A web services choreography scenario for interoperating bioinformatics applications

Remko De Knikker
Center for Medical Informatics
Department of Anesthesiology
School of Medicine
Yale University
PO Box 20800906520New HavenCTUSA

Youjun Guo firstname.lastname@example.com
Center for Medical Informatics
Department of Anesthesiology
School of Medicine
Yale University
PO Box 20800906520New HavenCTUSA

Jin-Long Li
Center for Medical Informatics
Department of Anesthesiology
School of Medicine
Yale University
PO Box 20800906520New HavenCTUSA

Albert Kh Kwan firstname.lastname@example.com
Department of Computer Science and Information Systems
University of Hong Kong
Pokfulam Road, Hong Kong

Kevin Y Yip
Department of Computer Science and Information Systems
University of Hong Kong
Pokfulam Road, Hong Kong

David W Cheung email@example.com
Department of Computer Science and Information Systems
University of Hong Kong
Pokfulam Road, Hong Kong

Kei-Hoi Cheung
Center for Medical Informatics
Department of Anesthesiology
School of Medicine
Yale University
PO Box 20800906520New HavenCTUSA

Department of Genetics
School of Medicine
Yale University
PO Box 20800506520New HavenCTUSA

BMC Bioinformatics

A web services choreography scenario for interoperating bioinformatics applications
BioMed Central Page 1 of 11 (page number not for citation purposes) Research article * Corresponding author
Background: Very often genome-wide data analysis requires the interoperation of multiple databases and analytic tools. A large number of genome databases and bioinformatics applications are available through the web, but it is difficult to automate interoperation because: 1) the platforms on which the applications run are heterogeneous, 2) their web interface is not machine-friendly, 3) they use a non-standard format for data input and output, 4) they do not exploit standards to define application interface and message exchange, and 5) existing protocols for remote messaging are often not firewall-friendly. To overcome these issues, web services have emerged as a standard XML-based model for message exchange between heterogeneous applications. Web services engines have been developed to manage the configuration and execution of a web services workflow.Results:To demonstrate the benefit of using web services over traditional web interfaces, we compare the two implementations of HAPI, a gene expression analysis utility developed by the University of California San Diego (UCSD) that allows visual characterization of groups or clusters of genes based on the biomedical literature. This utility takes a set of microarray spot IDs as input and outputs a hierarchy of MeSH Keywords that correlates to the input and is grouped by Medical Subject Heading (MeSH) category. While the HTML output is easy for humans to visualize, it is difficult for computer applications to interpret semantically. To facilitate the capability of machine processing, we have created a workflow of three web services that replicates the HAPI functionality. These web services use document-style messages, which means that messages are encoded in an XML-based format. We compared three approaches to the implementation of an XML-based workflow: a hard coded Java application, Collaxa BPEL Server and Taverna Workbench. The Java program functions as a web services engine and interoperates with these web services using a web services choreography language (BPEL4WS).Conclusion:While it is relatively straightforward to implement and publish web services, the use of web services choreography engines is still in its infancy. However, industry-wide support and push for web services standards is quickly increasing the chance of success in using web services to unify heterogeneous bioinformatics applications. Due to the immaturity of currently available web services engines, it is still most practical to implement a simple, ad-hoc XML-based workflow by hard coding the workflow as a Java application. For advanced web service users the Collaxa BPEL engine facilitates a configuration and management environment that can fully handle XML-based workflow.

Background

The nature of genome-wide data analysis often requires the use of multiple databases and programs in some coordinated fashion. For example, microarray data analysis typically involves a sequence of analysis steps, which may include filtering, normalization, cluster analysis, access to a variety of genome annotation sources, etc. To compound the problem of interoperation, there are different ways of analyzing the same dataset. For example, some microarray analyses such as clustering are exploratory in nature while others are more specific to nature of the biological problem at hand (e.g., genetic network modeling [1]). Also, there are different ways to perform the same type of analysis (e.g., there are different clustering approaches such as hierarchical methods vs. non-hierarchical methods).

Despite the fact that a large number of genome databases and bioinformatics applications have been made available through the web, it is difficult to automate interoperation because:

1) the platforms on which applications reside are heterogeneous, 2) their web interface is human-friendly but not machinefriendly, 3) they use a non-standard format for input and output data, 4) they do not make use of standards to define the application interface and message exchange, 5) existing protocols for remote messaging are often not firewall-friendly.

Web services now offer a single uniform method for application integration through the Internet. They provide a model for accessing software systems over the web by pointing to their web address (URI), while their public interfaces and bindings are defined and described using an XML standard format. The potential of web services in bioinformatics database/tool unification has been recognized [2]. Examples of bioinformatics web service projects include: BioMoby is a project involving biological data hosts, biological data service providers, and coders whose aim is to explore various methodologies for biological data representation, distribution, and discovery [3]; myGrid aims to design, develop and demonstrate higher level functionalities over an existing Grid infrastructure that support scientists in making use of complex distributed resources [4,5]; DDBJ has published a number of biological tools as web services like Blast, ClustalW, DDBJ, Fasta, and others [6]; Soaplab developed at EBI provides a web service interface to a variety of analysis applications [7]; Soap-HT-BLAST is a BLAST web service implementation by the Bio Informatics Institute (BII) of the Agency for Science, Technology and Research in Singapore (A*STAR) [8]; and the IBM Life Sciences project implemented web services for PubMed, GenBank, BLAST, Phylogenic Tree and ClustalW [9].

Overview of the web service technology

Three standards have emerged that comprise the web services model. The Basic Profile of the Web Services Interoperability model (WS-I) [10] describes the web services model as follows:

1) the Web Service Description Language (WSDL) [11] is an XML language that describes a web service in an abstract manner by defining the web service interface and the exchange of messages between the provider and requester.

2) the Simple Object Access Protocol (SOAP) [12] is an XML-based protocol for stateless message exchange. It is not bound to any transport protocol, but is in general built on top of HTTP, which makes it firewall friendly in contrast to protocols used by CORBA for instance.

3) Universal Description, Discovery and Integration (UDDI) [13] is a standard protocol designed to publish details about an organization and the web services. It provides a description and definition of web services in a central repository, which functions as yellow pages for web services.

WSDL and SOAP are W3C standards, while UDDI is an OASIS standard. For a client to use a web service it only needs WSDL with SOAP being commonly used as the default protocol. SOAP implementations like Axis by the Apache Software Foundation, The Mind Electric's (TME) GLUE or Systinet's WASP Server for Java provide an easyto-use method for publication and management of web services. These tools in general manage message exchange, register web services, generate WSDL files and implement functions like automatic error handling.

