Month: September 2014

09.22.14 by and

Share your ExRNA expertise with everyone!

Want to share your knowledge of a particular exRNA gene with the broader scientific community? Want to reach readers through both the “traditional” peer-reviewed literature as well as the sixth-most accessed website in the world? If so, continue reading about our three-way partnership between the journal GENE, the Gene Wiki project at Wikipedia, and the Extracellular RNA Communication Program (ERCP):

Gene_Wiki_logo

  1. What is it? The goal of the Gene Wiki is to create a comprehensive Wikipedia article for every human gene. To incentivize authors to improve Wikipedia content, GENE is now soliciting new gene-specific review articles under a new dual-publication model. ExRNA genes are especially desirable and authors are invited to create two separate versions of their review (one for the journal, and one in wikipedia). More on the partnership here: Gene Wiki Reviews: Marrying crowdsourcing with traditional peer review.
  2. How long should the review article be? The length of the review article is up to you! Since you are the expert on the exRNA gene you’re writing about, the length is based on whatever you think is necessary to describe the current state of the field.
  3. How long should the wikipedia article be? We are targeting a final length of approximately 1200 words (though longer and more detailed articles are certainly welcome)
  4. How are the two versions different? One version is targeted at professional scientists following typical academic and editorial standards. The second version is written for the Wikipedia audience and includes a slightly heavier emphasis on a general audience. Both versions will be peer-reviewed together, but for copyright reasons, these two versions must be separate works that have no substantial similarity. Some examples of review articles and wikipedia entries published under this model include:
  5. I am busy but intrigued, what is the time line? We generally suggest a 2-3 month deadline, but since this is an ongoing series in the journal, the time line is flexible and can be worked around your schedule. Don’t be discouraged from participating because you are busy now. Make the commitment to submit when your schedule permits.
  6. Do I have to go at this alone? Absolutely not! If you have colleagues who would make good co-authors for the review, feel free to solicit their assistance.
  7. Do I have to write the wiki article all at once? Nope. Our goal is to incentivize you, the expert, to make your knowledge about your exRNA gene accessible. If it’s easier for you to write the wiki article in pieces, go ahead and do so! As long as the wiki entry is complete by the time you submit your manuscript, we will be happy to accept your review article.
  8. The gene I work on doesn’t make much sense to write about alone, how should I contribute? Genes that work in concert can be tackled as a pair as with this example:
  9. Why should I do this? By publishing an exRNA gene-specific review article, you help your scientific colleagues stay abreast of the current literature on your favorite gene. By publishing under the dual publication model (ie- on wikipedia), you help make your favorite exRNA gene more accessible to everyone allowing more people to understand the importance of your field of research. Everyone wins!
  10. How do I get in on this? Check to see whether or not your favorite exRNA gene could use some serious contributions on wikipedia. If so, contact me. Include your exRNA gene of interest in the email, and your preferred deadline for the manuscript submission.

Looking forward to hearing from you!

09.09.14 by

Flow sorting extracellular vesicles

The isolation and characterization of subclasses of extracellular vesicles (EVs) has been an important goal of the field for many years. Measurements based on nanoparticle tracking analysis (NTA) (Dragovic et al, Nanomedicine 2011) and resistive pulse sensing (RPS) (Vogel et al, Anal Chem 2011) properties of these EVs are in common use in the field to measure various particle characteristics, including size and concentration. Flow cytometry is a well-developed technology that offers several unique advantages to characterize EVs, including high speed and quantitative analysis of individual cells or particles, with the added advantage of subsequent purification of discrete subsets of particles of interest. Our group has designed a robust flow cytometry-based technique capitalizing on recent improvements in light collection optics, electronic detectors, and computer sub-systems to develop FAVS (Fluorescence-Activated Vesicle Sorting). This technique requires at a minimum a modern digital sorter with highly efficient light collection optics. EVs need to be disaggregated into individual particles by shearing them sequentially through 22-, 27-, and 30-gauge syringes. Properly suspended EVs can then be stained, washed, and analyzed using sorter-optimized configurations. The sorter must be calibrated and set up so that the signals from the EVs are processed as fast as the electronics will allow, and the EVs must be diluted to no more than 10 μg/ml total protein to avoid swarm effects by which multiple particle events are measured together rather than individually (van der Pol et al J Thromb Haemost 2012). We have demonstrated the ability to purify vesicles below the diffraction limit of light to a post-sort purity of greater than 99% double-positive fluorescence and size range of 40 to 70 nm in diameter (Cao et al, Mol Cell Proteomics 2008). To achieve this result, we started with iodixanol gradient-enriched intracellular vesicles that averaged less than 70% double-positive fluorescence and a vesicle size range of 30-300 nm in diameter. We have gone on to use FAVS to analyze EVs in various contexts, including those secreted from cells in culture (Higginbotham et al, Curr Biol 2011 and Demory et al, Mol Cell Proteomics 2013) and to isolate specific subclasses of secreted vesicles in vivo (McConnell et al, J Cell Biol 2009 and Shifrin et al, Curr Biol 2012) to analyze their constituents. Other groups have employed similar strategies to measure secreted vesicles by flow methods (Nolte-‘t Hoen et al, J Leukoc Biol 2013 and Momen-Heravi et al, Front Physiol 2012). Strict adherence to the parameters described above and judicious gating can allow comparable results.

Negative-stain transmission electron micrograph (TEM) of vesicles present in sucrose gradient fraction 3. The considerable size variation observed before FAVS (i) is notably decreased after sorting (ii). Bars, 0.5 μm.

Negative-stain transmission electron micrograph (TEM) of vesicles present in sucrose gradient fraction 3. The considerable size variation observed before FAVS (i) is notably decreased after sorting (ii). Bars, 0.5 μm.

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