balapagos

Earlier, Pfeiffer et al. introduced a UAS construct that most notably contained the IVS and WPRE elements as upgrades to the bland looking 1.0 version (in retrospect). I actually jumped on the v2.0 wagon (which reads 10xUAS-IVS-GFP-WPRE-SV40term.) and quickly made UAS-dWGA:GFP and UAS-dPNA:GFP fly stocks towards developing a good trans-synaptic marker. I must say it paid off nicely! Well, just when I was thinking "OK, this should last a while", it got even better!

Upon RTFA it was easy to see why they have been trying to push the Gal4/UAS based transgene expression amplitude even higher. May be it also has to do with the weak driving capacity of many DSCP-Gal4 lines? I wonder... Anyway, I also applaud at the effort to overcome the endogenous level of Shibire activity with the robust level of transgenic UAS-Shibire(ts1) expression through the introduction of some really cool design changes. We noted this requirement of high Shibire(ts1) expression level as well recently for silencing larval sensory and motor neurons (bummer.. missed out on a cite).

In summary, if you want to express a cDNA of interest at an epic level using the Gal4/UAS system, then 10xUAS-IVS-syn21-cDNA-p10 is the way to go. For this, one could simply ligate a [RE1]-syn21-seq-AATCAAA-cDNA-[RE2] PCR product into a vector backbone that reads: 10xUAS-IVS-MCS-p10. I think such a backbone will be very useful to make.. may be it will also be useful to flank it with the Gypsy insulator elements to keep it extra quiet when there is no Gal4 around. I'd then call it v3.5! Finally, I must thank Barret for the e-mail discussions.

Direct link to Pfeiffer et al. 2012.

PS: This blog is now being mirrored at balapagos.wordpress.com. This is in case Twitter decides to gradually decommission Posterous, of which there have been some indications recently.

This Scholarly Kitchen blog post titled: The “Academic Spring” — Shallow Rhetoric Aimed at the Wrong Target" (by Kent Anderson) is a very interesting read. It critically examines an emerging socio-economic battle our time. Another reason why it is good to see such articles is because I think educators, academics and policy wonks also get paid for participating in open discourse on such matters. Anyhow, it was nice to see this quote in the post – “We have met the enemy and he is us”.

But one particular comment (made by Aalam Wassef in response to the criticism that the term "Academic Spring" is being misused) I believe deserves to be recycled, so here it is:

"the reference to the Arab spring came to me naturally and came to me even more easily as I happen to be Egyptian, and have lived the full intensity of the uprising and the long years preparing for it . Since 2006, many others and myself have been using social media to disseminate content, information and artwork exposing our regimes so we could raise awareness and, eventually, impact reality. You might recognize a pattern right here. Content, dissemination, impact.

"Returning to our main concern, could greater and cheaper access to knowledge have allowed scholars and civil servants living under such regimes to reverse, or at least slow down the many plagues of Egypt? I just had an amusing telephone argument with an Egyptian researcher (nutritionist) who started by telling me that not having access to research had absolutely nothing to do with Egypt’s current human and economic development. She continued by telling me that the real problem was that researchers were not seeking information and were not taught how and where to find it. When I asked her ‘why’, she replied that it was probably because libraries are poor and outdated. She added that the recent articles she used herself were those available in Open Access. Reading and copy-pasting abstracts from pay walls is
also something she does frequently."

"It’s maybe alright, after all, to compare the dynamics and motivations of the Arab Spring with what could be an ‘Academic Spring’ which, to me, is really about changing the way we fund, share and validate scientific results.

I don’t agree with blaming anyone in particular, or publishers, or universities and would gladly blame all of us. By us, I mean civil society, you, me, the publisher, the reviewer, the scholar, the parent, the librarian, your neighbours and mine. It’s always us, in all these different positions and places, doing the things that, overall, don’t serve us very well as a group."

I find it interesting that although there are some differences of opinion on how the "academic activism" is unfolding, there is a common realization that the practioners of academic research will do better by taking down the walls that have started to hinder global progress.

Here is an interesting IT article [computerworld.com] that brings to fore a very important aspect of how new breakthroughs actually happen. I hope that the funding bodies, that make the academic research happen, will get some insights from this and become more eager to support people who cannot help but chase high-risk ideas.  

Here is an excerpt from the article:

"In his more than 30 years in IT, Richter said he's seen three common barriers to innovation, all three of which were present at Kimberly-Clark at the time. The first was cultural resistance to change.

"I've seen many great ideas that didn't go anywhere because the company wouldn't let them do it," he said.

A second barrier is process. "I truly believe process kills innovation," he said. "I'd never come into an organization in my career outside of the government that was as process-bound as [Kimberly-Clark] was."

