6.poach (hunt illegally)：非法捕猎7.agog (eager)：Full of keen anticipation or excitement;渴望的8.on the sly (sneakily)：in a manner intended to avoid notice；秘密地9.lust (desired passionately)：An overwhelming desire or craving: 强烈的欲望10.on the track (close upon) :步入正轨，接近11.croon (sing in a low, soothing sound)：To hum or sing softly or soothingly 轻柔地哼，轻柔地唱12.wizard (magician): One who practices magic; a sorcerer or magician. 巫师：从事妖术的人；魔术师Unit 81. specified (stated exactly): To state explicitly, exactly or in detail: 明确说明或详明指明2. panorama (vista): An unbroken view of an entire surrounding area; 全景3. buoyant (cheerful)：Lighthearted; gay 轻松的，轻快的4. impairment (loss)：the fact that a part of your body is unable to do something fully 缺陷5. incredulous (dubious)：Skeptical; disbelieving；dubious 怀疑的；不相信的6.convolution (twisting)：a fold or twist in something which has many of them 盘旋结构7. pageant (spectacle)：a series of historical events that are interesting 壮观8. chronic (constant)：Of long duration; continuing:长期的；持续的Unit 91.tangible (perceptible)：Discernible by the touch; palpable；可感知的2.on the same plane (on the same level)在同一水平上3.sporadically (occasionally)：happening fairly often, but not regularly 偶然地4.paradoxical (self-contradictory)：a statement that seems impossible because it contains two opposing ideas that are both true；自相矛盾的5.in a position (well-grounded) : fully trained in an activity or skill; 精通的6.localize (locate)：To make local 使……具有地方性7.live (act out) : To practice in one's life 实行8.inhibition (restraint)：The act of inhibiting or the state of being inhibited；禁止9.primordial (primeval)：Being or happening first in sequence of time; original; 原始的10.i ntrigue (fascinate) : To arouse the interest or curiosity; 激起…的兴趣或好奇心11.s ynthesize (combine) : To combine so as to form a new, complex product: 综合Unit 101.agonize over (suffered painfully because of sth) : 为……所煎熬2.tied up (busy): not available 忙碌的3.meekly (gently and uncomplainingly) : very quietly and gently and unwilling to argue with people 温顺的4.give sb a black eye (beat sb very hard) : 揍某人一顿5.stark (bare) : blunt 生硬的：不加掩饰的6.plain (clear) : Free from obstructions; open; 清晰的，清楚的：毫无阻碍的7.to the letter (exactly) : 严格地不择不扣地8.zest (eagerness): Flavor or interest; piquancy; 兴趣：爱好或兴趣；刺激9.follow through (carry out exactly to the end)：坚持到底10.stall (delay) : to deliberately delay because you are not ready to do something, answer questions 拖延11.e xasperating (irritate): extremely annoying or irritating 使人恼怒的12.m ediocre (not very good)：Moderate to inferior in quality; ordinary; average 普通的：质量中等偏下的Unit 111.stricken (sorrowful): very badly affected by trouble, illness, unhappiness; 受打击的2.plod (trudge)：To move or walk heavily or laboriously; 沉重缓慢地行走arre (strange): very unusual or strange; odd 古怪的4.attire (clothes): Clothing or array; apparel. 服装，衣着5.evoke (produce): to produce a strong feeling or memory in someone 使人产生6.novice (inexperienced)：someone who has no experience ina skill, subject, or activity；beginner 新手7.demeanor (behavior)：The way in which a person behaves 举止，行为8.adroit (skillful)：Dexterous; deft; 熟练的；灵巧的9.spontaneous (unpremeditated): Happening or arising without apparent external cause; self-generated. 自然产生的，未计划的10.u pstage (overshadow)：to do something that takes people's attention away from someone else who is more important; 分散注意力；使……显得不重要11.g auge (estimate): To evaluate or judge; estimate 评估，判断12.d isconcerting (embarrassing): making you feel slightly confused, embarrassed, or worried 窘迫的13.b lank (expressionless): Lacking expression; expressionless; 茫然的，没有表情的Unit 121.wind (end): to bring an activity, meeting and so on to an end 结束2.bicker (quarrel)：to argue, quarrel especially about something very unimportant 发生口角3.malleable (easily train): Capable of being shaped or formed; easily trained or changed可锻炼的4.provoke (stir up)：To stir to action or feeling.煽动：激起行动或感情5.quest (pursuit): The act or an instance of seeking or pursuing something 追求6.trait (characteristics): A distinguishing feature, as of a person's character; quality 特征7.scrap (throw away): To discard as worthless; junk; 废弃：因为无用而丢掉；抛弃8.pool (collect): to combine and collect your money, ideas, skills etc. 聚集9.screen (examine): To examine (a job applicant, for example) systematically in order to determine suitability. 检查：系统地测试10.a ppraisal (evaluation): a statement or opinion judging the worth, value, or condition of something; 评价11.s trand (part): one of the parts of a story, idea, plan etc. 