New Publication: Clinical Overview of MDM2/X-Targeted Therapies

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Great news, we have a new Mini-Review published in Frontiers Oncology entitled “Clinical Overview of MDM2/X-Targeted Therapies“, which is apart of the Research Topic Human tumor-derived p53 mutants: a growing family of oncoproteins

Here is a little snippet from the Abstract to wet your appetite!

MDM2 and MDMX are the primary negative regulators of p53, which under normal conditions maintain low intracellular levels of p53 by targeting it to the proteasome for rapid degradation and inhibiting its transcriptional activity. Both MDM2 and MDMX function as powerful oncogenes and are commonly over-expressed in some cancers, including sarcoma (~20%) and breast cancer (~15%).

In this overview, we will review the current MDM2- and MDMX-targeted therapies in development, focusing particularly on compounds that have entered into early phase clinical trials. We will highlight the challenges pertaining to predictive biomarkers for and toxicities associated with these compounds, as well as identify potential combinatorial strategies to enhance its anti-cancer efficacy.

The article is Open Access, which means its free for everyone and anyone to read and download!

You can view and download it directly here [Link]

The Early-Mid Career Funding Crisis in Australian Research

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Its no secrete that Funding for Science in Australia, and around the world (see refs below), is in decline. The result is lower and lower success rates. While we wait for the #NHMRC to release the outcomes for 2015, the word on the street is that we can expect only 10-12% of grants to be successful. In other words, for 90% of Australia’s researchers they are wasting ~3 months of the year for nothing. Consequently as the funding pool decreases, funds naturally flow towards ‘sure bet’ senior researchers. This means that this wasted time is felt the hardest by early- and mid- career researchers (#EMCRs) who cannot compete with the long CVs of their senior peers, and are seen as a potential risky investment. Below is a graph I put together from the 2013 data, which is the most up to date information currently at hand. This trend of funding more senior researchers is clearly seen in the massive increase in average age of the CIA (chief investigator) on project grants over the past 30 years. It used to peak around 40 years, which perfectly aligned with the drop off in fellowships. So there was a very clear and clean transition from Post-Doctoral Funding for those that wanted to transition to a team leader role. However now, the average age has shifted to >50 years. This has created a significant 10-15 year ‘Funding Hole’ for EMCRs, where there are very limited number of fellowships on offer, and little to no chance of securing a project grant. While there has been a lot of talk about this black box, no solution or action has been taken to stem the loss of young, bright and talented researchers being forced out of research. Without action soon, we run the real risk that there will be no succession plan, and Australia’s ability to remain internationally competitive will be set back decades.

 

2013 NHMRC Data

 

Funding issues in the US system:
1. Alberts, B., Kirschner, M. W., Tilghman, S. & Varmus, H. Rescuing US biomedical research from its systemic flaws. PNAS 111, 5773–5777 (2014).
2. Cyranoski, D., Gilbert, N., Ledford, H., Nayar, A. & Yahia, M. Education: The PhD factory. Nature 472, 276–279 (2011).
3. Committee to Review the State of Postdoctoral Experience in Scientists and Engineers et al. The Postdoctoral Experience Revisited. (National Academies Press (US), 2014).
4. Powell, K. The future of the postdoc. Nature 520, 144–147 (2015).
5. Alberts, B., Kirschner, M. W., Tilghman, S. & Varmus, H. Opinion: Addressing systemic problems in the biomedical research enterprise. Proc. Natl. Acad. Sci. U.S.A. 112, 1912–1913 (2015).

 

AntiOxidants and Cancer: A complicated story

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A recent high profile publication in Science Translational Medicine proposed that antioxidants might increase the rate of metastasis in mice models of melanoma.

NAC and the soluble vitamin E analog Trolox markedly increased the migration and invasive properties of human malignant melanoma cells but did not affect their proliferation. Both antioxidants increased the ratio between reduced and oxidized glutathione in melanoma cells and in lymph node metastases, and the increased migration depended on new glutathione synthesis. Furthermore, both NAC and Trolox increased the activation of the small guanosine triphosphatase (GTPase) RHOA, and blocking downstream RHOA signaling abolished antioxidant-induced migration. These results demonstrate that antioxidants and the  system play a previously unappreciated role in malignant melanoma progression.

This goes against the common idea that anti-oxidants are cancer fighters!

So what is going on?