The third barrier is fear of failure. At Kimberly-Clark, this was the strongest roadblock. "There was a palpable fear that if you tried something and failed, it would damage your career forever. I never saw a single case where that occurred, but the perception was there."

"Everyone wants success, but by design the nature of innovation is that most ideas can be expected to be failures," he said. "Does that matter? Of course not. Failure is simply the opportunity to begin again, this time more intelligently. It's about what we learn from the failure. Not the failure itself. We celebrate that learning."

I have been in the academic research stream for quite some time now. I have managed to survive for this long despite the many hurdles and I am so glad that I still conduct my own experiments everyday using my favorite model system. But many others have not been as lucky as me. Due to the lack of institutional support or the wherewithal, such souls eventually consider alternative paths that will take them away from their original quest. In this regard, I suspect that there must be a large stream of well-trained talent out there that can be given a chance to become "liberated" (so to speak).

If I win a mega mega lottery I would establish system-wide micro-grants (30k per year + 1 free airline ticket to anywhere) to a large number of experienced postdocs (5 yrs+). I'd then go around and fund "core-facilities" that are interested in these "Research Entrepreneurs" (REs) to visit and hangout. REs will be required to update their experiments/results on a regular basis into an "open-access" "open-peer-review" website.

Since I will never win such a mega-lottery (because I'm not the lottery buying type), this will probably never happen. But may be someone who read this post will land one! Who knows!

Among the speakers of that recently held public debate on "Open science and the future of publishing" I think Prof. Lord Winston's take on this topic was perhaps the most substantive, in that I think it really does force some solutions for the future. The main reason for this is the tremendous amount of experience that Prof. Lord WInston carries with him. The man is a surgeon, a scientist, and a politician and I think his insights are just priceless. We must listen to him with full attention.

"I don't believe it [open access] really contributes to public engagement and I'd argue that it is about time we stopped talking about public understanding of science but much more about communicating science, and that means a two-way process. Communication is not done through Journals." - PLRW

"When it comes down to access I think clarity is more important than open access, I also think relevance is more important than open access too, I would argue that probably interaction is more important than open access." -- PLRW

BTW, he is not just a critique, he has actually tried to solve the problems he is talking about. For example, the "public engagement of science" issue, by creating the Reach Out Lab. For this alone, one must respect him greatly.

Having acknowledged that, I wonder whether he is underestimating the power of online communities that organize themselves around Facebook, blogs, and user-content driven sites like Reddit, Arstechnica and Slashdot to name a few. Anyway, I am quite sure that such communities will play a big role in the extraction of content and meaning, and then, recycle it, even from hardest of topics so that an intelligent lay person could understand. Here is an example on this related to the Higgs-Boson news.

In summary, I'd say that we need more criticisms of this type. Because they are most important in helping us navigate towards more optimal solutions. It seems we are heading towards stratifying the science landscape online. A layer of "open access data warehouses", and a separate layer of Journal publishers (open-access and closed-access types). The online "user" driven self-moderating open-communities will then most likely become an integral part of this landscape.

Because it didn't make sense to see an article discussing "open access" behind a subscription wall, I sent this e-mail yesterday to the author of the editorial:

"Dear Dr. Leptin,

I am a Drosophila biologist just like you. I would like to first of all say that I have been a big fan of your work on gastrulation events ever since the mid 90's.

Thank you for the very interesting Science Editorial. I think it would have been even better if the article itself was "open access" so that everyone could read it.

I don't think it is very expensive these days to publish interesting experiments online and receive useful peer reviews. Here is one of my recent projects through which I am experiencing  the "open science" movement.

http://figshare.com/authors/Balaji%20Iyengar/97222

I also actively blog about science related ideas through this website:

http://balapagos.posterous.com

I believe that open dissemination of scientific results and ideas will only encourage us to do better science. After all, the eyes of a 100 reviewers will always be better than two or three. That the transparency allows the tax payers see how biomedical research process works is a bonus.

Final point, the validity of this "open approach" I find is better appreciated in other fields like computer science, physics and mathematics.

Sincerely,
Bala"

--------------------

Dr. Leptin was very kind to acknowledge my e-mail promptly, despite her very busy schedule. I'm glad to hear that she has forwarded my comments to her colleagues in EMBO publications. I look forward to further discussions on this topic.

This video clip is from a very recent public debate on "Open science and the future of publishing" held at Univ. of Oxford. If you enjoyed this clip, here is more from Tim Gowers on this topic, and also views from a major funding agency that believes in open access, and interesting new ideas from a top-level publisher.