部分12.r emediate (make up for): To deal with a problem or make a bad situation better; 弥补13.b oost (increase): To increase; raise 增加；拔高Unit 131.warrant (justify): To provide adequate grounds for; 证明…正当：为…提供充足根据2.altruistically (in an unselfish way): showing concern for the happiness and welfare of other people rather than for yourself; selflessly 无私地3.inhibit (hold back): To hold back; restrain 抑制；限制4.groom (cleaning the fur and skin for): If an animal grooms itself or another animal, it cleans its own fur and skin or that of the other animal 把皮毛弄干净5.intriguing (interesting): very interesting because it is strange, mysterious, or unexpected 有趣的6.convention (generally accepted practice): General agreement on or acceptance of certain practices or attitudes: 惯例，习俗7.presumably (probably): say that you think something is probably true 可能，大概8.alleviate (ease): To make (pain, for example) more bearable; 减轻9.entwine (entangled): To twist around or together; entangle 缠绕：缠在周围或一起10.v alor (personal courage): Courage and boldness, as in battle; bravery. 勇气；胆量Unit 141.irrelevant (not related): Unrelated to the matter at hand. 不相干的，离题的2.succinct (concisely): clearly expressed in a few words; concise 简明扼要的3.mediocrity (commonplaceness): The state or quality of being mediocre; 平庸，平凡4.amateurish (not professional): Characteristic of an amateur; 业余的;非专业的5.abdicate (resignation): to give up the position；resign 辞职6.conversely (on the other hand): used when one situation is the opposite of another 相反地，另一方面7.effulgent (bright): Shining brilliantly; resplendent; bright 耀眼的；光彩照人的8.depravity (degradation): Moral corruption or degradation. 堕落：道德败坏9.salvation (deliverance): Preservation or deliverance from destruction, difficulty, or evil. 救助；解救10.o bdurate (obstinate): very determined not to change your beliefs, actions, or feelings, in a way that seems unreasonable; stubborn 固执的，顽固的11.i mmutable (permanent)：Not subject or susceptible to change; 永恒的：不可改变的Unit 151.wince (shrink): To shrink or start involuntarily, as in pain or distress; flinch. 畏缩，退缩2.savor (flavor): The taste or smell of something; 味道3.exertion (energetic use): the energetic use, especially a strenuous effort. 发挥，全力以赴的努力4.pronounced (conspicuous): Strongly marked; distinct; conspicuous 明显的：有很强烈的特征的5.sediment (settlings): Material that settles to the bottom of a liquid; lees. 沉淀物6.crevice (cleft): A narrow crack or opening; a fissure or cleft. 裂隙,裂缝7.remorseless (ruthless): Having no compassion or pity; merciless; 无情的，残忍的8.ravenous (gluttonous)：Extremely hungry; voracious;极饿的9.bellicose (pugnacious): Warlike in manner or temperament; belligerent 好战的10.e vict (force out): eject 武力驱逐；逐出11.g ermane (relate): an idea, remark etc that is germane to something is related to it in an important and suitable way; relevant 相关联的12.a ppall (shock): to make someone feel very shocked and upset; dismay 使……惊骇，震惊13.。
Maingot腹部手术学（第12版）_第29章肠梗阻---目录第五篇小肠和结肠┈┈┈┈┈┈383第十七章肠梗阻┈┈┈┈┈┈385第一节定义一、机械性肠梗阻二、功能性肠梗阻三、术后早期（机械性）肠梗阻第二节流行病学┈┈┈┈┈┈386第三节病理生理学┈┈┈┈┈┈387一、扩张、吸收和分泌二、肠道蠕动功能三、循环改变┈┈┈┈┈┈388四、菌群移位第四节病因学┈┈┈┈┈┈388一、粘连┈┈┈┈┈┈388二、疝┈┈┈┈┈┈389三、恶性肠梗阻┈┈┈┈┈┈390四、肉芽肿性疾病和克罗恩病五、肠套叠六、肠扭转七、其他病因┈┈┈┈┈┈390第五节诊断┈┈┈┈┈┈392一、病史与体格检查二、实验室检查三、影像学表现（一）立位腹平片┈┈┈┈┈┈393（二）对比剂检查┈┈┈┈┈┈394（三）CT检查┈┈┈┈┈┈395（四）超声检查┈┈┈┈┈┈395（五）MRE（磁共振肠动描记）检查（六）视频胶囊内镜检查四、缺血检测第六节治疗┈┈┈┈┈┈1275一、小肠梗阻┈┈┈┈┈┈1275（一）非手术治疗┈┈┈┈┈┈1276（二）何时中转手术治疗┈┈┈┈┈┈1278（三）手术治疗┈┈┈┈┈┈1279（四）旁路与切除决择┈┈┈┈┈┈1280（四）复发性小肠梗阻（五）粘连预防（六）术后早期小肠梗阻（七）放射性肠病（八）癌肿以及恶性肠梗阻二、大肠梗阻┈┈┈┈┈┈400三、肠麻痹┈┈┈┈┈┈401四、急性结肠假性便阻┈┈┈┈┈┈403 参考文献┈┈┈┈┈┈404第五篇小肠和结肠第十七章肠梗阻Hippocrates, the father of medicine, recognized, described, and treated bowel obstruction many years ago.医学之父希波克拉底在很多年以前就认识、阐述和治疗了肠梗阻。
Praxagoras appears to have performed the earliest recorded operation for bowel obstruction circa 350 BC when he relieved the obstruction of a bowel segment by creating a decompressive, diverting enterocutaneous fistula. 有记载的最早的治疗肠梗阻的手术是由Praxagoras 在公元前350 年完成的，他通过建立一个有趣的、减压的肠外瘘来缓解肠段的梗阻。