The answer, much like a Facebook relationship status, is “its complicated”. In fact anti-oxidants can have a wide range of effects on cells, including mitosis. Many “dietary antioxidants Resveratrol and Fisetin (found in red wine), inhibit Cdks, induce a G2 arrest and prevent entry into mitosis” (Burgess et al 2014). Furthermore, we recently showed that partial inhibition of Cdk1 can dramatically disrupt to mitosis. This caused increase cancer cell death… but also increased the rate of chromosome mis-segergations. (McCloy et al 2014). These mitotic errors can drive chromosome instability (CIN), which inturn can lead to the evolution of more aggressive, invasive tumours. Understanding the genetic background of each individual cancer will be key to determining why some cancer cells die and others thrive when given antioxidants.

If you would like to know more on how common stresses such as oxidation can disrupt mitosis, you can read our recent review Stressing Mitosis to Death.

Until then, if you have cancer and are thinking of taking antioxidants, make sure you consult your oncologist as they can significantly affect the efficacy of some chemotherapeutics, and hence maybe doing more harm than good.

 

 

 

New Review Article Published!! “Mechanisms Regulating Phosphatase Specificity During Mitotic Exit”

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Great News, we have a new review article that has just been published online today in Inside the Cell!
Its Open Access, so that means its free for everyone to read!

During mitotic exit, phosphatases reverse thousands of phosphorylation events in a specific temporal order to ensure that cell division occurs correctly. This review explores how the physicochemical properties of the phosphosite and surrounding amino acids affect interactions with phosphatase/s and help determine the dephosphorylation of individual phosphorylation sites during mitotic exit.

The Full Reference and link for the Article can be found below:
Samuel Rogers, Rachael McCloy, D Neil Watkins and Andrew Burgess Mechanisms regulating phosphatase specificity and the removal of individual phosphorylation sites during mitotic exit Inside the Cell [Link]

Position available: 2016 Honours Student Project in our Lab

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Great news we are currently looking for a new honours student for 2016.

The title of the project is “Developing novel biosensors to monitor DNA damage in cancer cells”.

Its a very exciting new project incorporating cutting edge microscopy and fluorescent biosensors.

If you think you have what it takes and are interested please feel free contact myself, or UNSW SoMS.
For more information on the UNSW honours program please visit: http://medicalsciences.med.unsw.edu.au/students/soms-honours/

Below is an example of the images that will be created during the project.

New Paper Published! More data on the global phosphorylation changes during early mitotic exit

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Great news, we have another publication. This time its some extra data left over from our large mass spectrometry study we published in August in Molecular & Cellular Proteomics.

This latest work we provide additional analysis of our large proteomics dataset and identify motifs that correlated strongly with phosphorylation status for each of the major mitotic kinases.

These motifs could be used to predict the stability of phosphorylated residues in proteins of interest, and help infer potential functional roles for uncharacterized phosphorylations.

If you would like more information you can check out the full paper here [Link]. And the great news is that its OpenAccess and FREE for everyone!

Rogers, S., McCloy, R. A., Parker, B. L., Chaudhuri, R., Gayevskiy, V., Hoffman, N. J., Watkins, D. N., Daly, R. J., James, D. E., and Burgess, A. (2015) Dataset from the global phosphoproteomic mapping of early mitotic exit in human cells. Data in Brief 5, 45–52

 

 

 

Using Thresholds to Measure and Quantify Cells in Image J

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I often get asked how to uses Thresholds to measure things in Image J.

There are some great guides on the web explaining how to use Thresholds in Image J, and here are a few that are well worth checking out [Link1][Link2].

Below are some of the Basic Steps for using Thresholds:

  1. Open your image and duplicate it (Image>Duplicate)
  2. On the duplicate go to Image>Adjust>Threshold
  3. Play with the sliders until all of your cells are red.
  4. Click ‘Apply’
  5. You should now have a ‘binary’ black and white image
  6. Now go to menu Process>Binary and select ‘fill holes’
  7. You may also want to select erode, dilate, open or close to optimise the binary image so that you have nice solid filling of your cells.
  8. Now go to menu Analyse>Set Measurements. Select all the things you want to measure.
  9. Critical steps: make sure that you select your original image (not the binary) in the ‘Redirect to:’ pull down Menu
  10. Also make sure the ‘Limit to threshold’ checkbox is ticked and also tick the ‘Add to overlay’ and ‘Display label’.
  11. Click ok to close the ‘Set Measurements’ box.
  12. Now go to Analyse>Analyse Particles
  13. Here you will need to play around with the size and circularity settings (bit of trial and error) in order to get accurate identification of your cells or ROIs. I suggest making duplicates before you start so that you can quickly try different things to see which one works best.
  14. Make sure you have the Display results tick box selected.
  15. Once you click ok you should have a the measurements box appear with all your measurements for each cell.
  16. You can copy and paste these into Excel or what ever program you like to use.
  17. Go get a coffee and cake you deserve it!