The "the unit of discourse" (UoD) point caught my attention because I've been thinking similarly for some time now. I think it will be fair to say that most experimentalists who have been working in a given area for a considerable extent of time (say 5 yrs or so) would love it if there was a method through which smaller "unit" contributions can be made more frequently and openly, followed by invited peer-reviews for each of those units. This will free up more time to do well-guided experiments!

Figshare is a great start in this direction. After all, in biological sciences, one or two figures can indeed capture a major movement or establish a new "benchmark" after many months of planning and execution. Smaller UoDs will also make the peer-reviewing process more efficient. 

Quite certain that if a publication model emerged around this idea, backed further by quick feedbacks from the expert-community (or invited peers), we will start hitting the science-ball a whole lot more closer to the sweet-spot, so to speak.

Ah! Last but not the least, I must link to this highly relevant xkcd comic.

It has been a very busy month since the last post. I'm still trying to find some rhythm here in Hammertown after a 6 month sabattical of sorts in CRG. Thanks to PHRI, I now have a bench where I can do some experiments off and on when I am not occupying a bench in Univ. of Toronto, this arrangement also helps me save some money to buy reagents.

It actually feels good to be washing my own fly dishes again! Well, may be more so now than before, because I now have a 13$ motorized tool to wash the fly tubes pretty efficiently (yes, I'm old school). Anyway, thanks to IKEA, for the FIXA 3.6 V! Attach a bottle scrubber to it and this gizmo has more than sufficient torque for the job.

I must say, it is good to hear that my friends in CRG haven't forgotten me, yet. It gives me more purpose to continue with the ideas that germinated in Barcelona. We have now submitted an interesting abstract to the FENS-2012 conference. The poster we will present there is on a brand new larval behavior assay that I think has a tonne of potential and I'm quite surprised that no one has followed it up before us. I'll save it for another day, besides, with Louis et al. powering the project as well I'm not worried about its progress. Now, it is the TSM project that needs more attention, especially because I'm flying solo on this one (self-funded etc.). So, to get back on track, I must start writing about it.

It was clear from the initial observations that dWGA:GFP expression in the Bolwig's Organ leads to a lot of background GFP-signal in the larval brain, especially at the periphery of the brain. Not only that, but the GFP profile near the Bolwig's Organ synaptic terminals (C, C') didn't look like post-synaptic dendrites, as one would expect, assuming that the transfer was indeed taking place to the lateral neuron dendrites. This made me suspect that whatever transfer was taking place is probably mostly on specific tracheal tubes that perhaps go through the larval optic neuropile. This makes sense because there is at least one previous study (Callaerts et al. 1995) where this affinity of WGA for trachea has been reported. 

Here is the figure and a note by Callaerts et al. on this observation made using Stage 17 embryos:

So, compared to WGA, the data for Peanut-Agglutinin in the same paper looked more promising for trans-synaptic labeling:

This was the basis for making a codon-optimized (d) UAS-dPNA:GFP construct using the same strategy that was used for making the UAS-dWGA:GFP fly stock.

I just posted the first piece of evidence for dPNA:GFP today in FigShare. In brief, the dPNA:GFP-label does appear to get transferred from the Bolwig's Organ presynapse to the RFP-expressing lateral neurons (LNs). A noteworthy point is that one can see a GFP-labeled fiber from the Bolwig's Organ terminal heading towards the RFP expressing LNs, that also co-localizes GFP-punctae. But there is one caveat here, I also have 3xP3-RFP expression in the background, this is part of the attP landing site where my attB containing construct was landed. Since this background signal does not interfere with the LN-RFP label (which is very distinctive and strong) it was easy to visualize the co-localization.

Well, that's it for this post. I have yet another panel in the works that I hope to post into my Figshare account sometime tomorrow.

Final point, don't hesitate to ask me any questions and if you wish to try out my TSM stocks send me an e-mail.

09 Mar. Update.

Made another Figshare post just now. May be the transfer of dPNA:GFP is telling me something interesting.

PS: Mark, the title-chewing bug is still alive.

I have now become more curious about the advantages of sharing experimental results openly well before a formal publication becomes a distinct possibility. I think such an approach will be particularly useful for tackling tricky research problems. Incidentally, at this very moment there seems to be a groundswell in progress to make "open-science" a reality, akin to the open-source software movement perhaps. The activism against the closed-access publication model on one hand, and the Panton fellowships in the other, can be taken as indicators that a new era may be upon us. I must add, it is also nice to see companies like Digital-Science emerge with new solutions for the future. So, in the spirit of exploring this frontier here is something that I have been tinkering for a while now.

The trans-synaptic marker (TSM) adventure began for me after I came back from the MiniBrain conference in late 2010. During Gerry Rubin's fantastic talk, titled: "Genetic tools for studying the anatomy and function of Drosophila nervous system", I had noticed that this particular tool was missing. Having asked a question about it at the end and receiving a negative response, I decided to take a swing at it.

My first aim is to try and deliver GFP across a well-known Drosophila central synapse, reliably. I have chosen the contact that Bolwig's Organ makes with the Pdf expressing lateral neurons in the larval brain as a model to identify a good trans-synaptic agent. 

Why is this important? For Drosophila functional-neuroanatomists who are interested in assembling neuronal wiring diagrams to understand how innate-behaviors are encoded in the CNS, such a tool will be very useful. For example, one can ask how many post-synaptic interneurons are in contact with one "identified" functionally important interneuron. The split-GFP approach is one alternative, but the scope for that appears to be limited, it is more suited for validations. So, I thought the use of plant lectins like Wheat Germ Agglutinin (WGA) is a reasonable place to start because this has been attempted before in Drosophila. The bright-side of starting with a route that is familiar but not so promising is that one can get all the pieces and the work-flow together. Anyway, going into this project, the first hypothesis was an obvious one - WGA sucked in flies because of poor transgenic expression levels.

To begin, I decided to remake a UAS-WGA:GFP transgenic fly strain with the main emphasis on producing higher levels of WGA:GFP expression when combined with a Gal4 driver. My starting point was a simple improvisation of the JFRC14 expression construct (Pfeffier et al. 2010). Now, since this is an on-going exploration I'm not going conclude anything until I test many different types of lectins (hopefully I can afford it). If you are very interested in this project, I have just started to publish my results in Figshare and will continue to do so. If you would like to contribute or test out my UAS-lectin strains on your favorite synapse, drop a line. Any comments, criticisms or suggestions are most welcome!

To end this post, here is the very first dWGA:GFP experiment that got me all excited. I was so impatient after setting up the cross that I dissected a first instar CNS to see if something interesting was going on in there.

Final point, many thanks to the members of the Matthieu Louis lab for tolerating my experimental obsessions. But for a few down-and-out days, it was by far the best 6 months of science-social-life I have ever experienced! I must also add that this project would not have come alive so easily for me without the excellent support and the atmosphere of CRG (it must be the "beach-state-of-mind"!) and its amazingly good core microscopy facility!

This is a deeply moving photo-essay by xdrtb.org based on James Nachtwey's photographs. It is likely that the drug-resistant-TB problem may become one of the most urgent ones to solve in the light of this worrisome news from India.

While poverty alleviation programs (combined with free-health-care) are fundamental in battling such outbreaks, to decisively eradicate such preventable infections, free biology-education of the masses and a role for NGOs in providing information on the best anti-TB drugs must be combined with it.

This quote from the Letter to the Editor by Udwadia et al prompts the necessity for a multi-level approach:

"The vast majority of these unfortunate patients seek care from private physicians in a desperate attempt to find a cure for their tuberculosis. This sector of private-sector physicians in India is among the largest in the world and these physicians are unregulated both in terms of prescribing practice and qualifications. A study that we conducted in Mumbai showed that only 5 of 106 private practitioners practicing in a crowded area called Dharavi could prescribe a correct prescription for a hypothetical patient with MDR tuberculosis [5]. The majority of prescriptions were inappropriate and would only have served to further amplify resistance, converting MDR tuberculosis to XDR tuberculosis and TDR tuberculosis."

As the year ends, a paper that I will carry in my mind is the one by Fried et al. In the video summary above, I thought the observation of the patient response - "I have an urge to move my hand!" upon the stimulation of a particular area in the brain was pretty neat! Fried et al thus had the unique opportunity to analyze the neuronal spike train data from 12 epileptic patients while running a behavioral-protocol previously established by Libet et al. 1983. Just to recap, the subjects were asked to press a laptop key "when they felt the urge to do so" while watching a clock hand sweep around (as described in the video). At key-press (P) the clock would stop and the subjects would indicate the time point when they felt the urge (W, "wanting") to press the key. Here is a schematic of the procedure:

An example of neurons in the SMA area whose firing precedes the W point (indicated by 0):

I wonder if we can detect such higher-order interneurons in the Drosophila brain using live-imaging methods. If they do exist, then it should be possible to bias the decision-outcomes predictably by opto-genetically triggering their activity at specific time-points during the behavior routine of an individual fly or the larva. May be the on-going larval and adult fly olympiads will stumble upon such candidate interneurons that can then be characterized further.

Oh, of course! this would make an excellent journal club paper as well, with the potential to trigger some very lively philosophical discussions that'd spill-over after the session has ended!