Disruption of type III secretion in Salmonella enterica serovar Typhimurium by external guide sequencesJeffrey S.McKinney 1,2,*,Haifeng Zhang 1,Tomoko Kubori 3,Jorge E.GalaÂn 3and Sidney Altman 11Department of Molecular,Cellular and Developmental Biology,Yale University,New Haven,CT 06520,USA,2Department of Pediatrics,Division of Infectious Diseases,Yale University School of Medicine,New Haven,CT 06520,USA and 3Section of Microbial Pathogenesis,Yale University School of Medicine,New Haven,CT 06536,USAReceived October 15,2003;Revised and Accepted December 16,2003ABSTRACTThe type III secretion system involved in Salmonella enterica serovar Typhimurium invasion of host cells has been disrupted using inducibly expressed oli-gonucleotide external guide sequences (EGSs)complementary to invB or invC mRNA.These EGSs direct single site cleavage in these mRNAs by endo-genous RNase P,and their expression in Salmonella results in invC mRNA and InvC protein depletion,decreased type III secretion and interference with host cell parison of these effects with those from studies of Salmonella invB and invC mutants suggests that invB EGSs have polar effects on invC mRNA.INTRODUCTIONExternal guide sequence (EGS)oligonucleotides target com-plementary mRNA for speci®c cleavage catalyzed by RNase P (1).EGS oligonucleotides require an accessible single-stranded region on their target mRNA to base-pair with and create the stem structure recognized as a cleavage substrate by RNase P (1).Using EGSs complementary to essential genes,Escherichia coli viability can be decreased in a manner which is EGS oligonucleotide sequence speci®c,dose dependent and dependent on time elapsed after EGS expression (2).Here,EGS studies are extended to Salmonella ,using EGSs complementary to two Salmonella pathogenicity island SPI-1genes (3),invB and invC ,neither of which are essential for bacterial viability (4).The invB and invC DNA sequences occur directly adjacent to each other in the multigene pathogenicity island SPI-1of Salmonella ,with the last nucleotide in the ®nal codon of invB also serving as the ®rst nucleotide in the ®rst codon of invC (4).Prior studies of Salmonella invB and invC mutants have shown that invC is required for host cell invasion and that the gene encodes a protein with ATPase activity (4).The ATPase encoded by invC is postulated to provide energy to power the type III secretion system involved in host cell invasion (4)andpathogenesis (5)by Salmonella .In contrast,invB does not appear necessary for invasion.InvB is a type III secretion chaperone speci®c for SipA,a translocated Salmonella protein which facilitates actin rearrangements within infected eukar-yotic cells (6).Mutations in invB do not alter the secretion of other type III secreted proteins (6)and do not disrupt invasion (4).Using a tightly regulated inducible EGS expression system in Salmonella (7),we show that EGSs complementary to either invB or invC mRNA can disrupt type III secretion and Salmonella invasion assayed in vitro .MATERIALS AND METHODS Plasmids and bacterial strainsThe EGSs listed below were cloned as previously described into high copy number EGS expression plasmids,derived from pUC19(2)or into low copy number plasmids derived from pWKS30(8).These plasmids were transformed into the Salmonella enterica serovar Typhimurium strain SB300A#1(7).SB300A#1has a T7RNA polymerase gene integrated with an adjacent araC-P(BAD)control element into the bacterial chromosome of parent strain SB300.SB300A#1allows tightly controlled arabinose-inducible T7promoter-driven transcription of our EGSs in Salmonella (7).The invA -de®cient Salmonella strain SB136(4),which is disrupted for type III secretion,was used as a control.An invC deletionmutant Salmonella (J.E.GalaÂn and Y.Akeda)was used as a negative control strain for studies of InvC intracellular protein level and of type III secretion.Salmonella was grown in 0.3M NaCl Luria±Bertani (LB)medium.Liquid culture incubation conditions and EGS induction with arabinose at 0.2%®nal concentration are as previously described (7).Following addition of arabinose for EGS induction,Salmonella liquid cultures were grown to late log phase prior to northern blot analysis,assay of Salmonella type III secretion or quanti®ca-tion of bacterial entry,as detailed below.Design of external guide sequencesEGS oligonucleotides were designed to be complementary to single-stranded regions of invB and invC mRNA,followed by*To whom correspondence should be addressed.Tel:+13142862912;Fax:+13142862895;Email:McKinney_J@Present address:Jeffrey S.McKinney,Departments of Pediatrics and Molecular Microbiology,Washington University,Saint Louis,MO 63110,USA848±854Nucleic Acids Research,2004,Vol.32,No.2DOI:10.1093/nar/gkh219Nucleic Acids Research,Vol.32No.2ãOxford University Press 2004;all rights reservedPublished online February 3, 2004an additional3¢-ACCA EGS terminal sequence.This strategy allows formation of a duplex EGS±mRNA molecule recognized as a substrate by endogenous RNase P with