From CellBiology

Lab Attendance

  1. Lab 1: --Z3289738 15:29, 8 March 2012 (EST)
  2. Lab 2: --Z3289738 14:10, 15 March 2012 (EST)
  3. Lab 3: --Z3289738 14:04, 22 March 2012 (EST)
  4. Lab 4: --Z3289738 14:51, 29 March 2012 (EST)
  5. Lab 5: --Z3289738 13:58, 5 April 2012 (EST)
  6. Lab 6: --Z3289738 14:24, 19 April 2012 (EST)
  7. Lab 7 - excursion
  8. Lab 9: --Z3289738 14:32, 3 May 2012 (EST)
  9. Lab 10: --Z3289738 15:20, 10 May 2012 (EST)
  10. Lab 11: --Z3289738 14:05, 24 May 2012 (EST)

Lab 1 - Introduction

  • 'External link' has one [ ]
  • 'Internal link' has two [[ ]]

External Link


QBI - Queensland Brain Institute

Internal Link

Lab 1

Lab 2 - Microscopy

Uploading a picture onto the wiki:

  1. Save picture to desktop
  2. 'Toolbox' - 'Upload file'
  3. Copy and paste the title into the wiki, put [[__]]

Dear Mark, I realized that although my original image was very pretty, it may be subject to copyright so I've updated my picture to this one.


File:Table 1. Human Diseases Associated with Mutations of the Wnt Signaling Components.png
Table 1. Human Diseases Associated with Mutations of the Wnt Signaling Components [1]

Homework Lab 2

Identify a reference article that uses the "superresolution" microscopy technique.

Brown TA, Tkachuk AN, Shtengel G, Kopek BG, Bogenhagen DF, Hess HF, Clayton DA. (2011). Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction. Molecular Cell Biology. PMID: 22006021

What did the paper show that normal microscopy could not show?

Superresolution fluorescence microscopy exceeded previous imaging techniques by allowing scientists to see within the small and highly compartmentalised mitochondria. Using PAML and iPALM techniques, scientists were able to visualize core dimensions and relative locations of mitochondrial nucleoids, discovering that they are much larger than previously anticipated.

Lab 3 - Fixation

* Tissue preservation for slides

Homework Lab 3

Locate a current SDS for one of the fixatives described in today's lab. Identify the properties and hazards associated with that chemical.

Fixative: Chloroform

The SDS for Chloroform can be found at the following website:

Properties and hazards associated with Chloroform


  • Colourless mobile liquid
  • Chloroform odour
  • Volatile
  • Miscible with water and ethanol


  • Highly flammable
  • Harmful if swallowed
  • Harmful: danger of serious damage to health by prolonged exposure through inhalation and if swallowed.
  • Limited evidence of a carcinogenic effect.
  • Carcinogen Category 3

Identify 4 papers required for your group work project. Cite on the Group Project discussion page and also on your own Individual page. Add one sentence for each as too why they are relevant to your group topic.

Paper 1

Taketo, M Mark. "Shutting down Wnt signal-activated cancer." Nature Genetics 36. (2004): 320-22. DOI: 10.1038/ng0404-320

In this article, New evidence suggests that Wnt signaling can be suppressed or further activated by upstream signals, even though the pathway seems to be constitutively activated by downstream mutations in cancer cells.

Paper 2

Macdonald, Bryan. "Wnt/β-catenin Signaling: Components, Mechanisms, and Diseases." Developmental Cell 17.1 (2009): 9-26. PMID: 19619488

This article highlights some key aspects of Wnt/β-catenin signaling in human diseases including congenital malformations, cancer, and osteoporosis, and discuss potential therapeutic implications

Paper 3

Luu, Hue. "Wnt/β-catenin Signaling Pathway as Novel Cancer Drug Targets." Current Cancer Drug Targets 4. (2004): 653-71. Doi: 1568-0096/04

This review discusses some of the strategies that are being used or can be explored to target key components of the Wnt/β-catenin signaling pathway in rational cancer drug discover.

Paper 4

Giles, R., Johan, H., Clevers, H. “Cought up in a Wnt storm: Wnt signalling in cancer”. Biochimica et Biophysica Acta, (2003). 1653 1-24.

This review considers the spectra of tumors arising from active Wnt signaling and attempts to place perspective on recent data that begin to elucidate the mechanisms prompting uncontrolled cell growth following induction of Wnt signaling.