Good luck!

 

Using ImageJ to Measure Cell Fluorescence

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Image J can be downloaded for free from here .
This guide can also be downloaded as a complete PDF here: Measuring Cell Fluorescence using ImageJ

Here is a very simple guide for determining the level of  fluorescence in a given region (e.g nucleus)

  1. Select the cell of interest using any of the drawing/selection tools (i.e. rectangle, circle, polygon or freeform)
  2. From the Analyze menu select “set measurements”. Make sure you have AREA, INTEGRATED DENSITY and MEAN GRAY VALUE selected (the rest can be ignored).
  3. Now select “Measure” from the analyze menu or hit cmd+m (apple). You should now see a popup box with a stack of values for that first cell.
  4. Now go and select a region next to your cell that has no fluroence, this will be your background.
    NB: the size is not important. If you want to be super accurate here take 3+ selections from around the cell.
  5. Repeat this step for the other cells in the field of view that you want to measure.
  6. Once you have finished, select all the data in the Results window, and copy (cmd+c) and paste (cmd+v) into a new excel worksheet (or similar program)
  7. Use this formula to calculate the corrected total cell fluorescence (CTCF).
    NB: You can use excel to perform this calculation for you.
    CTCF = Integrated Density – (Area of selected cell  X Mean fluorescence of background readings)

     
  8. Make a graph and your done. Notice that in this example that the rounded up mitotic cell appears to have a much higher level of staining, but this is actually due to its smaller size, which concentrates the staining in a smaller space. So if you just used the raw integrated density you would have data suggesting that the flattened cell has less staining then the rounded up one, when in reality they have a similar level of fluorescence.

How to Cite this if you wold like to:

We have used this method in these papers:

McCloy, R. A., Rogers, S., Caldon, C. E., Lorca, T., Castro, A., and Burgess, A. (2014) Partial inhibition of Cdk1 in G 2 phase overrides the SAC and decouples mitotic events. Cell Cycle 13, 1400–1412 [Link]

Burgess A, Vigneron S, Brioudes E, Labbé J-C, Lorca T & Castro A (2010) Loss of human Greatwall results in G2 arrest and multiple mitotic defects due to deregulation of the cyclin B-Cdc2/PP2A balance. Proc Natl Acad Sci USA 107: 12564–12569

But you can also find a similar method published here:

Gavet O & Pines J (2010) Progressive activation of CyclinB1-Cdk1 coordinates entry to mitosis. Dev Cell 18: 533-543

And here:

Potapova TA, Sivakumar S, Flynn JN, Li R & Gorbsky GJ (2011) Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited. Mol Biol Cell 22: 1191–1206

And my apologies to any others that I have not mentioned.

Our latest Publication has received a F1000Prime Recommendation !

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Great news our recent paper “Global phosphoproteomic mapping of early mitotic exit in human cells identifies novel substrate dephosphorylation motifs., Molecular & Cellular Proteomics, 2015 (DOI: 10.3410/f.725545508.793507630), has been recommended in F1000Prime as being of special significance in its field by F1000 Faculty Member Angus Nairn.

Really glad to see that all our hard work and research is being found useful for the research community.

You can checkout the recommendation here and our original publication here

Our Latest Publication Accepted and Now Online!

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Great news our latest publication “Global phosphoproteomic mapping of early mitotic exit in human cells identifies novel substrate dephosphorylation motifs” has been accepted by the top Proteomics Journal Molecular & Cellular Proteomics.

You can currently download the unformatted version for free here [link]

And here is an still image from the paper showing live HeLa cells undergoing forced phosphatase dependent mitotic exit. The red colour is Histone H2B tagged with the fluorescent mCherry protein, and the Green is tubulin tagged with GFP (green fluorescent protein).

 

HeLa cells undergoing phosphatase dependent mitotic exit
HeLa cells undergoing phosphatase dependent mitotic exit