SEARCH

SEARCH BY CITATION

Keywords:

  • Evaluation of computer-based learning systems;
  • molecular visualization;
  • interactive learning environment

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

A new computer-based molecular visualization tool has been developed for teaching, and learning, molecular structure. This java-based jmol Amalgamated Molecular Visualization Learning Environment (jAMVLE) is platform-independent, integrated, and interactive. It has an overall graphical user interface that is intuitive and easy to use. The application can be downloaded free from the internet at wabri.org.au/jamvle. A cohort of 28 third year undergraduate molecular biotechnology degree students evaluated the new application through an essay-style project. These were analyzed to identify themes expressed by students in the content of their evaluations. Most students were positive about the new jAMVLE learning environment, and five major benefits emerged from the analysis. In particular, the students perceived that jAMVLE has an appealing interface, is interactive, provides a useful integrated environment, is user friendly, and is an excellent learning tool. Overall, students found that the jAMVLE application stimulated their interest, was a more active learning environment, provided better guidance, and made learning fun.

Students of the chemical and biological sciences need to know how to work with molecular structures throughout their undergraduate studies and professional careers because molecular structure is inextricably linked to function. Learning molecular structure in three dimensions is a conceptually demanding subject for a student and requires approaches other than the traditional didactics [1]. One popular alternative approach is computer-based learning (CBL).11

CBL has been used as a teaching and learning strategy for many years and has been applied to many disciplines. Meta-analyses of CBL studies showed that student learning benefited from CBL when compared with traditional lectures [2, 3]. Computer-based learning incorporating visualization of three-dimensional molecular structure has been a particularly popular and useful application of CBL in both chemistry [47] and biochemistry [1, 814].

Table I shows that many good software applications, operating on a variety of computer platforms, are available for three-dimensional molecular visualization. These programs are used as stand-alone applications (e.g. Mage, RasMol, and Deep View), integrated within a web browser (e.g. Chime and Protein Explorer), used as stand alone Java-based applications (e.g. jmol, Cn3D), or used as Java applets integrated within the web browser (e.g. jmol, QuickPDB). Most of these programs were designed principally to help scientists visualize and analyze molecular structure and were not specifically designed for teaching.

Most of the stand-alone visualization programs suffer from a lack of integration with the written or web-based instruction. That is, written notes or notes on a web page are separate from the application. Web-based instruction also requires at least two separate components: a web browser and an appropriate helper application or plug-in for molecular visualization. Consequently, the user has to configure the web browser to work in conjunction with the helper application or plug-in. The Chime plug-in and some Java applets can also have compatibility issues with different web browsers or computer platforms. Further, some Java applets and other programs such as QuickPDB may offer only limited options for visualization or interactivity.

We felt that there was a need to develop a new platform-independent, integrated, interactive, and comprehensive molecular visualization tool for teaching and learning molecular structure. In particular, we have integrated a command line interface (for interactivity, flexibility, and creativity), a web browser (for instruction and interactivity), and a graphics viewer (for visualization, spatial development, and interactivity) within one Java application. This application has an overall graphical user interface environment that is easy to use and intuitive. A cohort of 28 third year undergraduate molecular biotechnology degree students evaluated the new application through an essay-style project. These were analyzed to identify themes expressed by students in the content of their evaluations.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The jAMVLE Application—

The jAMVLE (pronounced “jamvil”) application (version 1.0) was developed using version 1.4 of the Java programming language development kit (Java 2 JDK 1.4 at java.sun.com/), a Java build tool called Apache Ant (ant.apache.org/), and the jmol open source code (jmol.sourceforge.net). jmol is a Java-based application written with free open-source software and licensed under the GNU Lesser General Public License (www.gnu.org/licenses/lgpl.html) that contains Java classes specifically for molecular visualization. jAMVLE is also licensed under General Public License, and further details can be found on the web site. We used most of the graphics Java classes of jmol for molecular visualization. In our prototype version (version 1.0), we contributed to the development of the code for displaying molecular structure in the popular cartoon or “ribbon” format. This new development was included in the open source code of jmol. The information given in the pdb file summary window (see “Results”) is generated by a Protein Data Bank file parsing tool written exclusively for jAMVLE based on the file format described online at www.rcsb.org/pdb/file_formats/pdb/pdbguide2.2/guide2.2_frame.html.