Lab 4 - Immunochemistry

Class Activity

A researcher has a project for a student on a newly discovered protein called by the discoverer "Musashi".


Musashi-1 and Musashi-2 are RNA-binding proteins which play a role in asymmetric cell division of ectodermal precursor cells by regulating the translation of target mRNA. [2] Musashi contributes to the maintenance of neural stem cells. While Musashi-1 is frequently used as a marker for proliferating neural precursor cells, it is also expressed in epithelial stem cells including intestinal and mammary gland stem cells. Database: NCBI - Musashi

Musashi antibody #2541

  • Database: Cell Signaling Technology - Musashi Antibody
  • Source: rabbit
  • Applications: Western Blot, Immunoflourescence (IF-F) Endogenous
  • Species cross reactivity: human and mouse (determined by western blot)
  • Molecular weight: 30kDa
  • Polyclonal
  • Anti-rabbit secondary antibodies must be used to detect this antibody
  • Recommended Antibody Dilution: Wesetern blotting 1:1000; Immunofluorescence (IF-F) 1:25.
  • Specificity/Sensitivity: Musashi Antibody detects endogenous levels of total Musashi 1 and 2 protein.

Anti-rabit secondary antibody:

  • Database: Cell Signaling Technology - Alexa Fluor® 488 Conjugate
  • Alexa Fluor 488 goat anti—rabbit IgG.
  • Supplied by Life Technology
  • Size 0.5mL for $440.
  • It is a green flourescence.
  • Formulated at 2 mg/ml.
  • Specificity/Sensitivity: F(ab’)2 fragments are prepared from goat antibodies that have been adsorbed against pooled human serum, mouse serum, plasmacytoma/hybridoma proteins and purified human paraproteins.


  1. <pubmed>19619488</pubmed>
  2. <pubmed>16300654</pubmed>

Lab 5 - Cell Knockout Methods

See: 2011 Lab 5

The actin cytoskeleton together with actin binding proteins, such as trypomyosin, is an important regulator of cell function. Transgenic and knockout mice play a key role in studying the mechanism by which these structural proteins function.

List the key differences in the generation of transgenic and gene knock-out mice.

  • Transgenic mice are used to study over-expression of a gene product, and mainly focus on dominant alleles. Mice are generated by DNA microinjection of fertilized oocytes, which are then transfered to a pseudo-pregnant female. This results in random integration of the DNA, and the gene product is over-expressed. The advantages inculde a relative high rate of insertion of the injected gene into the genome, however the random insertion can lead to position effects.
  • Knock-out mice are used to look at recessive disorders by deleting the existing gene. Knockout mice are generated by the injection into a blastocyst of genetically modified ES cells. The blastocytes are transfered to a pseudo-pregnant female, producing Chimeric mice with the gene product missing or mutated. The advantages include specific insertion of a gene at specific location or removal of specific genes (KO) as well as the ability to mimic recessive disorders (loss of function mutations). The disadvantages are the low level of ES cells with wanted gene inserted, and that further breeding is necessary to obtain non-chimeric homozygous animal.

Describe how genetically modified mice have been used to study the biological function of tropomysin. In your answer include:

i. The type of cell used

Fat cell (adipocyte)

ii. The measurements used to determine whether any phenotypic changes were observed

  1. Organ weights (fat, brain, kidneys)
  2. Adipocyte proliferation
  3. Glucose uptake & insulin secretion was observed to have increased in the transgenic mice, which was measured by using a glucose intolerance test.

iii. A model to explain how actin changes in the levels of tropomysin can lead to the observed phenotypic changes.

Tm5NM1 increases filamentous actin in adipose tissue (evidence: F-actin staining). The over-expression of Tm in transgenic mice results in increased levels of actin filaments. This increase results in more GLUT4 transporters reaching the membrane surface, resulting in an increase in glucose uptake.

Lab 6 - Cytoskeleton Exercise

Z3289738 lab 6 table.JPG


1) Do you see a difference in phenotype (morphology) between Tm4 overexpressing and control cells?

  • Fan: Slightly more cell numbers in the TM4 group.
  • Broken Fan: The number of cells in the TM4 group is reduced by about 15%, indicating that TM4 may inhibit this cell phenotype.
  • Stumped: There is a slight increase in TM4 group of about 6% which may not indicate the role that TM4 played in this particular phenotype.
  • Pronged: The TM4 group expressed an increase about 6%, which may indicate that TM4 is actively involved in the events of neurites growth for this phenotypes.
  • Stringed: There is a slight decrease in TM4 group of about 2%. This may not indicate the role that TM4 played in this particular phenotype.
  • Pygnotic: Very small percentage of cells found for the TM4 group. This may indicate that TM4 entirely inhibits this phenotype.

2) If so, how could Tm4 over-expression lead to this difference?

TM4 is a striated muscle isomer of Tropomyosin, which is involved in the motile events of neurite growth and synaptic plasticity. This can be observed in the 'Over expression of Tm4' group, whereby the neurites involved have more processes branching than in the control group. These processes are necessary for cell-cell interaction and communication.

Lab 7 - Confocal Microscopy

Contributions to Group 5 Project

Hey guys, If we decide to go with notch signaling here's a really great diagram & description:

--Z3289738 14:28, 22 March 2012 (EST)

Hey guys, I found four articles relating to abnormal function of the pathway:

Paper 1

Taketo, M Mark. "Shutting down Wnt signal-activated cancer." Nature Genetics 36. (2004): 320-22. DOI: 10.1038/ng0404-320 <pubmed>15054482</pubmed> In this article, New evidence suggests that Wnt signaling can be suppressed or further activated by upstream signals, even though the pathway seems to be constitutively activated by downstream mutations in cancer cells.

Paper 2

<pubmed>19619488</pubmed> This article highlights some key aspects of Wnt/β-catenin signaling in human diseases including congenital malformations, cancer, and osteoporosis, and discuss potential therapeutic implications

Paper 3

<pubmed>15578921</pubmed> This review discusses some of the strategies that are being used or can be explored to target key components of the Wnt/β-catenin signaling pathway in rational cancer drug discover.

Paper 4

<pubmed>12781368</pubmed> This review considers the spectra of tumors arising from active Wnt signaling and attempts to place perspective on recent data that begin to elucidate the mechanisms prompting uncontrolled cell growth following induction of Wnt signaling.

--Z3289738 10:48, 29 March 2012 (EST)

Notes: Diseases associated with Wnt/β-catenin signalling

File:Table 1. Human Diseases Associated with Mutations of the Wnt Signaling Components.png
Table 1. Human Diseases Associated with Mutations of the Wnt Signaling Components [1]
File:Figure. Schematic representation of a colon crypt and proposed model for polyp formation.png
Figure 3. Schematic_representation_of_a_colon_crypt_and_proposed_model_for_polyp_formation [2]
File:The Wnt Signaling cascade, simplified.png
Figure._Schematic_representation_of_a_colon_crypt_and_proposed_model_for_polyp_formation [2]

Wnt/β-Catenin Signaling: Components, Mechanisms, and Diseases [1]

  • Signaling by the Wnt family of secreted glycolipoproteins is one of the fundamental mechanisms that direct cell proliferation, cell polarity, and cell fate determination during embryonic development and tissue homeostasis (Logan and Nusse, 2004). As a result, mutations in the Wnt pathway are often linked to human birth defects, cancer, and other diseases (Clevers, 2006). A critical and heavily studied Wnt pathway is the canonical Wnt pathway, which functions by regulating the amount of the transcriptional coactivator β-catenin, which controls key developmental gene expression programs.
  • Given the critical roles of Wnt/b-catenin signaling in development and homeostasis, it is no surprise that mutations of the Wnt pathway components are associated with many hereditary disorders, cancer, and other diseases (Table 1).
  • Association of deregulated Wnt/β-catenin signaling with cancer has been well documented, particularly with colorectal cancer (Polakis, 2007) (Table 1). Constitutively activated β-catenin signaling, due to APC deficiency or β-catenin mutations that prevent its degradation, leads to excessive stem cell renewal/proliferation that predisposes cells to tumorigenesis.
  • Mutations of β-catenin at and surrounding these serine and threonine residues are frequently found in cancers, generating mutant β-catenin that escapes phosphorylation and degradation (Table 1).

Caught up in a Wnt storm: Wnt signaling in cancer [2]

  • The Wnt signaling pathway, named for its most upstream ligands, the Wnts, is involved in various differentiation events during embryonic development and leads to tumor formation when aberrantly activated. Molecular studies have pinpointed activating mutations of the Wnt signaling pathway as the cause of approximately 90% of colorectal cancer (CRC), and somewhat less frequently in cancers at other sites, such as hepatocellular carcinoma (HCC).
  • Greater than 90% of all CRCs will have an activating mutation of the canonical Wnt signaling pathway, ultimately leading to the stabilization and accumulation of β-catenin in the nucleus of a cell.
  • Fig. Schematic representation of a colon crypt and proposed model for polyp formation. At the bottom third of the crypt, the progenitor proliferating cells accumulate nuclear β-catenin. Consequently, they express β-catenin/TCF target genes. An uncharacterized source of WNT factors likely resides in the mesenchymal cells surrounding the bottom of the crypt, depicted in red. As the cells reach the mid-crypt region, β-catenin/TCF activity is downregulated and this results in cell cycle arrest and differentiation. Cells undergoing mutation in APC or β-catenin become independent of the physiological signals controlling β-catenin/TCF activity. As a consequence, they continue to behave as crypt progenitor cells in the surface epithelium giving rise to aberrant crypt foci.

--Mark Hill 15:17, 3 June 2012 (EST) The images you have uploaded to this section are without copyright permission.

Lab 9 - Tissue Culture 1

Individual Assessment

  1. Identify a mammalian cell line in the ATCC catalogue (and add a link)
  2. Identify the original tissue of origin of that cell line.
  3. Identify the original paper that characterised the properties of that cell line. 
  1. Mouse cell line: ATCC: Mus musculus
  2. Tissue: subcutaneous connective tissue; areolar and adipose
  3. Earle WR, et al. Production of malignancy in vitro. IV. The mouse fibroblast cultures and changes seen in the living cells. J. Natl. Cancer Inst. 4: 165-212, 1943.

Lab 10 - Tissue Culture 2

3 major types of contamination in tissue cultures:

- bacteria. appearance: black dots. visible under phase microscope. Precipitation will be present at the bottom of the dish.
- fungal. filamentous expansion through media (strings of fungus) or yeast looks like circular structures. Precipitation will be present at the bottom of the dish.
- Microplasma = micro bacteria. Infect cells (live inside the cell cytoplasm). Not easy to identify because they are smaller than bacteria & grow inside the cell as opposed to the media. How to avoid? Regularly screen and check for microplasms in the cell line via PCR analysis or DNA screen or external Microplasm testing.

Peer Assessment

  • criteria
  • correct referencing (eg. copyright)
  • interesting?
  • brief paragraph of my assessment
  • paste all the assessments onto your personal page.

Group 1

  • Introduction: Very well explained, covers all relevant content and a good summary of testosterone. Maybe try add an interesting point about testosterone to draw the reader in.
  • History: The history is well researched and instead of just providing a list of discoveries, you outlined the importance of each one. It is clear that a lot of effort went into this section. There is a large gap between 1767 and 1849, as well as from 1994 until the present. What happened during these periods?
  • Biosynthesis: This section is very difficult to follow. A diagram illustrating each point would allow the reader to picture the structures and better understand the process. The image needs an explanation. Other than that the information is well presented as a table, and the key points are in bold which helps identify the the important information.
  • Regulation: The ideas are easy to follow and the information is easily understandable (as opposed to the Biosynthesis section). The only thing to improve here would be to add either spaces or headings between the paragraphs to break up the text.
  • Signalling Pathway: Although there is a lot of text in this section, the layout breaks it up nicely and provides structure to the information. The images are highly relevant and contributes to the readers understanding. This section was very well done!
  • Normal Function: This section is very overwhelming in terms of reading information and lacks structure. I suggest you have a look at the layout of the 'signalling pathway' section and add features such as headings, bullet points and images to make it more appealing to read.
  • Abnormal function: Each point is well researched and clearly explained. I think the addition of statistics involving the prevalence of each disease and the impacts they have could make the information more relevant and interesting for the reader.
  • Clinical uses: A very nice summary of clinical uses of testosterone. The video is very relevant and is a nice feature of this section.
  • Current and ongoing research: This section lacks an introduction and general layout structures such as headings. Links to the research papers would be useful, as well as more citations to show the depth of research involved.
  • Overall impression: Whilst some areas are very well done, there are a few sections which are letting you down. Mostly it is just to do with the layout of the information to make the page more appealing to read and easier to absorb the flow of information.

Group 2

  • Introduction: A good outline of the topic and introduction to the page. What is VEGF-A? How does it relate to VEGF?
  • History: I like how each line builds on the discovery before, which adds to the readers understanding of the steps involved. Were the last two clinical uses successful?
  • Normal function: This section lacks structure, images and references. The functions are described well, however it would be easier to understand the flow of event with the use of images and flow charts.
  • Signalling Pathway: The table provides a good summary of the receptors functions. I think you should summarize the written information below it and include it in the table as a new column. That being said it is not clear what the actual 'pathway' for VEGF is. Perhaps a flowchart or diagram would be necessary to clear this up. The image needs a caption.
  • Abnormal function: This section was very well done. The information is very relevant and links strongly to VEGF, and the pictures nicely illustrate the information and is correctly cited. It is clear a lot of effort went into this section.
  • Research: Therapeutic Applications This section showed an extensive amount of research. I particularly like the use of external links to related studies, and the pictures and table that breaks up the information.
  • Overall impression:The information is presented extremely well and the page is very appealing to read. The use of colours and images instantly drew my attention and the structure aided the flow of information. The information is well researched and covers all relevant content. Very well done.

Group 3

  • Introduction: The introduction is well written and easy to understand. Points to improve on: The details on which pathways will be focused on could be better presented in bullet points; I think that the second paragraph should be put into a 'compare/contrast' table in a separate section; the third paragraph very nicely outlines the main focus of your page "Signalling molecules, receptors, enzymes, inhibitors, genes and pathway", however where are these headings in the text??
  • History: This section highlights the importance of each discovery and attributes the discoveries to the researchers. Links to the research papers would be good for further reading.
  • Signalling Pathway: This section needs a diagram! The 'Fas-Mediated Apoptosis' would be much clearer with a flowchart to refer to. More details should be put into the proteins involved, such as their functions and relations to the pathway.
  • Function: This section is well researched, however lacks any structure. Headings, proper bullet points and pictures/diagrams/tables would draw the readers attention and highlight the key points in the text.
  • Abnormal Function? This section is very important as it highlights the importance of the pathway!
  • Current Research: You provided links to the journals and described the first and last paper very well. The importance of the research is clearly described. More detail is needed for the second research area.
  • Overall Impression: The current content is fairly well written however it lacks a depth of research and there are many sections that need to be expanded on. Pictures are essential to illustrate points, and long paragraphs could be better structured with the use of tables and bullet point. References are essential!

Group 4

  • Introduction: Easy to understand and a good outline for the rest of the page.
  • History: The table is well designed. The information is simple and well explained which shows a good understanding of the content (as opposed to paraphrasing complicated text). It is important to include the full name of the researchers (eg. Who is 'Lewis'?) and where possible links to the research articles. Surely there have been further discoveries since 1995.
  • Pathway: This section is well explained and I liked the summary paragraph. A flowchart would be ideal here; this would be a good place for the hand-drawn diagram.
  • Proteins and Receptors: I like the simple layout of the text, however more information is required. Perhaps set up a table to compare the structure and functions of each component, and highlight their importance to the pathway. The picture is really good, however I feel that it is more relevant for the 'Pathway' section. References are needed!
  • Normal Function: I don't feel that the specific role of Notch in CNS & cardiac development was well described. You have a really great flowchart illustrating the role in CNS development, so perhaps you should explain what is happening in that picture. This was done better in the role of Notch in kidney and pancreatic development, where you went into detail of what the Notch pathway is responsible for and the background information on how these discoveries were made.
  • Overall Impression: I really enjoyed reading the information on this page. The explanations are clearly written in your own words and are easy to follow and understand. The information is well formatted into subheadings and tables which makes it easy to navigate around key points. That being said, there is a lot of information missing and most sections need to be expanded on (however this has already been pointed out by many others).

Group 6

  • Introduction:The information is well explained, and the paragraphs are short and straight to the point. A picture in this section would make the page more eye-catching. Lacks referencing.
  • Structure: Good choice of content included. Perhaps add in a diagram of an isomer for comparison. The picture needs a caption.
  • History: Well researched, however next to each discovery should be the name of the researcher and where possible their research article.
  • Insulin Receptor: Well explained section and a good choice of diagram to illustrate the information.
  • Pathway: The information is well written, however it could be better interpreted with a listed format. Great diagram to illustrate the written information.
  • Normal Function: This section is well written and clearly structured. It lacks references.
  • Abnormal Function: The information is very clear, however I feel that it could be better interpreted in a table format. Pictures could make this section more interesting as well.
  • Current Research: Very well written. It would be nice to have links to the research articles for further readings.
  • Overall impression: I enjoyed reading through your page. For the most part the information is clear, interesting and thoroughly researched. Pictures and coloured tables would make the page more appealing and eye catching.

Group 7

The content in this page is well researched and demonstrates a lot of initiative with the hand-drawn images. The tables are bright and easy to read, and the images are well selected and are strongly linked to the written content. The amount of content shows the extend of research and it is clear that a lot of effort has been put into it.

Area of improvement: the structure and organisation of content makes the information overwhelming and exhausting to read. The long paragraphs could be summarized by the use of tables, diagrams (a picture says a thousand words!), bolded text to highlight key points and more subheadings to break up the paragraphs.


  • Introduction: Needs a sentence at the beginning to explain how the 3 receptors are related to each other.
  • History: Well researched and I like how you have included links to the research articles.
  • The Gene Description and Receptor Agonist sections could be merged into one table.

Group 8

  • Intro: Well written and easy to understand. A picture would make this section more eye-catching. Needs references!
  • Pathway: The use of numbering and bolded text makes the information very appealing and easy to read. The diagram is also clear and easy to understand. Try to connect the text and diagram by matching the numbers with the information (ie. 'point 1' in the text correlates to 'point 1' in the diagram). A video would also be great for this section, I remember seeing one in a biology lecture once and it was very interesting! This section needs references.
  • Normal Function: Excellent introductory paragraph, shows a good understanding of the topic area. You need to edit the spacing within the paragraphs, for example you have numbers listed within the paragraph which could be better read as a list going down the page. Needs references.
  • Abnormal Function: All the information is relevant, however could be better represented in a table format.
  • Proteins: The paragraphs are too long. They need to be broken up by subheadings, tables and lists to highlight key information. The first image is excellent, the only thing missing is a caption.
  • Overall impression: The choice of content shows a good understanding of the topic area. For the most part the information is very well written and easy to understand, however the use of lists, tables and diagrams could make the page easier for the reader to navigate around key information. More images would make the page more eye-catching and appealing to read.

Group 9


  • The first paragraph is very clear and well explained.
  • The second and third paragraph seem to be irrelevant for the introduction, however would be useful in other sections such as 'Abnormal Function' for the third paragraph.
  • A picture in the introduction would make the page more eye-catching


  • The information is well written, however there is too much writing in this section. The information would be clearer and easier to read with the use of subheadings and perhaps bullet points to highlight key information.
  • Relating the information to the flowchart would also make the information clearer.
  • The last paragraph provides a clear summary of the pathway.


  • The table format makes the information clear and easy to read.
  • The short sentences are clear and straight to the point. This highlights the key points and makes the information easier to read.
  • You need to go through and edit the grammar, especially for the use of sentence case.
  • Where possible, include the names of the researchers and/or project groups that made each discovery or breakthrough.

Current Research

  • I like that you added a link to the article
  • Rather than writing "a group of scientists", include the names of the researchers and the name of their research article

Normal Function

  • This section has the best use of subheadings, which immediately makes the information clearer and easier to navigate.
  • To make the information even clearer, try relate it to an image of a pathway
  • I think this section should be more towards the top of the page.

Abnormal Function

  • Really good diagram
  • I can see this section is incomplete, however some ideas would be to add information about the effects of mutations and associated diseases.

Overall Impression: Although the page seems largely incomplete, the completed sections are well written and could be improved with the use of subheadings and images to illustrate information.

--Mark Hill 13:02, 17 May 2012 (EST) Well done, this is a detailed critical analysis of each project including comments for each sub-section. You could have also applied the project assessment criteria, I like the "overall impression" section for each.

  1. 1.0 1.1 <pubmed>19619488</pubmed>
  2. 2.0 2.1 2.2 <pubmed>12781368</pubmed>