Monthly Archives: May 2007

The salt lakes in the Egyptian dessert, The Sargasso sea and the cutting edge in the optical querying of neuronal circuits

I recently went to a talk given by Karl Deisseroth of Stanford University whose lab has been in the forefront of developing tools that allow neurons to be activated or de-activated using pulses of light in combination with expressed opsin transgenes. The opsins are basically photoactivated proteins ( like the rhodopsin in our retina) and they are activated by a single photon of light . Deisseroths lab had a while ago developed the use of a Channel rhodopsin- ChR2 which showed a light activated cationic (positive ion) current . Thus, when ChR2 was expressed in neurons a flash of blue light triggered a current of sodium and potassium ions which resembled the neuron firing or the action potential. The group set out looking for opsins that had the opposite effect , i.e they accelerated the suppression of the neuronal action potential ( or brought the neuron back to rest) in response to a light photon. The protein had be functional in neurons and also bring about the light activated depolarization at a time scale of the action potential .

After trolling through a few opsins the Deisseroth group in close collaboration with the Georg Nagel lab at the University of Wuerzburg in Germany zeroed in on the opsin from Naturomonas pharaonis (NpHR or Halo) an archaebacteria that lives in transient salt lakes in the Egyptian dessert in salt concentrations as high as 3.5 M NaCl and a pH of 11. The bug possibly uses the opsins to capture the energy from sunlight and uses the energy to drive the uptake of chloride which allows it to survive in the very salty lake it lives in.

This new opsin now was able to drive a chloride current on photoactivation which effectively turned the neuron off. Fortunately for the group the NpHr opsin was also activated at a wavelength entirely different from the “on switch” ChR2 opsin.

Concurrently with the publication in Nature the group released a video of a worm expressing both opsins which could be paralysed and tickled into movement by pulses of light that stimulated NpHr and CpHr respectively. In the talk Deisseroth also spoke of developments to move the experiments into a mouse model where a transgenic mouse would have a fibre optic probe inserted into its head that would then allow light to stimulate a precice area of the brain ( like a pulse of light would cause the neuron that controlled whisker movement to turn on and the mouse would twitch its whisker).

All of this clearly opens the road to other such opsins that can possibly respond to other wavelengths and drive excitatory and stimulatory currents with different properties. Interestingly way back in 2004 the metagenomics initiative by Craig Venter (the Sorcerer II expedition) published details of several novel opsins from the sargaso sea mass sequencing samples. Who knows , maybe the next such opsin may come some deep sea archaeon and have totally different spectral and kinetic properties which would allow an added level of control to the optical querying of neuronal circuits.

References and additional material

  1. Video of light stimulated supression of worm twitching upon NpHr (Halo) activation
  2. NpHr genome project page
  3. Free full text PlosBiology paper on NpHR by Xue Han and Edward Boyden
  4. Image link from an article in the MIT technology review
  5. MIT technology review article on ChR2 and NpHr ( worm video link )
  6. Request the ChR2 and NpHR (Halo) plasmids deposited by the Boyden lab at addgene
  7. The TED talks have an interesting talk by Craig Venter : around minute 3:46 he talks about the opsins
  8. Full text ( free access) PLOS Biology article on Sorcerer II data.. one of the figures contains an analysis of the spectral characteristics of proteorhodopsins from the metgagenomics dataset

University-knowledge beyond authority

Knowledge Beyond Authority at Harvard Law SchoolI have recently started attending the Berkman Centre Thursday Blog group meetings. The meetings which are open to the general public were started many years ago by Dave Winer and colleagues and are loosely structured to discuss topics related to blogging, the social web and also recently ( since I have been attending) entrepreneurial activity in the web 2.0 space from local startups .

At the meeting I heard from Mike Walsh of a conference to be organized by the center titled , University- knowledge beyond authority.
The conference aims to “generate questions, insight and solutions from diverse perspectives across the landscape of University, with a focus on the role of University as an institution.We seek to establish University as a collective force much like ‘Government’ or ‘Private Enterprise’ in its ability to negotiate and compromise for our needs in the digital environment.”

The conference has several sessions dealing with topics ranging from fair use , open-access to discussing the relationships between “University” and library and the RIAA.

The concept of University it seems is to organize thought and opinion on these topics from an educational perspective. Since I know very little about the complex world of licensing , and the DMCA is almost a four letter word to me, it will be fun to listen to how this concept of University evolves at University.

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Raindance and Genomics 2.0

An interesting article in BioIT world
talks about Jonathan Rothbergs ( the founder of 454 Life Sciences) keynote address to
the Bio 2007 conference in Boston , where he talked about his future
venture, called  Raindance technologies
a “nanoreactor” company that seems to use a combination of
microfluidics and “454 style” oil emulsion and quantum dot based optical analysis to create a
highthroughput assay platform.
The impressive set of videos at the raindance site explain the various steps of the assay platform. The adoption of quantum-dot based detection systems incorporated into their nano-reaction vessels makes it possible to conduct a wide variety of assays at an amazing throughput of several thousand reactions per second.
The article talks of the possibility for
moores law like advancements in such platforms bringing the genomics world closer and closer
to the IT world and are all part of a revolution, Rothberg dubs as
“genomics 2.0”.

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Academic plasmid sharing made easy

While I was researching an article on opsins that are revolutionizing neuronal investigation, I came across a small blurb on the Boyden lab homepage with a link to addgene which said ” Request Boyden Lab plasmids through addgene”.

Addgene as its webpage states is a non-profit research support service dedicated to archiving and distributing plasmids that appear in published articles.The plasmids in addgenes database are classified on the basis of the Principal investigator deposting them and gene name. Also each plasmid has a lot of detailed information available making its use in other experiments quite easy.

It was very heartening that several leading labs had deposited their plasmids for academic use at this service. In the world of open science , services such as this are invaluable. I don’t know how long addgene has been around, but it will be quite something if the NIH made it mandatory to have all plasmids used in published work deposited at such a service.
While it is still a little involved to get plasmids from addgene, since it does involve some paperwork exchange, there is no denying the fact that a central managed repository such as addgene.org will ease the load for both end user and innovator labs as well as tech transfer offices.

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The Resistome – the superbug arsenal characterized

I have heard of many large scale omics studies and their resultant “omes”, but it was only last week when I was reading a review in the Journal Cell on drug resistance in bacteria did I chance on a reference to the soil bacteria resistome , which was published by De Costa et. al. almost a year ago.
The paper deals with mapping the spectrum of antibiotic resistance among 480 streptomyces , the bacteria that produce several of the classes of antibiotics to kill other soil dwelling microbes. These microbes also encode a myriad of resistance mechanisms to make sure they survive the battle themselves. The resistance mechanisms brought into play by these soil dwelling bugs mimic those seen in clinically relevant bacteria. Given that its more a question of WHEN and not IF most of these mechanisms will surface in the clinical world.Therefore characterizing the diversity and density of resistance mechanisms prevalent in the environment is of extreme importance makes their study extremely relevant given the emergence of many super-bugs worldwide. The most surprising finding of the study was the diversity and density of resistance mechanisms present across all the strains.

The paper tested resistance to 20 different antibiotics belonging to every known class of antibiotics produced. Shockingly several of the bacteria were resistant to all 20 of them , with the average bacterium resistant to at least 7 different classes of antibiotics. Some of the antibiotics tested, were ineffective in almost 100% of the cases . Surprisingly the newly launched daptomycin which is effective against some multi-drug resistant microbes found in hospitals etc, was inactive in almost all of the soil isolates.
Resistance profile diagram from  www.science.mcmaster.ca
The authors also tested the resistome for possible mechanisms of inactivation and offered the possibility that possible novel mechanisms as well as variations of known mechanisms were operational and present in the resistome. The resistome illustrates how clever micobes are at outsmarting even the most well thought out antibiotic. Before we even think of creating an antibiotic to rule over all antibiotics , the chances that resistance to it is lurking in some niche in the microbe world seems to be quite likely. Although the resistome cannot predict how likely these resistance mechanisms are to transfer from the soil to a bug that can create problems when it sweeps through a hospitals ward: But it does make a case for using modern tools to address possible drug discovery in this class of drugs that indeed introduced chemotherapeutics to the everyday people.
Superbugs have been the stuff of many a popular cover story. Drug resistant tuberculosis , cholera , malaria etc continue to wreak havoc in the developing world. Recent articles in Nature and other journals spoke of the trials and tribulations of platensimycin , the only new class of antibiotic to be discovered in nearly two decades. Even that was hardly “resistance proof” in that over-expression of its target protein was able to confer resistance to the bug. Although the resistome, you could argue makes the case for the absence of a resistance proof antibiotic it definitely underscores the importance of improving the diversity of our arsenals against infectious bacteria. Given that our pace of discovery of antibiotics seems to be slower and slower, the resistome puts some quantitative muscle behind the cries for renewed drug discovery efforts in this area.

Image courtesy  Dr Gerry Wright Lab homepage