This tool essentially extracts all the header information from the Protein Data Bank (PDB) file. The integrated web browser of jAMVLE uses basic HTML code and protocols but does not presently support scripting languages such as JavaScript and Perl or the ability to authenticate on secure sites.

Java may have some compatibility problems, but this is usually when a Java applet is to be used within a browser. The integrated standalone application we have developed circumvents browser problems but is still subject to a suitable Java software engine (Java Virtual Machine) being installed on the computer platform. Some PCs may also require the installation of a simple batch file to launch the program, and this is provided, with explanation, on the web site. We have run the application on Macintosh, PC, and Linux platforms without issue. The application only takes up 2 megabytes of hard disk storage and should run on computers that have Java Virtual Machine 1.4. The recommended hardware is a 750 MHz CPU with at least 256 megabytes of RAM. No special graphics hardware is required to run the application as it utilizes a software implementation of a Z-buffered graphics engine specially developed for visualizing molecular structures. The application (with some sample tutorials) can be downloaded free from the internet at wabri.org.au/jamvle

Student Cohort and Survey—

The student cohort for this study was a third year undergraduate class of 28 students studying structural bioinformatics in the B.Sc. (Molecular Biotechnology) degree in the School of Biomedical Sciences at Curtin University of Technology. There were 10 males and 18 females in the class with ages ranging from 22 to 43 (mean = 24 years and S.D. = 6). The students were asked to evaluate the newly developed software application and compare it with an older, web page only, version of a protein structure tutorial. In particular, the students were asked to: 1) evaluate jAMVLE and include in their evaluation its purpose, its design, its content, their ability to use the software, and how it helped (or did not help) them learn the subject and 2) compare and contrast the new protein structure tutorial (with jAMVLE) with the older (web page only) version of the tutorial. Students were asked to submit their evaluation as an essay style project because the learning outcomes for the unit included developing skills in: independent learning; critical analysis and evaluation; research; written communication; and problem solving. The students were advised on the assessment criteria and were assessed based upon their knowledge, critical evaluations, honest comments, attention to detail, thought provoking analysis, presentation, and English expression. Students were advised to provide a comprehensive, honest, analysis and were encouraged to submit negative comments (where “negative” also includes comments and suggestions for improvement). Two of the authors (S. B. and E. H.) were responsible for assessing the student submissions. Students were encouraged to discuss the project between themselves and to form study groups, but they were also strongly advised to submit their own work. Their evaluation was worth 30% of their total semester mark.

We felt that the essay was an appropriate qualitative survey instrument for our particular study for three reasons. 1) It was inappropriate to directly measure each student's incremental learning of content by a “pre and post” summative assessment because students were comparing two versions of the same content (protein structure tutorial) presented in different ways. Consequently, the result of such an assessment would only reflect the consolidation and frequency of learning the same thing. 2) It was inappropriate to directly measure the efficacy of a CBL approach over more traditional didactic approaches because the student cohort was comparing a web-based tutorial with a software-based tutorial, and both of these approaches are non-traditional approaches. 3) We felt that by giving students the freedom to express themselves, they would provide us with more informative feedback when compared with, say, having them simply answer a questionnaire. In particular, we analyzed student responses based on the qualitative grounded research model [30] to identify themes expressed by students in the content of their evaluations.

jAMVLE and the Older Web-based Tutorial—

An older web page tutorial was used as a comparison with the new jAMVLE tutorial and was a series of web pages with hypertext links to appropriate diagrams and structures. The PDB structure files in the web page tutorial had to be viewed with either Chime (as a plug-in) or RasMol (as a helper or standalone application). Web page navigation in the jAMVLE application was by hypertext links to other sections of the tutorial on the bottom of each page. Navigation on the older web page tutorial was by a contents (or index) page as well as contents, forward, and back buttons on each page. Otherwise, much of the content, formatting, and hypertext links was the same for both tutorials. The essential and principal difference between the tutorials was the user interface.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The jAMVLE Application—