resultant cleavage of target mRNA(9).The individual EGS oligonucleotide sequences were named according to their predicted site of target mRNA cleavage.For example, invB108EGS(5¢-AAUGCAAAUAAAUCCacca-3¢)is complementary to invB mRNA nucleotides108±122 (5¢-GGAUUUAUUUGCAUU-3¢)and will result in RNase P cleavage of invB mRNA at nucleotide number108. The other invB or invC EGS sequences were:invC98 EGS(5¢-GGCGUGAUUUCACAAacca-3¢),invC269EGS (5¢-ACCGCGCCUAAUACCacca-3¢)and invC293EGS (5¢-ACGAUUUUCCCUGUCacca-3¢).Two previously char-acterized EGSs which are not complementary to invB or invC were also used:synthC5EGS21and synthC5EGS45(2).The EGSs synthC5EGS21and synthC5EGS45are complemen-tary to,and can guide the RNase P cleavage of,mRNA used for the recombinant synthesis of the C5protein subunit of the RNase P holoenzyme of E.coli,but are not complementary (containing at least®ve unpaired nucleotides per EGS)to the mRNA encoding C5in Salmonella.Herein,the EGSs synthC5 EGS21and synthC5EGS45are referred to as synthC5EGS1 and2,respectively.Partial RNase T1digest mapping of invB and invC mRNASingle-stranded regions of invB and invC mRNA were identi®ed using RNase T1digestion(1).Two mRNAs were digested:(i)a joint in vitro transcript containing invC mRNA 3¢to invB mRNA,transcribed from the plasmid pSB553(4) DNA after digestion with BamHI;and(ii)an invC mRNA in vitro transcript alone,expressed from plasmid pIC001(a pSB553derivative,with invB coding sequence removed via KpnI and BspEI excision)DNA after digestion with EcoRI. In vitro RNase P assaysAssays of mRNA cleavage in vitro by RNase P were performed as previously described(10),using the EGS sequences and the invB and invC mRNA targets detailed above.RNase P M1RNA was folded in a buffer containing 10mM magnesium,using a heat block to®rst heat the sample at65°C for5min and then slowly cool the sample to room temperature.For conditions of substrate excess,reagent concentrations were:11fmol labeled substrate(1100c.p.m.), 1,5and10pmol EGS,and1pmol of enzymatically active recombinant E.coli RNase P M1RNA.For conditions of limited substrate,10fmol of labeled substrate RNA (1000c.p.m.)and50,100and500fmol of EGS were used. Samples were electrophoresed in5%polyacrylamide±7M urea gels.Northern blotsNorthern blots were performed on total RNA extracts of Salmonella,using previously published techniques(11).Each lane of a2.5%agarose gel was loaded with4m g of total RNA. The UV transillumination pattern of rRNA bands after separation of each sample on an agarose gel revealed similarity in rRNA band patterns in terms of both gross quantity and quality.Northern blot probes were5¢-end-labeled DNA oligonucleotide probes.They included probes comple-mentary to invC mRNA,5S rRNA and each of the four invB and invC EGS oligonucleotides listed above.In each case, 8pmol of oligonucleotide labeled with4pmol(30m Ci)of [g-32P]ATP was used per40ml of rapid hybridization buffer (Amersham).Signal was detected using a phosphoimager (Fuji)and quantitated using image analysis software(Fuji ImageGauge).Quantitative results are reported as the ratio, expressed as a percentage,of northern blot invC mRNA band signal intensity for the matched culture specimens of a given Salmonella strain with EGS induction versus without EGS induction.Assay of Salmonella type III secretionSalmonella culture supernatant proteins were prepared and analyzed as previously described(12).Western blots were probed with polyclonal antibodies against SipB and SipC and chemiluminescent signals produced using ECL Plus western blotting detection reagents from Amersham Biosciences. Signal was detected using autoradiograph®lm(Kodak)and quantitated using image analysis software(Fuji ImageGauge). Quantitative results are reported as the ratio,expressed as a percentage,of western blot band signal intensity for the matched culture specimens of a given Salmonella strain with EGS induction versus without EGS induction.Matched culture specimens were employed as internal controls for western blots.Speci®cally,a given Salmonella transformant was grown to early log phase in a single liquid culture,then split into paired cultures which were grown simultaneously either with or without arabinose induction of EGS expression.Equal volumes of paired cultures were harvested for protein preparation at the same point of their late log phase growth,as assessed by optical density.Equal volumes of these protein preparations were loaded per well for western