Web Services Choreography

While interlinking between web services is relatively simple, in reality applications need to work together in order to offer more advanced functionality. Thus to make full use of the advantages that the web services model offers, like advanced automation and application integration to group a set of applications together, we need more than a simple point-to-point connection. That is, we need a language that allows us to create a more complex composition of interdependent web services that easily operate together. This concept of interrelated web services that are linked together in a functionally coherent and repeatable process is called "Web Services Choreography".

There are more than a dozen languages that coordinate messaging and transaction for web services available but currently there are mainly two dominant open standards: 1) the Business Process Execution Language for Web Services (BPEL4WS) [14] was co-written by IBM, MicroSoft and BEA, and is (royalty-free) submitted as a standard to OASIS. SUN Microsystems and Oracle recently joined the WS-BPEL Technical Committee [15], 2) the Business Process Markup Language (BPML) [16] is defined by BPML.org and extends the Web Services Choreography Interface (WSCI) that was developed by SUN.

The W3C published in August 2003 a draft of its specification for Web Services Choreography language (WS-CHOR) [17]. BPEL4WS and BPML are the leading emerging standards. They are compatible languages and both offer a rich set of workflow options. BPEL4WS seems most promising at this moment to emerge as a widely supported standard for web services choreography since it has the broad industry support from companies like IBM, MicroSoft and BEA, while SUN recently also joined the Technical Committee. In addition, BPEL4WS is well documented and tools like editors and choreography engines are already available. Both BPEL4WS and BPML allow complex scenarios of choreography like concurrent processes, synchronous and asynchronous messaging, roll back mechanisms, data manipulation and error handling. These features benefit certain classes of biomedical informatics applications that involve sensitive data and the use of parallel programming to speed the complex analyses of large datasets.

Choreography engines

A choreography engine is the "smart center" of a web services choreography, which executes and manages workflow, process automation, data mapping, error handling and security. These engines significantly improve the management of the web services workflow. Currently there are a few web services choreography engines available. IBM offers its Business Process Execution Language for Web Services Java Run Time (BPWS4J), which implements a choreography as an RPC-style web service (for Document vs. RPC style see next section). Another project of interest is myGRID [3], which developed Taverna, a tool that integrates Choreography into a graphical workbench, for creating, editing and browsing workflow. Taverna uses a simple non-standard choreography language, called XScufl, and is an ongoing collaborative project between EBI, IT Innovation and the Human Genome Mapping Project (HGMP) of the Medical Resource Center (MRC). The Collaxa BPEL Server [18] implements the choreography as a document-oriented web service, is easyto-manage with an advanced graphical interface (which works for IE6 only) but is available under a commercial license. The Mind Electric (TME) is to release Gaia, and other engines are being developed at the moment. Most of these engines support either BPML or BPEL4WS.

Document vs. Remote Procedure Call (RPC) styles

There are two ways of exchanging data between web services: document-oriented vs. remote procedure call (RPC). The document-style exchanges data as XML documents, while the RPC-style web service describes the interface in the format of a method-signature and takes input and output in a programming language specific data type. A data type-mapping interface (called a proxy in dot Net or a Serializer in Apache Axis) is used to converse data between a client and a web service.

While some vendors (e.g., Microsoft) adopt the document-oriented method, others (e.g., IBM, Apache Axis and SUN) use the RPC-oriented method. In practice, a mixture of document-style and RPC-style web services will exist. While it might be easier to wrap an RPC-style web service interface around existing applications, newly developed applications could be more inclined to use documentstyle web services. Currently however, most tools that are available initially have focused on RPC-style web services.

Results and discussion

In our web services choreography scenario [19], a user obtained a set of microarray spot IDs (GenBank accession numbers, Affymetrix chip probe set IDs, and Unigene Cluster IDs) corresponding to the genes (both known and unknown) being studied. The user then wants to create a hierarchy of Unified Medical Language System (UMLS) concepts formatted in XML and group the result per MeSH root category.

Three web services have been created to execute this scenario: one that maps a set of microarray spot IDs to their corresponding GenBank IDs, and if available their corresponding PubMed IDs and UMLS concept IDs (GetHAPI); the second web service builds an XML formatted hierarchy of UMLS concepts for a set of UMLS concept IDs (GetUMLS), while a third web service generates an XMLbased report of UMLS concept ID occurrences per MeSH root category from a hierarchy of UMLS concept IDs (GetUMLSReport). A similar functionality as the choreography of GetHAPI, GetUMLS and GetUMLSReport is provided by the High-density Array Pattern Interpreter (HAPI) [20,21] developed at the University of California, San Diego through a web-based (HTML) interface.

When using the traditional web interface of HAPI [21], the user has to engage in a three-step process. In step 1, the user provides a name for the result set and uploads a file containing a list of spot IDs. Figure 1 shows step 2 of HAPI in which the user needs to select a column that contains the GenBank IDs and can enter a name for the HTML summary page. In step 3 the result file is being generated and the user has to click the link to view the HTML summary page.

The result, shown in figure 2, is an HTML table containing a hierarchy of keywords from literature associated with the genes that were provided. The HTML table displays the occurrences per category and the split up for each category of the keywords hierarchy. To retrieve information from GenCard, Entrez or PubMed the user can click links to these databases per UMLS keyword.

While the traditional web approach provides the user with an easy-to-use interface and easy-to-read (HTML) output, it lacks a flexible programmatic interface, making automatic tool linking and interoperation difficult. The WSDL files of the choreography and of the web services used in the choreography on the other hand describe the interfaces and message format that provide the equivalent functionality of the HAPI application. This allows the client program to access and interpret the output data programmatically. In addition the user can choose to access additional web services in his choreography, like XEMBL [22] to retrieve GenBank information for genes of interest, based upon the outcome of the GetUMLSReport or choose to access only part of the web services choreography that comprises the HAPI functionality, for instance in order to retrieve the complete hierarchy of UMLS concepts associated with the submitted genes or to retrieve only the PubMed IDs for the associated genes. The result is that the remote functionality of the HAPI utility can easily be integrated in a local workflow, data can be programmatically processed and the individual modules can be utilized more dynamically and flexibly.

Hard coding the implementation of a BPEL workflow through a Java application allows for the easy implementation of a workflow and the full use of the power of the Java programming language with the ease of altering the workflow by simply configuring the BPEL file. Extending the BPEL workflow can be achieved by adding the invoke tag of the new web service to the BPEL sequence as long as the workflow consists of synchronous web services and a straightforward sequence of web services. Mapping of data can be achieved by XSL transformations implemented in Java. While it is fairly simple to implement a Java application and execute the BPEL Workflow, implementing the XML-based scenario in either Collaxa or Taverna is more arduous.

