You probably recall our conversation on Friday concerning a recent paper which claims that fruit flies (Drosophila melanogaster) are capable of distinguishing between deuterated and undeuterated compounds.
Franco, M. I. et al., (2011), Molecular vibration-sensing component in Drosophila melanogaster olfaction, www.pnas.org/cgi/doi/10.1073/pnas.1012293108
This seemed like a tall order at the time, and still does, but after reading the paper I have to conclude that the authors were exceedingly thorough in ruling out as many experimental variables as possible: it definitely appears that the effect is completely real, and vibration sensing is a proposal which seems to be in keeping with the experimental results.
The paper is cogent and laid out in a thoroughly logical manner, systematically eliminating possible sources of bias. All the relevant data are given too.
Before we even get started, the authors point out that deuteration doesn't appreciably change bond length, stiffness or angle.
Potential bias #1 - humans have become accustomed to different scents throughout their life.
Solution -- use fruit flies: they have such short lives that you can ensure that they haven't become accustomed to a smell, and they are generally more sensitive to odotopes (odourant molecules) than humans to boot. Other studies have shown that humans can't perceive a difference between deuterated compounds and their regular isotopes, but the authors believe that (a) humans can't smell well and (b) we probably could tell the difference if the odourants were purified well.
The basic idea of the rest of experiment is simple: run two fragrances down either arm of a t-junction `maze' and see which direction the flies pick most often.
Potential bias #2 -- Some visual, auditory or other cues may lead to flies picking one arm over the other, even in the absence of olfactory stimuli.
Solution -- run a control: one with wildtype flies and no odours, and another with mutant fruit flies which cannot smell at all. It turns out that no significant differences are seen in either of these cases.
Big question #1 -- do flies show spontaneous (naive) responses to odourants?
Answer -- yes: no surprises here really.
Big Question #2 -- can flies distinguish between different isotopes of acetophenone (apparently a common perfume base)?
Answer -- yes! The hydrogen-bearing version is attractive, and as deuterium is added (3, 5 or 8 deuterium substitutions) the acetophenone becomes less attractive and then more and more repulsive. The same goes for octanol and benzaldehyde.
Big Question #3 -- can we teach flies to recognise a deuterated compound?
Answer: yes -- this was done by foot-shock, which apparently is a well-tested method of conditioning fruit flies. If conditioned to deuterated compounds the flies consistently chose that arm of the maze, even if it went against their naive reaction.
Potential bias #3 -- maybe it depends on the type of fruitfly chosen.
Solution -- try a different wildtype strain... which gives the same results.
Potential bias #4 -- perhaps the flies are only recognising and responding to different impurities left over from synthesis and purification of the different deuterated compounds.
Solution -- try conditioning them with one compound (deuterated or undeuterated), but then test them with a different type of compound that is also either deuterated or undeuterated (respectively). Amazingly, the flies can still differentiate the deuterated compound from its regular counterpart.
Potential bias #5 -- could there be residual odours?
Solution -- completely replace all of the tubing after every trial.
Big Question #4 -- if the mechanism underlying this discrimination is based on molecular vibrations we should be able to train fruit flies using deuterated/undeuterated compounds, but then test them using completely unrelated compounds that happen to have similar IR spectra in the region of 2200cm^-1 (C-D stretch vibration). Do we see similar effects?
Answer -- yes! It turns out that the CN triple bond has a similar vibrational frequency to C-D, so this was used to test against. Flies again could be conditioned with deuterated/undeuterated compounds, then select completely unrelated molecules with similar regions of the vibrational spectrum.
I have to admit that it sounds pretty conclusive: the flies are, in some way, sensing molecular vibrations, or something very closely related to them. The only really obvious way in which they could be wrong is if the whole thing were fabricated (I'm not suggesting it is though)!
The authors go on to outline the very core ideas behind inelastic electron tunneling spectroscopy, which they believe is a reasonable model for olfaction. An electron acceptor sits near a donor molecule, but electron transfer only occurs when a suitable intermediary is able to accept an amount of energy, becoming vibrationally excited. Perhaps this is how smell works: after all, we see light because the absorption of a few quanta of light are enough to cause a conformational change in a protein. Why shouldn't smell have a related mechanism?
I can see two obvious ways forward: the first is to selectively knock out each of the 62 different olfactory sensors that drosophila is endowed with, and test which is most important in the apparent discrimination of deuteration. With this determined, electrophysiology would be a must, probably along with gene expression analysis.
The second is to selectively deuterate different portions of materials, to see if the effect depends on the vibrational mode or just the frequency. This could involve some intense chemistry! Finally, perhaps some more dramatic investigations could be carried out: how about if tritium is used?
I'm interested to hear your thoughts!
Monday, 10 October 2011
A Brief Review of Mechanical DNA Models
On Friday some of us wondered why Nelson included such a large amount of material on DNA when he could have happily truncated the discussion much earlier (from a pedagogical standpoint). The answer is simple... Nelson is into DNA stretching!
I have been doing some reading around to try and resolve just what the precise definition of the stretch (u) is, but have had little success because the majority of studies don't bother to include it! I strongly suspect that it is simply the elastic strain, given as a function of the applied stress by some appropriate constitutive relation (e.g. Hooke's Law relates the stress F/L to the strain x/L, where x is the change is the difference between the actual and equilibrium lengths (L), by the spring constant).
As I mentioned, most studies appear to completely neglect the stretch and focus on the wormlike chain (here's a nice blog on this topic), freely-jointed chain or discrete persistent chain models. The first two are discussed in the text, whereas the last is a generalisation of the freely-jointed chain in which there is an energy cost associated with introducing an angle between neighbouring segments.
The discrete persistent chain is a mathematical beast! This paper, one of Nelson's own, gives a lot of detail on this model, and demonstrates the use of the transfer matrix and variational methods (this link goes through many different variational methods e.g. mean-field approach, Bayesian estimation, graph theoretical approaches, etc.). It seems to give good results in the study of B-to-S DNA transitions: S-DNA being an overstretched state.
It also seems that Nelson was a little disingenuous: he didn't actually start with the most general model and simplify down to the FJC... the beginning model was actually already dramatically simplified! Extensions include sequence-dependent parameters (bend and twist persistence length, etc, which can even be asymmetric about the axis to account for the pitch of the molecule) which account for possible mechanical differences between different base arrangements (believed to be important in protein binding to dsDNA, even more so than the actual chemical properties of the bases), cyclisation (making small loops of DNA), phase-dependent parameters, finite-length effects and the influence of histones and other DNA-binding and packing proteins.
It turns out that cyclisation occurs in much shorter segments than people expected, and no-one knows why yet. There's also a whole great big world of knot theory and DNA which was left unexplored, and different types of nucleic polymers (dsDNA, ssDNA, RNA, etc).
There's still a lot of research to be done, and a lot of reading in the meantime!
I have been doing some reading around to try and resolve just what the precise definition of the stretch (u) is, but have had little success because the majority of studies don't bother to include it! I strongly suspect that it is simply the elastic strain, given as a function of the applied stress by some appropriate constitutive relation (e.g. Hooke's Law relates the stress F/L to the strain x/L, where x is the change is the difference between the actual and equilibrium lengths (L), by the spring constant).
As I mentioned, most studies appear to completely neglect the stretch and focus on the wormlike chain (here's a nice blog on this topic), freely-jointed chain or discrete persistent chain models. The first two are discussed in the text, whereas the last is a generalisation of the freely-jointed chain in which there is an energy cost associated with introducing an angle between neighbouring segments.
The discrete persistent chain is a mathematical beast! This paper, one of Nelson's own, gives a lot of detail on this model, and demonstrates the use of the transfer matrix and variational methods (this link goes through many different variational methods e.g. mean-field approach, Bayesian estimation, graph theoretical approaches, etc.). It seems to give good results in the study of B-to-S DNA transitions: S-DNA being an overstretched state.
It also seems that Nelson was a little disingenuous: he didn't actually start with the most general model and simplify down to the FJC... the beginning model was actually already dramatically simplified! Extensions include sequence-dependent parameters (bend and twist persistence length, etc, which can even be asymmetric about the axis to account for the pitch of the molecule) which account for possible mechanical differences between different base arrangements (believed to be important in protein binding to dsDNA, even more so than the actual chemical properties of the bases), cyclisation (making small loops of DNA), phase-dependent parameters, finite-length effects and the influence of histones and other DNA-binding and packing proteins.
It turns out that cyclisation occurs in much shorter segments than people expected, and no-one knows why yet. There's also a whole great big world of knot theory and DNA which was left unexplored, and different types of nucleic polymers (dsDNA, ssDNA, RNA, etc).
There's still a lot of research to be done, and a lot of reading in the meantime!
Friday, 7 October 2011
Thursday, 6 October 2011
Optical Thermal Ratchets
Whilst searching for pictures for my talk on Saturday I discovered a paper which discusses a quasi-one-dimensional `thermal' ratchet using holographic optical tweezers. Lee and Grier develop a simple "two-state" ratchet in 1D and demonstrate a "three-state" ratchet in 2D.
The ratchet is based around a simple idea: some spatial or temporal symmetry must be broken in order for Brownian motion (which is isotropic and time invariant in a thermal system) to be rectified. This is the same idea that we were exposed to last year. In this case the potential energy landscape in which the ratchet operates is not due to some mechanical constraint (e.g. the teeth on a cog for a Feynman ratchet, etc.) but is an externally-imposed optical potential. For more details on optical trapping see some of my earlier posts, or ask.
Holographic optical tweezers use devices known as spatial light modulators (SLM) to dynamically alter the phasefront of the laser used to trap particles. In the focal plane this phase patterning results in interference between different parts of the beam, with the net result being a change in the irradiance pattern. This can be used to split a single laser trap into an array of sub-traps.
Lee uses this ability to create arrays of lines of Gaussian trapping potentials, where the distance between traps within a strip is much less than the distance between strips. The positions of these lines may also be dynamically altered.
Brownian motion was rectified simply by turning the traps on in one arrangement and leaving them for a short time before translating the entire array one-third of the strip separation rightwards. How does this work? Particles first become localised in the traps, where they still undergo Brownian motion. As the traps are displaced this region goes from low-potential to being essentially force-free, so the Brownian motion takes over and the particle diffuses. It is essentially equally likely to head in either direction, but on average it takes much less time to reach the traps to the right than the left (especially seeing as the traps will be shifted back to their original positions). By tuning the time the traps spend in each configuration it is therefore possible to control the net flux through the system (within statistical limits, of course).
Lee and Grier also looked into a radial version of the same experiment: this allowed size-dependent sorting of particles (the optical forces are sensitive to particle size).
The paper is reasonably straightforward and contains some interesting maths, which I would recommend. It really highlights the similarities between the formalisms of quantum mechanics, diffusion and electromagnetism (Green's functions, for those who have heard of them)!
The ratchet is based around a simple idea: some spatial or temporal symmetry must be broken in order for Brownian motion (which is isotropic and time invariant in a thermal system) to be rectified. This is the same idea that we were exposed to last year. In this case the potential energy landscape in which the ratchet operates is not due to some mechanical constraint (e.g. the teeth on a cog for a Feynman ratchet, etc.) but is an externally-imposed optical potential. For more details on optical trapping see some of my earlier posts, or ask.
Holographic optical tweezers use devices known as spatial light modulators (SLM) to dynamically alter the phasefront of the laser used to trap particles. In the focal plane this phase patterning results in interference between different parts of the beam, with the net result being a change in the irradiance pattern. This can be used to split a single laser trap into an array of sub-traps.
Lee uses this ability to create arrays of lines of Gaussian trapping potentials, where the distance between traps within a strip is much less than the distance between strips. The positions of these lines may also be dynamically altered.
Brownian motion was rectified simply by turning the traps on in one arrangement and leaving them for a short time before translating the entire array one-third of the strip separation rightwards. How does this work? Particles first become localised in the traps, where they still undergo Brownian motion. As the traps are displaced this region goes from low-potential to being essentially force-free, so the Brownian motion takes over and the particle diffuses. It is essentially equally likely to head in either direction, but on average it takes much less time to reach the traps to the right than the left (especially seeing as the traps will be shifted back to their original positions). By tuning the time the traps spend in each configuration it is therefore possible to control the net flux through the system (within statistical limits, of course).
Lee and Grier also looked into a radial version of the same experiment: this allowed size-dependent sorting of particles (the optical forces are sensitive to particle size).
The paper is reasonably straightforward and contains some interesting maths, which I would recommend. It really highlights the similarities between the formalisms of quantum mechanics, diffusion and electromagnetism (Green's functions, for those who have heard of them)!
Monday, 3 October 2011
Chirp chirp chirp
Bats now have two unique characteristics: the only mammal that can fly and a larynx that flexes at a very fast speed. Their 'chirping' is made at an accelerating rate, increasing their calls to 160 - 190 chirps per second.
Bats start out with shorter-rate chirps, increasing their frequency as they approach their prey and leading to a hypersonic pulse called the 'terminal buzz'. A group of scientists investigated how bats produced this buzz. They also wanted to determine whether the upper buzz limit is a function of how quickly the bats can hear the return signals that bounce off their prey, or whether it's because of the bats' own call-producing abilities.
They set up a chamber with 12 microphones and recorded the activities of five different free-flying Daubenton's bats, little bats found in woodland areas from Britain to Japan. The bats hunted mealworms that were suspended in the chamber. The animals' chirp rate was so rapid that the researchers knew they couldn't be using normal skeletal muscle.
They attached the bats' vocal muscles to a motor and a force monitor, and stimulated the muscles to flex. The researchers monitored how long it took a muscle to twitch, and determined the muscles were able to contract and relax at frequencies up to 180 Hz and, in one case, up to 200 Hz.
They also noticed that echoes from individual calls ended before the start of the next call, so the bats don't confuse themselves. But a bat could theoretically produce calls at a greater frequency than 200 Hz - up to 400 Hz before echo interference would become a problem. The reason they don't? The superfast muscles are only so fast.
The muscle performance is said to be equated to a car engine. As quoted from a researchers: "It can be tuned to be efficient, or tuned to be powerful depending on what you want it to do."
These laryngeal muscles contract at a rate 20 times that of the fastest human eye muscles, and about 100 times faster than typical skeletal muscles, the researchers say.
Previously, scientists thought these ridiculously quick muscle contractions were only found in the sound-producing organs of rattlesnakes and some types of fish. This suggests that these special muscles are more common than previously thought.
Source:
Elemans, C. P. H., A. F. Mead, et al. (2011). "Superfast Muscles Set Maximum Call Rate in Echolocating Bats." Science 333(6051): 1885-1888 %R 1810.1126/science.1207309.
HIV Cure?
The cure for AIDS has been a puzzle for a very long period, until recently, someone may have found a way to halt the progression of HIV to AIDS. The HIV infection basically sends the immune system to overdrive, thus exhausting it, which eventually leads to AIDS. But removing the cholesterol from HIV seems to cripple the virus' ability to over-activate part of the immune system, so it could potentially lead to a vaccine that lets the adaptive immune system attack and destroy the virus - just as it would if HIV was any other pathogen.
Dr. Adriano Bosso, an immunologist from the Imperial College London, describes the 'cure' as follows:
"Think of the immune system as a car. HIV forces the car to stay in first gear, and if you do that too long, the engine is not going to last very long," he said in an interview. "But if we take the cholesterol away, HIV is not capable of attacking the immune system quite as well. Practically, what we've done is turn HIV into a normal jump-start of a car."
Of course, there are drawbacks to this proposal due to lack of evidence on the de-cholesterolised virus as an effective form of vaccine. Cholesterol removal leads to less access of the HIV virus into the dendritic cells, which means there's less of the virus for the cells to detect, which leads them to produce fewer interferons (proteins that allow communication between cells to trigger the protective defenses of the immune system that eradicate pathogens or tumors) and can be potentially harmful to the body. Thus, more research needs to be done on this matter.
Source:
Adriano Boasso, Caroline M Royle, Spyridon Doumazos, Veronica N Aquino, Mara Biasin, Luca Piacentini, Barbara Tavano, Dietmar Fuchs, Francesco Mazzotta, Sergio Lo Caputo, Gene M Shearer, Mario Clerici, and David R Graham
Over-activation of plasmacytoid dendritic cell inhibits anti-viral T-cell responses: a model for HIV immunopathogenesis
Blood 2011 : blood-2011-03-344218v1-blood-2011-03-344218.
Dr. Adriano Bosso, an immunologist from the Imperial College London, describes the 'cure' as follows:
"Think of the immune system as a car. HIV forces the car to stay in first gear, and if you do that too long, the engine is not going to last very long," he said in an interview. "But if we take the cholesterol away, HIV is not capable of attacking the immune system quite as well. Practically, what we've done is turn HIV into a normal jump-start of a car."
Of course, there are drawbacks to this proposal due to lack of evidence on the de-cholesterolised virus as an effective form of vaccine. Cholesterol removal leads to less access of the HIV virus into the dendritic cells, which means there's less of the virus for the cells to detect, which leads them to produce fewer interferons (proteins that allow communication between cells to trigger the protective defenses of the immune system that eradicate pathogens or tumors) and can be potentially harmful to the body. Thus, more research needs to be done on this matter.
Source:
Adriano Boasso, Caroline M Royle, Spyridon Doumazos, Veronica N Aquino, Mara Biasin, Luca Piacentini, Barbara Tavano, Dietmar Fuchs, Francesco Mazzotta, Sergio Lo Caputo, Gene M Shearer, Mario Clerici, and David R Graham
Over-activation of plasmacytoid dendritic cell inhibits anti-viral T-cell responses: a model for HIV immunopathogenesis
Blood 2011 : blood-2011-03-344218v1-blood-2011-03-344218.
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