blot analysis.Assay of intracellular InvC protein levelSalmonella cultures in liquid media were pelleted and resuspended in one-twentieth volume of phosphate-buffered saline(PBS)±Tris(77mM Tris±HCl,pH8.0).Samples were denatured by boiling with SDS±PAGE loading buffer and separated on a9%polyacrylamide±SDS gel.Western blots were probed with a polyclonal antibody against InvC(J.E. GalaÂn and Y.Akeda),and chemiluminescent signals produced using ECL Plus western blotting detection reagents from Amersham Biosciences.Signals were detected using auto-radiograph®lm(Kodak).Quantitation of results,using matched culture specimens,was performed as described for type III secretion assays,above.Quanti®cation of bacterial entryEntry of different Salmonella strains into Henle-407cells in a gentamicin protection assay of bacterial entry into host tissue culture cell monolayers was performed and quanti®ed as previously described(13).RESULTSDesign of EGSs for invB and invC mRNA in vitroTo design the EGSs reported here,mRNA transcripts of invB and invC made in vitro were mapped using partial RNase T1 Nucleic Acids Research,2004,Vol.32,No.2849nuclease digestion to suggest EGS-accessible single-stranded mRNA regions.The ®rst nucleotide of the start codon of each gene is labeled as nucleotide 1;single-stranded guanine residues of invB mRNA were identi®ed via partial RNase T1nuclease digestion at invB nucleotides 108and 217.For invC mRNA,single-stranded guanines were identi®ed at invC nucleotides 98,237,269and 293.Given uncertainty about whether invC mRNA exists in cells independently from invB mRNA or as a joint transcript with invB ,both possibilities were examined in RNase T1digestion in vitro .RNase T1digestions were performed on two in vitro transcripts:invC mRNA alone,as well as a tandem transcript of invC mRNA immediately 3¢to invB mRNA.Single-stranded regions of invC identi®ed in the joint invB ±invC in vitro transcript were notably also found for the invC in vitro transcript alone (data not shown).EGS oligonucleotides were designed to be complementary to the RNase T1-accessible mRNA sequences invB 108±122,invC 98±112,invC 269±283and invC 293±307,and were named for their predicted nucleotide cleavage sites by RNase P:invB 108,invC 98,invC 269and invC 293,respectively.RNase P-speci®c cleavage of mRNA in vitroRNase P hydrolyzes the phosphodiester bond (in the target mRNAs)that precedes the ®rst base pair in the 5¢end of thetarget mRNA±EGS complex,akin to the site-speci®c cleavage reaction the enzyme catalyzes in the 5¢processing of precursor tRNA (9).The reaction ingredients for RNase P assays in vitro were E.coli RNase P,internally radiolabeled invB and invC mRNA target transcribed in vitro ,and EGSs complementary to portions of invB or invC mRNA.All four EGSs guide RNase P to cleave the mRNA at the predicted sites of EGS mRNA hybridization,yielding appropriately sized 5¢and 3¢cleavage products (Fig.1).RNase P cleavage of mRNA increases with increasing EGS dose,with the EGSs invB 108and invC 293most ef®cient at guiding mRNA cleavage in vitro in conditions of limited substrate (data not shown).Speci®c mRNA targeting in SalmonellaInduction of expression of invB or invC EGSs in Salmonella is followed by a decrease in invC mRNA compared with identical Salmonella transformants lacking EGS induction.Northern blots of equal microgram amounts of total RNA isolated from various Salmonella liquid cultures are shown in Figure 2,where northern blot signals for invC mRNA in matched Salmonella cultures decreased between 27and 50%following relevant EGS induction.The invB or invC EGS expression effects appear speci®c,in that there is no similar change detected in the level of constitutive 5S rRNA after EGS induction (Fig.2).Arabinose was used to induce Salmonella EGS expression.It did not have as pronounced an effect on invC mRNA in the absence of EGS expression plasmids,either in the case of Salmonella which was not transformed with an EGS expression plasmid,or in the case of Salmonella for which the EGS expression plasmid was presumably lost after prolonged culture (Fig.2).PlasmidFigure 1.RNase P±EGS cleavage of mRNA in vitro .Target substrates for cleavage include a joint transcript including both invB and invC mRNA (lane 1),and a transcript of invC mRNA alone (lane 6).The former was incubated with increasing amounts of the invB EGS 108(lanes 2±4);the lat-ter as a target for invC EGSs invC 98(lanes 7±9),invC 269(lanes 10±12)and invC 293(lanes 13±15).Lanes labeled N (lanes 5and 16)lack any EGS,but do have active RNase P with the invB and invC joint transcript (lane 5)or the invC transcript (lane 16)and show no non-speci®c target cleavage in the absence of EGS.Cleavage products were separated by size using electrophoresis in a 5%polyacrylamide±7M urea gel.Cleavage prod-uct sizes are consistent with RNase P cleavage occurring at the predicted site at the 5¢end of the mRNA region to which each EGS hybridizes.Predicted sizes of reaction products following RNase P enzymatic cleavage of in vitro transcripts are listed on the left and right of the image (e.g.invB and invC joint transcript mRNA cleavage products of 585and 123nucleo-tides for EGS invB 108;invC transcript mRNA cleavage products of 485and 198nucleotides for EGS invC 98,etc.).Figure 2.Northern blots in Salmonella with inducible expression of EGS molecules.RNA was isolated from Salmonella ,electrophoresed in a 2.5%agarose gel,and probed for invC mRNA or for constitutively produced non-targeted 5S rRNA.The partial invC in vitro transcript expressed from plas-mid pIC001DNA after digestion with EcoRI (as described for T1digest mapping)serves as a size marker (683nt).The invA mutant is SB136(also used for invasion assays).SB300is the parent Salmonella strain,from which SB300A#1was constructed.Other lanes are paired by SB300A#1Salmonella transformant type,either with (+)or without (±)the addition of arabinose for the induced expression of the EGS molecules listed.Longer term induction (++)was suspected to be accompanied by the loss of EGS expression plasmid invC 293,and an accompanying lack of effect on target mRNA.Note the decrease in invC mRNA after the induction of EGS expression,whereas non-targeted constitutive 5S rRNA levels are independ-ent of EGS expression.850Nucleic Acids Research,2004,Vol.32,No.2maintenance in transformants was assessed by parallel quan-titative plating on LB or LB ampicillin plates as previously reported (2,11).Plasmid loss was considered to have occurred when colony counts on LB plates were greater than on LB ampicillin plates by at least an order of magnitude.As previously demonstrated for other EGS transcription in this Salmonella system (7),northern blot probes complementary to EGS oligonucleotides detected EGS expression only after arabinose induction (data not shown).The effect of expression of EGSs on type III secretion by SalmonellaA standard functional assay of InvC-dependent type III secretion was employed,in which proteins secreted by the type III secretion system were measured in cell culture supernatants (12).These secreted proteins,SipB and SipC,are also encoded in the Salmonella pathogenicity island I gene complex and are reviewed elsewhere (14,15).A panel of Salmonella transformants,containing EGSs in various inducible expression plasmid vectors,was used for InvC-dependent type III secretion assays.The concurrent expression of two EGSs using any one of four high copy number plasmids derived from pUC19(2)consistently decreased SipB secretion by b 65%from that detected for the same Salmonella transformant grown in parallel under non-EGS-inducing conditions,based on the relative signal intensities of SipB western blot bands for a given Salmonella strain with versus without EGS induction (Fig.3).Using the same method of quantitative analysis,induction of the same set of EGS pairs in Salmonella transformed with a low copy number plasmid derived from pWKS30showed a 20±30%decrease in secretion (data not shown),as did any of the four EGSs when expressed alone from pUC19-derived plasmids (data not shown).These ®ndings are consistent with prior studies of EGS dose±response features in E.coli comparing EGS expression plasmids encoding single versus multiple EGSs (2)and comparing EGS expression plasmids with strong versus weak promoters (10).To assess sequence speci®city,the negative control EGSs,synthC5EGS 2and 1,against the synthetic C5component of E.coli RNase P,described above and in McKinney et al .(2),was also used.Induction of this negative control EGS was accompanied by a <10%decrease in SipB secretion as compared with non-induced parallel cultures (Fig.3).In addition,the loss of an invB or invC EGS expression plasmid from a bacterial strain was accompanied by a loss of previously observed inhibitory effects on secretion (data not shown).The effect of EGSs on intracellular InvC protein in SalmonellaA polyclonal antibody (J.E.GalaÂn and Y.Akeda)raised against recombinant InvC protein was used for western blot analysis of cellular InvC protein level as an assessment of EGS effects (Fig.4,top).A band consistent with the 47kDa InvC protein is found in the Salmonella strain SB300A#1with no EGS.An SB300strain (constructed by Y.Akeda)in which InvC expression has been disrupted via an invC deletion mutation serves as a negative control for InvC expression.Intracellular expression of InvC was also assessed in SB300A#1transformed with the EGS expression plasmid for the concurrent expression of either the EGSs invB 108/invC 98or the negative control EGSs synthC5EGS 2and 1(also shown in Fig.3).After the induction of EGS expression,the relevant protein band is not detected for SB300A#1with the invB /invC EGSs.No effect on the detection of this protein band is seen after the induction of the negative control EGS.Figure 4is representative of repeated assays in that the decrease in putative ~47kDa InvC signal cannot be explained by a general decrease in the intensity of other bandsdetectedFigure 3.Western blots of cell culture supernatants from Salmonella with (+)or without (±)the induced expression of EGS molecules listed.Western blot detects the proteins SipB and SipC,secreted into cell culture superna-tant by the type III secretion system and subsequently electrophoresed in a 9%polyacrylamide±SDS gel.The transformants shown contain a pUC19-derived high-copy number plasmid (2)from which two EGSs are concur-rently expressed.InvBB denotes inducible expression of invB 108and invB 108EGSs in tandem,InvBC denotes invB 108and invC 98EGS expression,InvCB denotes invC 293and invB 108EGS expression,and invCC denotes invC 293and invC 98EGS expression.The control concurrently expresses two EGSs targeting the mRNA used for synthetic C5protein over-expres-sion in E.coli .Migration patterns of SipB and SipC bands relative to protein molecular weight 66and 45kDa markers are as shown in Figure4.Figure 4.Western blots for Salmonella intracellular InvC (top)and for SipB and SipC secreted into Salmonella cell culture supernatants (bottom,as for Fig.3).SB300A#1without an EGS expression plasmid (no EGS)and a SB300invC deletion mutant which does not express InvC (D invC )(pro-duced by J.E.GalaÂn and Y.Akeda)are shown to the left of protein mol-ecular weight markers.At right:SB300A#1transformed with the high copy number plasmids for arabinose-inducible expression of either invB 108and invC 98EGSs (InvB/C EGS)or the control EGSs (Control EGS)described for Figure 3.EGS induction status by arabinose addition is shown as (+)or (±).Arrowheads denote the predicted locations of InvC,SipB and SipC after electrophoresis in 9%polyacrylamide±SDS gels.Nucleic Acids Research,2004,Vol.32,No.2851by the polyclonal antibody.Indeed,for the two cases of EGS induction shown here,invB/invC EGS induction is accom-panied by decreased~47kDa InvC signal in the context of strong signals for other bands,whereas negative control EGS induction is accompanied by strong~47kDa InvC signal in the context of relatively weak signals for other bands.Cell supernatants from the same experiment were used for type III secretion assays(Fig.4,bottom)as above.In each case,detection of the band representing intracellular InvC protein correlates with type III secretion.Type III secretion is decreased when intracellular InvC protein levels are de-creased,either by the static invC deletion mutation,or by the dynamic disruption of InvC expression following the induc-tion of the invB/invC EGSs.While the presumptive InvC protein band disappears in both situations,the decrease in type III secretion is more pronounced following the static deletion of the invC gene than following the induction of the invB/invC EGS.This functional assay of InvC cellular activity suggests that our invB/invC EGS system inhibits InvC-dependent type III secretion less completely than does the invC deletion-mediated ablation of InvC expression.Wild-type levels of secretion are observed for SB300A#1with no EGS plasmid, without induction of invB/invC EGSs,or with negative control EGS expression.EGS impact on Salmonella invasion into host cells Salmonella strains containing EGSs in various inducible expression plasmid vectors were also tested for their ability to invade Henle-407cells in tissue culture.In each case,a given Salmonella strain was grown in parallel liquid cultures,either with arabinose added to induce EGS induction or without the addition