Fig. 1 shows a screenshot of the complete window interface of the jAMVLE application. The jAMVLE application window can be resized to suit the screen (and the user) and is comprised of three general sections: the graphics molecule viewer on the left, the web browser in the top right, and the command line interface in the bottom right. The graphics section is comprised of the graphics window, a general menu (above the graphics window), and functional icons (below the graphics window). The graphics window has nearly all of the molecular visualization options available in RasMol and Chime. The web browser section is comprised of three tabbed windows: the web page, a text window for displaying the PDB file summary, and a text window for displaying the raw structure file. A simple click on each tab brings the appropriate window to the foreground. The command line section is comprised of an Open button and two tabbed windows labeled Command Line and Measurements. The Open button opens any script file, records the script in the command line window, and then modifies the graphic display of the molecule according to the script. The Command Line tabbed window is used for entering commands that act upon the molecule in the graphics window. The Measurements tabbed window displays measurements made on the molecule in the graphics window. Contextual help menus are provided for most options within the application, and when the user points to a particular option, a brief explanation is displayed. Help is also available from the top menu. PDB files can be loaded directly into the graphics window in three ways: 1) by using the Open URL command (not shown) in the File menu and connecting to the Protein Data Bank using an explicitly stated web address, or URL, for a particular PDB file; 2) by opening a PDB file located on a user's own computer using either the top File menu or the file icon on the bottom menu; and 3) by clicking on a hypertext link (pointing to a PDB file stored on the same server as the tutorial) within the tutorial section of the browser window.

The application is designed so that the student can read, view, manipulate, and interact with the tutorial on molecular structure (in this case protein structure) in one single learning environment. The text on the web page is complemented by the three-dimensional visual information of a molecule in the graphics page. The graphic display can also be manipulated by appropriate commands in the command line interface. This allows an integrated learning environment and work flow with no need to open separate applications, configure browsers, or open additional windows. The web page can also contain static pictures and animations to further illustrate particular aspects of molecular structure. Hyperlinks within the web browser section can be used to immediately load a molecule into the graphics window, whereas simultaneously loading the text into the pdb file Summary and Structure File window. The web browser integration presently provided within the application is rudimentary.

The molecule in the graphics window can be manipulated from the top menu, the contextual menus within the window, or the command line interface. The command line interface is an especially important part of the application. Students can manipulate the molecule in the graphics window by typing appropriate commands (e.g. color cpk) independently of, and without losing any reference to, the information on the web page window.

The menu provides drop-down options for basic input and output as well as molecule representations such as ribbon, cartoon, and “ball and stick.” This is also reflected by the graphic icons in the menu window.

Qualitative Analysis of Student Responses—

The students were asked to evaluate jAMVLE and compare it with an older, web page only, version of a protein structure tutorial. Students made 305 individual comments encompassing the design and tutorial content of both jAMVLE (251 comments) and the older, web page only, tutorial (54 comments). This included 86 negative comments about jAMVLE and 44 negative comments about the older, web page only, tutorial. Most of the negative comments about jAMVLE included suggestions for improving the design of the program (outlined later) and about “missing links” (hypertext links that did not connect with the appropriate page) within the tutorial content. Most of the comments about the older web page tutorial were about the difficulty in setting up different programs (browser and molecular visualization programs), missing links, the tedious nature of the tutorial, window clutter, difficult navigation, and the additional work involved in using the tutorial (such as using different programs and downloading the necessary PDB files from the internet).

Five major themes emerged from the analysis of content in students' evaluations of jAMVLE. These were that jAMVLE had an appealing interface, was interactive, was integrated, was user friendly, and helped learning. Almost all students were positive about jAMVLE, and only two students expressed dissatisfaction, with the following comments. One student said, “The software lacks the power of a sophisticated program such as DeepView, but has similar functionality to the RasMol package, with a few extra features.” Another student said, “I personally find the program to be a waste of time unless otherwise for beginners. At this level, I am familiar with RasMol and DeepView therefore do not see the need for jAMVLE.”