Taverna provides a user-friendly interface and RPC-style web services can quickly and easily be implemented in Taverna without advanced knowledge of web service technologies. It simply involves adding the URL location of the WSDL to the available services and adding the chosen operation or method of the web service to the model. By creating variables and data links the model is completed. It is very easy to quickly add services to the model, and reroute the workflow by altering the data links. To execute the workflow, switch to the Workflow Input Panel and press the run-button while the result can be viewed in the result tab (Figure 3). But as Taverna is an ongoing, open source effort not all Document-style WSDL implementations appeared to be supported at this moment, while mapping output to input options are still limited, restricting the applicability at this moment.

Collaxa is committed to Document-style web services and it has created a BPEL designer called bpelz and a browserbased (IE6 only) workflow management console that allows instance inspection amongst others (Figure 4). The designer's interface may require still some hand coding of the source files to make corrections, while most WSDL files will need to be extended by a partnerLinkType definition. But Collaxa offers full support of BPEL with advanced support for extensions to the model. Data sources can be mapped using XPath and BPEL extension for Java coding is supported.

Conclusions

Web services facilitate bioinformatics software interoperation based on a standard XML-based protocol. While the development of web service technologies is as much ongoing as rapid. Many bioinformatics applications have been published as web services and more initiatives are being developed. With more web services for bioinformatics becoming available, there will be an increasing need to use metadata and ontologies to facilitate description, tracking, classification, and constraining of web services based upon UDDI or domain-specific standards as developed by BioMoby.

While it is relatively straightforward to implement and publish web services, the interoperation of bioinformatics web services through the use of choreography engines is in the early stage. Documentation and examples for most choreography engines are scarce. For example, IBM's choreography engine BPWS4J can only be deployed as an RPC style web service. MyGrid's Taverna offers an advanced GUI and can process most WSDL files, but support for document-oriented web services is still limited. Collaxa's orchestration engine offers full BPEL4WS support, fully HAPI, the High-density Array Pattern Interpreter  Figure 4 Collaxa BPEL Server Screenshot of Collaxa's bpelz design tool, building a choreography of GetHAPI and GetUMLS web services supports document-oriented web services and provides an easy-to-use and advanced GUI, but it is only available under a commercial license and its console runs only under IE6. To sum up, there is a need for further development of user-friendly choreography engines that support choreography standards like BPEL4WS and both document-style and RPC-style choreographies, as well as broader manipulation of data. Of the two web service engines, only Collaxa seemed to support our example XML-based workflow, providing XPath and full BPEL support. Collaxa requires working knowledge of all web services technologies, whereas Taverna is transparent to use. For an RPC-based web service and some XMLbased workflow that does not require complex mapping, Taverna can be used. In addition, it offers advanced support for additional services, which we were not able to fully explore, and is more than Collaxa focused on the scientific community. While transparent engines fully supporting BPEL might be available soon, for most simple workflow implementations it is currently still most convenient and flexible to use hard coded implementations. For more advanced workflow Collaxa can be used while workflow requiring specific services or RPC based workflow Taverna is available.

Collaxa BPEL Server

Methods

Although it is possible to invoke a web service using any other protocol, we use WSDL and SOAP. Since we already know the location of the target web service and its functionality, publication and discovery with UDDI is not our focus here, although it is part of the WS-I Basic Profile. We refer to web services projects such as BioMoby that extensively use UDDI for more information.

In exchanging messages between web services, we use the document-oriented method since it has the following advantages.

1) It uses a language independent and self-describing data format.

2) It allows for validation based on DTD or XML Schema.

3) It provides a better separation of data and functional logic.

It allows us to exploit the various bioinformatics-related XML standards (including BSML [23], BIOML [24,25], MAGE-ML [26,27], etc) to further facilitate interoperability.

Our web services choreography scenario involves the use of BPEL4WS to define a workflow that consists of several document-oriented web services. To execute the choreography, we have created a client that functions as a chore-ography engine that is implemented as a documentoriented web service. For demonstration purposes the choreography engine can be accessed through a web interface that calls the client engine. The user configures a BPEL file describing the web services choreography and a WSDL file that describes the choreography engine's web service interface and message format. The client executes the preferred workflow of web services as described in these two files. Figure 5 illustrates the central elements of the choreography code that integrates the GetHAPI, GetUMLS, and GetUMLSReport web services. This web services choreography returns an XML document that represents a report of UMLS concept occurrences per category from the hierarchy of UMLS keywords that correlate to the submitted set of microarray spot IDs.

Authors' contributions

KC initiated and directed the research project. RdK performed the technical research and documentation, and the overall design and implementation of the project. YG provided XSLT Transformations and assisted in implementation. JL assisted in the data analysis and database optimization. AK, KY and DC collaborated with the YCMI team on the web services interoperation. All authors read and agreed with the final manuscript. Figure 5 BPEL Choreography An extract from the BPEL file describing the choreography of GetHAPI and GetUMLS web services.

BPEL Choreography

Figure 1
1HAPI, the High-density Array Pattern Interpreter. Step 2 of 3 when using HAPI utility's web-based (HTML) interface provided by UCSD HAPI, the High-density Array Pattern InterpreterFigure 2 HAPI, the High-density Array Pattern Interpreter. Summary page of HAPI utility provided by UCSD Taverna, the Scufl Workbench Figure 3 Taverna, the Scufl Workbench Screenshot of Taverna implementation of RPC based workflow retrieving EMBL information from DDBJ.
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AcknowledgementsThis research is supported in part by NIH grant K25 HG02378 and by NSF grant DBI-0135442. The authors are grateful to Dr. Dan Masys (at UCSD) for allowing us to download the current HAPI dataset. We would like to acknowledge Tom Oinn at the European Bioinformatics Institute and head of the Taverna project, for his feedback with regard to Taverna.
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Hypocoagulation induced by broad- spectrum antibiotics in extensive burn patients

Jian Liu
†
Yiqing Liu
†
Shengjun Liu
†
Qin Zhang
Jiexin Zheng
Yiwen Niu
Xuefeng Wang
Hypocoagulation induced by broad- spectrum antibiotics in extensive burn patients
10.1186/s41038-019-0150-7C A S E R E P O R T Open Access
Background: Patients with extensive burns usually develop pro-coagulation soon after the injury if there is no sepsis occurred. We describe the case of an extensive burn adult suffering from hypocoagulation not related to sepsis, but secondary to antibiotic treatment. Case presentation: Here, we report a case of an adult male patient suffering from flame burns of 45% total body surface area (40% full thickness) combined with inhalation injury. Hypocoagulopathy with soaring prolonged activated partial thromboplastin time value occurred on third week post-burn while systemic infection had been under control by application of broad-spectrum antibiotics. Investigations showed that not the infection but vitamin K-related coagulation factor deficiency were responsible for unexpected bleeding. However, supplemental vitamin K was not the key as we expected, which prompted us trying to decode the underlying cause of coagulation disturbance in this patient and pick out the most effective treatment for live-saving. After the withdrawal of highly suspected broad-spectrum antibiotic, Meropenem®, disturbed vitamin K related coagulation factors gradually restored to their optimal levels so as to maintain normal coagulation status. Therefore, surgical procedures without further risk of bleeding could be carried out in time for wound recovery. The patient was discharged on post-burn day 67 and transferred to a secondary hospital for his rehabilitation. Conclusion: Hypocoagulopathy may be devoted to different reasons other than sepsis in extensive burns. Early recognition of the cause for coagulation disturbance is critical to make appropriate treatment and save patients' lives. This case illustrated the importance of unveiling the mist cause for coagulation disturbance occurred in extensive burn patient, which paved the way for optimal life-saving treatments. And we also recommend burn surgeons to be alerted to antibiotic-induced vitamin K deficiency-related coagulopathy among critical burn patients.

Background

Patients with extensive burns usually develop pro-coagulation soon after the injury if there is no sepsis occurred. Hypocoagulation seldom comes upon critically burned patients, except in those with severe infection or sepsis with dissemination intravascular coagulation (DIC). We describe the case of an extensive burn adult suffering from hypocoagulation not related to sepsis, but secondary to antibiotic treatment.

Case presentation

A 40-year-old male, weighing 65 kg, suffered from flame burns of 45% total body surface area (TBSA) (40% full thickness) combined with inhalation injury, affecting his face, neck, trunk, upper extremities, and right lower extremity. He was rescued by the firemen from the accidental site and directly sent to the local hospital, which provided sufficient fluid resuscitation during hypovolemic stage post-burn. Negative personal medical history and family history were confirmed while the patient was transferred to our hospital for further treatment on the post-burn day (PBD) 3. Topical usage of 1% silver sulfadiazine (1%SD-Ag cream) was served as wound management together with surgical procedures of escharectomy (upper extremities and right lower extremity) and heterograft on PBD 4, consecutively 2 autografting on PBD 10 and 16. Broad-spectrum antibiotics, Meropenem® (1.0 g three times daily), was applied intravenously since PBD 3 as the empirical therapy for extensive burn patients and continued for ensured Pseudomonas aeruginosa-positive of the wound in this patient (Fig. 1). Calorie intake of Fresubin® 1000 mL/day was commenced on PBD 5 via gastrointestinal (GI) tube as a supplemental nutrition other than oral intake of normal food, except on those days of surgical procedures. Not until PBD 13 did the patient show the signs of infection, such as chill, high fever, wounds infiltration, delirium, and neutrocytosis (Figs. 2 and 3), while his hemodynamic status remained stable, all coagulation criteria within normal range. Wound and blood culture were both reported Klebsiella pneumoniae-positive (multi-drug-resistant strain, tigecycline medium). Hence, treatment with tigecycline (0.5 g three times daily) was initiated intravenously on PBD 13, together with Meropenem®, and worked well in controlling those infections till PBD 18. All lab findings coordinate with the severity of the patient during that period of time, including his coagulation status.

Nevertheless, concurrent with remission of signs of severe infection, there came the abnormal oozing of blood on the donor site during the third autografting and uncontrolled bleeding while removing the central venous line on PBD 23, which related to the prolonged activated partial thromboplastin time (APTT) 61.5 s, normal range 25.1-39.5 s, while prothrombin time (PT) 15.6 s, normal range 10.0-16.0 s, and the international normalized ratio (INR) 1.32 were normal. An intravenous bolus of 10 mg vitamin K1 was applied during the procedure and continued in the following 5 days. Further investigation of the patient's coagulation status was launched after the operation. There was no sign of DIC at this time, with normal value of platelets (227 × 10 12 /L), fibrin degradation products (FDP) 4.7 mg/L, normal range 0-5 mg/L, D-dimer 1.68 mg/L, normal range < 0.55 mg/L, and slightly reduced fibrinogen (Fg) 1.8 g/L, normal range 1.8-3.5 g/L. Complete screening of patient's coagulation factors was carried out on PBD 26 (Table 1). von Willebrand factor (vWF) level and activity were within the normal range, lupus anticoagulant (LAC) was negative, and coagulation factor VIII and V were normal, deficiency of multiple coagulation factors (Table 1: coagulation factor II, coagulation factor VII, coagulation factor IX, and coagulation factor X activities were 39%, 35%, 45.1%, and 28% respectively on PBD 26) that related to vitamin K deficiency (VKD) was indicated as the reason for coagulopathy in this patient. Unfortunately, specific detection of serum vitamin K concentration and protein induced in vitamin K deficiency (PIVKD) were not available in our hospital. However, what really mattered was to relief further risk of bleeding and coagulation crash as quickly as possible. Thereafter, supportive therapies, such as 200 ml fresh frozen plasma (FFP), was administered daily for consecutive days and 600 IU prothrombin complex was infused on PBD 26 when the patient was diagnosed of hypothrombinemia. Although the coagulation status remained abnormal on PBD 27, surgical debridement of the neck, trunk, and right lower extremity and grafting were performed. There was no abnormal bleeding either on the donor site or burn wounds, and wound healing of any sites was not interfered. At the same time, application of vitamin K1 intravenously increased to 20 mg daily for another 15 days, but showed mild effect to reverse the surging APTT level.

Nothing should be considered as the first priority, except finding out the exact reason for VKD. Concerning no short of vitamin K intake, no impairment of liver function, and vitamin K recycling occurred in this case, but scarcity of endogenous vitamin K generated by intestinal bacterial flora soon came to our view. Meropenem® application was discontinued on PDB 34. Interestingly, 2 days after, APTT improved to 47 s and came back to the normal range on PDB 39 along with normal value of coagulation factor II, VII, IX, and X (Figs. 1 and 4), even with combined usage of tigecycline (0.5 g three times daily) and fosfomycin sodium (4.0 g three times daily) intravenously, started on PBD 38, dealing with multi-resistant strain of Klebsiella pneumonia (tigecycline resistant). Another two operations were performed for grafting on PBD 40 and 52. Tigecycline and fosfomycin sodium were applied in the same dosage as previously used for multi-drug-resistant Klebsiella pneumonia still detected in wound culture. The patient was discharged on PBD 67 with all burn wounds recovered and no abnormality in coagulation status (Fig. 5).

Discussion

Patients with severe burns usually develop pro-coagulation soon after the injury. Elevated fibrinogen levels are often present immediately following burn injury, owing to the shift in hepatic protein synthesis to acute-phase proteins including complements, interleukins, fibrinogens, and etc. [1], while dilution effects might explain the temporal reduction in the levels of coagulating proteins during the early resuscitation phase with no coagulation function affected. For those combined with inhalation injury, studies confirmed that hypercoagulability occurred among patients after PBD 4 [2,3]. Furthermore, extensive endothelial impairment ignites coagulation cascade, which intimately intertwined with inflammation reaction following the thermal injury and during infection [4]. And along with the wound recovery, coagulopathy is less related to burn injury but more likely owe to sepsis [5]. In our case, patients' coagulation status was kept normal at the time of transferring to our burn center and the following 2 weeks, even if the patient had been suffering from severe wound and bloodstream infection since PBD 13. There were no signs of DIC, and coagulation criteria were all within normal range (PT 14.1 s, APTT 34.9 s, fibrinogen was 4.9 ng/L and normal fibrinogen degraded product level). When the first coagulation abnormalities developed on PBD 23, signs and symptoms of severe infection had already been remitted (relief of high fever, delirium, and wound infiltration, also blood culture was reported negative on PBD 24), attributed to wound debridement accompanied with effective systemic antibiotic treatment, and white blood cell counts were elevated within accepted level (10.38 × 10 9 /L, neutrophils 85.1%). Platelet (PLT) counts (277 × 10 12 /L) and precalcitonin (PCT) (0.5 ng/mL) were normal, so the possibility of thrombocytopenia and septic coagulopathy was ruled out. There was no over-transfusion or enormous blood loss during surgical procedures, and serum calcium level was occasionally slightly decreased (fluctuated from 1.82 to 2.05 mmol/L), which indicated no relationship between surgical procedure, blood loss, transfusion, and coagulopathy. On the other hand, acquired coagulation factor deficiency might be associated with systemic amyloidosis, respiratory mycoplasma infection, and is extremely rare among extensive burn patients but sporadically reported in the literature [6][7][8][9]. It could due to the presence of an inhibitor, anti-cardiolipin antibody, or be related to antibiotic treatment, such as Meropenem®, of which the mechanism still remained unknown. In the present case, the patient had a negative medical or family history. The plasma-mixing test, with resultant normalization of PT and APTT contradicted the presence of antibody or inhibitor. Furthermore, not just factor X activity was at an extremely low level (28%, normal range 50 × 150%), but other coagulation factors related to vitamin K metabolism (coagulation factor II, coagulation factor VII, coagulation factor IX, and coagulation factor X activities were 39%, 35%, and 45.1% respectively) were all impaired significantly.

Vitamin K acts as the cofactor for γ-glutamyl carboxylase that is responsible for the post-translational modification of glutamate residues in of coagulation factor II, VII, IX, and X [10]. VKD may reduce biological activity of these factors presenting with increased PT or APTT and clinically manifested with bleeding. Serum concentration of phylloquinone (vitamin K1), vitamin K1 2,3-Epoxide (K1O), and protein induced by vitamin K absence or antagonist II (PIVKA-II) can be measured; however, the diagnosis is usually clinical and by exclusion [11]. Dietary insufficiency and long-term broad-spectrum antibiotic therapy could be the reason for VKD among critically ill patients. For our patient, the first possibility was easy to be ruled out since normal diet and supplemental GI nutrition out-reached 65 μg/day, the recommended nutrient intake of vitamin K by the World Health Organization for adult males [12] or the daily requirement of 100 mg phylloquinone recommended for the maintenance of hemostasis [13]. Vitamin K can be obtained not only from digestive food, but also synthesized by intestinal bacterial flora colonizing in the colon or the distal ileum (vitamin K2, menaquinone-MK), that is activated in the liver [14]. In our opinion, patients' hepatic function tests kept normal when he was hospitalized. No cholestasis or hepatic pathological entities found, which rejected the hypothesis of vitamin K recycle interdiction. Therefore, came the high suspicion of broad-spectrum antibiotics as the reason for secondary VKD that led to hypocoagulopathy in this case.

Antibiotic-induced secondary VKD can develop by the following possible mechanisms: the first is the inhibition of vitamin K metabolism in the liver. Cephalosporins, which include the N-methylthiotetrazole (NMTT) side chain (e.g., cefmetazole, cefotetan, cefamandole, cefoperazone, and moxalactam) or the 2-methyl− 1,3,4-thiadiazole-5-thiol side chain (e.g., cefazolin), may inhibit vitamin K epoxide reductase in the vitamin K metabolism cycles and makes it accumulated in the liver. Usually, patients on parenteral nutrition are at stake of the  [15]. Low plasma vitamin K1 concentration can ensure the co-relationship between antibiotic application and secondary VKD. It was reported that the average time from the initiation of antibiotic therapy to diagnosis of hypoprothrombinemia was 5.7 days (range, 2-15 days), and patients' prognosis was well after being administered vitamin K supplements [16]. Obviously, this was not the key in our case since no cephalosporins had been applied. The second mechanism of antibiotic-induced VKD is that broad-spectrum antibiotics suppress intestinal flora that synthesize vitamin K2 (menaquinone-MK), which might play the causative role in our case leading to the clinical manifestation of bleeding. Mero-penem® is the bactericide of which the anti-bacterial spectrum covers Gram-positive, Gram-negative bacteria (including Pseudomonas), and anaerobic bacteria. There was the report that Meropenem® induced the transient coagulation factor X deficiency of a 12-month-old boy suffering from 25% TBSA and underwent hypocoagulability 3 days after the initiation of the antibiotic. And it is also considered to be responsible for drug-induced immune thrombocytopenia (DITP) that shows acute thrombocytopenia in patients [17]. The mechanism of Meropenem® to initiate different ways of coagulopathy still needs further investigation; however, in present case treatment, long-term usage of Meropenem® might eradicate vitamin K producing intestinal microflora, interdicted the vitamin K2 pathway and made it deprived of vitamin K storage in the patient. Moreover, the combined application of tigecycline might aggravate the clearance of intestinal microflora. Protein induced by vitamin K absence or antagonist II (PIKA-II) is the predictor of VKD in such cases. However, tests investigating specific vitamin K concentrations that might be helpful to the diagnosis were not available in our hospital. Indirect evidences such as recovery of vitamin K-related-coagulation factor values after efficient dosage of supplemental vitamin K bolus (20 mg q.d), clinical and laboratory improvement after discontinuation of Merope-nem® gave us the hint that long-term and high dosage of Meropenem® application could be the cause of hypocoagulopathy in this patient.