of arabinose.For invasion,these Salmonella strains were®rst incubated with Henle cells for45min in Hanks buffered salt solution.This was followed by a2h treatment with gentamicin to kill extracellular bacteria,and subsequent washes with tissue culture buffer.Invasion was quanti®ed as the percentage of bacteria inoculated into the tissue culture wells which were recovered from lysed Henle cells.As shown in Table1,a control Salmonella strain,SB136, with a null mutation in invA and a previously documented functional defect in type III secretion and host cell invasion (4),achieves<1%invasion into Henle cells,independent of arabinose addition.Salmonella SB300A#1transformants which express an EGS complementary to mRNA encoding synthetic C5(also shown as a negative control for type III secretion assays,above)have a b20%rate of invasion,with a small decrease in invasion observed after arabinose addition. Salmonella SB300A#1transformants which inducibly express EGSs against invB or invC from a high copy number pUC19-derived plasmid exhibit an8±12%rate of invasion without arabinose addition,which decrease to2.8±3.5%after arabi-nose induction of EGS expression.The same EGS constructs, expressed from low copy number pWKS30-derived plasmids, did not affect invasion rates after EGS induction.DISCUSSIONThe pathogenicity island genes invC and invB of Salmonella provide intriguing targets for gene product disruption.In the case of invC,mutagenesis studies clearly show that the ATPase encoded by invC is required for type III secretion in assays in vitro and is important for pathogenicity in animal models(4,5).Following appropriate EGS expression,we observe a decrease in invC mRNA,InvC intracellular protein, InvC-powered type III secretion and type III secretion-dependent host cell invasion.The inhibition of type III secretion and of Salmonella invasion using EGSs to disrupt invC mRNA is less complete than that resulting from invC deletion mutagenesis.This suggests that a certain critical level of mRNA disruption is able to partially inhibit type III secretion and host cell invasion.The level of mRNA disruption required for phenotypic changes probably varies for different target mRNAs,depending on factors such as the ratio of EGS to target mRNA(2,11),the relative ef®ciency of various EGSs and the functional reserve capacity a cell has for a given target mRNA and the protein that mRNA encodes.The EGSs reported here show greater phenotypic effects when expressed from high copy number,rather than low copy number, plasmids.This is consistent with prior EGS dose±response observations in bacteria.Phenotypic effects in E.coli are greater following EGS expression driven by a strong promoter as compared with a weak promoter(11),and the concomitant expression of different EGSs in E.coli results in phenotypic effects exhibiting additive synergy(2).The application of EGS technologies to regulate gene expression in bacteria in in vivo models of infection may bene®t from EGS expression plasmids which can be stably maintained in bacteria within an animal.We produced the low copy number plasmids reported here in an initial effort toward this end.The fact that EGSs expressed from our low copy number plasmid system did partially inhibit type III secretion but showed no apparent effect on host cell invasion suggests a possible threshold effect,in which inhibition of type III secretion must reach a critical threshold to result in inhibition of cell invasion.In contrast to the static effects on gene product disruption produced by mutagenesis techniques,our techniques of geneTable1.Invasion of Henle-407cells by Salmonella strains Salmonella strain%Invasion%InvasionNo arabinose Plus arabinoseSB136(invA-)0.08T0.040.1T0.06 SB300A#127T4.7SB300A#1transformantspUC synthC5EGS12620pUC synthC5EGS2and129T2.1pUC InvB/B EGS10T1.5 2.9T0.2 pUC InvB/C EGS9.6T1.2 2.9T0.5 pUC InvC/B EGS8.8T1.0 2.8T0.3 pUC InvC/C EGS12 3.5 pWKS InvB/B EGS2519pWKS InvB/C EGS2323pWKS InvC/B EGS1818pWKS InvC/C EGS1313Invasiveness of different Salmonella strains into Henle-407cells,in a standard gentamicin protection assay.The numbers of internalized bacteria are shown as a percentage of bacteria input.Means T SDs are shown for triplicate well assays,with no standard deviations shown for single-well assays.Salmonella strains include SB300A#1,SB300A#1transformants expressing various EGSs described in the text,and the invA-de®cient SB136.852Nucleic Acids Research,2004,Vol.32,No.2。