Most students perceived that jAMVLE had an appealing interface. That is, it looked good (had an appropriate combination of colors and text) and had a simple, clear, and intuitive layout. Indicative comments to support this theme included the following. One student said, “I was amazed with the interface. It is much better than the old tutorial.” Another student said, “Being quite appealing and professional, the way in which the tutorial is displayed provides a great sense of integrity and reliability greatly suited for users wanting to learn or research.” Another comment was, “I like the layout of the program, it is very clear and neat.” Also, another student said, “The interface makes the user feel very comfortable with the visualization. It makes the user feel interested in using the program.”

Students also found jAMVLE to be more interactive than the web page tutorial. The interactivity can principally be attributed to the integrated format of jAMVLE when compared with the older web page tutorial. It is interesting that students perceived jAMVLE to be more informative than the older web page tutorial despite the fact that much of the content was the same in both learning environments. Indicative comments to support this theme included the following. One student said, “… the new tutorial is more informative and more interactive than the old tutorial.” Another student commented, “The interactive features of jAMVLE make it more fun to explore proteins.” Also, another comment was, “In general, the jAMVLE tutorial was more interactive and this enabled me personally to grasp each concept better.”

Students valued the integrated nature of the jAMVLE application. In particular, five subthemes were evident. Students liked the amalgamated aspect of the application because it meant less work, it was easier to use, and it had everything in “one place.” The PDB raw structure file could be viewed in its own tabbed window, and this was identified as a very useful feature of the integrated application. This enabled the student to refer to additional and relevant information when studying a molecule. Students appreciated the fact that they did not need to switch windows between different applications. Essentially, this avoided window clutter, saved time, and helped them focus on the subject. A structure could be immediately displayed in the graphics window after clicking on a hypertext link in the web page notes, and this was considered another significant benefit of the tight integration of the application. Thus, students did not need to spend time downloading the appropriate PDB file, use another application to open the file, or configure a web browser.

Students indicated that the integrated application was intuitive. This observation is potentially biased by the fact that most of these students had prior experience with other molecular visualization programs, but it still indicates that even experienced students thought that the application was logical. Students considered jAMVLE to be a much more user friendly program than the older web page tutorial. Indicative comments to support this theme included the following. One student said, “jAMVLE in my opinion is much user friendly, with the tools and display functions pretty much self-explanatory..” Also, another student commented, “jAMVLE is a very user friendly program. The whole design of the program is very smart.” A program judged user friendly by the students indicates that it makes it easier for the student in some way. The term actually incorporates aspects of all of the previous comments made about the integration, interface, and interactivity of the jAMVLE application. Finally, and more importantly, the survey clearly demonstrated that jAMVLE had helped the students learn the subject and had even made learning fun. Indicative comments to support this theme included the following. One student said, “jAMVLE is both comprehensive, easy to use and provides the user with a lot of relevant information, which I believe, is helpful in learning the subject.” Another student commented, “jAMVLE definitely support active learning. … being more interactive (with the assistance of technology) makes learning and understanding much easier and sometimes less confusing than reading words and referring it to pictures..” Another comment was, “Overall, it is obvious that jAMVLE is an easier to use, more fun and powerful protein structure learning tool.” Also, another student said, “This is more comprehensive & conducive to Learning than the WebCT Tutorial. It's far better than a textbook!” Generally, students found that the application provided a more active learning environment, better guidance, and better focus and that it stimulated their interest.

Students indicated that there were some areas for improvement in the jAMVLE application and willingly made suggestions. In particular, almost all students asked for a “back button” for the web browser section of jAMVLE. We had only implemented a rudimentary web browser in the prototype (used by the students) and did not initially include a back button. Students also wanted more menu commands and buttons, despite the fact that there was an abundance of commands available in the graphical contextual menus. The ability to resize each window within the integrated application, save structure files as displayed in the graphics window, and display multiple structures was also requested. Some students commented that a small tutorial on how to use jAMVLE (in particular, the command line window) would be needed for students new to molecular visualization. We aim to modify the application to implement these and other improvements.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The efficacy of computer-based molecular visualization to teach molecular structure concepts is based on at least three premises: 1) knowledge of chemical structure is essential to understanding the mechanism and function of both simple and complex molecules (the structure-function paradigm); 2) visual aids enhance learning [31, 32]; and 3) three-dimensional visualization of molecules helps students understand structural concepts and mechanisms that would otherwise be difficult with traditional methods [1, 5, 6, 11]. We developed the jAMVLE computer-based application not only with these premises in mind but also to provide the student with a single, user friendly, integrated, interactive, and visual learning environment.