The role of tigecycline treatment in this case also merits careful consideration. Tigecycline is a tetracycline-class bacteriostatic agent that acts against gram-positive, gramnegative, and anaerobic microbes, but not Pseudomonas spp. or Proteus spp. [18]. Coagulation disturbances were observed only as an interaction between tigecycline and warfarin. Few, but severe cases of hypofibrinogenemia that related to tigecycline treatment were reported and high dosage of the drug or combination with warfarin could be the risk factors [19,20]. The mechanism by which tigecycline provokes coagulation disturbance could either relate to the reduction of intestinal microflora or direct on vitamin K activation in the liver. In our case, tigecycline was used to act against the multi-drug-resistant Klebsiella pneumonia with recommended dosage, and later combined with fosfomycin sodium when the wound culture proved that the strain shift from medium to resistant to tigecycline. It could have acted synergic with Meropenem® in eradicating intestinal micro-organisms, also according to Naranjo scale [21], which investigates the probability of adverse drug reactions (ADR), indicated that tigecycline could be the doubtful cause for hypocoagulopathy, while Meropenem® might be the possible one. No coagulation disturbance occurred after the re-administration of tigecycline in the case even supplemental vitamin K bolus had been withdrawn at that time.

Fig. 1
1Chronological relationships of antibiotic treatment, vitamin K1 administration, surgical procedures, and coagulation time fluctuation of the patient. APTT prolonged activated partial thromboplastin time, PBD post-burn day, PT prothrombin timeFig. 2 Daily maximal temperature and heart rate (HR) of the patient during his stay in hospital. PBD Post-burn day

Fig. 3
3Infection related indicators (blood cell count and Precalcitonin (PCT)) levels of the patient during his stay in hospital. PBD Post-burn day, PLT Platelet, WBC White blood cell risk

Fig. 4
4Coagulation factor level fluctuation of the patient before and during his treatment of hypocoagulation. PBD Post-burn day

Fig. 5
5Relationship between intravenous vitamin K1, meropenem, fosfomysin sodium, tigecycline, and levels of factors related to hypocoagulopathy. Meropenem, 1.0 g three times daily; tigecycline, 0.5 g three times daily; fosfomycin sodium, 4.0 g three times daily. PBD post-burn day

Table 1
1Coagulation factors values on post-burn day (PBD) 26 vWF von Willebrand Factor, LAC Lupus AnticoagulantActivity

AcknowledgementsNot applicable.FundingNot applicable.Availability of data and materialsAll list authors agreed data sharing to this article.ConclusionsHypocoagulopathy may be devoted to different reasons other than sepsis in extensive burns. Early recognition of the cause for coagulation disturbance is critical to make appropriate treatment and save patients' lives. Extensive burn patients are vulnerable to secondary VKD related to broad-spectrum antibiotic treatment, but treatable if early supplemental vitamin K is applied. We recommend coagulation parameters be closely monitored and to withdraw antibiotics application as soon as possible. The exact mechanisms of Meropenem® to impair activation of coagulation factors and why there was only endogenous pathway impairment in this case remained to be further elucidated; the potential role of genetic susceptibility could be one way out. Ethics approval and consent to participate Shanghai Jiaotong University School of Medicine Affiliated Ruijin Hospital Ethics Committee guidelines for Case Reports states that ethical review is not required if the patient has provided written consent.Consent for publicationWritten informed consent was obtained from the patient for the publication of this case report.Competing interestsThe authors declare that they have no competing interests.
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Resurrection of DNA Function In Vivo from an Extinct Genome
2008

A J Pask
R R Behringer
M B Renfree
Resurrection of DNA Function In Vivo from an Extinct Genome

PLoS ONE
3522402008Editor: Erik I. Svensson, Lund University, Sweden Received January 4, 2008; Accepted April 16, 2008;Competing Interests: The authors have declared that no competing interests exist. *
There is a burgeoning repository of information available from ancient DNA that can be used to understand how genomes have evolved and to determine the genetic features that defined a particular species. To assess the functional consequences of changes to a genome, a variety of methods are needed to examine extinct DNA function. We isolated a transcriptional enhancer element from the genome of an extinct marsupial, the Tasmanian tiger (Thylacinus cynocephalus or thylacine), obtained from 100 year-old ethanol-fixed tissues from museum collections. We then examined the function of the enhancer in vivo. Using a transgenic approach, it was possible to resurrect DNA function in transgenic mice. The results demonstrate that the thylacine Col2A1 enhancer directed chondrocyte-specific expression in this extinct mammalian species in the same way as its orthologue does in mice. While other studies have examined extinct coding DNA function in vitro, this is the first example of the restoration of extinct non-coding DNA and examination of its function in vivo. Our method using transgenesis can be used to explore the function of regulatory and protein-coding sequences obtained from any extinct species in an in vivo model system, providing important insights into gene evolution and diversity.

Introduction

Extant species represent less than 1% of the genetic diversity that has existed in the animal kingdom [1]. Extinction rates are increasing at an alarming rate, especially of mammals [2,3]. Many efforts, such as that of the Frozen Zoo (San Diego Zoological Society Conservation and Research for Endangered Species) are working to cryo-archive cell and tissue resources from a diverse range of threatened species, to protect their genetic information. However, for those species that have already become extinct, access to their genetic biodiversity may not be completely lost.

The Tasmanian tiger or thylacine (Thylacinus cynocephalus) was a large, carnivorous Australian marsupial. Often described as the most striking example of convergent evolution in the mammalian lineage, the marsupial thylacine was morphologically almost indistinguishable from the eutherian canids, apart from the presence of a pouch where its young developed ( Figure 1) [4]. Thylacines were hunted to extinction in the wild in the early 1900s and the last known animal died in captivity in the Hobart Zoo in 1936 [4]. Fortunately, some thylacine pouch young and adult tissues were preserved in alcohol in several museum collections around the world (Figure 1b).

With improved techniques for the isolation of ancient DNA, it is now possible to access the genomes of extinct species [5]. There have been several recent papers published examining the genomes from a diverse range of extinct species from plants and bacteria to mammoths and Neanderthals [2,[5][6][7]. While most studies to date have examined the mitochondrial DNA of these species for phylogenetic purposes, the focus is now shifting to the analysis of genomic DNA. Recent advances in sequencing technologies, such as direct high-throughput parallel pyrosequencing, are expected to expand the ancient DNA data bank exponentially over the coming years [5]. Most studies have used the sequence data to examine divergence times and population structure of extinct species. However, more recently this information is being used to examine how the function of these genes may have evolved. The function of the Melanocortin 1 Receptor (MC1R) gene has recently been investigated from the extinct mammoth and Neanderthal [1,7]. Isolated MC1R sequences from ancient DNA samples were cloned and transfected into cell lines to examine the function of the receptor in activation assays in vitro. The results of these experiments suggest that variation in skin and hair pigmentation may have occurred within these species [1,7] and describes a technical platform for examining extinct protein function in vitro.

While many adaptive changes throughout evolution have been ascribed to changes within the proteins themselves [8,9], the high conservation of protein coding regions between mammalian genomes suggests that changes in the open reading frames are unlikely to be the primary cause of the vast differences observed in both form and function [10][11][12]. With the recent release of the ENCODE pilot study [13] it is clear that the majority of the noncoding mammalian genome is transcribed. It appears that rather than changes in the genes themselves, it is subtle differences in their non-coding regulatory elements that control their spatial, temporal and quantitative expression that underpins the variation in the animal kingdom [10][11][12]. Therefore, the non-coding regions of extinct genomes are likely to hold the most important genetic information that defined a species. We have therefore developed a technique for examining the function of non-coding regions from extinct genomes in vivo. To gain a greater insight into the function of these elements we isolated a regulatory sequence from the genome of the extinct thylacine using transgenesis and examined its function in mice.

Results

Extinct DNA Isolation and Fragment Characterisation

To resurrect the function of non-coding DNA from an extinct mammal, we isolated genomic DNA using the technique described by Pää bo [14]. DNA was obtained from four 100-year-old specimens: three alcohol-fixed pouch young (one shown in Figure 1b) and one dried adult pelt, obtained from Museum Victoria (Melbourne, Victoria, Australia). The isolated DNA was fragmented as expected [6], and ranged from 300-500 bp. We obtained 200 ng to 1 mg from each sample. We chose to isolate the well-characterised transcriptional enhancer element of the proa1(II) collagen (Col2a1) gene [15][16][17]. This element was chosen because it is relatively conserved among mammals and directs chondrocyte-specific expression in the mouse [15,17]. Primers were designed to span the core enhancer from regions conserved across all described mammalian species using a Clustal-W alignment. Primers were engineered with Spe1 (forward primer) and Xba1 (reverse primer) restriction sites to allow for multimerisation of the resulting product (see Materials and Methods). We performed PCR amplification from each DNA sample independently to reduce the risk of cross contamination, and to ensure the sequence was identical across all individuals, confirming it was thylacine in origin. This approach was also used to ensure sequence obtained was not harbouring any genetic changes caused by spontaneous hydrolysis or oxidation of the preserved DNA [6]. The resulting 264-bp PCR product from each sample, encoding the enhancer was independently subcloned and sequenced. The sequence obtained from each of the four independent thylacine samples was identical and a BLAST search confirmed it was different from all those available in the database and from the most likely sources of contamination in the lab (human and mouse) (Figure 2a). Phylogenetic analyses of the thylacine sequence with that of eutherian and marsupial species confirmed its identity as the thylacine Col2a1 enhancer orthologue (Figure 2b), as it was most closely related to the tammar wallaby (Macropus eugenii, an extant marsupial species) than to human, mouse or rat.  The morphology of the head shows remarkable convergent evolution. However, there are some differences: in marsupials, the lacrymal extends outside the orbit and the angle of the dentary is medially inflected (c). The thylacine palatine has the vacuities characteristic of marsupial skulls (e). The teeth also show striking convergent evolution but the muzzle of the thylacine is quite narrow compared to that of the dog (e,f). Scale bar = 5cm. doi:10.1371/journal.pone.0002240.g001

Transgenesis and Functional Analyses

The strength of the Col2a1 regulatory element can be enhanced by using multiple copies of this region upstream of a reporter gene [16]. Four copies of the thylacine sequence were multimerised and ligated to the human b-globin basal promoter fused to lacZ and followed by a polyadenylation signal. The 4.75 kb TcyCol2a1-lacZ-pA construct (Figure 3a) was purified from the vector and microinjected into the pronuclei of mouse zygotes [18]. LacZ expression was examined in founder mice at 14.5 days post coitum, by staining with X-gal [18] (5-bromo-4-chloro-3-indolyl-b-Dgalactopyranoside) (Figure 3b-e).

Whole mount staining was observed in all 4 founder fetuses created in a pattern indicative of developing cartilage (Figure 3be). This pattern was identical to that of the endogenous mouse Col2a1 gene and mouse enhancer transgenes [15][16][17]. Histological cross sections of the developing limb confirmed that lacZ expression, directed by the thylacine enhancer element, was restricted to the developing chondrocytes (Figure 3g). Nontransgenic littermates were used as negative controls (Figure 3f) and showed no staining.

Discussion

A non-coding DNA fragment from an extinct mammal was able to drive expression of a reporter gene construct in a developing fetus. While the intensity of the reporter gene expression seen in the transgenic fetuses varied, the sites of expression did not. Variations in transgene expression levels are common, and can be affected by the site of integration and by the effect of epigenetic silencing mechanisms on the transgene [18]. Despite the level of variation, the expression of the thylacine Col2A1 enhancer recapitulated that of the endogenous mouse Col2a1 gene [15][16][17] and was restricted to the developing chondrocytes. This confirms that the thylacine Col2a1 gene had a conserved developmental role in cartilage formation, and that its promoter directed expression in chondrocytes in this extinct marsupial mammal. It also suggests that the associated transcription factors and the minimal enhancer element required for inducing expression from this promoter have remained sufficiently conserved between mice and marsupials to direct expression [19,20] despite approximately 148 million years of divergent evolution between these species [21][22][23].

The minimal 18-bp element [15] required for chondrocyte specific expression is only 17-bp in the thylacine and differs at 4 nucleotides positions compared with the mouse. Furthermore, the sequences of the inverted repeats thought be to essential for the function of this region that flank the minimal element are completely complementary in mouse, rat and human, [15]. However, in the thylacine, the outer repeat is highly conserved but the inner repeat differs by 2 nucleotides and is not complimentary. Despite these differences, the enhancer element was able to function in the developing mouse, suggesting that these differences are not critical for the function of this element. Therefore, the examination of extinct DNA can provide important information about gene function in extant species. Taken together, the results of this study confirm an ancestral role for the thylacine Col2A1 gene in cartilage formation and demonstrate that it is possible to examine non-coding DNA function from extinct animals in vivo in the mouse.

These observations advance previous studies that have so for only examined extinct protein function and only in in vitro systems [1,7]. However, both systems (in vitro cell cultures and in vivo whole organism models) rely on the availability and compatibility of cofactors within the host cell or embryo in order to examine gene or regulatory sequence function. It is possible that the variations in receptor signalling strength seen in the mammoth and Neanderthal MC1R gene analyses in vitro [1,7] did not occur or affect gene function in their native organisms. The native ligand in the mammoth and Neanderthal may have varied to the one used in culture, as could the downstream activation strength of the signalling pathway. Similarly it is possible that the thylacine Col2A1 enhancer had a different pattern of expression in the developing thylacine embryo to that observed in the mouse, due to the different availability of cofactors required for its activation. The precise function of an extinct gene or regulatory element is impossible to determine without examining every part of the pathway in one system. Results from extinct DNA analyses therefore should be cautiously interpreted.

Thus extinct genomes can be examined by the in vivo method described here, in conjunction with in vitro techniques, to enable functional analyses of the genes and regulatory elements. This could provide important insights into genome evolution by defining functional features unique to each species. We have demonstrated that non-coding genetic information from an extinct species can be resurrected in vivo and in doing so, we have restored to life the genetic potential of a fragment of this extinct mammalian genome.

Materials and Methods

Tissues, DNA isolation, amplification, and transgene construction and analyses Tissues were obtained from three 100-year-old thylacine pouch young specimens fixed in ethanol and one dried 100-year-old adult skin from Museum Victoria, Melbourne. Tissue samples were stored in sterile cryo-vials and transported to the University of Texas. DNA was extracted according to the method described by Pää bo [14] with a control extraction performed on an empty vial. All processing was done in a UV-sterilised bio-containment hood to prevent any contaminating DNA from entering the samples. DNA samples were kept separate and used as separate templates in subsequent PCR reactions. Negative controls (blank-DNA extraction, and no template) were included in each PCR reaction to ensure no contamination of samples. Primers were engineered with Spe1 (forward primer) and Xba1 (reverse primer) restriction sites to allow for multimerisation (forward 59 NNNACTAGTG-CATTGGGAGATTGGCAGCGAT 39; reverse 59 NNNTCTA-GAGCTACCTCTTTCGGGGAACTG 39). The resulting 264bp PCR product was subcloned and sequenced in both directions for at least three clones from each tissue sample. The sequence was compared to that from possible sources of contamination within the lab using genome databases (human, mouse and tammar wallaby) and was shown to have consistent differences with each species clearly indicating it was derived from a unique source. The fragment was also subjected to phylogenetic analysis to confirm its marsupial origin. Phylogentic relationship of the thylacine PCR fragment was compared to the orthologous region from tammar wallaby, human, mouse and rat using the PHYLIP 3.63 program (University of Washington; http://evolution.genetics.washington. edu/phylip/doc/main.html) using maximum-likelihood, maximum parsimony and neighbor-joining analysis with 1000 replicates. The resulting data was viewed with TREE-view 1.6.6 (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html).

Once sequence identity was confirmed, the clone was digested with Xba1 and Spe1 and incubated with T4 ligase at 16 o C for 2 hours. After ligation, the reaction was digested with Xba1 and Spe1 such that only products multimerised head to tail (ablating the restriction sites) would remain intact. Ligation products corresponding in size to 4 mutimerised copies (1056 bp) were purified. The multimer was ligated to a human b-globin basal promoter fused to lacZ followed by a polyadenylation signal. Embryos were stained for LacZ activity [17]. The human b-globin basal promoter-lacZ-pA reporter construct on its own does not have tissue-specific activity in transgenic mice [24]. For histological analyses, X-gal stained embryos were postfixed in 4% paraformaldehyde, embedded in paraffin, sectioned and counterstained with eosin [17].

Figure 2 .
2Analyses of the thylacine Col2a1 enhancer element. (a) Sequence alignment of the thylacine PCR cloned Col2a1 enhancer. Identical alignment between species is shown by black boxes. The thylacine sequence was most similar to, but distinct from that of another marsupial, the tammar wallaby, Macropus eugenii. The black box outlines the minimal 18-bp element. Inverted repeats (arrows) surrounding the minimal element are shown with complementary nucleotides in red. The outer repeat is highly conserved between all mammals, but the inner repeat differs by 2 nucleotides in the thylacine. (b) Phylogenetic analysis of the thylacine Col2a1 enhancer. The thylacine sequence groups with the tammar wallaby, both of which are more similar to the human sequence than to mouse or rat. Numbers indicate bootstrap values based on 100 replicates. doi:10.1371/journal.pone.0002240.g002

Figure 1 .
1The thylacine, Thylacinus cynocephalus. (a) Young male thylacine in Hobart Zoo in 1928, photograph (Q4437). (b) One of the preserved pouch young specimens (head length 34 mm) from which DNA was extracted, from the Museum Victoria collection. (c-f) The skull of the thylacine (c,e) compared with that of the domestic dog Canis canis (d,f).

Figure 3 .
3From extinction to gene expression. Functional analysis of the thylacine non-coding DNA fragment. (a) Diagram of transgene construct. 4 copies of a 264-bp fragment containing the Thylacine Col2a1 enhancer (TcyCol2a1) region was ligated to the human b-globin minimal promoter (black box) and ligated to lacZpA. (b-e) X-gal stained 14.5 dpc TcyCol2a1-lacZpA transgenic mouse embryo showing varying levels of reporter gene expression within the developing cartilage (blue). (f) Non-transgenic littermate, negative control fetus. (g) Top panel; Magnified image of forelimb from fetus in (b) black line indicates the plane of section shown in (g) bottom panel. Bottom panel; Histological section of transgenic forelimb digit, showing lacZexpressing chondrogenic tissue (blue) counterstained with eosin (pink). doi:10.1371/journal.pone.0002240.g003
May 2008 | Volume 3 | Issue 5 | e2240
PLoS ONE | www.plosone.org
AcknowledgmentsWe thank Dr. Joan Dixon and Lina Frigo and the Museum Victoria for allowing us to sample rare thylacine material. We thank George Adebayo for pronuclear injections, Ying Wang for histology. We also thank Dr. Deanne Whitworth, Dr. Chris Cretekos and Richard Moyle for assistance and Professor Roger Short for helpful comments. We especially thank the Tasmanian Museum and Art Gallery for the kind permission to reproduce photograph (Q4437) by Benjamin A. Sheppard, David Paul for the photograph of the thylacine pouch young and Associate Professor Geoffrey Shaw for the photographs of the dog and thylacine skulls.Author ContributionsConceived and designed the experiments: RB AP MR. Performed the experiments: RB AP. Analyzed the data: RB AP. Contributed reagents/ materials/analysis tools: MR. Wrote the paper: RB AP MR.
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