Molecular visualization applications such as RasMol [27], Chime (Table I), and Deep View [17] have been used or integrated with web browsers to create freely available web-based tutorials on molecular structure. Indeed, we have used RasMol and Chime programs for a web-based tutorial on protein structure for second year undergraduate students in biomedical science (www.biomed.curtin.edu.au/biochem). Other excellent examples of web-based tutorials using these programs are the protein structure tutorial by Gale Rhodes using Deep View (www.usm.maine.edu/∼rhodes/SPVTut) and by Eric Martz using Protein Explorer [25] (proteinexplorer.org). However, web-based tutorials can have issues with compatibility (both computer platform and browser), slow internet access speed, window clutter, the need to use more than one application, and the need to configure browsers to accept the programs as either a helper application or a plug-in.

jAMVLE was designed to integrate the web browser, PDB file information, the three-dimensional graphics page, command line interface, and molecular measurements into one Java application. This application was platform-independent and removed the need to use separate applications, configure web browsers, or open additional windows. Students can also be instructed to write and develop their own script files (which can then be loaded at the command line interface) to further encourage creativity. Script files (simple text files containing a series of commands to manipulate the molecule), used in RasMol, are an excellent vehicle for creating specific views and animations of a molecule. Advanced students can also be encouraged to develop their own molecular structure tutorials incorporating web page design and scripting. These features of the jAMVLE application give students the freedom to explore, analyze, and learn molecular structure in a way that is self-paced, self-directed, empowering, and student-centered.

Indeed, students perceived the jAMVLE tutorial to be a much more compelling environment for learning protein structure when compared with an older tutorial that used web pages only. This was despite the fact that much of the content for each tutorial was the same and that molecular visualization software could still be used with the older web page tutorial. Students found the fact that jAMVLE could only display one web page window at a time to be an advantage. This contrasted with the older web page tutorial, which would open a new window every time a hyperlink was selected. This obviously created window clutter, which could further confuse students. Students also seemed to find navigating the older web page tutorial cumbersome. Both of these issues are a common experience with most web-based tutorials and were a major design fault in our own older web page tutorial. However, these do not seem to be sufficient to explain the almost unanimous preference for the jAMVLE learning environment and indicate that interface design may also play a significant role.

Previous studies have shown that interface design in computer-based learning is crucial for improved student understanding and learning [5, 6, 33]. The reasoning is that a student cannot concentrate on the subject if they are simultaneously distracted by a difficult or complex interface [5]. Good interface design effectively decreases visual searching of integrated information and reduces this “split attention” effect [31]. Furthermore, the indicative comments made by our student cohort support the results of Evans et al. [33], who showed that the presentation of biology subject material in an interactive and integrated multimedia tutorial was much better received by students and demonstrated improved learning when compared with the same material presented as web pages.

Additionally, jAMVLE allows the instructor to design a variety of tutorials, assignments, and exercises to explore a wide range of structural concepts that would not normally be covered by traditional textbook or lecture approaches. For example, π non-covalent interactions in biochemistry [1, 34] can be investigated within the context of a didactic or exploratory tutorial. New tutorials can easily be developed in hypertext markup language (html) by using programs such as Dreamweaver™. jAMVLE can also be used to explore aspects of molecular structure in many other disciplines including pharmacy, molecular biology, and chemistry.

The efficacy of computer-based molecular visualization in teaching molecular structure has been questioned [35], and others have pointed to the lack of any definitive experimental data on the effectiveness of molecular graphics tools to teach three-dimensional concepts of molecular structure [36]. However, a study involving surveys both before and after the use of molecular visualization software (in both lecture and laboratory environments) has shown that the software can improve a student's understanding of protein structure and function [14]. Our study provides further qualitative evidence for the efficacy of molecular visualization in teaching and learning molecular structure concepts in a computer laboratory environment. Indeed, our study shows that, at least from the students' perspective, the integrated molecular visualization learning environment of jAMVLE empowers students to ask questions, seek their own answers, improve their own learning experience of molecular structure, and have fun at the same time.

thumbnail image

Figure FIGURE 1.. The learning environment of jAMVLE, showing the integration of graphics, command line, contextual menus, and web browser windows. The application can be downloaded free from the internet at wabri.org.au/jamvle.

Download figure to PowerPoint

Table Table I. Molecular visualization applications
ApplicationPlatformaTypebReference or web sitec
  • a

    aM = Macintosh, W = Windows, L = Linux, U = Unix. For the sake of brevity and clarity no distinction is made between versions or distributions of the operating system. Thus, Macintosh could refer to either Macintosh operating system 9.x or 10.x. Windows could refer to Windows 98, Windows XP, Windows 2000, or Windows NT. Unix could refer to BSD, Solaris, IRIX, X-Windows, and others. Linux could refer to Red Hat, Debian, Yellow Dog, and others.

  • b

    bSA = standalone, PI = plug in for a web browser, JA = java applet, J3D = Java3D. A standalone application, not labeled with Java or Java3D, could be compiled from C, C++, Python, or other programming language. A standalone application could also be configured as a web browser helper application. Eric Martz and Trevor Kramer also maintain a useful web page listing molecular visualization applications at: molvisindex.org.

  • c

    cA web site URL, reference, or both are given wherever possible.

jAMVLEM,W,L,UJSAwabri.org.au/jamvle
BiodesignerWSAwww.pirx.com/biodesigner/index.shtml
ChimeM,WPImolviz.org
ChimeraW,L,USA[15] www.cgl.ucsf.edu/chimera/
Cn3DM,W,L,USA[16] www.biosino.org/mirror/www.ncbi.nlm.nih.gov/Structure/cn3d
Deep ViewM,W,L,USA[17] www.expasy.org/spdbv/
DinoM,L,USAcobra.mih.unibas.ch/dino/intro.php
FPVW,L,UJ3DSA[18] www.cs.ucsb.edu/∼tcan/fpv/
iMolMSAwww.pirx.com/iMol/
JavaMAGEM,W,L,UJAkinemage.biochem.duke.edu/software/javamage.php
JmolM,W,L,UJSA,JAjmol.sourceforge.net
JMVM,W,L,UJ3DSAwww.ks.uiuc.edu/Research/jmv/
KiNGM,W,LJSAkinemage.biochem.duke.edu/software/king.php
MAGEM,W,L,USA[19] kinemage.biochem.duke.edu/software/mage.php
MICEW,L,UJ3DSA[20] mice.sdsc.edu/
MOLMOLW,USA[21] hugin.ethz.ch/wuthrich/software/molmol/index.html
MOLVIEM,W,L,UJA[22] www.cs.ucsb.edu/∼mli/Bioinf/software/
MolView MolViewXMSA[23, 24] www.danforthcenter.org/smith/MolView/molview.html
Protein ExplorerM,W,L,UPI[25] proteinexplorer.org
PyMolM,W,L,USApymol.sourceforge.net/
QmolW,USA[26] www.mbg.cornell.edu/ShallowayLabQMOL.cfm
QuickPDBM,W,L,UJAcl.sdsc.edu/QuickPDB.html
RasMolM,W,L,USA[27] openrasmol.org
VMDL,USA[28] www.ks.uiuc.edu/Research/vmd/
WebMolM,W,L,UJA[29] www.cmpharm.ucsf.edu/∼walther/webmol.html
XmolUSAwww.msc.edu/msc/docs/xmol/XMol.html
YasaraViewW,USAwww.yasara.org/yasaradl.htm

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We thank Associate Professor Sue Fyfe and Professor Alma Whitely for discussions and information on the qualitative grounded research approach.

  • 1

    The abbreviations used are: CBL, computer-based learning; jAMVLE, jmol Amalgamated Molecular Visualization Learning Environment; PDB, Protein Data Bank.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES