Physiology physics woven fine

Studying Genes the Ophthalmic Route by MRI, and That too in Living Subjects

2010-09-19T22:28:00.005+05:30

It is said that the eyes are the windows to the soul, though science is yet to prove that given the elusive nature of ‘soul’. But researchers has now been able to probe genes in a traumatized brain using the eyes as a gateway.

The brain is normally ‘secured’ from the circulating blood directly, so that endogenous and exogenous toxic substances, macromolecules can not gain entry easily into (and out of) the brain. More importantly, this ‘firewall’ like barrier, called the ‘blood brain barrier’ maintains the constancy of ions inside the brain such as K+, H+, Mg++, Ca++, which is vitally important for the neurons to function normally.

The ‘blood brain barrier’ (BBB: see picture) results from the ‘relative’ impermeability of both the capillaries supplying the brain as well as that of the ‘choroid plexus’ covering the brain. Actually, the endothelial cells of the capillaries are tightly packed (tight junctions) and they are non-fenestrated too. In addition, end feet of astrocytes, a type of glial cells (cells that support and aid neurons), cover these capillaries.

But there are disease conditions in which the BBB becomes leaky. For example, in traumatic brain injury, cardiac arrest, stroke and multiple sclerosis the blood brain barrier is breached, to different extents. In Alzheimer’s disease too, there is thinning of the capillaries as the disease progresses. As expected, the supporting glial cells, particularly the astrocytes, jump into action to seal the leaks. They proliferate, resulting in ‘gliosis’. Gliosis is also found in a tumorous condition of the glial cells called ‘glioma’.

These glial cells contain a protein in them called the glial fibrillary acidic protein (GFAP). Naturally, there is an mRNA for it that ‘translates’ its formation in the cytoplasm. Scientists target this mRNA molecule because tagging it will track the GFAP and consequently the astrocytes in whom GFAP is expressed.

Previously scientists had to inject MR contrast agents intra-cerebro-ventricularly or by other invasive techniques to map these leaking areas. Scientists at Harvard embarked on a novel idea. They produced a short cDNA sequence ‘complementary’ to the mRNA of GFAP. This short stretch of this ‘antisense’ oligodeoxynucleotide (ODN-gfap) would latch onto the GFAP mRNA just as a lock would to its key. They then tagged it with a paramagnetic molecule that they designed, called superparamagnetic iron oxide nanoparticles or SPION, a magnetic resonance susceptibility contrast agent. The SPION-ODN ‘report’ any inhomogeneity in transverse magnetization in ‘T2 star’weighted MRI scan, due to the paramagnetic properties of iron oxide. Liu et al also used a sequence complementary to the mRNA of beta-actin as well (actin is the most abundant protein in mammalian cells and its mRNA is found in all types of cells) to act as a ‘control probe’.

They then anesthetized the mice, the animal model they selected; and caused BBB leakage by inflicting a small puncture or by performing bilateral carotid artery occlusion (BCAO) for 60 minutes. They also tried other methods (see reference). They subjected another group of mice to a sham (=false) operation (no puncture or vessel occlusion but the same operation) at the same time. BBB leakage was checked by T1 weighted Gadolinium-DTPA contrast MRI scan. Gd-DTPA was injected into the jugular veins of the mice. Leakage would show up as enhanced areas on T1 weighted scan (normally Gd-DTPA does not cross the BBB). Due to repair process to seal the leak, glial cells would be recruited and gliosis would result.

The telltale signature of gliosis (and BBB breach) may be found in postmortem tissue samples of the brain. Previously, the GFAP antigen was detected by immunohistochemical methods. But the Harvard team was looking for a non invasive method to detect GFAP. They instilled ‘SPION-ODN gfap’ reporter into the conjunctival sac of the mice by means of eyedrops. They then measured the ‘T2 star’ values in MRI scan and transformed the values to ‘R2 star’ maps (R2 star = 1/T2 star). Areas of leakage showed up as elevated (hyperintense) signals in R2 star maps. It corroborated well with Gd-DTPA scans and also on post mortem examination. SPION-beta actin, the control probe, got bound to the endothelial cells of the vasculature as expected.

The eye drop was absorbed by the lymphatics draining the palpebral (eyelid) and bulbar conjunctiva. The lymphatics then transferred the reporter probe into the veins which finally found their way into the brain. Since the BBB was breached, it finally came out of the circulation into the brain parenchyma. As the probe is detecting mRNA which is ‘transcribed’ from the DNA of the cell, it may be said that they are, in a sense, detecting the genes for GFAP.

Thus we may hope to detect gliosis, a pathology that occurs in a variety of diseases already mentioned, non invasively, the ophthalmic way.

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Reference: Liu, C., You, Z., Ren, J., Kim, Y., Eikermann-Haerter, K., & Liu, P. (2007). Noninvasive delivery of gene targeting probes to live brains for transcription MRI The FASEB Journal, 22 (4), 1193-1203 DOI: 10.1096/fj.07-9557com

fMRI, BOLD and the Beautiful

2010-09-11T01:34:00.006+05:30

When we want to examine the brain of a person noninvasively by Computed Tomography (CT) or MRI, we get a ‘snapshot’ of the anatomy (or pathology, if any) of the subject’s brain. We are however clueless as to its functional aspect. fMRI or Functional Magnetic Resonant Imaging allows us to do just that. The difference is not unlike a ‘still picture’ versus a ‘video of a moving train’. PET scans, previously described, also can asses the functional state of the brain.

Whenever we do a task, think, dream, memorize, speak or see things, the brain is not activated as a whole; but only certain portions of it are activated. Activation, here, means increased metabolic activity of neurons in certain areas of the brain. Naturally, these ‘metabolically active’ neurons would demand more energy which would power them. The blood supply to these areas increases as a result of this metabolically driven vasodilation. The arteries then bring in glucose and oxygen with them, with Oxygen being transported in the form of Oxyhemoglobin (oxygenated hemoglobin or HbO2). Neurons on the other hand use up the oxygen contained in the blood, thereby reducing it to de-oxyhemoglobin or simply Hb. However, the alteration in tissue perfusion exceeds the extraction of oxygen by the neurons, so the concentration of deoxyhemoglobin within ‘the areas’ decreases. This causes molecular inhomogeneities in the magnetic field.

Oxyhemoglobin is diamagnetic, meaning that they align perpendicularly to magnetic field lines. On the other hand, deoxyhemoglobin is paramagnetic, i.e. it aligns parallely and proportinately with the intensity of the magnetic field. This causes the inhomogeneity within the magnetic field (magnetic susceptibility) in the tissue sampled. This inhomogeneity is exploited in fMRI in terms of decay of transverse magnetization, T2*, with longer T2* values in HbO2 blood and shorter values in Hb (paramagnetic) blood.Since this stems from the oxygen content in blood, fMRI is also known as the BOLD ((blood oxygenation level dependent) effect.

The machine is essentially the same as the MRI machine with echo planar imaging technology that permits faster imaging due to faster gradient switching, improved algorithm and faster CPU processing power. The patient/subject is placed inside the magnetic chamber and MRI signals are acquired, Fourier transformed and corrected for artifacts. Finally the computer reconstructs a 3D fMRI image out of this.

As is obvious, we can learn about the motor areas of a patient by asking him to grasp an object or giving him any motor task and noticing which area(s) of the brain lights up. A neurosurgeon can then be cautious about not hurting these areas. Similarly, the mapping will help spare motor and other vital areas like auditory, visual and language areas from damage in radiotherapy procedures, in addition to neurosurgery. It can also detect occult Alzheimer’s disease and cognitive deficits including those of the autism spectrum and dyslexia (reading disorder).

fMRI can also be employed to ‘read peoples’ minds’, thoughts, intentions including lie detection. Watch the video below which explains how an FMRI scan is done and interpreted. Thus the legal and forensic implications are obvious. However, in fMRI, correlation doesn't always mean causation. Whatever it may be, it seems that fMRI is very much here to stay, both in the clinics as well as in cognitive neuroscience research. It may also be combined with tractography, MRI or other diagnostic radiologic modalities.

Hardenbergh et al combined Tractography techniques with fMRI, using a technique capable of rendering multiple color-coded functional activation volumes and fiber tract bundles. Many pharmacologically active drugs have effect on memory impairment, which can be seen in ‘telltale’ fMRI scans. Sperling et al studied the effects of lorazepam (a benzodiazepine) and scopolamine (an anticholinergic drug once used as ‘truth serum’ by the CIA) on healthy volunteers and found that they did impair memory and their functional coordinates could be reproducively mapped on fMRI scans (see figure on the left). I still shudder at the thought of what happened during my PG exam when I took a benzodiazepine.

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Reference: Integrated 3D Visualization of fMRI and DTI tractography
Gore, J. (2003). Principles and practice of functional MRI of the human brain Journal of Clinical Investigation, 112 (1), 4-9 DOI: 10.1172/JCI200319010

The World of Tractography Where The White Matter Tracts Appear Colored

2010-09-07T23:47:00.012+05:30

The Central Nervous System (CNS) communicates with the exterior (gets visual, tactile informations etc. on the one hand; and performs limb movement, posture regulation etc. on the other) via the peripheral (somatic) nervous system. It also connects with the interior (our viscera or organs) via the Autonomic Nervous System. That is, it does its job in a bidirectional way: by the motor or the actuator arm, and sensory or the receptor arm. For such ‘actions’ to occur, cables of nerve fibers are laid within our body. Wouldn’t it be nice if we could visualize them, their dispositional anatomy or any pathology that could afflict them?

The brain and spinal cord constitute the CNS. We also know that there are about a hundred billion neurons in the CNS. Each neuron has a cell body (soma), an axon wrapped by myelin, and many dendrites. (See figure). It is the axon that carries the information in the form of action potential. These cables (bunch of axons, called tracts) are not laid haphazardly. Nature tries to conserve space, minimum length, conserve energy and so on and the axons form into tracts. They run up and down (also front-back and sideways) the cord to the brain; the organ we will now concentrate upon.

Your electrical wiring to your ceiling fan would include a switch and the fan itself. The wire (cable) from the switch would ascend vertically up the wall, make a 90 degree angle, and then reach the fan horizontally up in the ceiling. Likewise, in our brain, which is made up of two hemispheres, would connect. Three broad fiber types may be visualized: from one hemisphere to the other (commisural fibers), restricted to one hemisphere (front to back or antero-posteriorly are association fibers) and finally vertically (up down orientation go the projection fibers).

Exploring the tracts can now be done in live animals including humans. Improvement in MRI technology has enabled us to see the tracts (tractography). Improved gradient coils, faster processors and superior software have shortened the scanning time, thereby reducing blur due to organ movement (e.g. diaphragm) and patient movement. This procedure called Echo Planar Imaging (EPI) has given birth to Functional Magnetic Resonance Imaging (fMRI), Diffusion Weighted Imaging (DWI), tractography and many other diagnostic and research procedures.

Consider the neuron shown in the above picture. Water molecules in the axon (yellow) are constantly in Brownian (random) motion due to thermal energy within. Hence they tend to diffuse. In most of the cerebrospinal fluid spaces these microscopic motions are equal in all directions. This is called isotropic diffusion. But in myelinated neurons, as in the white matter fiber tracts, water motion is constrained due to the fatty nature of the myelin sheath (in blue) which hinders water flow across it. This anisotropic diffusion allows more water molecules along (parallel) the direction of the nerve fiber. The apparent diffusion coefficient (ADC) is thus more along the nerve fiber. Diffusion Weighted Imaging (DWI) can capture this microscopic water flow and delineate anatomically the orientation of nerve fiber tracts.

Pyogenic abscesses hinder diffusion by virtue of their increased viscosity. In the early stages of acute cerebral infarction there is reduced diffusion too, giving rise to high signal intensity. However, in most pathologies of the brain the ADC is increased. Diffusion Tensor Imaging (DTI), a diffusion MRI technology, tracks fiber orientation by assigning values in ellipsoid voxels (VOlume piXEL). Ellipsoid because unlike isotropic diffusion where molecules diffuse equally in all directions, the anisotropy in the white matter tracts does not permit them to move with equal ease in all directions, and hence the pattern is that of an ellipsoid and not a sphere. By connecting the long axes of all the ellipsoids, the trajectory is deduced.

Colors are added to it with respect to the three principal axes (x, y and z) and the result is a stunning tractography! (see left)

The YouTube video below describes the colorful realms of tractography and how they are used.

When fMRI and DTI are combined together, a whole new world emerges. But, I prefer to keep it on hold till I discuss Functional Magnetic Resonance Imaging.

NB: The picture of the neuron shown is not representative of CNS neurons. Oligodendrocytes that form myelin in the CNS does not encircle so many times as the Schwann cells in peripheral neurons do. It has been represented thus for the sake of clarity.

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Reference:Diffusion Tensor Tractography: Exploring the Cost-Benefit Ratio of Incorporating CSF Suppression into Fiber Tracing Algorithms

William P. Dillon. Neuroimaging in Neurologic Disorders. In: Harrison's Principles of Internal Medicine, 17th Ed., Volume 2, McGraw Hill; 2008. p. 2491-2497.

P. Mukherjee,, J.I. Berman,, S.W. Chung,, C.P. Hess, & R.G. Henry (2008). Diffusion Tensor MR Imaging and Fiber Tractography: Theoretic Underpinnings AM J Neuroradiol DOI: 10.3174/ajnr.A1051

Relaxation in the Nuclear Microcosm

2010-07-21T21:17:00.012+05:30

All of us want to give themselves a hard earned ‘rest’ after a “hard day’s night”, don’t we? So do the protons, perturbed by the destabilizing magnetic component of the radio-frequency pulse [which previously ‘happily’ aligned themselves to the externally applied magnetic field; one way (parallel) or the other (antiparallel)] applied at the Larmor frequency. It is like slapping an individual in a “merry go round” each time he came near a person who is paid just for slapping that person. But, when we call ‘spin’, we do not mean ‘spin’ the way we see them in a classical world. [We’ve given various names to the ‘quarks’: up, down, strange, bottom etc. depending on ‘something’ called ‘flavor’; and red, green and blue depending on ‘something’ called ‘color’.However, spin, flavor, color etc.‘in the quantum world’ have ‘no relevance’ to what we usually attribute to them in our everyday life. Things are a bit crazy in the quantum world, but I will take recourse to some ‘classical world’ analogies to make the description lucid.]

Thus, the already aligned nuclei (parallel or anti-parallel to the applied steady external magnetic field B0), has now been perturbed owing to the ‘knocking’ by the ‘magnetic component’ (B1) of the electromagnetic RF pulse. The nuclei gain energy and sway away from the perpendicular to the horizontal (90 degree) depending on how long the RF pulse is applied. So, now the nuclei behave like ‘punch-drunk’( like a person who’s been reeling due to a strong blow to the head!). Remember, that this new angular momentum is also a vector quantity having magnitude and direction. It can be resolved in terms of a horizontal component (Mxy) and a vertical component Mz. Anyway, the proton does recover from this situation, after some time, once the external RF field has stopped. Typically, Mxy component decays faster than the recovery of Mz.

The excited proton recovers in two ways and both forms occur simultaneously: (1) The excited nuclei which now have been ‘forced’ to lie horizontally (90 degree), ‘re-align’ themselves back to their ‘original position’ as they were before the RF pulse (perpendicularly towards the field of externally applied field B0); and (2) the energized protons dissipate their energies to the surrounding nuclei (horizontally) at their level. The first example, obviously, is called the (spin-lattice, or longitudinal) relaxation; while the second one, transverse relaxation (T2). There is little energy loss due to RF emission.

T1 relaxation, also known as, longitudinal relaxation or spin-lattice relaxation can be best understood if you see the following Youtube video. [The spiral trajectory, in this case, reminds me of the laser experiment I did to satisfy my lesser friends. Analogically, the trajectory would be such, if the power supply were switched off.] In T1 relaxation, the proton loses energy to the surrounding lattice, by interacting with nuclei in the lattice which are in vibrational, translational and rotational motion. Clearly, the surrounding nuclei (lattice) having the same (or nearly same) Larmor frequency will efficiently absorb energy of the excited proton, resulting in a tiny rise of temperature.

T2 relaxation (transverse or spin-spin relaxation) on the other hand, does not involve exchange of energy with the lattice.
The magnetic moments of the protons merely changes phase. Here, the nuclei exchange “quantum states” (kind of, what Einstein called ‘spooky action at a distance’): an excited nucleus (proton) will transfer its energy and relax, while the neighboring nucleus in the lower energy state that absorbs it becomes excited. This loss of phase coherence of spins can be clearly seen in this beautiful video.

It can be understood easily that T1 and T2 values would depend on the surrounding molecular environment (tissues, for example). Hence, the values differ in different tissues. Again, since Mxy decays faster, as described, it may be understood why T1 is greater than T2 (usually, T1=5T2). Both T1 and T2 contribute toward contrast in tissues. T1 relaxation time is the time needed for 63% of protons to return to their previous equilibrium state. Likewise, T2 relaxation time is the time needed for 63% of protons to become dephased owing to their interaction with nearby protons. The contrast, naturally depends on the water content of the tissues. Grey matter has about 10% more water than white matter and this creates a contrast. We can also create contrast by varying TR and TE times.

TR (Repetition Time) refers to the time gap at which consecutive RF pulses are applied; while TE (Echo Time) refers to the time delay between the applied RF pulse and its reception (echo). T1 weighted images (T1W) are produced by keeping TR and TE relatively short, while T2 weighted images (T2W) are produced by keeping TR and TE relatively long. Water molecules being relatively light spins much faster than the Larmor frequency, making energy transfer rather tough (exchanging of packets of energy becomes more efficient as the relative angular velocity narrows). Consequently, water has a long T1 time. Proteins and nucleic acids being rather heavy, spin slowly. They also have problem with energy exchanging, and thus have a long T1. Cholesterol, a medium sized molecule, precesses near the Larmor frequency, efficiently absorbing the energy and giving a small T1 value.Thus (fat) liquid cholesterol in craniopharyngiomas, a benign tumor, appears bright on T1W images (T1 being small, the rate at which RF energy is released is fast. Hence, the signal intensity in NMR is high).

Subacute hemorrhage also has shorter T1, due to the presence of paramagnetic iron in methemoglobin present in the tissue, hence high signal intensity. Cerebrospinal fluid (CSF), edema (collection of fluid in tissue space or ECF) having more water content have both long T1 & T2 relaxation time. They give low signal intensity in T1 (dark) but higher signal intensity (bright) in T2W images. T2W images are superior to their T1 counterparts in case of infarction, edema, demyelination etc. Contrast agents like the heavy metal Gadolinium, a paramagnetic substance, has been used to reduce both T1 and T2 times by introducing inhomogeneity in the magnetic field. Gadolinium is complexed (chelated) with a substance called DTPA to prevent toxic build-up inside body tissues. This gives high signal in T1W but a low signal in T2W. It (the complex) does not cross the blood brain barrier (BBB); but disruption in the BBB or parts of the brain where it is deficient (circumventricular organs), take-up the substance and affects relaxation properties.

Below is an MRI showing changes in Subacute Sclerosing Panencephalitis, a complication of measles. Note: Panels A and C are T1-weighted images; B and D are T2-weighted images. The hypointense (darker) signal on the T1-weighted image (arrow in A) and a hyperintense (bright) signal on the T2-weighted image (arrow in B) can be clearly seen.

Given all these, it can be said that relaxation parameters of nuclei have enabled us in visualizing biological tissues nonivasively, identifying chemicals spectroscopically and a lot more as we shall see later.

References: Magnetic Resonance Imaging: David D. Stark, William. G. Bradley, Jr.

NMR spectroscopy
Magnetic Resonance Imaging
Principles of NMR
William P. Dillon. Neuroimaging in Neurologic Disorders. In: Harrison's Principles of Internal Medicine, 17th Ed., Volume 2, McGraw Hill; 2008. p. 2491-2497.
Ian L. Pykett, Ph.D., Jeffrey H. Newhouse, M.D., Ferdinando S. Buonanno, M.D., Thomas J. Brady, M.D., Mark R. Goldman, M.D., J. Philip Kistler, M.D., & Gerald M. Pohost, M.D. (1982). Principles of Nuclear Magnetic Resonance Imaging Radiology
Time constants

Last Modified: Aug 19, 2010

Understanding the Basic Principles of Nuclear Magnetic Resonance Imaging

2010-06-24T02:41:00.092+05:30

Nuclear Magnetic Resonance Imaging (NMRI), better known as Magnetic Resonance Imaging (MRI) in medical parlance, is an invaluable tool in the study of the neurological system, soft tissue and musculo-skeletal system disorders. The word “Nuclear” was intentionally dropped later, as the procedure could then be wrongly interpreted by patients in relation to “ionizing radiation”, which certainly is not the case. However, the term Nuclear Magnetic Resonance (NMR) continues to be used in other (non-medical) fields of science, such as analytical chemistry, physics, biochemistry, petroleum industry, analysis of biological samples etc. In either case, the procedure and the basic principles remain the same. Paul Lauterbur was one of the pioneering inventors of this seemingly tough technological field.

Matter is made up of atoms, which in turn, are composed of negatively charged electrons orbiting around the nucleus (look at the animation of a Helium atom on the left), consisting of positively charged protons and charge-less neutrons (with the exception of Hydrogen 1H nucleus, which contains a single proton and no neutron). These subatomic particles (electron, proton etc) somehow, can not be understood in terms of shape or color; instead they are denoted by their charge, mass or spin (angular momentum). An even number of them will cancel each other’s spin [just like two revolving spheres, in touch with each other would in a ‘classical world’ (if one rotated clockwise, the other would anticlockwise canceling any resultant spin)].

Hence, a net resultant spin would result in the nucleus only if it contained an odd (unpaired) number of protons, an odd number of neutron or both. [The concept that certain nuclear species had angular momentum was first suggested by Wolfgang Pauli, while explaining the fine structures in the Atomic spectra. In the presence of an external magnetic field, the spectral lines got split, depending on the strength of the field (Zeeman Effect).]

Since nucleons bear a net charge (owing to the protons contained), the spinning nuclei will generate a magnetic field (since moving charges generate magnetic field). Each of these charged spinning ‘spheres’, hence, may be thought of as a tiny bar magnet having a magnetic dipole (that is a north-south orientation). [Electrons, similarly, have their own angular momentum though, responsible for molecular structure which nature uses, but they are not used by humans (Milestones in Spin podcast)] When we talk about “MRI” in humans, we mean proton nuclear magnetic resonance; i.e. NMR that detects the presence of hydrogen (proton) nuclei.

Our bodies have a plentiful of Hydrogen atoms: from the water within us (and less commonly) to the adipose tissue (fat). These charge-carrying ‘unpaired’ protons (Hydrogen nuclei) rotate around their axes, but since all are spinning in a random fashion (as there’s no coordinator of any sorts); their net spin is zero, or in other words, their net magnetic moment is zero (as shown on the left).

Understanding spins aren’t easy either. But, Prof. Stephen Hawking made it quite simpler for us using the real classical world analogy of ‘playing cards’ in his famous book A Brief History of Time (follow the link to learn more about ‘spin’). Having said that, the unpaired, positively charged protons having half integer (1/2) spins, behave like magnetic dipoles; it may now be understood easily that the spinning protons (nuclei) would align themselves to an externally applied magnetic field.

Thus, in a static magnetic field, the randomly oriented ‘tiny bar magnets’ align themselves up according to the applied magnetic field. These spinning protons (nuclei) also precess (make an angle) with the applied magnetic field (Bo). An animation of a proton precessing around a field is shown on the right.

[The magnet used for this purpose employs superconductivity. In a superconductive magnet, the electromagnet coils are immersed in liquid Helium at minus 269 degree Centigrade. At such a low temperature, the coils loose ‘resistance’ to the flow of electrons, resulting in a highly stable and a very strong magnet. (However, any minute vibration in the superconducting magnet can lead to runaway Eddy current leading to a phenomenon called 'quenching', that happened in the Large Hadron Collider at CERN.) Normally, 1.5 Tesla magnets are used, though nowadays 7 Tesla magnets have arrived. A 1 Tesla (1 Tesla=10,000 Gauss) magnet is 20,000 times stronger than the earth’s magnetic field)]. Also, note that we are considering magnetic moments along the axis of the external field only, as far as the sum-total alignment of individual magnetic torque contributing to a 'macroscopic' magnetization (M) is concerned. This is because the transverse components of the individual spins cancel out, as is seen in the 'cone' of the above picture. 600 persons of equal power, each pulling a rope either 30 degrees Northwest or 30 degree Northeast (in a 2 dimension), will certainly cancel out the 'east-west' vector, while the Northward vector will add-up. [It is this M that produces the induction current in the receiver coil].

The protons have two choices. Either they have to align parallel or anti-parallel to the applied magnetic field (known as spin-up and spin-down position respectively). In any case, the protons only ‘partially polarize’ since they tend to ‘make an angle’ with the applied static magnetic field. Spin down position is the higher energy state while spin-up state is the lower energy state of the spinning protons (in the case of 23Na, there can be 4 spin-states instead of 2 as in 1H!). (Obviously, a swimmer swimming upstream has more energy than his antiparallel counterpart.) The protons revolve (precess) around the direction of the magnetic field (Bo) at an angle, while at the same time they rotate around their own axis. Just as what happens in the solar system. [However, the upper (-1/2) and lower energy (+1/2) spin states are almost equally populated with only a very small excess in the lower energy state at room temperature. Since, there are so many of them that we finally make some headway].

Let me clarify a bit. You’ve seen a spinning-top rotating around its own axis. Due to Earth’s gravitational field, the top ‘maintains’ an angle (with the perpendicular/vertical), more visible when its angular momentum (speed) decreases, as it continues spinning. The top may be seen to revolve around “the perpendicular” at an angle (=‘precess’), (in addition to its rotation around its “own axis”) during its course of revolution. [Watch the Video "Introductory NMR & MRI Video 01 Precession and Resonance" to see what precession & NMR is]. This is what precession is about.

The frequency of precession is given by the Larmor relationship:
f=w/2*pi=yBo/2*pi (2*pi=360 degree)
w=angular freq. in radians per second; since there are 2*pi radians (360 degree) in a circle; we can find f, the frequency of rotation.
y is the magnetogyric (gyromagnetic) ratio, nuclear constant characteristic of every isotope. For 1H it is 42.5 MHz/T;
Bo=static magnetic field

The above equation is important, as we shall see later. Now let’s summarize what we learned so far.
Protons (nuclei) spin randomly in an atom. They tend to align with respect to an external magnetic field. These protons make an angle with the magnetic field as it goes about the magnetic field (while it also dutifully goes around itself), some parallel, and some antiparallel.

In MRI, our objective will be to disturb this alignment of protons with a dose of radio frequency pulse, in a similar way I discussed in my radio transmitter article but in a much, much bigger way. But since the ‘target’ (proton) is moving (precessing) around the field, we better ‘punched’ the target as if we were moving at the same angular velocity (so that the relative velocity was zero). Thus, when we apply the RF frequency pulses at the Larmor frequency, perpendicular to the magnetic field; the magnetic component (B1) of this electromagnetic wave temporarily knocks the protons out of alignment (see picture). If energy is absorbed by the nucleus, then the angle of precession will change. Assuming the field strength to be 1 Tesla, the protons are revolving 42.5 million times per second; it is at this frequency we give the pulse (i.e. at the Larmor frequency).

The protons are pushed out of alignment and as the pulse ends, they ‘relax’ (more on how they ‘relax’, later) back to their undisturbed ‘equilibrium’ position. This causes emission of an RF signal (the Echo) that can be picked up by the receiver coil (the same transmitter coil that produced it, in most cases); a damped oscillating wave generated, as the ‘disturbed’ magnetic moments coming back to realign with the magnetic field. Now, the problem begins. We have applied a uniform/homogeneous magnetic field (Bo). There are a lot of protons but we don’t know who’s who and residing where. That is why we also apply orthogonal magnetic field ‘gradients’ along the three (x, y, z) axes. [In a classroom, spray gradually ‘more’ yellow color in the front row and to the left than the back and to the right. In a similar way, spray blue color; hope your students don’t object. Now, every one of your students has a unique color: yellow, blue or green and with different hues]

Now that we get a decaying signal, which of high frequency; we mix it with a low frequency signal, in much the similar way as in heterodyning, to produce an ‘interferogram’. This interference map is digitized, which is called the Free Induction Decay (FID). Thus, we do find too many frequencies in ‘the low frequency map’ which occur in ‘almost’ the same time. So, what can we do?

Waka Waka! In this football World Cup 2010 at South Africa, audience seems to have a deafening organ, what they call ‘vuvuzela’. How are we going to analyze so many vuvuzelas when they are blowing at the ‘same time’? Just plot them in ‘frequency domain’ instead of ‘time domain’. Here’s Discrete Fourier Transform (DFT) which will do happily for you. [Simply, it samples the different frequencies and plots them; not all vuvuzelas have the same frequency]

Now, that fuzzy picture of multiple frequencies has a 'spatial information' (owing to its orthogonal gradient magnetic field), contrast information (due to its ‘relaxation’ parameter), and foremost that it can be analyzed visually by humans, have enabled MRI to be a indispensable tool for the medical professional, as much as NMR has to the physicist or the discerning chemist. In MRI (NMR) it is not that important where or how energy is absorbed, but how quickly the excited protons revert back to its previous position is much more important, and hence the relevance of T1 and T2 relaxation times.

By the way, contrast depends on the t1 and t2 relaxation, the surrounding chemical environment affecting relaxation, and of course the water content of the tissues [gray matter contains 10-15% more water than white matter.]

Finally, the article wouldn't be resourceful enough if I do not post some MRI scans of the brain, this time, that of an epileptic patient (below).

(A sagittal section is obtained as the 'slice' takes a 'left to right' view (and vice versa); a coronal section means a 'front to back' view (or vice versa), and an axial slice means a virtual transverse section through the head.) Here's the picture of an actual MRI Machine below:

Naturally, the small tunnel may induce claustrophobia; the whirring acoustic noise from switched gradient coils may be troublesome to the patient; any implanted pacemaker may be subjected to interference from the electrical field resulting in dislodgement or malfunction (as in other ferromagnetic objects such as wrist watch, key rings etc.). Moreover, sudden movement by the patient may induce voltage in semicircular canal producing vertigo, a sensation of giddiness. Advances in MRI technology is happening fast. Claustrophobia may now be ameliorated with a wide bore MRI. A newly developed MRI scanner with Total Imaging Matrix (TIM) technology patients don't feel as claustrophobic, the imaging time is quick, quality of picture is better and even the acoustic noise is less (watch the YouTube video here). Whatever be the shortcomings of MRI, the benefits far outweigh the risks and it is here to stay and evolve.

References:
Magnetic Resonance Imaging: David D. Stark, William. G. Bradley, Jr.
NMR spectroscopy
Magnetic Resonance Imaging
Principles of NMR
William P. Dillon. Neuroimaging in Neurologic Disorders. In: Harrison's Principles of Internal Medicine, 17th Ed., Volume 2, McGraw Hill; 2008. p. 2491-2497.
Ian L. Pykett, Ph.D., Jeffrey H. Newhouse, M.D., Ferdinando S. Buonanno, M.D., Thomas J. Brady, M.D., Mark R. Goldman, M.D., J. Philip Kistler, M.D., & Gerald M. Pohost, M.D. (1982). Principles of Nuclear MagneticResonance Imaging Radiology
P.S. We will discuss T1 and T2 relaxation, fMRI, tractography and NMR spectroscopy later.

Created: Jun 24, 2010; Last modified: Jul 21, 2010

Mobile Phones' Impact on Health

2010-02-09T23:21:00.013+05:30

Mobile phones have drastically transformed our lives. Also known as cellular phones or cell phones, these gadgets not only incorporate a phone, as the name suggests, but also a lot of other technologically advanced features. They include a camera, a sound recorder cum music system, a Bluetooth device and many more depending on the model and the maker of the phone. They are called mobile phones since they can be used while on the move.

A mobile phone maintains a two way (transmit and receive) communication with the nearby tower within a cell. Even when you are not talking on your mobile, it is constantly in touch with its ‘cell’. A cell may be thought of as the operational unit of a ‘base station’. A city or area may be likened to a bee hive, each hexagon representing a ‘cell’ having its own tower. As you move from one honeycomb to other, your mobile will change contact from one tower to another (another cell).

Cell phones radiate high frequency (hence, microwave, as wavelength is inversely proportional to the frequency) electromagnetic radiation as a means of communication. One could easily demonstrate this electromagnetic emanation by putting a mobile flashing sticker close to a phone when it’s being used. This radiation can pierce our body tissues, particularly the head. But they could also microwave our scrotum if we keep them inside our pant pocket, as the phone is constantly in touch with the tower and emitting radiation unceasingly.

The rapidly alternating electromagnetic field makes the polar molecules in our body move back and forth, as a tiny magnetic compass would move if the external magnetic field was allowed to change. This molecular movement results in heating of the tissues. Scientists were curious if this could harm us.

Previously, it was thought that they could cause brain cancer but it was later found out that there was no significant relationship. There were some unconfirmed reports suggesting an association between mobile phone usage and an increase in the incidence of acoustic neuroma, a benign tumor of auditory nerve. The thermal effects arising out of the to-and fro effects of the polar molecules could give rise to the increased production of a class of proteins, called ‘heat shock proteins’ or stress proteins.

Some drugs (like glucocorticoids, estrogen and progesterone) enter inside the cells where they combine with molecules called receptors, in the cytosol. This drug-receptor complex then translocates to the nucleus and commands the DNA into producing protein molecules by transcription. The resulting proteins typically account for the actions of these steroidal drugs. Heat shock proteins (like Hsp90) cover the DNA binding domain of the cytosolic receptors, preventing interaction with the DNA. When a steroid molecule attaches with the receptor, a conformational change occurs in the receptor releasing the Hsp, thereby freeing the DNA binding domain. Naturally, more stress proteins would mean more blocking of steroid receptors.

Studies have also shown that microwave radiation at doses considered harmless caused DNA damage after two hours of exposure. All these led authorities in some countries advice Bluetooth usage and to keep your head away from your mobile. Read the next few lines if you really should keep your head away!

Having said all those, let me state that the WHO, the American Cancer Society and the National Institute of Health have concluded that there was no scientific evidence that cell phone use had any adverse health risks.

A University of South Florida research team wanted to find out any association of Alzheimer’s disease with cell phone usage. In the past, several studies have hinted at a possible increased risk of Alzheimer’s disease in humans with low frequency electromagnetic radiation, such mains power line frequency. But as they went on with their research, they were surprised at what they saw.

They employed about 100 mice and subjected them to a daily radiation of 1 hour by an antenna that was kept in the center of the cage as shown. Some of these mice were 2 month old, which were genetically programmed to develop Alzheimer's disease like symptoms and signs with age; and some 4 month old, which already had the symptoms. They also placed normal healthy mice in the same cage. The electromagnetic field was made to emulate the radiation received by a man as he talked on his mobile phone and the wavelength was the same as that of the mobile phones. The mice’s memory was checked by maze tests.

They were astonished to find that the electromagnetic field (EMF) not only boosted memory in both healthy and transgenic mice (compared to other mice who did not receive radiation) but also they actually reversed symptoms of Alzheimer’s. EMF seemed to break up tell-tale beta-amyloid plaques, a histopathologic marker of Alzheimer’s disease in mice which already expressed them. There was no evidence of increased tumour/cancer formation, DNA damage or behavioral changes.

While what exactly cleared the plaques was not certain, but to quote Gary Arendash of the University of Southern Florida: "One thing is clear, however -- the cognitive benefits of long-term electromagnetic exposure are real”. Arendash also wondered if the preferential use of one of our ears in holding the phone could have asymmetric outcomes in the brain in terms of the plaques. He also observes: "It might also be useful in traumatic brain injury, which is also characterised by plaques, or just to improve cognitive performance”

While it’s too premature to use your phone too close to your head to get a boost in your exams in the near future, its possible use as a “nootropic” is certainly encouraging.

Mobile phones have a lot of other usable paraphernalia, some of which I pointed earlier. These features and the availability of inexpensive high-efficiency light emitting diodes (LEDs) inspired Breslauer et al to construct a microscope that would be helpful in developing countries.

They have developed a high-resolution microscope attachment that is meant for camera- phones (picture on the left; click to enlarge). Their microscope can capture colour images of the malignant malaria causing parasite Plasmodium falciparum, red blood cells sickling in peripheral blood smear in homozygous sickle cell anemia (hemoglobin SS ) using brightfield microscopy. When fluorescence microscopy was performed with the sputum of tuberculosis patients using Auramine-O stain, the device captured Mycobacterium tuberculosis as well. The resolution was sufficient for the identification of single TB bacterium. The contraption was also good enough to highlight the rod shaped morphology of the acid-fast aerobic bacteria. In addition, epidemiological studies could be easily performed given that the individual mobile cells had their own identification codes and was under GPS location monitoring.

Thus, we can create a cheap and efficient brightfield and fluorescent microscope out of a simple mobile phone (they used Nokia N73 camera phones, equipped with a 3.2 megapixel CMOS camera) and some easy to obtain components. To end up, there seems to be more to cheer than fear.

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References:
Gary W. Arendash, Juan Sanchez-Ramos, Takashi Mori, Malgorzata Mamcar, Xiaoyang Lin, Melissa Runfeldt, Li Wang, Guixin Zhang, Vasyl Sava, Jun Tan, & Chuanhai Cao (2010). Electromagnetic Field Treatment Protects Against and Reverses Cognitive Impairment in Alzheimer's Disease Mice Journal of Alzheimer's Disease, 19, 191-210

Breslauer DN, Maamari RN, Switz NA, Lam WA, Fletcher DA (2009) Mobile Phone Based Clinical Microscopy for Global Health Applications. PLoS ONE 4(7): e6320. doi:10.1371/journal.pone.0006320

Cell Phone Radiation Reverses Alzheimer's and Boosts Memory in Mice

Cellphone radiation is good for Alzheimer's mice

Mirror Neurons: Resonant Circuitry in Brain?

2010-01-18T21:49:00.010+05:30

Back in the time of the “black and white” motion picture days, when “talkies” weren’t even born, we still could make out the essence of what Charlie Chaplin had to “say”. We understood his unspoken words, courtesy a system of neuronal networking, called the mirror neuron system. Another example: you observe a man kissing ‘his’ girlfriend, ‘your’ neuronal network that would otherwise activate when you ‘actually’ kissed her, would fire! Mirror neurons are at work. Seems to me a bit like ‘mechanical resonance’, where the string of a guitar resonates (vibrates at the fundamental or overtone frequency of its chord's natural frequency of vibration) when a second guitar/chord is strummed nearby.

It all began with the experiment led by Giacomo Rizzolatti, a neuroscientist at the University of Parma. His team wanted to locate regions in the brain which controlled hand and mouth actions in monkeys, such as grasping or licking of an object. So, they had placed electrodes in the ventral premotor cortex, a part of the brain, [see fig] of a macaque monkey with the hope that whenever ‘that part’ of the brain were activated, the electrode would activate an electronic circuitry and give an audible beep. But all hell broke loose when a student entered the lab with an ice cream in his hand. Every time he was raising the ice cream to his lips, the system responded with a beep! Thus, although the monkey wasn’t having the ice-cream himself (and not moving his limbs), the mere observation of ‘the act’ fired the neurons that would otherwise be stimulated if the monkey ‘actually’ indulged in ‘the act’. The mirror neuron area, ventral premotor cortex, is also known as ventral premotor area F5.

Mirror neurons are defined as ‘those’ neurons that fire when an animal performs some work and also when the animal observes the ‘same work’ being performed by others. In humans, the activity has been traced down to the ‘premotor cortex’ and ‘inferior parietal cortex’ regions of the brain. When a part of the brain ‘fires’ (discharges), it becomes metabolically active and the areas of this enhanced activity may be mapped by a procedure called fMRI (functional Magnetic Resonance Imaging). In a study by Iacoboni et al, 23 right-handed participants were shown different types of image clips (figure on the left). The pictures consisted of a teapot, a mug, cookie jar and related objects in different contexts, action and intention. At the same time the subjects were shown the pictures, the participants’ brains were also being mapped by fMRI to assess the regions of the brain that lit up during the procedure. The premotor cortex and some other parts of the brain showed a significant signal increase on fMRI scans in the action and intention clips. But the signal increase in the Intention condition was much higher compared to the Action condition, with high activity recorded in visual areas and in the right inferior frontal cortex, they noted. Thus the mirror neuron areas of right inferior frontal cortex were involved in understanding the intentions of others, in addition to action recognition.

This ‘sniffing’ of intention behind action is essential to social animals like humans and a deficit in understanding this is seen in autism, a developmental disorder where there is lack of social smile, aloofness, absent eye to eye contact and marked impairment in interpersonal interaction. Autistic children can see sad or happy faces but they fail to ‘read’ the underlying emotions (sadness or happiness). Normally, children acquire mirror neuron activity by the time they are 1 year old. Exactly how they ‘program’ their neurons into being mirror neurons is not known. Learning by Hebbian association has been proposed. Mirror neurons are also involved in language acquisition, empathy and even possibly mind reading, giving credence to the ‘theory of mind’. Telepathy and clairvoyance now seems plausible (psychologists frequently employ transference and counter-transference, kind of ‘feeling’ a patient by their ‘mirror neuron systems’ and consequently ‘filling’ the patient with his own thoughts to remedy patients, in clinical practice.)

Considering their importance in social communication, our brain would have sufficient number of them. Here, I would like to wonder if pedestrian neurons could spontaneously organize into ‘mirror neuron system’ as a person watched say, an action film. Certainly, this can not happen in real-time, as there will be a delay due to visual processing and synaptic passage within the brain. But, given the plasticity of the brain and the dynamicity of dendritic spines, the idea seems conceivable. Mirror neurons also respond to sound. Breast milk ejection of a mother in response to her baby crying is an example. In cases of postoperative urinary retention, sound of running water has helped the patient to pass urine (1). This may be another example in point. It may also shed light about how ‘suggestion’ works in Hypnosis.

Given the diverse range of inputs, the brain must manage (compress) its database as space within the skull is limited. It certainly can not afford to have different sets of mirror neurons for red oval tea cups or green cylindrical ones and so on. So, what the brain does is pattern matching by some ‘fuzzy logic’ or it may simply analyze the scene; break down the signal by some kind of Fourier analysis into simpler functions and then compare resulting signal with its prior database.

Mirror neurons may explain the elusive LSD Flashback phenomenon. It occurs in LSD abusers who are NOT currently taking the drug, but find themselves in a situation reminiscent of a previous drug spree. The person gets a ‘kick’ even though he may have taken it days ago. Clearly, psychedelic lights may trigger a flashback (and watching violent TV programs has been found to activate mirror neurons in children). We should also ask ourselves if dreams, at least some of them, were the handiwork of some of these neurons.

In her fantastic article ‘Cells That Read Minds’, Sandra Blakeslee ponders and exculpates all men from voyeurism:
“In yet another realm, mirror neurons are powerfully activated by pornography, several scientists said. For example, when a man watches another man have sexual intercourse with a woman, the observer's mirror neurons spring into action. The vicarious thrill of watching sex, it turns out, is not so vicarious after all.”

In a lighter vein it may be said that the search engine Google has developed 'mirror neuron like' properties. Just type, “how can i get my girl” in Google search box and watch: Google would ‘ping’ your intention and come up with some real smart choices.

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Reference: (1) Bailey & Love; A Short Practise of Surgery,18e, page 1230
Mirror neurons and the simulation theory of mind-reading
Cells That Read Minds
THE MOTOR CORTEX
Iacoboni M, Molnar-Szakacs I, Gallese V, Buccino G, Mazziotta JC, & Rizzolatti G (2005). Grasping the intentions of others with one's own mirror neuron system. PLoS biology, 3 (3) PMID: 15736981

Fourier Analysis: The Art and Science of Finding The Needle in a Haystack

2009-12-13T23:21:00.004+05:30

Every time I listen to the heavy metal band Pantera my wife would invariably wonder aloud why I listen to all this ‘noise’. True, many music lovers would rather refer bands like Pantera as quintessential noise than music; there are persons like me who can dissect the melody from the apparent chaos of runaway frequencies of guitars, drums and so on. I can even analyze and follow individual instruments over time. This is what Fourier is about, or stated otherwise, my ear & brain can be said to be doing a Fourier transform on the said musical piece.

Joseph Fourier, a French mathematician, realized that all periodic waves could be ‘synthesized’ by mixing sine waves of right frequency, amplitude and phase. For example, a square wave could be prepared by ‘adding’ the fundamental frequency (the lowest frequency; say 70Hz) with an infinite number of its odd harmonics (e.g. 210Hz, 350Hz, 490Hz and so on. Harmonics are multiples of the fundamental frequency.) This is Fourier synthesis. Similarly, you could break down a periodic signal in which the amplitude varies over time into one of a frequency versus time graph. This is Fourier analysis, and it can be seen that here we are actually ‘decomposing’ the ‘signal’ into its frequency spectrum, over time. The process of decomposing a function into its constituent frequencies is known as Fourier transform. You can have a ‘hands on experience’ at what a square wave ‘looks’ or ‘sounds’ like and how a periodic wave is decomposed into its constituent parts here. Do experiment on the sine, cosine, triangle wave and square wave functions as well and turn on the sound of your PC while you are at this site!

While Fourier originally devised this to solve the problem of heat propagation, the impact of Fourier analysis can now be felt in almost every field of science, instrumentation, entertainment and telecommunications, and even arts. Whenever you use your audio graphic equalizer to suit a piece of musical performance to your taste, you are doing a Fourier. Here you are boosting some particular audio frequencies while suppressing others, obtained by a Fourier analysis of the audio signal. You are assigning relative weights to the frequencies by sliding those sliders. Likewise, when you compress a picture (graphic) file using software such as JPEG, an inbuilt program does a Fourier transform,--> eliminates the weaker components from the analysis and--> then saves the information in a compact way.

In Nuclear Magnetic Resonance Imaging (NMRI), the emitted radio frequency is Fourier transformed to give frequency versus time, throwing valuable information about nuclear spins. Fourier analysis may also be employed to remove mains AC hum frequencies, in mobile telephony and many other situations.

One day, we may expect, that Fourier analysis may be used to pick up the ‘right frequency’ in the brain EEG waves and may put the study of ‘mirror neuron’ and ‘thought controlled devices’ into a whole new domain.

Finally, I would like to tell you that this site was featured as one of the best 50 physics blogs at "Accredited Online Colleges". Whether I deserve it or not, I certainly like the idea!

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Reference: Fourier analysis (Wikipedia),
Explained: The Discrete Fourier Transform

A Tale of a Microprocessor, RISC and a Few Loops of miRNA

2009-11-16T18:31:00.005+05:30

The word ‘microprocessor’ is generally used to designate VLSI and SLSI (Very/Super Large Scale Integrated circuits) devices which accept, decode and execute instructions presented in binary coded forms. They may be called the heart of the computer. RISC (Reduced Instruction Set Computer), on the other hand, is a type of microprocessor architecture that uses a simplified, yet highly-optimized set of instructions to deliver good performance. However, like ‘cell’ and ‘nucleus’, they too have been adopted in biology, and not without reason!

Proteins are essential for cells as they perform various functions as enzymes, ion channels, receptors and so on. They are manufactured in the ribosomes, organelles present in the cytoplasm, under the instruction of messenger RNA (mRNA). This instruction code is encoded in the sequence of nucleotides that make the mRNA molecule. However, the sequence of nucleotides in mRNA is dictated in turn by the DNA that is present in the nucleus. Messenger RNA carries this message from the nucleus into the protein production units. But what would happen if we interfered with the ‘message’?

RNA interference (RNAi) would occur affecting the regulation of gene expression. Micro RNAs (miRNA) are one of the small RNAs that regulate the expression of protein-encoding-genes, after the mRNA strand has formed. miRNAs have partly or fully complementary sequence to one or more mRNAs. This enables them to latch on to the mRNA molecule masking the ‘instruction codes’ in the mRNA strand, interfering with protein formation (translation). In other words, the gene has been silenced!

miRNAs are first transcribed from DNA by the enzyme RNA polymerase II into primary miRNA (pri-miRNA). pri miRNA is then cleaved by another enzyme, RNAse III, called Drosha, into precursor miRNA (pre miRNA) (see the picture on the left). However, Drosha (an RNAase III endonuclease) is assisted by Pasha (partner of Drosha), another enzyme, in this task. Later, it was found out that these two resided in a 500 kilo Dalton complex, called the microprocessor (micro RNA processor). So far, all these have been happening in the nucleus of the cell. The pre miRNA then moves into the cytoplasm through the exportin 5 pathway. Next, Dicer, another RNase III endonuclease, makes a mature miRNA duplex, which is then ‘uploaded’ into a complex called RISC (RNA induced silencing complex). RISC then prevents translation of the mRNA strand, as the ‘partially’ complementary miRNA strand interferes with the translation of the mRNA molecule into specified amino acid sequences can not occur. We can compare complementarity of nucleotide bases in terms of a pair of gloves and its corresponding fingers. The information of the gloves' coordinates gets obliterated by the occupying fingers. This RISC dependent mechanism occurs in parts of the cytoplasm, called P bodies (‘p’ for processing).

RNAi is very important for plants as they lack an immune system. Invading organisms can not dictate foreign protein formations as their RNAs are destroyed, not merely inhibited, as is usually seen in higher animals (animal miRNAs exhibit only imperfect homology to the mRNA in contrast to plants, and thus they only inhibit translation). Some of the tumor suppressor genes inhibit tumor formation by the action of miRNAs and not through protein formation. In humans, exploiting RNAi may be a useful tool in combating diseases such as cancer, AIDS etc. So it remains to be seen whether the microprocessor can bring a revolution in medicine and research as its counterpart in electronics did in the field of computing.

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Reference: Saumet, A., & Lecellier, C. (2006). Anti-viral RNA silencing: do we look like plants ? Retrovirology, 3 (1) DOI: 10.1186/1742-4690-3-3
Processing of primary microRNAs by the Microprocessor complex. doi:10.1038/nature03049
Wikipedia
The Macro World of MicroRNA (pdf)

Metallica Goes The Stem Cell Way

2009-10-18T20:00:00.002+05:30

I had previously written a little about stem cells. While researchers still don’t yet know exactly how the four factors transform the fully differentiated fibroblast cells back into pluripotency, possible explanations are pouring in.

Pluripotency (by which the stem cell may become any tissue; muscle or nerve, for example) and “self renewal” (cells should not only differentiate, some ready stock of stem cells must be there for future need) are important determinants for stem cells.

According to Shinya Yamanaka, the steps could be somewhat like this: c-Myc first confers the open chromatin state and immortality to the skin fibroblasts. But it also induces apoptosis by acting on the p53, “the guardian of the genome”. Apoptosis or cellular senescence causes the cells to die. Klf4 inhibits p53 induced apoptosis. Again, if we added only Klf4 and c-Myc we would get tumor cells (both being oncogenes). Oct4 here acts and makes ES like cells (ES= Embryonic Stem) out of what was destined to be tumor cells. Sox2 confers pluripotency and you’ve got what you wanted.

Now, we just have to hand pick the right cells from the petridish. Scientists can do it either by looking for Fbx15 expression or the expression of nanog in the treated sample. Both Fbx15 and Nanog are targets of Oct3/4 and Sox2; but Nanog is found to be more closely associated with pluripotency, as is evidenced by adult chimera formation (chimera is a monstrous fire breathing creature like dragon of ancient mythology).

There have been some important modifications. Researchers have shown that one could still get human induced pluripotent stem cells (iPSC) without the need of the c-myc oncogene. The mode of delivery of these four factors could also be undertaken by plasmids, rather than the traditional retroviral vector approach. Retroviruses (like c-Myc) could potentially induce cancer. You may like to hear this Nature Podcast where both Yamanaka and Rudolph Jaenisch give a very good summary. As a bonus, you may also appreciate another way of creating iPSC. Replace the genome in “early embryonic cells” or zygotes (fertilized eggs) during cell division. During cell division, the nuclear membrane disappears and the factors are no longer in the nucleus. They are in the cytoplasm. Dieter Egli explains that if you replaced the genome of this zygote with another (genome) while the cell was still dividing, the new genome would adapt to the new cytosolic environment and get instructions from the factors in the cytosol. It will go ‘back in time’ and become a stem cell.

Now, a bit of refreshment. Watch this awe inspiring Metallica video called 'All nightmare long'. It portrays the Tunguska event, A-bomb, Soviet Revolution, American supremacy (?) and ‘revival of organisms’. Some key phrases are:

“like a split worm, a part of the organism can reconstitute the whole”. Check about Planarians (flat worms, picture on the left), they not only reconstitute but also become separate individuals!
“Instead of offspring, they become skin cells, nerves and muscle”- just as we described! Seems Metallica is well informed! Do see this wonderful video in YouTube (Metallica All Nightmare Long (Official Music Video))

Reference: hyper-links and

Okita, K., Ichisaka, T., & Yamanaka, S. (2007). Generation of germline-competent induced pluripotent stem cells Nature, 448 (7151), 313-317 DOI: 10.1038/nature05934

Developmental reprogramming after chromosome transfer into mitotic mouse zygotes, doi:10.1038/nature05879

Atomic Force Microscopy: Feels The Atoms, Sees The Bonds

2009-10-13T19:42:00.006+05:30

When it comes to viewing things on the atomic scale, one has to be very careful and innovative. To understand how an atomic force microscope works, we should better discuss a bit about its predecessor: the scanning tunneling microscope (STM). STM was invented by Binnig and Rohrer for which they got the Nobel Prize in Physics. Binnig and colleagues later went on to develop the first Atomic Force Microscope (AFM). Both AFM and STM are types of Scanning Probe Microscopy, which employs a probe that scans the sample.

STM consists of a sharp probe tip, which scans over the specimen as the adjoining picture shows. First, the probe tip is brought near the sample manually, and then the finer adjustment of maintaining probe sample distance (height) is done by piezoelectric control. A voltage applied between the tip and the sample causes electrons to tunnel from the tip to the sample. As we have seen in Ohm’s law, the tunneling current will depend on the applied potential difference (voltage bias); and the height of tip-sample separation and the local density of states (factors determining ‘resistance’). If we know two of the three unknowns, we can calculate the other, which is actually done by the computer by data acquisition.

We can do STM in two ways. We can keep the tip position (height) fixed as it scans the specimen topography (constant height mode). Here the voltage and height are both held constant, while the tunneling current varies. In constant current mode, the tip is always at a specific height over the specimen. That is, as the tip hovers over the rugged terrain of the sample surface and comes close to a raised spot, the tunneling current will increase. The increased current will be sensed, amplified and passed to the feedback electronic circuitry which will ‘lift’ the probe-tip by applying a voltage to the piezo crystal. Hence the electronic servomechanism maintains a constant tip sample distance in constant current mode. Piezoelectric crystals translate pressure changes into electricity (and vice versa) as we see in oven gas lighters and in mobile phone speakers.

But STM has its inherent drawback: the sample has to be a conductor or a semiconductor, in order for tunneling to occur. Hence, biological tissues, non conducting polymers can not be imaged. So, the need for atomic force microscopy arose. Here again, Gerd Binnig played a pivotal role.

Atomic Force Microscopy operates on a similar principle. First, let’s discuss how the music on gramophone record grooves is translated. The stylus (which overlies a piezo crystal) feels the groovy surface of the vinyl LP disc which revolves on a turntable. The mechanical vibration sets in a voltage in the piezo via the stylus. We get the surface topology in the form of music. Another great way of viewing how the AFM scans and interprets a sample is how a blind person ‘feels’ a surface by using a stick (see picture).

In AFM, a very sharp tip (made of silicon or silicon nitride) is scanned over a surface with similar feedback mechanisms that maintain the tip at a constant force (to get height information), or height (to obtain force information) above the sample surface. This sharp tip is mounted on a cantilever, a rod like structure whose other end is fixed and unmovable. As the probe tip raster scans (i.e. scans in a zigzag fashion as done in TV scanning) the surface of the sample, a laser light is made to fall on the back of the cantilever. The light gets reflected off from this side and is detected by one of a dual photodiode. The cantilever ‘tilts’ as it slopes ‘down a bump’, causing another reflection that hits the other of the dual photodiode. A differential amplifier then calculates the difference output between the two light intensities and this is proportional to the cantilever deflection. We can calculate the force (acting between the cantilever tip and the sample) using Hooke’s law (F=-kx), where F is force, k is constant for a particular cantilever, and x is the deflection of the cantilever. The amplifier difference output may be used to keep the tip in either at a constant force or at a constant height above the sample, through piezoelectric servo mechanism (feedback).

AFM is very versatile and allows many user specific modifications. It can be done in contact mode, where the tip remains in contact with the surface as it scans over the specimen. In tapping mode, the cantilever oscillates over the sample, touching the surface intermittently. Interatomic forces like van der Waals forces and electrostatic forces cause a deflection of the cantilever when the tip comes closer to the sample. In the non contact mode, the cantilever does not touch the sample, and is oscillated at slightly above its natural resonance frequency. Any long range force like van der Waals force will decrease the frequency of the vibrating cantilever, when the tip approaches the sample. There are many other variants of AFM and combination of AFM with other imaging modalities like optical microscopy, Raman spectroscopy and so on.

But Leo Gross and colleagues at IBM Research Zurich wanted another, so they went on to develop another variant of AFM. They used a tuning fork like probe. One end of the probe was near, while the other end was away from the sample. When the fork was vibrated, the prong next to the sample experienced a minute shift in frequency due to forces acting upon it. This frequency drift when compared to the other prong, gives the molecular picture.

To get the finer detail of a molecule, one had to use a sharp AFM probe tip. They linked a molecule of carbon monoxide to the tip in such a way so that the lone oxygen atom became the de facto tip. It gave the structure of pentacene in unprecedented detail. You can clearly see even the bonds between carbon atoms, just as we read in our textbooks. However, the experiment must be vibration free, thermal noise free (at a very low temperature), and should be done in vacuum.

References: Scanning tunneling microscope (Wikipedia), Atomic Force Microscope (Wikipedia),
Single molecule's stunning image (BBC)

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Reference: hyper-links, unless specifically mentioned

To Unfold The Secret of Protein Folding, Foldit!

2009-09-24T02:28:00.006+05:30

Genes in living cells dictate the cellular machinery to form proteins, the ultimate product of genetic information that is encoded in the DNA. These proteins perform various functions in the body. Some maintain the structure of the cells, some act as enzymes thereby catalyzing reactions, some act as pumps and ion channels thus maintaining ionic equilibrium and events like muscle contraction and action potentials in neurons, some act as receptors which recognizes ligands and binds them and so on.

However, genes merely determine the sequence of amino acids in the protein. These amino acids form the primary structure of proteins by joining themselves by peptide bonds, just as different colored beads make up a string. Some amino acids in the protein undergo post-translational modification such as carboxyllation, phosphorylation, once the primary structure has been determined. Then the protein folds in such a way that it is most stable in the tissue conditions like pH etc. Indeed, living tissues try to make order (stable protein configuration) out of seeming disorder (random amino acids) in an apparent violation of the second law of thermodynamics.

Folding also saves valuable space. But why and how should the protein fold? There are interactions between amino acid residues in the form of covalent bonding such as disulfide bonds; non covalent interactions like hydrogen bonding (between hydrogen and oxygen atoms in the peptide backbone), electrostatic or salt bonds between oppositely charged residues, and hydrophobic interactions whereby hydrophobic (water hating) portions of the molecule stay away from water. So, the protein folds to a conformation where the conflict is kept to a minimum. X-ray crystallography, NMR spectroscopy, computational biology and atomic force microscopy are useful tools in elucidating protein structure. Although the way it folds has been simulated in the computer, having humans do it as a computer game and then trying to figure out how the computer did so is surely worth trying. That’s where Foldit comes in.

I first knew of Foldit about a week ago in the print version of the August edition of HHMI Bulletin. After a user downloads the program and installs it, he can see proteins as multicolored structures. All he has to do is to grab the mouse, then pull, twist and wiggle the structure so that it has the most optimal position using the mouse. The program will give you a hint should the atoms be too close or if the hydrophobic ends are sticking out. The program relies on the pattern recognition ability and visuospatial scratchpad (of the working memory) of individuals. Intuition plays a big role and thus scientists may not be much good at this game. The ABCs of Foldit are Apart(sidechains), Buried (hydrophobic domains) and Compact (protein).Users could also play online so that their scores were kept on the servers, and collaborated with each other evolving the game further (Online Darwinism?) Persons having exceptional folding solving abilities are aptly called 'foldit savants', possibly deriving its name from 'idiot savants', persons belonging to the autism spectrum but having extraordinary abilities in certain subjects like mathematics. Albert Einstein was thought to be autistic.

Previously I have used Wolfram’s Mathematica and NanoCAD written by my friend Will Ware. NanoCAD is indeed an outstanding tool, given that it was programmed more than 10 years ago. The basics of NanoCAD and Foldit look rather similar to me, only the complexity and online participation is differing.

Anyway, you always win because you are playing for a cause. A definite and stable protein structure prediction might help researchers the right antibody, the right vaccine, develop better drugs with little side effects and so on. Who knows if this paves the way for the treatment of Alzheimer’s disease, cancer or AIDS?

Since this game exploits intuition rather than intellect, we could perhaps also measure hemisphere dominance in the participants. The effects of psychotropic drugs on game performance could also perhaps be measured.

As of me, I could not play above a certain level. My CPU usage, as shown in the task manager was 100%.
P.S. I still use a Celeron 1.2 GHz CPU and have only 256 MB SD RAM. I could not connect with the server as well.

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Reference: hyper-links, unless specifically mentioned

Of Twinkling Nanostars and the Possible Application of Stroboscopes in Biological Imaging

2009-08-16T12:00:00.006+05:30

Imagine a strong crowd, as you see in a Manchester United versus Liverpool football match and you wished to concentrate on a particular person. How would you do it? Make him wear a fluorescent shirt and dye his hair (don’t do it in the middle of the crowd, I can’t guarantee your safety).

Purdue University researchers have been successful in focusing at the cell of interest among a background of equally noisy and boisterous biomolecules and other metabolically active cells. Currently, researchers use immunological techniques to create an antibody to a molecule and then visualize the ‘molecule of interest’ by tagging the antibody to a radioisotope or a fluorescent dye; and flow cytometry can sort out different types of cells.

The Purdue University team used gold coated nanoparticles with an iron oxide core that was impregnated in the cell they wished to see. They then subjected the specimen to a periodically changing magnetic field. The superparamagnetic cores (superparamagnetic nanoparticles have no net magnetization, but an external magnetic field can magnetize them) responded by rotating as the magnetic field rotated around them. The rotation could be seen in the ‘near infra-red’ light spectrum, as the incident light bounced off (scattered) the specially designed arms of the gold nanostar as it revolved. The rate (rpm) of this gyromagnetic (gyros means to rotate) twinkling could be externally controlled by varying the rate of the externally applied field. You now could identify the cell by its characteristic ‘twinkling’ (lighthouse type) effect.

I am tempted to go beyond what’s been achieved so far. Here I go. I guess you are all familiar what happens to the rotating ceiling fan blades when you turn on a fluorescent lamp. Don’t you see a momentary snapshot of the three blades (some have 4)? That’s what where stroboscope comes in. It consists of a Xenon lamp (ordinary fluorescent lamps could do, but incandescent lamps won’t work as the glowing filament takes time to extinguish) flashing at a controllable rate. The electronic circuitry may be had here.

Suppose that the fan is revolving at 1200 RPM and it is not changing. Set your stroboscope to flash at this rate. You’ll ‘see’ that the fan blades are absolutely not moving, which is certainly not true! But be there any mechanical defect in the fan, it will stand out as the centrifugal force widens it (provided that the fault is more or less tangential to the axis of rotation). Here also we are looking at our object of interest, aren’t we?

Now lets look what implication it might have in biological imaging. We now know that the gamma subunit of mitochondrial F type ATP Synthase ‘actually’ rotates when it is synthesizing ATP (reverse rotation occurs when ATP is hydrolyzed). There are other locomotive units within the cell as well. They comprise of actin and myosin based molecular motors. Could we study them using an externally adjustable stroboscope? The optical (electromagnetic) signals so obtained may then be similarly broken down into simpler trigonometric (sine and cosine) functions by Fourier analysis (Fourier transform) as was done in the ‘twinkling nanostars’ experiment. At least, we expect to get rid of some 'noise' and some good still photos. But if we wanted better resolution and used higher frequency (electromagnetic) for it, some extraneous error will be introduced. It's a trade-off!

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Reference: hyper-links, unless specifically mentioned
Principles of Biochemistry, Lehninger, 4th ed
http://en.wikipedia.org/wiki/ATP_synthase
Wei, Q., Song, H., Leonov, A., Hale, J., Oh, D., Ong, Q., Ritchie, K., & Wei, A. (2009). Gyromagnetic Imaging: Dynamic Optical Contrast Using Gold Nanostars with Magnetic Cores Journal of the American Chemical Society, 131 (28), 9728-9734 DOI: 10.1021/ja901562j

The Versatile GABAa Chloride Channel Receptor Complex

2009-08-13T20:36:00.010+05:30

In today’s industrialized society we are constantly exposed to work related stresses. Consequently, anxiety and insomnia (sleeplessness) have become quite common. No wonder, we are using anxiolytics and sedatives more often; to get relief from the anxiety and insomnia respectively.

Benzodiazepines such as diazepam (Valium), chlordiazepoxide (Librium) can effectively treat anxiety and insomnia. They do so by binding with a receptor (called Benzodiazepine-GABAa-chloride ion channel complex [henceforth to be referred to simply as GABAa receptor]) in nerve cell membranes. It is known that most drugs (medicines) exert their actions by combining with receptors: macromolecular complexes present in the cell membrane or within the cytosol or the nucleus.

The GABAa receptor is a very versatile receptor complex (a hypothetical model is shown at the bottom). Its main action is to inhibit transmission along neurons in which they are present. Normally, proper functioning of the brain is ensured by a balance between the action of excitatory and inhibitory neurotransmitters [henceforth to be referred to simply as NTs]. Simply put, excitatory NTs (for example, glutamate) give a green or go signal; while inhibitory NTs (such as GABA) tell the nerve not to fire (red or stop signal). In this connection, it must be said that GABA (gamma amino butyric acid) is the most important inhibitory NT. When GABA binds with the GABA receptor ionophore complex, the receptor changes shape (conformation); and then a centrally located chloride channel, that is a part of the receptor itself, opens. Since the concentration of the chloride ions (Cl-) is much more on the outside of the cell than on the inside, Cl- now rushes in due to the increase in chloride conductance. The cell voltage goes further down and the interior of the cell becomes more negative (hyperpolarized) with respect to the outside. The cell becomes less excitable and is thus inhibited.

Apart from maintaining the much needed critical balance already mentioned, they also ensure that the brain works in a relatively noise free environment. Billions of neuronal units are always firing in the background creating a constant ‘noise’. A constant release of GABA by the brain drowns out this noise thus improving the ‘signal to noise’ ratio, making the brain’s task of finding the proverbial ‘needle in a haystack’ a lot easier.

The GABAa receptor not only binds with GABA, but it is a binding site for various other ligands. But before we discuss them, let us briefly analyze its structure first. The receptor has a pentameric structure which means that it consists of five subunits, and each subunit has four membrane-spanning (transmembrane) domains (see picture). And there are many of the polypeptide subunits to choose from a vast array consisting of alpha, beta, gamma, delta, pi, rho and so on. (In addition, there are six different forms of alpha, 4 beta and 3 gamma subunits). Thus, it’s no wonder that a great variety of GABAa receptors will be found, given the possible permutations!

This receptor heterogeneity explains actions of various pharmaceuticals on the receptor. One major form of GABAa receptor (found throughout the brain) consists of two alpha1, two beta2 and one gamma2 subunits. In this isoform, GABA ‘somewhere’ between alpha1 and beta2 subunits, and benzodiazepines bind with the BZ1 (also called omega1) pockets located between alpha1 and gamma2 subunits. Benzodiazepines act only when the receptor isoform has one of the following alpha subunits: 1, 2, 3 or 5 and the subunit should have a conserved histidine residue in the N-terminal domain. In ‘knock-in’ mice where histidine has been replaced by arginine in the alpha1 subunit (alpha1H101R; H for histidine and R for arginine in the 101st residue of alpha1 subunit) there was no sedation or amnesia (as evidenced by their unchanged ‘energy’ and memory to electric shocks). It may be mentioned at this moment that the so called ‘date rape’ pills exploit the amnestic properties of benzodiazepines. The drug plays tricks with the victims’ memories. However, the anxiolytic and muscle relaxant properties were retained in these mice.

These mice also do not respond to the hypnotic effects of zolpidem and zaleplon, non-benzodiazepines that act at GABAa receptors containing alpha1 subunits. But in mice with selective histidine arginine mutation in the alpha2 subunit of GABAa receptors, resistance to the antianxiety action of benzodiazepines has been seen. Based on these observations, it is thought that alpha1 subunit mediates sedative and amnestic effects, while alpha2 takes care of the anxiolytic and muscle relaxant ones. It also seems that we are poised to make better benzodiazepines in future (like one that works in anxiety but doesn’t wreak the patients’ memory).

Lastly, the versatility. The GABAa receptor also binds barbiturates (urea derivatives used as anesthetics, anticonvulsants, Marilyn Monroe supposedly died of its overdose) in addition to the benzos. Alcohol, alphaxolone (a steroid anesthetic), etomidate (a short acting anesthetic), propofol (diprivan, Michael Jackson supposedly used it), volatile anesthetics like halothane, anticonvulsants like gabapentin and vigabatrin, anthelminthics like ivermectin, and neurosteroids (metabolites of androgen and progesterone) exert part or all of their actions by acting through this receptor, thereby hyperpolarizing the neuron. Conversely, convulsants picrotoxin blocks the chloride channel directly, while bicuculline blocks the receptor’s GABAa binding site causing depolarization and convulsion. There's a lot more than this mere exegesis, and I hope to discuss about it furher later.

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Reference: Bertram G. Katzung, Basic and Clinical Pharmacology, ninth edition
Pharmacology: Rang, Dale, Ritter, Moore
Wisden, W., & Stephens, D. (1999). Pharmacology: Towards better benzodiazepines Nature, 401 (6755), 751-752 DOI: 10.1038/44482

Errors, terrors, statistics and a confession

2009-06-29T00:42:00.008+05:30

Past three months have been a bit too hectic for me. I had to finish the whole 3-year-course of physiology during that period, since by nature, I am a late starter. Now that the results are out and I passed my MD, I can breathe more freely.

During those periods of forced 'bibliomania', I did not write any article for my blog, though I checked mails and sometimes I checked Sitemeter stats for my blog. I do this sometimes to see where my visitors were coming from, what they look for and for how long and so on. Almost everyone does it. It gives me pleasure when I see people from many different countries and educational institutions visit my blog. I noticed how the visits per day dropped from about 70 to below 35 during the period. I also noticed some errors in some of my pages, I'll rectify them as early as possible. Meanwhile, please point out errors so that I may correct them.

I was in for surprise when I found a US military site (.mil domain) as one of my hallowed visitors. What on earth have I done? I clicked on the landing page; it was "Show me the enemy and I'll take action". The reason was now obvious, it was just some kind of 'code sequence identification algorithm' (pattern recognition) giving a 'false positive'. One puzzle I am yet to crack is what my friend from Ljubljana, Slovenia find so interesting in "cerebellum, electronically speaking". He (she?) visits my site at least 3-4 times per day. I guess, he has configured his browser this way so that this was the opening page each time he started the browser. Anyway amigos, have a good day and do visit.

While I did commit some inadvertent errors in some of my pages (of my blog); the mistake I committed on the examination (viva part) eve was unpardonable and was least expected of a medical person, to be very honest. After finishing a summary of my thesis work, I sat down to prepare a powerpoint presentation on "water reabsorption in the kidneys". After finishing the two, the clock ticked 4 AM. I took 5 mg of Valium (diazepam) orally. (Diazepam, a member of benzodiazepines family interacts with the GABAa receptor-chloride channel macromolecular ionophore complex and help us to get sleep.) Then I went on to burn them onto a CD. But sleep wasn't really coming. I took another 5mg of valium, this time chewing the bitter drug and keeping it inside my oral cavity (this route would bypass the portal circulation) for about 5 minutes [THIS ROUTE FOR DIAZEPAM IS NOT RECOMMENDED IN ANY TEXTBOOK I KNOW]. I increased the buccal pressure (for better absorption) by blowing my mouth, while keeping my mouth shut (resembles Valsalva maneuver) during this time. Then I took a cup of hot tea to facilitate its entry across the mucosa (also hoping it would induce diuresis so that I would have less amount of of the drug next time I voided urine). It was 5:30 AM when I finally went to sleep, and I were to wake up at 7:00 AM!

Thus you see how many mistakes I committed at one go: sleep deprivation, intake of a drug for sleep that causes psychomotor slowing and impaired cognitive performance, being fully oblivious of its half-life of 20-80 hours! It takes 4-5 half-lives for a drug to be 'considered' eliminated from the body. Next, if a person was aroused from REM sleep while on diazepam, he could become irritable and anxious. Moreover, there is the specter of anterograde amnesia (inability to remember lessons learnt during the drugs' duration of action). (I should have taken Alprazolam/xanax or Zolpidem instead, for they have less half life and are quicker acting. Even better would be to take nothing at all, sleep or no sleep!)

Given those, you can well imagine the difficulty I had when I faced the examiners. Thought processing, information retrieval and even articulation was not at their peaks. Despite all odds, things luckily went well, as I was able to answer some key questions well and I got through. One should take lessons from it: have adequate sleep and completely abstain from benzos the night before their exams. Hope you learn from my mistakes.

Capturing Thought, in Real Time

2009-04-05T00:29:00.003+05:30

Wouldn't it be nice if we mapped how the thought processes traveled across our brain, in real time? That's exactly what Mazahir Hasan et al of Max Planck Institute for Medical Research in Heidelberg, have enabled us to view, when an action potential (AP) is underway in the central nervous system (CNS). The researchers introduced fluorescent calcium indicator proteins (FCIP) into the brain cells of mice by means of viral gene vectors. Each time an AP was underway, a lot of ionic phenomena happened. For example, the fast Sodium channels (Na+) opened (letting positive charges to the interior of the cell) leading to depolarization, Potassium (K+) channels opened (to bring back the resting membrane potential to normal, since K+ egress out of the cells) and so on.

Next , the impulse is transmitted to the post-synaptic neuron through the agency of neurotransmitters. But, for this 'coupling' between the presynaptic and postsynaptic neurons to occur; Calcium ion (Ca++) levels in the synaptic knobs of the presynaptic neurons must rise for effective degranulation of the presynaptic vesicles. And that's precisely these researchers were banking upon.

Just before the degranulation of synaptic vesicles begins; calcium ion concentration surges. Such short calcium currents peak within milliseconds, making them the appropriate ions for studying fast neuronal activity. Previously scientists had measured such currents by using microelectrodes implanted within the brain; but this method was quite unsuitable in studying moving animals or for a longer time period. So, they went on to produce stable transgenic mouse lines responding to functional calcium indicators; (including 'inverse pericam' and 'camgaroo-2') using viral vectors. These transgenic mouse lines were under TET inducible promoter (tetracycline, a broad-spectrum antibiotic) control. The TET system offered the advantage of targeting combination of different neuronal cell assemblies. The other side of the Ptetbi (bidirectional promoter tetracycline) promoter was attached to the firefly luciferase gene. They were also sensitive to doxicline (another antibiotic belonging to the same category as tetracycline) in terms of regulation of luciferase, as well.

They then used a heteromeric sensor protein called D3cpv, which was made to produce in the nerve cells of the transgenic mice. Two subunits of this protein reacted to the binding of calcium ions in a way that when the yellow-fluorescent protein (YFP) lit up and the cyan-fluorescent protein (CFP) intensity diminished. When calcium was bound to the D3cpv complex; CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein) came closer together bringing about FRET, in such a way that there was a visible color change, 'visually' or optically indicating the progression of action potential in real time. CFP and YFP are spectral variants of GFP linked together by a Ca++ sensitive linker.

They used 'two-photon imaging microscopy' to study this phenomenon. They excited thinned out rat skulls using two-photons simultaneously using 'mode-locked' Titanium-sapphire laser. They then amplified the signal using photomultipliers and analyzed them.

The resolution of the experiment was limited to less than 1 Hz (frequency of action potentials). They conferred that human thought processes might be mapped in much the same 'opto-physiologic way', in contrast to the usual electrophysiologic approach. Not only does the experiment throw light on the thought processes in real-time, but also, it is expected that it will be useful in the pathophysiology and treatment of Alzheimer's disease, Parkinson's disease and Huntington's chorea.

FCIP-positive cells were found in the hippocampal CA1 and CA3 regions, mossy fiber areas of the dentate gyrus, neocortical pyramidal cells and olfactory receptor neurons, they remarked. They studied cortical pyramidal cell, olfactory and optical responses in the mice in their experiment.

Hasan, M., Friedrich, R., Euler, T., Larkum, M., Giese, G., Both, M., Duebel, J., Waters, J., Bujard, H., Griesbeck, O., Tsien, R., Nagai, T., Miyawaki, A., & Denk, W. (2004). Functional Fluorescent Ca2+ Indicator Proteins in Transgenic Mice under TET Control PLoS Biology, 2 (6) DOI: 10.1371/journal.pbio.0020163
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Reference: Damian J Wallace, Stephan Meyer zum Alten Borgloh, Simone Astori, Ying Yang, Melanie Bausen, Sebastian Kügler, Amy E Palmer, Roger Y Tsien, Rolf Sprengel, Jason N D Kerr, Winfried Denk & Mazahir T Hasan. doi:10.1038/nmeth.1242

Brains of Guitarists in Unison Harmonize Too

2009-04-04T20:06:00.002+05:30

During the 80's, I listened to heavy metal bands like Iron Maiden and Metallica, although I couldn't follow their lyrics always. What used to captivate me in awe was how the guitarists synchronized themselves together so well. It apparently seemed as if only one guitar was playing in the background, which on closer scrutiny revealed the actual truth: it was really a duet. It is only now that scientists are beginning to find the secret behind this 'time and phase synchrony'.

Scientists at the Max Planck Institute for Human Development in Berlin, have shown that musicians playing the same tune have their brains 'coupled' together. They started off experimenting with 8 such musician pairs. They first recorded the brain activity of each
'duetter' by taking their electroencephalographic recordings (EEG). The musicians kept the EEG set-up atop their heads throughout the experiment.

After taking the baseline EEG recordings, the researchers then made the guitarists to listen to metronome beats. Metronome beats are beats of sound that occur periodically and are used to keep track of time. They found that the EEG activities of the players were synchronized to that of the metronome beats. Next, the lead guitarist of the pair had to tap his guitar in a gesture to signal his partner as to when and at what speed they would begin. At this point, the researchers looked at the brainwaves of the guitarists again and found that the EEG of both the guitarists were in synchrony to each other (and no longer to the metronome beats). Curiously, this happened even before the actual performance began. This oscillatory synchronization was found to be especially stronger at the frontal and central electrode sites (of the EEG leads). This may indicate simultaneous firing of the motor and somatosensory neurons.

This experiment also throws light as to how empathy and the 'mirror neuron network' might be working. These inter-personally coordinated behaviors will only result if they happen fast and both the sensory and the motor actions are coordinated. Certainly, there has to be some kind of a feedback between the pair for effective harmonization to occur.

It has been previously seen that in addition to the EEG coupling; magnetoencephalography (MEG; measures the magnetic field around the skull) and electromyography (EMG: measures the muscle activity) related well between neuronal activity of a person to the voluntary activity of the same person. The new finding may help us probe the basis of social interaction but it also poses a question: how do the performers synchronize and through which media? You can find videos of duetting guitarists and the corresponding EEG recordings at Biomedcentral.

P.S. Finally, let me allow to propose 2 mechanisms which may be responsible for this apparent 'phase lock'. Firstly, the performers have a very clear idea about the piece they were about to perform, since they are well rehearsed. Naturally, the guitarists are in tune with the next intermezzo and if they were to strum chord C major, their corresponding motor planning areas would become active. It is known that the motor planning areas become electrically active even before the execution of actual action (1). Secondly, we can also assume that they, being emphatically coupled to the music, get connected across by mirror neurons. The mirror neuron system then does the rest: driving the players in a rapturous synchrony.

Lindenberger, U., Li, S., Gruber, W., & Müller, V. (2009). Brains swinging in concert: cortical phase synchronization while playing guitar BMC Neuroscience, 10 (1) DOI: 10.1186/1471-2202-10-22 Last modified: Jan 19, 2010
Reference:(1) William F. Ganong, Control of Posture & Movement, 22nd Ed, Review of Medical Physiology, Page: 202

An Anatomy of Noise And Its Implications

2009-04-02T23:26:00.007+05:30

Noise is something we dislike, because by definition, noise means unwanted sound. But this definition is subjective, for what is music to my ears (say the heavy metal band Metallica) is noise to most people. In fact Iraqi prisoners were forced to listen to Metallica songs as a means of torture (culture shock and noise) by the American soldiers. Perhaps a better definition is, wrong sound at the wrong place at the wrong time.

Apart from acoustic noise; there is visual noise as found in television as ‘snow’, electronic noise (e.g. thermal noise or Johnson noise), cosmic noise and so on. Speaking of acoustic noise, one can’t help but think about the dreaded ‘noise pollution’ that seems to envelop us all. In addition to the nuisance it poses, it also causes anxiety, insomnia, increased blood pressure (hypertension), deafness and a hell lot of other bad things. So, it seems that noise is all bad. It’s not always so!

There is a disease called otosclerosis. In this disease, the footplate of stapes (a small bone in the middle ear) gets fixed to the oval window of the internal ear, producing conductive deafness. The patient can not hear normally as the ossicular (bony) conducting chain is at fault. But surprisingly, such persons hear well in noisy places (market, railway station). This phenomenon called Paracusis Willisii is said to occur due to the fact that one has to speak out real loud (over and above the background noise) in such places; thus making this loud voice cross the patients’ threshold of hearing. However, it may also be possible that the amplitude of the voice (in decibel) might ‘ride’ (summate) on the background noise amplitude, and this combined sound amplitude is heard by the ears. The brain then does some kind of fuzzy logic (or acts as a differential amplifier); and the ‘information’ is decoded. So, it seems that noise isn’t all that bad.

In ‘information theory’ even noise is said to contain information in it. One fine example that illustrates how visual noise might contain information is random dot stereography (and autostereogram). So, noise could be meaningful.

In diabetes mellitus, a very common disease across the globe, the blood glucose level rises. This and other metabolic products causes a condition called diabetic neuropathy, among other things. The person’s sense of touch is diminished and this results in inattention to sustained pressure(causes decreased circulation) or trauma to the affected area. This, along with the increased blood glucose and infection may then cause gangrene of the limb which might require an amputation of that limb. Cloutier et al have resorted to noise in an attempt to address the issue.

They applied mechanical noise directly over sensory neurons and have found that both vibration and tactile perception in these patients improved. This mechanical noise was christened as ‘stochastic resonance’ (stochastic means random or probabilistic; this particular term is coined since the frequencies are not tuned to match any particular frequency), and was applied at an imperceptible level. They applied this noise to the great toe of some of the affected individuals, while the controls received none (i.e. no SR). The effect was studied by measuring the vibration perception threshold (VPT). VPT was significantly lower in patients receiving SR compared to the controls (no SR). As the threshold was low, the patients’ sensitivity to detect vibration and tactile sensation improved. They hoped that a continually vibrating shoe insert could improve nerve function in these cases.

In another instance, Toshio Mori and Shoichi Kai of the University of Kyushu, Japan, showed that noise might improve brain function. They shone periodic signals (of 5 Hz flicker) onto the right eyelids and noisy signals onto the left eyelids of the subjects when they were at rest, and measured the intensity of their brain waves. Brain waves are electrical signals that occur in the brain due to the firing of neurons and are detected by electroencephalography (EEG). They found a sharp peak at 5 Hz, the frequency of the periodic varying signal. As they increased the strength of the noise signal relative to the periodic signal, a ‘harmonic’ peak emerged in the alpha wave band at 10 Hz. As the noise signal gained strength, this peak first increased and then diminished. The researchers believe that this harmonic peak is indicative of stochastic resonance in the cerebral visual cortex. Stochastic because of the non-linear way the brainwave behaves in response to the external stimulus. They argue that naturally occurring background electrical noise in the brain (from electron transport chains, neuronal activities) may play important roles in cognition and behavior.

However, not everything about noise is healthy as researchers from the University of California at San Francisco, USA suggest. They exposed healthy young rats to ‘white noise’, (random audio frequencies covering the full spectrum with randomly assigned amplitudes) and found that the development of their auditory cortex was delayed. They used electrophysiology tools to explore this. They also suspected that everyday environmental noise, also a type of white noise, could harm children by interfering with language acquisition and speech.

The story doesn't end here. Researchers have shown that noise has an important role in eukaryotic gene expression. When messenger RNAs (mRNA) are transcribed in the nucleus of a cell, they do so in a 'quantal' way; meaning that mRNAs are produced in spurt, in a stochastic (random) manner. The transcription process needs energy; as the promoter sequence have to be activated and for other biochemical reactions. This transcriptional noise may have implications in phenotypte diversity and cell differentiation process. Alternatively, bacterial pathogenicity may be increased by this 'noise' in gene expression.

The question is: should we scold our children when they continue with those awful noises? I am confused. But one more thing; it was this noise (in the microwave spectrum) that gave scientists the experimental proof that the Universe was expanding.

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Reference: Prolonged Mechanical Noise Restores Tactile Sense in Diabetic Neuropathic Patients.
Cloutier R, Horr S, Niemi JB, D' Andrea S, Lima C, Harry JD, Veves A.
Int J Low Extrem Wounds. 2009 Jan 6.

Noise in eukaryotic gene expression, doi:10.1038/nature01546

Noisy signals strengthen human brainwaves
T Mori and S Kai 2002 Phys. Rev. Lett. 88 218101

White Noise Delays Auditory Organization in the Brain

Noise, Wikipedia

Mori, T., & Kai, S. (2002). Noise-Induced Entrainment and Stochastic Resonance in Human Brain Waves Physical Review Letters, 88 (21) DOI: 10.1103/PhysRevLett.88.218101

The Circle Of Life And Soul

2009-04-01T17:49:00.003+05:30

Before embarking on this arcane topic, let us talk about death first. Clinically, death is said to occur when the heart and the lungs stop working (cardio-respiratory failure). However, with the advent of modern life support systems (such as cardiopulmonary resuscitation, artificial ventilation), quite a few such 'dead' persons have been brought back to life. Legally, death is now defined as when the activities in the brain stop: brain death. The EEG signals may cease completely or fall to undetectably low levels. Body organs may be taken from the 'dead' person and transplanted onto a 'living' recipient. Certainly, death does not mean that all the body tissues and organs die at once. However, death is considered an 'all or none' process and is irreversible. That some tissues remain alive in a dead person, it may be assumed that some as yet unexplained binding energy that keeps track of the living system, is amiss.

If you worked with computers, you may have noticed that there's a 'registry' which keeps note of the hardwares, softwares and other machine configurations vital to the computer's health. These informations are kept in the form of a 'tree' and are referred to as 'hive keys' (e.g. HKLM, HKCU etc). Should anything go wrong in the registry, the computer won't work; its dead. The hard disk is OK, the RAM is fine, even the CPU is intact; but the computer is dead. A similar analogy may be drawn with the BIOS (basic input output system) flash memories of the computer.

Do we have anything like this in our bodies that coordinate functions among various tissues (separated at a distance)? Could it be something like covalent or electrostatic interaction or some form of quantum entanglement between the tissues that works in an analogous way the system registry in computers does? Perhaps, interactions like the one observed among microtubular assembly (interactions at 'hydrophobic pockets') might be involved in a broader scale.

Looking from a different perspective, living systems may be thought of as a combination of different compounds and elements. They can be broken down into molecules, which may again be divided into atoms--> the so called 'elementary particles' like electrons, protons and neutrons. These can again be broken down into quarks and gluons and finally into the 'vibrating strings' of string theory.

Thus the whole gamut of living and non-living things may be construed in terms of a vast network of vibrating strings. This reminds of 'cosmic consciousness' of Carl Jung. Living beings may 'tap' onto this 'server' network by some form of electromagnetic resonance or quantum phenomena (disregarding decoherence for the moment). Life stops when we are 'off resonance', as if we get a DNS server error 769 or destination unreachable. May be there is some self sustaining oscillation that runs amok and cause a kind of 'thermal runaway' and entropy rises unmanageably.

Honestly, any endeavor to delve deeper into the topic will be futile at this point. First, we are bound by our senses and interpret things in the light of our past experiences. Second, we are prone to introduce Heisenberg's uncertainty errors, the closer we get to it. The more accurately we determine the location of 'soul' (if there's any) the further off we are to notice its properties. Thirdly, you can't really judge the velocity (not speed!) of a moving bus from inside (and blindfolded). You need to look from the outside.

Persons with 'near death experiences' (out of body experiences) and those who have undergone dissociative anesthesia (using ketamine) have reportedly 'seen' their bodies from a different dimension. Finally, intuition and not intellect, should be invoked to address this delicate issue as the former's approach is holistic, while the latter breaks an event down into its component parts (in the light of present knowledge) and analyzes them (and thus inherently error prone).

May be the souls is indestructible; it just relocates into another 'braneworld' having a few more dimensions and hence hidden from our views. As long as we can not definitively answer what life is, we certainly can not hope to speculate on what 'spirit' or soul is. A few more interesting points to ponder upon: Does a pregnant woman have 2 souls? Do individual cells and molecules have their own soul equivalents?

Disclaimer: While the facts described are true (hyperlinks given), this article is mostly a speculative 'synthesis' of sorts and largely reflects my own view.
Related: Is Science Killing the Soul? by Richard Dawkins, Steven Pinker

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Reference: hyper-links, unless specifically mentioned

Build Yourself This Cheap Tuned Radio Frequency Receiver

2009-03-31T23:54:00.003+05:30

Building a simple tuned radio frequency (TRF) receiver is an easy job. TRF receivers work by tuning to the transmitting carrier frequency (duly amplitude modulated, of course) and amplifying them before feeding this RF to the detector stage. Three major types of AM receivers are:

1. The Crystal Radio Receiver: The crystal was actually an impure form of lead sulphide, called 'galena'. A spring was made to contact this crystal through a fine tip; thus as if working as a point contact diode. This was known as 'cat's whisker' in the past era. Acharya Jagadish Chandra Bose (and G. Marconi) devised this contraption. You will be surprised to learn that this radio consumed no external power supply. The induced current was sufficient to drive a high impedance crystal headphone. You can learn more here.

2. Superheterodyne set: Here we have two oscillators; one tunes with the incoming carrier signal while the other, 'the local oscillator' generates a frequency so that there is always a difference of 455 kHz (470 kilo Hertz in some countries). This constant difference is achieved by 'ganged' variable capacitors, i.e. rotating the shaft will cause movements of both (tuner and local osc) vanes together. For example, if the tuner freq was 500 kHz, the local osc freq would be 500+455 kHz. So, you see that the intermediate frequency (IF) is always 455 kHz no matter what the tuner freq is. In other words, those 2 freqs would produce 'beat formation' in much the same way two tuning forks of different frequency does. (Should you try to simulate 'beats' in your very own computer, want to use your computer as an oscilloscope and study various waveforms, then this software by Christian Zeitnitz might be of interest to you.) Now, since this beat is not occurring in the audible range, the phenomenon is referred to as 'superheterodyning' (for supersonic heterodyning; ultraheterodyning would have been a better term as supersonic relates to the speed of sound) and the receivers are called superhet sets for short. Anyway, the IF is amplified and demodulated before being fed to the loudspeaker.

3. Tuned Radio Frequency (TRF) receiver: This is the one I'm going to talk about. Here in the adjoining figure you can spot our good old LC tank circuit. This is our tuner that detects the amplitude modulated carrier frequency. The RF signal is then fed to the input pin (pin 2) of IC ZN 414 (or MK484; YS414 in India). The IC originally produced by Ferranti, has 10 transistors that has RF amplifier stages, a detector circuitry and an AGC (automatic gain control) built in. The current consumption is extremely low and the IC itself operates in 1.2 to 1.6 volt range. Now follow the procedure of cleaning and soldering described in the radio transmitter article and populate the components on the stripboard. Solder them as shown in the figure keeping these points in mind:

1. the output decoupling capacitor (0.1 MFD) should be soldered as close to the IC as possible.
2. keep all leads short.
3. the tuner assembly should be kept as far away from the battery, headphone and their leads as possible.

The amplified RF signal is first 'half wave rectified' by diodes inside the IC and the bypass capacitor smooths out the carrier waveform. The preset (5 k ohm pot) is adjusted for better response. You may want to have a look at the ZN 414 datasheet and different connection diagrams here.

Nowadays, FM transmission is in vogue across the globe for its unmatched audio quality which includes stereo reception. The concept is quite complex which I don't understand well. Briefly, just as we consider force or velocity a vector quantity; the waves are also quite like them. They have magnitude and direction, and they are called 'phasor's. FM receivers work out the signal by a mechanism called the 'phase discriminator' or a 'ratio detector' circuit. The way FM sets do this analysis has a lot in common the way doctors analyze your ECG/EKG (vector analysis).

Last modified: never
Reference: hyper-links, unless specifically mentioned

Build Yourself a DIY AM Radio Transmitter

2009-03-30T11:04:00.010+05:30

Introduction: Understanding the fundamental principles of radio waves, their generation, propagation and reception is essential not only for the electronic enthusiasts, but also in other disciplines of science which employ radio-frequency (RF) in scientific investigation and even medical treatment. Here, the construction of a cheap and simple DIY (do-it-yourself) transmitter (Tx) circuit is described and its operations explained in simple terms.

Materials you'll need: A microphone, a transistor (ask your friends to help with the emitter, base and collector terminals. alternatively, look up the web), a radio frequency coil (choke, RFC), a variable capacitor (365 or 500 pF; pF = pico farad), 2 resistors, 3 condensers , an aerial (antenna), a soldering iron, solder wire, a stripboard (also known as veroboard) and a power supply (9V).

Assembly: Clean the leads (wire terminals) free of oxides/ enamel coatings by rubbing with fine grained sandpaper. Next, in accordance with the accompanying diagram, wire the components together in one of the following ways:
1. Connect the components directly and cover these joints by insulating tapes such as black tapes, cello tapes or scotch tapes (it is said that the scotch tape emits X-rays when pulled off, well it's unimportant here) . This will prevent accidental shorting with similar joints.
2. Or use a breadboard and press fit the leads in the slots. The breadboard has prewired connections within it, which will take care of the rest. Make sure that these prefixed connection layouts are followed. You may need to make some extra connections by using external hook-up wires/jumpers.
3. Or use a stripboard/veroboard: Put the components into the drilled holes according to the circuit layout. Now, solder the leads using a 20 watt soldering iron. It is the best option among the three mentioned.

When the iron is sufficiently hot, apply the hot tip to the component lead (not to the solder wire!) to be soldered, and bring the solder wire near it. Within a few seconds, solder will melt and flow evenly around the joint. Withdraw the iron (take as less time as possible).
Check with the circuit again. Cut any extra strip of copper track which might act as a potential short. You may need to make some extra connections by using external hook-up wires/jumpers.

Turn on your radio: Tune in to any 'empty' medium wave frequency on your radio receiver. Switch on your newly assembled wireless Tx. Turn the spindle of the variable condenser of 'your set'. At some point you'll hear a 'smooth hissing carrier tone' on the receiver. Keep the variable capacitor there. You're almost done! Speak on the mike, you'll hear the same on the radio receiver. Now, press the mike on your left chest, you will hear your heart sounds on the receiver set. You can make a 'phonocardiogram' too this way.

How it works: The Tank oscillator produces the carrier frequency (freq= 1/{2*pi*sq.rt.[LC]}; L=inductance of the coil in Henry, C= capacitance in Farad, as dictated by the variable capacitor). To see an animation of how a 'tank circuit' works visit this page. The amplitude of the freq. of the carrier wave is modulated by the input from the microphone. As we speak on to the microphone, the diaphragm vibrates. This makes a coil of copper, wound around a powerful magnet, vibrate too, making induced current in the coil, the magnitude of which depends on the speech/audio frequency. The mike is connected to the base of the transistor via a capacitor. This capacitor allows AC (alternating currents) to pass, but blocks any DC component. The second feature thus, does not let the microphone change the bias voltage conditions of the transistor. The biasing of the NPN type transistor is done by the two fixed resistors.
When some audio signal is present, collector current will increase. Since it is the tank circuit at the collector load, the amplitude of the 'carrier frequency' will thus be modified. Amplitude Modulation has been achieved! You will notice that one end of the output capacitor has been connected to the antenna, while the other end is connected to the ground (earth, ground of your wall power outlet, or safer still the lead water pipe). This arrangement makes the capacitor plates look like, as if, they are placed far apart; earth and sky! This causes dissipation of the energy as electromagnetic waves.
Frequency Modulation (FM) transmission is better, since the static (electrical noise from man made appliances, lightnings etc) is almost absent and stereo signal can be transmitted, to name a few.

Experiments and lessons on it:
1. Move a magnet at the back of the radio receiver (where the RFC choke is located) with the receiver tuned to the short wave band. You'll notice that the frequency changes: many different stations come in (without even having to turn the tuner knob!). This happens as the magnet alters the local oscillator of the receiver.
2. Put the radio inside a metal container and try to catch a station. You won't succeed. This is 'shielding'; the metal does not allow electromagnetic wave to pass in. Co-axial cables exploit this phenomenon to avoid noisy interferences. Sensitive instruments used in medicine (e.g. EEG) or radioastronomy were often built underground.
3. Just touch two different metals in front of the radio receiver: the radio will make noise. This is due to passage of electrons between the metals.
4. Short two ends of a battery (cathode and anode), the radio hisses and makes noise. Obviously, electromagnetic fields are created.
5. The telltale undulations heard in shortwave, is due to the reflection (actually total internal reflection, a type of refraction) of radio waves from the Kennely-Heaviside layer of the ionosphere, a charged layer in the higher atmosphere. If you put a laser light or any light on the ground and put a mirror higher up, you would get a reflection. Moving the mirror up and down will put the reflected beam closer or further away (light is also an electromagnetic wave). This layer is absent in night time and Appleton layer still higher up reflects then. This is why SW broadcasting stations are best heard at different frequencies during different parts of the daytime.
6. You can listen to cosmic noise, falling meteors etc.
7. Take a radio and tune it to a station. Now take a second radio and turn the tuner knob till that same radio station is obtained. You'll notice a drop in volume of the former radio (and vice versa). This illustrates energy transfer by resonance.

Uses of Radio waves: Telecommunications, radar, radioastronomy, medical investigations such as MRI (NMR), medical therapies (radio frequency ablation in ectopic pacemakers of the heart (electrophysiology), research procedures such as thought broadcasting etc.

N.B. : 1. This is a very simple, low cost transmitter, but many countries might have legal restrictions on it. A crystal controlled version of the set is more likely to be approved.
2. Other forms of electromagnetic waves may be broadcast in much the same way e.g. our good old TV remote uses infrared waves.

Last modified: never
Reference: hyper-links, unless specifically mentioned

The Sabbatical: A Forced Hibernation

2009-02-15T22:11:00.003+05:30

It is painful to stay away from the addictive internet and my newfound blogging obsession. But I have to maintain a distance from the digital world for a couple of months. No, the current economic recession has nothing to do with it. A greater danger, called examination, is looming ahead. So, all I know now are books, books and more books! The desire to 'ping' the cyberspace is always there. Therefore, I wan't to break free as soon as I can; and when I do that, I'll really be back in black. Hoping to see you in full throttle when I come back as the enlightened one!

Quantum Biology: The Spooky NanoWorld of Molecules

2009-01-29T00:06:00.010+05:30

We are quite adept in solving numerical problems in our everyday ‘analog world’ using decimal rules developed by us. Digital computers, on the other hand, calculate using binary or Boolean (0, 1) rules, and then convert the result in decimal format with the help of dedicated binary to decimal converter ICs. In the molecular world, calculations ‘happen’ in a strange way.

Take for example the case of Fluorescent Resonant Energy Transfer or FRET. Also known as Forster Resonant Energy Transfer, this phenomenon is characterized by the emission of a photon of one frequency (upon stimulation) which, in turn, activates an acceptor molecule to emit a photon of another wavelength. There’s one clause that says that the first photon (from the donor molecule) will only be emitted when it can definitively be coupled with the ‘acceptor’. But in the first place, how is this ‘virtual photon’ to know whether its bride was waiting or not when it hasn’t even visited her? Yet FRET doesn’t fret, and the process goes on.

All plants use chlorophyll to trap sunlight and convert it to chemical energy in the form of carbohydrates by photosynthesis. The efficiency approximates 100%. The predominant classical approach was that the photons hopped from light capturing pigment biomolecules to the ultimate reaction center where the actual conversion was taking place. But this ‘first choose and then pick’ approach that classical physics suggested would mean considerable loss of energy as heat, as photons wasted time as they hopped down the energy ladder. Quantum mechanics bypassed this by allowing simultaneous sampling of all energy states at one go by its unique properties of ‘superposition’ and ‘entanglement’. Graham Fleming and researchers at Lawrence Berkeley National Laboratory and the University of California at Berkeley showed the existence of a process of ‘quantum beating’, (a phenomenon akin to 'heterodyning’ in radio sets that is used to obtain intermediate frequencies for amplification) occurred which allowed sampling of all energy states by interference of the propagating wave. They used two-dimensional electronic spectroscopy in order to probe the sequence of events that occurred.

That the RBCs (erythrocytes), actomyosin complexes use quantum mechanics for system optimization has been established. Cellular respiration in the mitochondria, DNA, and the brain too might exploit quantum computing.

Counting without disturbing the molecule may be achieved by quantum mechanics, for it allows a molecule to know as if ‘intuitively’, the state of another molecule placed at a distance. Erwin Schrödinger, in his book 'What is Life?', opined that biological systems could be using the principles of quantum theory to maintain biological order. Sir Roger Penrose along with Stuart Hamerhoff proposed that the brain could be working as a quantum computer. In reaction to this, Max Tegmark showed that environmentally induced decoherence would foil any quantum interaction taking place. But Tegmark assumed the average kinetic energy (temperature) of the brain as 310 K (273+37). While this is true in a macroscopic world, Koichiro Matsuno has shown, using black body radiation measurements, that actomyosin complexes which are abundant in the axons of nerve cells, can reach local temperatures as low as 1.6*10-3K. It is as if nature has evolved ways to ensure decoherence free subspaces where entanglement and quantum interaction were possible. Stephen Hawking in his book 'A Brief History of Time' observed that quantum mechanics was the basis of modern biology and chemistry and the only area where quantum mechanics was not properly integrated were gravity and the large-scale structure of the universe (page 60).

To quote Ogryzko "Indeed, if it has taken Humankind only few decades to approach the use of entanglement in quantum information technology, one can wonder why Life, in billions of years of evolution, could not also learn to take advantage, finding in entanglement an alternative resource for stabilizing biological order." It seems we need an entirely different approach if we wanted to probe the mysteries of life and quantum theory is poised to help us in this regard.

P.S. I am glad that the prestigious multidisciplinary journal "NeuroQuantology" published this article with the title "The Spooky NanoWorld of Molecules" and archived it in their "arNQ Eprints and Repository". I thought I could share this with you, my readers!

Last modified: Jun 29, 2010
References:
Quantum Biology
Vasily V Ogryzko (2008). Erwin Schroedinger, Francis Crick and epigenetic stability Biology Direct, 3 (1) DOI: 10.1186/1745-6150-3-15

Period Concatenation in The Brain, And The Synthesis of Beta 1 Rhythm

2009-01-28T23:41:00.007+05:30

The principles of generation of EEG waves in the brain are still ill understood. Although the general mechanism of cortical dipoles and thalamocortical oscillations behind the generation holds true; there has been speculations that the alpha waves could actually be originating in the heart- the cardiac electromechanical hypothesis, which states that the arterial pulse ‘shocks’ the skull-brain mass (and interacts electrically and mechanically) to oscillate at its naturally resonant frequency of approximately 10 Hz.

Now, Kramer et al propose that beta 1 rhythm could be the result of a process called period concatenation (concatenation means chain forming or serial addition). Beta rhythms (18-30 Hz) were thought to be harmonics (integer multiples of the fundamental frequency) of alpha rhythms (8-12 Hz). Kramer et al observed that application of 400 nanomolar kainate to rat somatosensory cortex produced gamma rhythm in the superficial cortical layers and beta2 rhythms in the deep cortical layers.

They observed that after an initial interval of simultaneous gamma (~25 ms period) and beta2 (~40 ms period) rhythms in the superficial and deep cortical layers respectively, a resultant, synchronous beta1 (~65 ms period) rhythm in all cortical layers occurred. They concluded that the time period (the inverse of frequency, or 1/f) of gamma wave (25ms) concatenated with that of beta2 (40ms), to form the time period of 65 ms (40+25). That was the time period of the beta1 rhythm, which resulted as a consequence of this concatenation. They concluded that neural activity in the superficial and deep cortical layers of the brain could combine over time to generate a slower oscillation.

Frequency synthesis would, naturally, have both energy and space saving implications for the system concerned. That the brain economizes is not new in computational biology and electronics. For example, in the simplest and realistic model of the 40 Hz gamma rhythm, only 2 neurons (one excitatory and the other inhibitory) interconnected by reciprocal paths are required. The excitatory neuron will ‘charge’ the inhibitory neuron. The inhibitory neuron will suppress (inhibit) the activity of the excitatory neuron as a result, and any oscillation will be dampened. Hence, a decay in the inhibitory synapse will not inhibit the excitatory neuron anymore and thus cause oscillation; and clearly, the frequency of rhythm will depend on the decay time. This “gamma-motif” resembles a lot with the ‘flip-flop’ circuits in digital electronics.

Its not surprising that the human brain which had evolved as a result of nature’s selection process will learn to compute things so that the metabolic costs of additional neural pacemakers were curtailed to the bare minimum.

Last modified: never
References: Mark A. Kramer, Anita K. Roopun, Lucy M. Carracedo, Roger D. Traub, Miles A. Whittington, Nancy J. Kopell (2008). Rhythm Generation through Period Concatenation in Rat Somatosensory Cortex PLoS Computational Biology, 4 (9) DOI: 10.1371/journal.pcbi.1000169

A Cardiac Hypothesis for the Origin of EEG Alpha
Castillo, Horace T.
Digital Object Identifier: 10.1109/TBME.1983.325080

Phase Alignment of Neocortical Gamma Oscillations by Hippocampal Theta Waves

2009-01-19T17:09:00.009+05:30

An empty brain is the devil’s workshop, goes the proverb. Actually, the brain is never empty. Even in our deepest slumber, the brain continues to weave waves of electrical rhythms that can be seen with the aid of electroencephalogram or EEG. When we place electrodes on the scalp or on the cortex (inside the skull), and amplify the faint signals via bioinstrumentation amplifier, we can lay our hands on these fluctuating rhythms. (More on the electronics of EEG may be found at the OpenEEG project site).

We have as many as 100 billion neurons in the brain. In the superficial layers of the cortex, the neurons have numerous dendrites branching out from the soma or cell body (shown in grey oval in this picture).These neurons have been compared to a forest of trees where the branches are the dendrites and the trunk the axon. These dendrites make extensive connections among each other. They also get connections from the axon collaterals of neighboring axons (i.e. the 'trunks' of other trees connect to these 'twigs' by offshoot from the trunks). Since there are a lot of axons converging on the dendrites of each neuron, and given the fact that these axons can be excitatory (red) or inhibitory (green) depending on the neurotransmitter, the sum of input may be either negative or positive (with respect to the cell body). Thus an alternating current (cortical dipole) will flow between the shifting dendrites and the soma. This along with thalamocortical oscillations produces the EEG waves.

The brain doesn’t churn out the rhythm just like that. Had the neurons fired randomly the oscillations would have cancelled out.EEG waves occur due to synchronous discharge of neurons producing the alpha, beta, theta, gamma and other telltale waves. Like all other electrical waves, they too have a frequency and amplitude. Alpha waves, for example, have a frequency of 8-12 Hz (cycles per second) and an amplitude ranging from 50-100 microvolt when recorded from the scalp, and it is found when a person is resting comfortably with eyes closed and the mind wandering. On the other hand, gamma rhythm has a frequency of 30-80 Hz, and it is found when a person is deeply engrossed on some work.

It was known for a long time that the hippocampus exerted a role in learning by fostering long term potentiation (LTP) by aligning the neocortex, where memories are stored. The mechanisms behind this are now emerging. Sirota et al and Siapas et al have analyzed rat brains and found out that there were many localized gamma oscillators within the brain that gave rise to neocortical gamma bursts. These oscillators had varying frequencies but they phase aligned themselves with the arrival of hippocampal theta waves. A large fraction of pyramidal cells and interneurons too were phase aligned to the hippocampal theta rhythm.This is similar to a bar magnet aligning iron dust or other ferromagnetic materials by virtue of its magnetic field. Apart from the cerebral cortex, the cerebellar cortex and the hippocampus too can generate brain waves. Such a mechanism may explain the orchestration of many parts of the cortex (and hence the memory engrams they contain); and data synchronization and downloading to the hippocampus for memory retrieval. It also shows how hippocampus does the ‘indexing’ of cortical contents. These experiments throw light on neuronal plasticity and information flow, and may be someday they could help clinicians in fighting memory loss as it occurs in neurodegenerative diseases like Alzheimer’s disease.

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References:

Prefrontal Phase Locking to Hippocampal Theta Oscillations
Athanassios G. Siapas, Evgueniy V. Lubenov and Matthew A. Wilson. doi:10.1016/j.neuron.2005.02.028
A SIROTA, S MONTGOMERY, S FUJISAWA, Y ISOMURA, M ZUGARO, G BUZSAKI (2008). Entrainment of Neocortical Neurons and Gamma Oscillations by the Hippocampal Theta Rhythm Neuron, 60 (4), 683-697 DOI: 10.1016/j.neuron.2008.09.014

Visualizing Viral Kinetics Using Fluorescence and Bioluminescence

2009-01-11T23:58:00.007+05:30

It would be nice if we could see an individual virus particle, a virion, in real time within a mammalian tissue starting from its attachment to the host cell and entry, to its assembly and budding and release. The dynamics of viral production has been studied using computational models by noting the response of the virus to exogenous administration of reverse transcriptase and protease inhibitors. It was noted that a mind boggling 10^10 to 10^11 virions are produced each day by using this mathematical model. Now, Jouvenet et al have been able to fluorescently label a molecule called Gag protein (for group specific antigen), the major structural component of HIV. With the aid of fluorescence resonance energy transfer (FRET) and other techniques on these fluorescently tagged virions in living cells, they have been able to see the biogenesis of HIV virions in real time; from viral assembly to release by budding. The assembly rate accelerated as the Gag protein accumulated inside the cells. Typically, the time required for the assembly was just 5-6 minutes.

In fluorescence resonance energy transfer (FRET), an external light source shines on a donor fluorescent molecule. The donor molecule gets excited and emits light of a different frequency (fluorescence), which activate the acceptor molecule. The acceptor fluorophore then emits a photon of yet another wavelength (or a quantum, as we are referring to the particle nature of light here). Both donor and acceptor fluorophores are nothing but color variants of green fluorescent protein or GFP. The whole process (FRET) is noisy as the incident light messes up with the emitted light. The incident light may also activate the acceptor fluorophore directly, leading to error.

Recently, Asokan et al used bioluminescence from Gaussia luciferase to study adeno associated virus (AAV) kinetics in living mammalian cells. By using bioluminescent molecules, the external light source as used in FRET was no longer needed. This way, direct activation of acceptor molecule was avoided and background noise was kept to a minimum. They first amplified gLuc (Gaussia luciferase) in a plasmid by polymerase chain reaction or PCR, using primer sequences. They then fused the resulting protein to that of an adenoviral subunit of AAV, called Vp2. The resulting gLuc/AAV construct was then injected into the left hind limb of rats. They could clearly notice the AAV vector dynamics. The importance of such dynamics is realized when the use of AAV as a vector in gene therapy is considered. They opined that such a technique would be ideal in studying viral dynamics in peripheral tissues such as the eye and the brain.

Bioluminescence is used to study virus tropism and viral kinetics. Tropism refers to the different populations of host cells a virus can attack. Retroviruses have a narrow tropism meaning they can infect only a few types of cells such as CD4+ T cells and macrophages. Previous studies employed Gaussia luciferase reporter gene as a tool for studying viral dynamics. Recent experiments promise a better future for the study of viral behavior.

References:
A Asokan, J S Johnson, C Li, R J Samulski (2008). Bioluminescent virion shells: new tools for quantitation of AAV vector dynamics in cells and live animals Gene Therapy, 15 (24), 1618-1622 DOI: 10.1038/gt.2008.127

Human Immunodeficiency Virus Disease: AIDS and Related Disorders: Anthony S. Fauci, H. Clifford Lane, Harrison’s Principles of Internal Medicine, 17th Ed.

Imaging the biogenesis of individual HIV-1 virions in live cells
Nolwenn Jouvenet, Paul D. Bieniasz, & Sanford M. Simon
Last modified: never

AIDS,Virology and Nobel Prize in Physiology or Medicine 2008

2008-12-31T21:18:00.005+05:30

In the summer of 1981, the United States Centers for Disease Control and Prevention (CDC), reported occurrence of Pneumocystis jiroveci (previously known as P. carinii) pneumonia in five otherwise healthy homosexual men in Los Angeles. At about the same time, 26 cases of Kaposi’s sarcoma were noted in New York and Los Angeles, in previously healthy homosexual individuals. Soon such cases came to be found in intravenous drug abusers. Though HIV disease (AIDS) was officially recognized in the US in 1981, the virus had traveled most of the world by 1971. AIDS (Acquired Immunodeficiency Syndrome) is a slowly progressing disease as it has a median latency period (incubation period in AIDS) of 10 years.

In AIDS the number of CD4+ T cells, a subpopulation of T lymphocytes, diminishes drastically. Since these cells are concerned with fighting microorganisms, the immunity takes a severe beating, making the body vulnerable to foreign invaders. Clinically the 4H mnemonic was coined: Homosexuals (and heterosexuals), Heroin addicts (and other intravenous drug users), Hemophiliacs (and those requiring blood transfusions) and Haitians.

The pattern of spread pointed it clearly to the scientists that it had something to do with blood and body fluids. But the etiologic agent foxed the researchers. In the early 1982 Robert C Gallo with Max Essex proposed that it could be the handiwork of a virus, a retrovirus. Their assumption was based on another virus, feline leukemia virus, which caused T cell depletion in addition to leukemia.

They searched for similarities between DNA and RNA of some tissues of AIDS patients, and HTLV-1 and 2 (Human T cell Lymphotropic Virus; related to HIV). Away in France, Luc Montagnier was stimulated in part by their ideas. In early 1983, Dr Gallo sent Montagnier IL-2 and antibodies against the HTLV's. Montagnier and his co-workers had found a new retrovirus in a patient with lymphadenopathy, and they could distinguish it from the HTLV-1 and with the antibodies Gallo provided. While the French called it the LAV (Lymphadenopathy Associated Virus), the Americans called it HTLV3. It is now called Human Immunodeficiency Virus (HIV).

The virus has two important glycoproteins, GP120 and GP41. GP120 binds with CD4 molecules present in the host CD4+ T lymphocytes and some other cells. This causes a conformational change in the GP120 molecule, allowing it to draw the virus near the host cell. The membranes fuse and the viral core material gains entry. The materials comprise of 2 identical RNAs, an enzyme called reverse transcriptase, and another key enzyme called integrase. Normally, DNA is transcribed to form RNA, a process called transcription. Reverse transcriptase does just the opposite. It makes DNA out of RNA (reverse transcription). Integrase then integrates this viral DNA with the host DNA. The virus thus, not only reproduces with each division, it usurps all control from the host cells. The cells now do what they are told to do. The virus remains for the lifetime of the cell, making AIDS an incurable disease.

Professors Luc Montagnier and Françoise Barré-Sinoussi were awarded the Nobel Prize in Physiology or Medicine for their 1983 identification of what was later named the human immunodeficiency virus1 (HIV1). They shared the prize with Harald zur Hausen of Germany, who discovered that human papillomavirus (HPV) caused cervical cancer, the second most common cancer in women.

Robert Gallo wasn’t among them. Like the Nobel Prize in Chemistry this too is disheartening. Anthony S. Fauci, director of the National Institute of Allergy and Infectious Diseases said "Gallo deserves enormous credit…It's a shame you can't give it to four people, because Gallo's contributions were enormous". It really is and this leaves us in a sad note.

A splendid Youtube video depicts how the virus gains entry, replicates, the areas where pharmacological intervention is carried out and many more. Watch this.

References:The discoveries of human papilloma viruses that cause cervical cancer and
of human immunodeficiency virus

Robert C Gallo (2006). A reflection on HIV/AIDS research after 25 years Retrovirology, 3 (1) DOI: 10.1186/1742-4690-3-72

An Overview of Gene Therapy

2008-12-28T23:19:00.008+05:30

Ashanthi, a four year old girl, was suffering from an immune deficiency disorder called SCID (Severe Combined Immune Deficiency). Due to the lack of a healthy immune system, she was susceptible to infections even from germs which otherwise would not affect healthy persons. She was confined to her room, met no one outside her family, and had to take heavy doses of antibiotics to fight the microbes on behalf of her dilapidated immune system. A team of doctors from the National Institutes of Health, in the United States, drew blood from the patient’s body, and separated the WBCs (white blood cells; cells which fight infections). They then cultured the WBCs, inserted the missing gene and then infused the blood back into Ashanthi’s bloodstream. The girl survived, she no longer lived a recluse life and antibiotics were no longer a ritual. That was the first approved gene therapy (ex vivo, as the engineering was done outside the body) procedure carried out in a human.

Gene therapy is the procedure of replacement of faulty genes (nucleic acid sequences) by healthy ones. Frequently, a normal gene is added to an existing faulty allele, rather than a replacement of the gene at fault. Genes consist of stretches of deoxy-ribonucleic acids (DNA). The nucleic acid sequences in the DNA dictate the formation of proteins via the mediation of ribonucleic acids (RNA). Information contained in the DNA is passed on to the RNA by a process called ‘transcription’, which occur in the nucleus of the cell. RNA then goes to the cytoplasm of the cell where it forms a protein, in a process called ‘translation’; the functional product of that gene, its spokesman! The DNA sequence determines the sequence of amino acids in the protein, which is important in that any mistake in having the right amino acid in the right place may yield a non-functional protein with an abnormal configuration. Thus, an abnormal DNA sequence might (not always) produce a non functioning enzyme (a protein), causing diseases of immunity, metabolism and cancer.

We can ‘insert’ a normal functional gene into the genome containing an abnormal one; exchange an abnormal gene for its normal counterpart by homologous recombination; we could even ‘regulate’ the ‘expression’ of a particular gene. Inherited genetic diseases like thalassaemia, sickle cell anemia and cystic fibrosis could best be tackled by manipulating the ‘germ cells’ (sperms and ova) and this not only would ensure that the progeny was healthy but would also be passed (this new gene) onto the next progeny. Such heritable ‘germ line therapy’ despite sounding promising, is prohibited due to ethical concerns and the lack of expert technical knowhow. ‘Somatic cell gene therapy’, the gene therapy practiced these days, however, is not heritable.

Now that we know the basics, we should find a suitable carrier (vector) to deliver the goods inside the cell. Viral vectors are the most commonly used. Retroviruses, for example, take with them 2 identical copies of single stranded RNA (ssRNA); an enzyme called ‘reverse transcriptase’ and ‘integrase’, another enzyme, when it enters a cell. Reverse transcriptase or RNA dependent DNA polymerase converts the RNA sequences into DNA. The double stranded DNA then integrates with the host genome by the mediation of ‘integrase’. A therapeutic gene could now express itself in the form of a usable protein, via the integrated viral genome. Since viruses may cause disease, researchers must ensure that the disease causing genes of the virus are deleted. For example, AIDS is caused by a retrovirus (HIV). Another cause for concern is that retroviruses integrate randomly in the human genome. If they sat close to a proto-oncogene, or in the middle of a tumor suppressor gene (this might disable the suppressor gene), it might cause cancer.

Adenovirus is another option. A double stranded DNA (dsDNA) virus, adenovirus, does NOT integrate with the host cell, hangs free in the nucleus and just carries out transcription. Frequent administration is necessary, as the gene does not replicate with the host cell. Adeno-associated virus (AAV), an ssDNA virus, may also be used as a vector. The recombinant type (rAAV) carries NO viral gene & does NOT integrate. But they can infect quiescent (non-dividing) cells, hence may prove useful in neural/neurodegenerative diseases.

Non viral vectors include:
Naked DNA: Transfection (using phosphate-DNA mixture), Electroporation (use of electrical pulse for better membrane permeability), Sonoporation (using ultrasound for facilitation of DNA delivery), gene gun (DNA coated gold nanoparticles ejecting out along with high velocity gas) are some techniques for delivering DNA fragments.
Oligonucleotides: Antisense nucleotide sequences for the target gene. Being antisense, the nucleotides will latch onto the sense strand, just like the opposite poles of a magnet, thus preventing its translation. Fomivirsen is one such drug which is used in cytomegalo virus (CMV) retinitis.
Short interfering RNA (siRNA); Small nucleotide sequences which tell the cell to cleave faulty mRNA.
DNA-lipid complexes (lipoplexes): here the DNA molecule is covered with an arrangement of lipids in the form of a micelle. Using a nonionic surfactant such as Tween 80 in addition, gave a better yield.

The challenges are still great. Our immune system and the genome do not take these pieces of DNA easily. For example, the gene transfer frequency (in hematopoietic stem cells of dogs and monkeys) for adenosine deaminase, the deficiency of which causes SCID, was only 3%. Still scientists hoped that the healthy cells would outgrow diseased cells as they had distinct survival advantages. But the efficacy of delivery didn't improve.
As of today, most major trials on gene therapy are on pluripotent hematopoietic stem cells (PHSC) and cancer cells. It is only natural to assume that genetic manipulations on blood stem cells (PHSC) would cure a variety of diseases affecting the blood cell lineages. And the quest goes on.

1. Mark A. Kay*,, 2. Dexi Liu, and, 3. Peter M. Hoogerbrugge (1997). Gene therapy PNAS , 94
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References: Gene therapy
Gene therapy PNAS November 25, 1997 vol. 94 no. 24 12744-12746

Fantastic Fluorescence:Brainbow and The Nobel Prize 2008

2008-12-09T02:43:00.000+05:30

In my childhood, I used to be fascinated by the mysterious glow of fireflies. Later I learned that it was due to a reaction between a substance called Luciferin and an enzyme, luciferase, a phenomenon called bioluminescence. This kind of glow is not limited to land creatures. Creatures living at the bottom of oceans too emit light.

Osamu Shimomura of Japan was given the task of isolating the substance which let the marine mollusk Cipridina glow when it was crushed and mixed with water. He succeeded, and on the wings of his publication, was recruited by the Princeton University, in the United States. There he began studying Aequorea Victoria, a marine jellyfish which glowed green when agitated. The jellyfish had an umbrella like shape and its outer rim glowed green. He chopped off the outer edges, crushed it, and filtered it to obtain a ‘squeezate’. He noticed one day that the ‘squeezate’ glowed blue when he poured some into the sink. He understood that it was the calcium ions (Ca++) in the seawater present in the sink that made it glow blue. It was christened aequorin.

During the extraction process they also chanced upon another protein called GFP, for green fluorescent protein. It glowed green when excited with ultraviolet light or light in the blue spectrum. The structure of GFP is barrel shaped (also likened to beer can shape), and consists of 238 amino acids. The chromophore or light emitting part is in the interior of the ‘beer can shaped’ molecule, in a region called the alpha helix region (of the molecule); while the exterior of the molecule was comprised of beta pleated sheets of the GFP molecule. Shimomura and colleagues showed that the blue color emitted by aequorin donor excited the GFP acceptor in an energy transfer process. The photons in the blue wavelength were absorbed by the GFP chromophore and photons of green wavelength were emitted - a phenomena called (bio)fluorescence. Fluorescence differs from luminescence from the fact that in fluorescence light of another wavelength is emitted than the one absorbed and luminescence means emission of light.

The whole story struck a chord in Martin Chalfie’s ears. He thought what if he could harness the gene that codes for GFP and bind it to the segment that coded for a protein of interest? He worked with a roundworm, Caenorhabditis elegans, a simple organism with only 959 cells and yet a complete organism for it could procreate, had a brain and even one third of its genes were related to humans. It was translucent and hence studying its interior was easier. He contacted Douglas Prasher who was also hot in the trail for the GFP genes. Douglas Prasher did as he promised. He sent the GFP gene to Chalfie once he got hold of the gene. Chalfie introduced it behind the promoter of the gene that coded for proteins in C elegans’ touch receptor neurons. The neurons were cleanly delineated, that too in a live worm and ‘real-time’! GFP, being a natural gene product, is non toxic.

Roger Tsien wanted more. He knew from earlier studies that the ‘chromophore’ had 3 key amino acids: serine, tyrosine and glycine in position 65, 66 and 67 respectively in the 238 amino acid long GFP molecule which formed the chromophore. He used DNA technology to alter the amino acid sequence so as to obtain GFP variants that would absorb and emit light in different part of the electromagnetic spectrum. This way he obtained cyan, yellow and blue. He obtained DsRED, a red GFP-like protein extracted from coral, from two Russian researchers and modified it so that it was stable and of desired molecular weight.

It was all set by now. Researchers now modified mice genetically and introduced the gene for red, cyan and yellow GFP. They expressed the corresponding proteins in their brain and what we got is a riot of colors, the ‘brainbow’, short for brain and rainbow. Like the elementary colors, these colors when combined in different proportions, produce many colors, just as a color printer does using them (cyan, yellow and red). One could now visualize the neural circuitry in much the same way as seeing electronic circuits. Disease detection and progression in Alzheimer’s disease, cancer and Parkinson’s disease are some potential clinical applications. Watching biogenesis of HIV1 (the virus that causes AIDS) in live cells in real time is now an easy meat. GFP can also be engineered to recognize heavy metals like cadmium (a cancer causing chemical), explosives like TNT and Arsenic (a water pollutant causing Arsenicosis).

Osamu Shimomura, Martin Chalfie and Roger Tsien were awarded the Nobel Prize in Chemistry, 2008. Sadly, Doug Prasher was left out, despite his outstanding contribution in this field. He is now driving a van at $10 an hour to meet his living expenses. He is not alone. This year’s Nobel in Physiology or Medicine too left out Robert Gallo, an HIV pioneer. So, not totally a happy ending.

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References:

The Nobel Prize in Chemistry 2008
The Man Who Missed the Nobel Prize
Stuart Cantrill (2008). Nobel Prize 2008: Green fluorescent protein Nature Chemistry DOI: 10.1038/nchem.75

PET Scan: Particle Physics And Electronics in Medical Imaging

2008-12-05T19:41:00.007+05:30

Numerous imaging modalities are there to view anatomical structures in our body. They include X rays, Ultrasound imaging, MRI and many other procedures where we can see normal or diseased tissues in our body. They tell us ‘the location’ (position) in the body the image corresponds to. If we wanted to see what were happening in these locations, we would then need to perform functional imaging techniques like PET scan or fMRI.

In Positron Emission Tomography or PET, a radioactive isotope that decays by positron emission is introduced into the body. Positron emitting radioisotopes are prepared by bombarding stable atomic nuclei by protons. Protons are speeded up in a particle accelerator called cyclotron which then impinge upon the stable nuclei, and knocks out one neutron from its nucleus. The proton now occupies the position where the ousted neutron once stayed. But this atomic configuration is unstable, so the proton now decays. It decays by emitting positron, a particle resembling an electron in all aspects except that the charge is positive and not negative. In other words, a positron is an antimatter: an anti-electron.

Many different radioisotopes are there, such as Fluorine18, Oxygen15, and Carbon11. 18F is the most commonly used isotope. It replaces hydroxyl (OH) group in molecules of interest. We can use 18F Fluorodeoxyglucose (18FDG) an analog of glucose for probing the activity of brain. Our brain uses glucose for its metabolism, so when it encounters 18FDG, it stores them. The FDG in the brain begins emitting positrons. These particles travel only a short distance before they meet nearby electrons and annihilate. Two gamma ray photons, each having 511 keV of energy are produced. Photons because they are electromagnetic waves, and gamma ray because the frequencies correspond to the gamma ray spectrum of electromagnetic waves.

So by detecting these photons, we can find out where they came from, since we know that these photons are emitted back to back, 180 degrees apart. (They aren’t exactly collinear as their initial velocity is not zero, and some computational error always creeps in). For the detection part, we need a detection array which will convert these photons into electrons. This is done by scintillators. Bismuth germanate, Luterium oxyorthosilicate (LSO) are some of them. Photons which are incident on them produce electrons by photoelectric effect. These electrons are then guided through a vacuum tube, which has many positive electrodes (dynodes) held at successively higher voltages. These dynodes of this photomultiplier tube accelerate these electrons, which in turn knock-off more electrons from the dynode plates. Thus we get more electrons than what we started with. The signal has now been amplified and we now have a measurable current.

With advancement in detection technology, silicon avalanche photodiodes (silicon APD) has now shown promise to replace the vacuum technology (photomultiplier tube). As the name suggests, APDs work in a similar way an avalanche gains its momentum as it descends from the mountain-avalanche effect. Detecting photons aren’t sufficient. We need to detect only co-linear (coincident) photons. Each collinear photon pair (i.e. 180 degrees apart) will constitute an event. All other photons (noise) must be rejected. About 10^7 to 10^8 or more ‘events’ must be registered in order to have a good signal to noise ratio. Image faithfulness varies proportionally with the square root of the number of events.

By acquiring a large number of events, the computer software is able to determine exactly where these radioactive tracers are located. This, in our case, means the locations where the neurons are accumulating (accumulation is a function of utilization of glucose) 18FDG. Thus we get a functional map. In order to know ‘what’ these structure were, we need to combine anatomical imaging like MRI or CT with PET. This combined PET-CT or PET-MRI let us know what structures are doing how much.

PET scan is very useful in neuroscience researches, clinical diagnoses like cancer detection, receptor analyses and even watching gene expression in molecular biology.

References: A good site with animation: PET animation

Physicsworld

Peripheral Clocks Synch With The Master Zeitgeber

2008-12-03T23:07:00.006+05:30

In our bodies there are clocks in addition to the Master clock located in the suprachiasmatic nucleus. In computers, there are multiple clocks too, and they are tightly coordinated. For example, Integrated circuits like AV 9155 generate multiple clock frequencies for different portions of a computer (e.g. bus clock, CPU clock, keyboard clock etc.). All these clock frequencies are well regulated, since ICs like AV9155 use 2 quartz crystals (14.318 MHz) which generates of all these frequencies (they have inbuilt circuitry for dividing/multiplying these frequencies to create other necessary frequencies).

Our bodies have their own version of these ‘crystal oscillators’, the BMAL1/CLOCK heterodimer. Since genes are present in all cells (leaving aside germ cells for a while, since they are haploid, and chiasma formation gives rise to gene rearrangement), theoretically all cells also has the machinery for BMAL/CLOCK generation. Thus in the periphery, where these genes are expressed, circadian oscillating mechanisms are automatically incorporated.

The role of peripheral circadian clocks is still uncertain. But it is known that the peripheral clocks regulate cell division, estrous cycles and glucose and lipid homeostasis. Lamia et al knocked out the BMAL1 gene in mice liver and observed that the liver was no longer able to pour sufficient glucose into the blood circulation for cellular activity, resulting in hypoglycemia. Normally, the liver produces glucose from lipids and amino acids in a process called neoglucogenesis; and from glycogen, a glucose polymer, by glycogenolysis, in the fasting phase, to make up for the dwindling blood glucose level. In liver specific BMAL1 deletion, this did not happen and the animal suffered from hypoglycemia, indicating the important role of the liver peripheral clock.

These peripheral clocks certainly need to be regulated too in order to achieve physiological harmony. The master clock in the suprachiasmatic nucleus might regulate these peripheral clocks by hormones and hemodynamic cues.

Gatfield et al used two groups of mice and inactivated BMAL1 in all their cells in one group (BMAL1-/-); and only in liver cells in the other group (L-BMAL1-/-) [the 2 minus signs indicate homozygous, or in both alleles, deletion/inactivation]. The mice in which all BMAL1 were deleted did not show any problem which glucose homeostasis, whereas those with only liver specific BMAL1 deletion had problem maintaining normal sugar level in the inactivity (fasting) phase. Thus the role of liver clock is undeniable. The hepatic oscillator synchronises on feeding cues, since feeding is related to circadian metabolism. In the L-BMAL1 knockout mice, both neoglucogenesis and glycogenolysis operated adequately, but the machinery for the pouring of glucose into the circulation, the final step that is carried out by glucose transporter 2 (GLUT2) is suboptimal. GLUT2 expression in L-BMAL1-/- rats is inadequate.

In BMAL1-/- mice, the master clock in the SCN was inactive along with all other peripheral clocks. This presumably abolished the circadian feeding responses and thus glucose homeostasis was minimally affected. It is as if both the SCN (master) and liver (slave) clocks gone wrong and they were fully asynchronous. But in the L-BMAL1 knockout mice, the SCN was OK and it expected the desired blood glucose level in the habitual feeding time, but the liver lacked GLUT2 to supply the required glucose in the bloodstream. UNITED WE STAND, we better synch!

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References:
Physiological significance of a peripheral tissue circadian clock. Katja A. Lamia, Kai-Florian Storch, and Charles J. Weitz doi:10.1073/pnas.0806717105

BMAL1 and CLOCK, Two Essential Components of the Circadian Clock, Are Involved in
Glucose Homeostasis. R. Daniel Rudic , Peter McNamara , Anne-Maria Curtis, Raymond C. Boston, Satchidananda Panda, John B. Hogenesch, Garret A. FitzGerald doi:10.1371/journal.pbio.0020377

D. Gatfield, U. Schibler (2008). Circadian glucose homeostasis requires compensatory interference between brain and liver clocks Proceedings of the National Academy of Sciences, 105 (39), 14753-14754 DOI: 10.1073/pnas.0807861105

A Crazy Way To Explore Respiratory Aerodynamics

2008-11-26T22:25:00.005+05:30

We need to breathe in order to live. So respiration is a vital part in sustaining life. The tissues take up Oxygen for burning of ‘fuels’ such as carbohydrates and fatty acids, for the production of energy; and produce CO2 or Carbon di Oxide as a result. This ‘internal respiration’ is actually ‘cellular respiration’.
CO2 then goes to the lungs and exhaled out. Fresh Oxygen from air gets in. This is ‘external respiration’ and the lungs acted as an organ of gas exchange.

We can’t really see any air coming out of our nose or mouth; this is because the optical density of exhaled or inhaled air is somewhat similar to the surrounding air. Wouldn’t it be nice if we could actually see them? The way ‘air’ came out, the 'aerodynamics' part; and the way it smoothly mingled with the surrounding air, the 'diffusion' part?

Well, tobacco smoke could be suitable for this rather crude experiment. There is NO DOUBT that tobacco in any of its forms is injurious to health, and I am in no way advocating its use for any purpose, not even here. But there are guys who do smoke. If you look at the expired ‘air’, you can ‘see’ how the thick dense smoke that exits from the mouth gets thinner, as it expands, as the ‘air’ goes further away from the smoker. In a sense, we are 'watching' how exhaled air would actually come out from us, its trajectories and its aerodynamics!

Over a certain period of time, we can see that the density of the smoke diminishes and we can no longer see that wisp of smoke separate from the ambient air. This is due to diffusion of smoke particles down the concentration gradient; surrounding air may be assumed to have zero concentration of smoke, for all practical purposes.

However, one thing that is still ‘smoky’ to me is how the smoke rings, that 'artistic smokers' can produce, behave.
As shown in the picture, one such smoker is seen making circular rings of smoke. Both the outside and the inside of the rings are 'not visible' and we may assume them to be consisting of air rather than smoke. Now as the ring move further away, both the internal and external diameter of the exhaled smoke-ring increase. Provided that the procedure is carried out in a room where there is hardly any air flowing, and stray electrostatic charges are absent; extraneous artifacts can then be minimized and faithful aerodynamics may be expected. If one plots the trajectory of the smoke-ring over time, he will get the result as shown in the picture.

If we now joined the outer peripheries of consequetive rings, we would get a cone. Quite expectedly, the narrower end of this ‘outer cone’ will correspond to the inner margin of the artistic-smoker’s lips. Problem occurs when we construct the inner cone, by joining the internal circumferential points of those rings. Where does the narrower end correspond to now? Honestly, I’m not sure. Clearly, it is somewhere within the mouth (oral cavity). I have asked a few such ‘annular performers’ about how they achieved this feat. Gathering and analyzing data from these hallowed ‘Lords of the rings’ have suggested that the actual ‘point of origin' corresponded to somewhere in the middle of the oral cavity. They used the bases of their tongues to maneuver the smoke by apposing it to the hard palate, and then gently pushed the smoke out. The pharynx and the larynx were automatically shut off in the process.

What is your opinion? Quite some feat anyway!

Statutory Warning: Smoking is injurious to health
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Reference: hyper-links, unless specifically mentioned

Molecular Basis of Genetic Switch In The Circadian Clock

2008-11-25T23:44:00.006+05:30

It is said that the early bird gets the worm. So what is it that makes them rise early? Scientists have questioned it for long. It was in 1995, David Welsh, then a graduate student, discovered that individual cells dissected out from the 'suprachiasmatic nucleus' of rats' hypothalamus showed spontaneous oscillations. And this set the ball rolling!

All organisms from simple unicellular to humans have their own clock mechanisms. We for example, have not one but many oscillators. The master clock that oversees all the other clocks is located in a part of the brain called hypothalamus, the suprachiasmatic Nucleus or SCN for short. The clock circuit is based on transcription and translation of a genetic switch that resides in the SCN. In the nucleus, a gene, called the Per1 gene, produces a protein called PER (for period). Like other proteins, its production is regulated by a promoter sequence of DNA, which is known as E-box. A heterodimer (dimer because it consists of two molecules; hetero because the molecular weights/size is different) consisting of proteins BMAL1 (also known as MOP 3) and CLOCK sit atop the E-box sequence. Together they regulate the Per1 gene (other clock genes like AVP or arginine-vasopressin genes are also regulated) resulting in the production of PER1 protein. So, in a way the E-box may be considered as the genetic switch and the heterodimer of BMAL1 and CLOCK the regulator.

Lets suppose that Per1 gene is producing PER1 protein. So, the concentration of this protein in the cytoplasm will rise. This PER1 protein will now combine with other clock proteins namely, PER2 protein, CRY 1 and 2 proteins (CRY for cryptochrome) in the cytoplasm; and will finally reach the nucleus. In the nucleus, they inhibit the Bmal1 and Clock heterodimer transcription factor, which will lead to a drop in PER production. Thus, the positive feedback of BMAL1 and CLOCK on Per1 gene; and negative feedback of PER and CRY protein on the BMAL1 and CLOCK heterodimer keep the clock running. See the adjoining figure. Other proteins like TIM (timeless) and CK1e (casein kinase 1 epsilon; it degrades PER proteins) may also play some role. New research however suggests that CRY proteins, particularly CRY1 protein is a stronger repressor of the said heterodimer.

Research by Leloup et al showed that the mRNA of Bmal1 was in antiphase with that of Per and Cry. This was expected, because they are negatively correlated. Else both the proteins would peak at the same time and the periodicity would be lost. They also observed that the phase of the spontaneous circadian rhythm did not lock. This is because, circadian rhythm is very flexible. In humans, the cycle repeats about every 24.2 hours. The circadian clock is reset by light and our circadian apparatus is exquisitively sensitive to lights falling on the retina. The retina sends this light (for synchronization) to the SCN via the retino-hypothalamic tract. This synchronization or entrainment can now 'phase lock' the circadian rhythm.

Clinical implication of circadian (circa=about; dian=day) rhythm is enormous. Our sleep-wake cycle, growth hormone and cortisol secretion are only a few example. A person in whom the circadian period is short will rise early (early bird?) and a 'night owl' will have his/her circadian period short. Curiously, our sleepiness, tendency to sleep and occurrence of REM sleep peaks (resulting from endogenous circadian rhythm) when we are about to rise; and our endogenous clock reaches its peak about 1-3 hrs before our habitual bedtime. They say that it is a natural homeostatic mechanism, so that we fell less sleepy as daytime passes on and etc. But I not convinced.

But one thing I am sure to abide by is this that I won't deprive my SCN its daily dose of sunlight. I will also not expose myself to undue light (from computer monitor etc) at night and go to bed at a reasonably fixed time. Fiddling with these may result in insomnia or excessive somnolence as in night shift workers and in jet lag (due to latitude/time-zone changes).

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Reference: BMC Molecular Biology 2008, 9:41 doi:10.1186/1471-2199-9-J.-C. Leloup (2003). Toward a detailed computational model for the mammalian circadian clock Proceedings of the National Academy of Sciences, 100 (12), 7051-7056 DOI: 10.1073/pnas.1132112100

Hearing Involves Sound Physics

2008-11-20T23:25:00.013+05:30

The way we hear sound is complex. The different attributes of sound (namely, intensity, frequency, the direction from which it is coming etc.) are faithfully perceived in the auditory cortex. The whole procedure may seem rather straightforward, but it is far more complicated than what looks so deceptively simple.

The sound waves (say from an orchestra) impinge on our eardrums. Sound waves are mechanical waves consisting of condensation and rarefaction, things we learned in our school days. These waves then vibrate our eardrums (Tympanic Membrane; TM). The vibrating TM then transfers its mechanical energy to the oval window, in the membranous labyrinth of the cochlea, via an ossicular chain consisting of three (3) very small bones.

The membranous labyrinth consists of three adjoining tubes coiled side by side (as shown in the figure). If we were to make a section through it, we would find 3 separate compartments within it: Scala vestibuli, Scala media and Scala tympani. Scala vestibuli is connected to Scala tympani at the apex of the cochlea, a place called helicotrema. While Scala tympani contains a fluid called endolymph (a fluid rich in K+ or potassium ions); the other 2 tubes contain perilymph (a fluid very similar to plasma, rich in Na+ and low in K+).

As the oval window vibrates, the fluid in Scala vestibuli (perilymph) also vibrates. Sound was traveling in air before it struck the eardrum, but here, we see that they are now propagating in a fluid medium, which has far more inertia than air. The possible impedance mismatch that would happen is compensated by the eardrum itself and the ossicular chain. The lever system of the ossicular chain amplifies the force of sound waves about 22 times, so that the total force at the oval window is 22 times than what the TM experienced originally.

Now, vibrations have set up in the Scala vestibuli form the oval window. These vibrations find their way to the Scala media, as the two tubes are separated by only a very thin membrane (Reissner’s membrane). Hence, fluid in the Scala media (endolymph) vibrates whenever the oval window is vibrating.

The vibrating endolymph sets up a wavy motion in the basilar membrane (BM). The ‘real analysis’ of sound waves starts here! The BM performs real time spectral analysis of sounds it is presented with (analysis of frequencies below 200Hz is skipped though). We normally hear in the frequency range of 20Hz to 20 kHz.

The hair cells in the organ of Corti, our hearing apparatus, are arranged in such a manner along the BM that those near the base of the cochlea will respond to high frequencies; while as we go up to the apex of the cochlea, the BM reacts best at low frequencies. In other words, each part of the BM has its own unique maxima, the frequency at which the BM responds most well. Below 200Hz, there is no such place encoding.

Our ears follow another principle. The ‘traveling wave’ spreads quicker near the base of the cochlea and its speed diminishes fast as it goes up. This ensures that a longer stretch is available for the higher frequencies; else the higher frequency part would have been bunched together, creating a loss in the HF range. This non linearity in traveling wave propagation is thus needed.

Generator Potential
The genesis of generator potentials in the hair cells is also interesting. Imagine that a group of persons of varying height are standing on a carpet. A thread is attached from the top button (of his shirt) of the smaller person to the top button of his taller counterpart. If the carpet is now tilted by pulling it up from the short person’s end, the thread will now be stretched snapping the taller person’s button. The hair cells also have threads (Tip Links) extending from shorter to their taller cousins. A traveling wave will cause pulling of tip links, resulting in the opening of a mechanically sensitive cation channel. Since potassium is the predominant cation in the endolymph, K+ will then enter the taller hair cells, creating depolarization.

Frequency Discrimination
When we listen to music, our ears pick up the frequencies in a number of ways. Firstly, the place on the BM where maximum excitation takes place is actually a function of frequency. As a matter of fact, there is a frequency map along the BM. Secondly, at frequencies below 3 kHz, the nerves fire in synchrony with the incident sound waves. This is called the ‘volley principle’. The ‘phase locking’ of the two frequencies that occurs below 3 kHz, is highly analogous to the ‘phase locked loops’ in electronic circuits (NE565). Thus ‘volley principle’ allow us to discriminate frequencies. Actually, volley effect is more important in ‘loudness’ assessment. ‘Pitch’ (the subjective/psychological dimension related to frequency) can also be moderated by factors such as loudness and the duration of sound. At low frequency (below 500 Hz) pitch seems lower and at higher frequencies (above 4 kHz) pitch seems higher, as the loudness increase, when the frequency is kept constant. Again, when the duration of sound increase from 0.01 second to 0.1 second, the pitch will rise too, for a particular frequency. Sound of less than 0.01 sec duration does not evoke appreciation of any pitch by us.

Loudness Discrimination
Loudness is the perceived intensity of sound, a subjective psychological dimension. The interpreted sound sensation is proportional to the cube root of the actual sound intensity. As such, the ear works at the top of its limit, at a point analogous to Hopf bifurcation, beyond which instability in oscillations occur.
As the sounds become louder, the amplitude of BM movement is more, resulting in more excitation of hair cells. Secondly, with greater loudness, the hair cells around the ‘maxima’ fire too. This causes spatial summation. Thirdly, the outer hair cells are stimulated at loud sounds. The brain will automatically infer loudness levels when cells corresponding to the outer hair cells fire.

Locating Sounds
Then there is location of the direction of sound. We can locate whether the drums are on the left and the lead guitar is to the right. This is achieved by calculating which ear gets the sound first (time lag) and/ or which ear gets it louder (intensity). The time lag method works below 3 kHz; while intensity method works at higher frequencies.
Front/ back discrimination is done by the pinna (auricle) of our ears due to their particular shape.

Auditory nerve
It is interesting that each auditory nerve fiber has its own characteristic frequency, the frequency at which it responds most well. However, it is true only at low intensity. At higher intensities, this specificity is lost and they then respond to a wider spectrum of frequencies. The auditory nerve produces a flurry of action potentials, the frequency of which depends on the intensity of the sound stimuli, it is exposed to. This is very much similar to ‘voltage to frequency converter’ ICs (LM331 is one such VFC IC), where a change in voltage at the input of VFC will cause a change in frequency at the output.

The auditory nerve then goes to the: cochlear nucleus in the medulla-->Superior olivary nucleus-->Inferior colliculus (via lateral lemniscus)-->Medial Geniculate Body (in the Thalamus)-->Auditory cortex.
The nerve synapses with higher order neurons in the above places, which then relay to the nerves upstream. Everywhere in its course, including the nuclei, there are clearcut ‘tonotopic maps’, representing definitive frequency layouts. There are also extensive crossing of nerve fibers to the opposite side. Before the fibers reach the auditory cortex, they connect to many reflex pathways vital to life.

The auditory cortex (Brodmann’s area 41) is the portion of the cerebral cortex in the superior temporal gyrus. Its anterior part is mainly concerned with low frequency and the posterior part tackles the higher frequencies. Thus area 41 also has its own tonotopic map. Secondary auditory cortex (auditory association area) is vital for the interpretation of sounds. A person, in whom Wernicke’s area (part of auditory association area) is damaged, will hear normally but will fail to understand its meaning, leading to aphasia.

The sheer complexity of the auditory circuitry is really mind-boggling: the logarithms (we hear in a log scale, not a linear one), cube functions, phase locking are only some of them. The range of sound intensity (from whisper to the roar of a jet plane) we hear is about 1 trillion fold; but surprisingly, the auditory nerve fibers have a much less dynamic range. Yet we hear the full range. It’s really amazing.

P. Martin (2001). Compressive nonlinearity in the hair bundle's active response to mechanical stimulation Proceedings of the National Academy of Sciences, 98 (25), 14386-14391 DOI: 10.1073/pnas.251530498

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Reference: Textbook of Medical Physiology, 17e, Guyton and Hall

Sound Tactics

2008-11-19T18:56:00.000+05:30

I always wondered, I do even now, as to how the diaphragm of a loudspeaker vibrates, when sounds of different frequencies, as in a musical excited it. Everyone knows how the diaphragm vibrates in response to an alternating current. Just wind a few turns of enameled (insulated) wire around the tapering end of a cone of paper, place it on a permanent magnet and touch both the ends (the ends should be rubbed off of their enamel at their ends) to the positive and negative terminal of a battery, you will see it vibrate. Its response to a pure sine wave is easily predictable; it will vibrate back and forth as a function of simple harmonic motion.

The case becomes problematic when many different frequencies are present at the same time. Will the diaphragm vibrate ‘on the whole’ as an algebraic summation of those mechanical waves, or will different coordinates (parts) of it vibrate independently?

I personally think that the latter choice is the actual response. When a person beats a drum, it will produce different frequencies depending on where it is struck. Thus some parts of the loudspeaker will be more sensitive than others, for a particular frequency. In a similar way, the vibrations in the eardrum produce a wave on the basilar membrane (BM) of the internal ear; the location of peak amplitude of that wave being a function of frequency.

In electronics, sound to electricity transducers is called microphones. The loudspeaker described above can also function as a microphone: a dynamic or moving coil type microphone (see picture). Electret or condenser type microphones (picture given) employ two capacitor plates, mechanical vibrations bring one plate of this capacitor near the other making a change in capacitance. This is mechanical to electrical transduction is done by the BM in the cochlea of our internal ear.

Our body is a gadget par excellence. We hear music or words when those sound waves strike our eardrums. Like the diaphragm, it has excellent damping action, the oscillation stops almost as soon as sound stops. This ensures that it is ready for the next wave. I do not know how the tympanic membrane (TM) vibrates to those complex sounds, topology wise. Anyway, the TM then transfers its kinetic energy to the ‘oval window’ via an ossicular chain (in the middle ear) consisting of 3 tiny bones: malleus, incus and stapes. Stapes transfers this vibration to the cochlea (in internal ear), a spirally shaped tunnel consisting of 2.75 turns. What happens next is high level electronics, physics and mathematics, the body resorts to for processing and interpreting sounds. It performs cube roots, logarithms, compares phases and time lags and many complex functions.

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Reference: http://hyperphysics.phy-astr.gsu.edu/hbase/Sound/oss.html
http://www.lifesci.sussex.ac.uk/home/Chris_Darwin/Perception/Lecture_Notes/Hearing2/hearing2.html#RTFToC14
http://www.cs.indiana.edu/~port/teach/sem08/hearing.for.linguists.final.html

A DIY Attempt In Telemedicine

2008-11-13T02:53:00.007+05:30

Summer of 2006, but not quite Bryan Adams' Summer of 69. A hot and humid day typical of the tropics. A man in his 40's was trying hard to beat the 'sands of time'. He had chores he needed to complete within a short deadline. He finished them successfully, but was exhausted and sweating profusely.

On reaching home he felt an uneasy sensation. He was acutely aware of his heart beating irregularly, what we call palpitations. No one was home at that moment, what would he do now? He thought a way out, whereby, he could transform his subjective feelings (symptom) into an objective rendering, so that he could share his sensation with his medical colleagues. Clearly a ECG (EKG) was the best option, but he didn't have a device at home. So, he went on his own ingenious way.

He pulled out the microphone jack off his computer, and negotiated its 'male plug' into a funnel
and pulled it out through the apex (figure shown), approaching from the base to the apex. After the plug came out of the funnel, he pulled the wire gently out till the microphone was fitting snugly (no visible air-gap) within the funnel wall ( a plastic or rubber funnel is the best). He then plugged the jack back in to the computer and switched the computer on.

Next, he checked (enabled) microphone option in the sound configuration and opened Windows Movie Maker. Clicked 'tools' then chose 'narration' mode and placed the rim of the funnel on his chest. As expected, the program faithfully digitized his aberrant sounds (through its analog to digital converter, built-in within the computer) and recorded them. Upon completion of the recording, he played it back using Windows Media Player. Hell, the sounds suggested he definitely had some problem, and a real 'objective' one at that. In the visualization option, 'bars' even 'showed' his sounds. He decided to email 'the file' to his colleague as an attachment.

An ECG was done.

The guy was having Premature Ventricular Complex as the ECG revealed. Perhaps anxiety and stress coupled with electrolyte disturbance had their toll. No antiarrhythmic medications were given (in fact, they are often avoided in this situation). Just a sedative-anxiolytic for a couple of days cured the condition completely.

Premature ventricular complexes arise due to ectopic generation of beats and faults in cardiac conduction pathway. I have covered them in detail elsewhere. As of now, the sound files are here (1MB) and here (597K). Some unwanted sound due to mechanical friction is present. But our purpose is served well.

I'll let you in with a little secret. That guy was me :-)

Last modified: July 11, 2009

N.B. [This is JUST an experimental device. Undue emphasis may be avoided]
Reference: hyper-links, unless specifically mentioned

Do We Really Forget? Fathoming The Esoteric Realms of Memory

2008-11-08T22:34:00.013+05:30

“Smells like teen spirit”, but it could be true that we never really loose any memory in our lifetime. Our memories are stored in the synapses (junction, more specifically, gaps between adjoining neurons) as a function of synaptic strength, in the nerve cells like dendrites as proteins, and some other processes which mostly encompasses a chemical interaction. I am excluding memories such as T cell or B cell memories here; memory, here, will refer to neural ones that occur in the CNS.

We know that in dementias such as global multi infarct dementia, Alzheimer’s disease; there are diffuse losses of neurons and losses of cholinergic neurons in particular, respectively. In surgical cases of epilepsy or brain tumor, there are losses of neurons too. In these cases, memory loss may be irrecoverable, though cases are on record which points to shifting of those memories into some other safe havens. But what about the rest of the population? Does an established long term memory vanish completely?

Let’s consider some facts. The numbers of synapses and their strengths are finite, though both can change in response to stimuli. Even the number of neuron themselves can increase, contrary to the belief held earlier. Neuronal stem cell pool has been identified in the brain. Memories stored in the brain are finite too. Memories are inherently dynamic in nature. Even long term memory stored in the neocortex (medial temporal lobe, on the other hand, stores memories as a buffer, like a D RAM chip, a temporary storage) can change location, as much as transferring itself to the other hemisphere (intercortical transfer), when needed; via the optic chiasm and corpus callosum. So, we see that the number of synapses, though finite, can rise to the demand of an enhanced input from sensory cues which are finite too, leading to memories that can jump across their own allocated territories. A finite brain capacity (say C) can certainly contain a finite memory (say M) as long as C is greater than/ equal to M. Certain computer softwares even trespass this limit; a zip file of 2 MB may deliver 3MB of contents on unzipping! Who knows if the brain isn't using this for the past thousand years.

Synapses, simplistically, may be thought of in binary terms: 1, when it is on; 0, when it is off. Both 1 and 0 is a bit in Boolean terms. We leave aside the synaptic strength part here for the sake of simplicity. In addition, memories may shuttle between synapses in such a way so that it is present in the brain, but not represented by any synapse. I will explain. We all have seen those jugglers juggling those colorful balls too many at a time using only their two hands. A similar thing like dipole dynamics may occur in the brain. Added to this is quantum superposition, which allows the situation of BOTH 1 and 0 state at the same time at the synapse. That the brain can be in a quantum state at the core body temperature and the brain can effectively avoid ‘decoherence’ in the background thermal noise has been discussed by Roger Penrose and Stuart Hameroff. We also know that memories aren’t kept as such, but they are fragmented into individual elements, which are mostly matched to existing elements and are associated. This is economic as it saves space, and useful for indexing and contextual retrieval.

Thus it seems that we ought to have immense memory storage. Haven’t we encountered long forgotten memories in our dreams? Electrical stimulations in some parts of the hippocampus (deep brain stimulation or DBS, figure shown) during routine surgical procedures have given rise to ‘deja vu’ phenomena. The patients remembered things considered long forgotten. We may not be aware of the vast database of memories and are liable to infer that we have “killed ‘em all”, but in reality this may not be the case as Norio Ota et al clearly points it out in their paper. It smells like another chapter from your favorite science fiction novel, but it could be true.

Last modified: never; N.B.There is a substantial amount of speculation in this paper. Please exercise your own judgment and enlighten me about any possible error.
Reference: hyper-links, unless specifically mentioned.

Scientists Simulate Learning In Amoeba Using Memristor

2008-11-08T03:02:00.011+05:30

It is surprising how small insects get energy from a wide range of food (not merely petrol or diesel), crawl, fly, reproduce and do so many maneuvers. Now it has been seen that amoeba, a unicellular organism, can learn and memorize too. We are far from creating devices of such versatility, let alone making them as compact as they are.

Amoebae can move, and they do this by changing the physical state they are made of: sol-gel state. The interior of amoebae contains endoplasm, which is in sol state; while the surrounding ectoplasm remains in gel state. The ectoplasm, being in gel state, is more viscous than it’s inside counterpart. When the organism moves, its contractile elements made of actin myofilaments contract, pulling the inside of the amoeba. This causes tension in the endoplasm, creating a change in the sol-gel state. If you squeezed a sponge ball that had been dipped in water, you would notice that water would spurt out from the pores of the sponge. Likewise, the increased tension inside, will create channels through the more viscous ectoplasm, courtesy some parts of ectoplasm (gel state) giving away (to sol state).

We know that reptiles hibernate in winter, when the humidity and temperature is low (we too are no exception). Amoebae too, slow their locomotion in response to these conditions. There are inherent oscillations within the amoeba (alternate sol gel transformation, changes in ionic flux etc) which are continuously adjusted with external signals like temperature and humidity. We, complex multicellular organisms, too have our own master oscillator (circadian clock) in the suprachiasmatic nucleus, which also continuously adjusts by lights falling on the retina.

Yoshiki Kuramoto of Kyoto University and colleagues subjected Physarum polycephalum, an amoeba, to three regularly-spaced dips in temperature and humidity, and found that its locomotive activity decreased. Thereafter, they noticed that a single dip was sufficient to elicit this response. It seems they adjusted their oscillations to the external cue and developed a conditioning later. The study implied that the amoeba anticipated that other such dips might be forthcoming, from the memory it learned. Such response did not occur when the temperature and humidity changes were irregular.

Memory in this case occurs due to the persistence of the channels etched by the organism in the ectoplasm. But this ‘memory’ did not persist for long, if we continued giving them a single dip instead of a regular triplet. This plasticity (change due to reorganization as a function of a stimulus) in amoeba has now been simulated with the aid of electronics by Massimiliano Di Ventra et al.

They used a capacitor, a resistor, an inductor in series and connected a ‘memristor’ in parallel with the capacitor. Memristors (for memory resistors) are devices which consist of two layers of titanium dioxide (often present in medicine coatings and chewing gums). When current is applied to one layer, the resistance of the other changes. Leon Chua, of the University of California at Berkeley, predicted it long time ago; and now R. Stanley Williams and colleagues at Hewlett Packard have developed it. It can store memory like DRAM, but unlike DRAM it doesn’t forget when a current is no longer flowing. They hold promise as energy efficient chip for computers and we can also expect faster ‘booting’ of computers, since memory will already have been stored there. The adjoining figure shows ‘memristors’ in a row, as seen by atomic force microscopy (AFM).

Now when a current, fluctuating (AC) in a non periodical manner or a stable DC, was made to pass through the circuit, the memristor went to a low resistance state, virtually short circuiting and dampening the oscillation. However, with a regularly fluctuating current, whose frequency matched the resonant frequency of the circuit, the memristor went into a high resistance state, strengthening the oscillation. What connects electronics to amoeba is the memory that both the circuit retain. The memory of memristor, called memristance, is due to atomic rearrangement in the device. The high resistance state lingers for quite some time, so that next time one single pulse was necessary to put it into oscillation. This phenomenon is quite akin to the protozoal response.

It seems that those days are certainly not far when we will just need to jack-up a USB device in our head to boost up our memory.

Last modified: Nov 13, 2008
Reference: http://arxiv.org/abs/0810.4179?context=q-bio Tetsu Saigusa, Yoshiki Kuramoto (2008). Amoebae Anticipate Periodic Events Physical Review Letters, 100 (1) DOI: 10.1103/PhysRevLett.100.018101

Unlearning Memories: You Have Been Erased!

2008-10-28T11:09:00.008+05:30

In the movie 'Eraser', John Kruger, played by Arnold Schwarzenegger, removed the identities and all relevant information of persons-at-risk and gave them brand new identities, to save their lives. The title of this topic derives its name from the famous dialog: "You have been erased!”.

In reality, memories can be erased too, even long term ones! In humans, diseases like Alzheimer’s dementia, cause defects in short term memory limiting further memory acquisition; which finally progresses to erasure of previously stored memories. In experimental animals, long term memories can be prevented if they are subjected to electrical shock, anesthetics (possibly work by disrupting London forces operating in hydrophobic pockets in dendrites thereby causing 'unbinding' of memory elements), hypothermia and other insults within 5 minutes of learning a specific task.

There are guardians of memories keeping a constant vigil so that ‘memories are forever’. For example, we have seen masons at work standing on those makeshift scaffolds. In our bodies too, a protein called PKM zeta constantly ferries across the synapse, to give its healing touch. A protein called PSD-95, when phosphorylated, takes the role of the scaffold, in this analogy. Inhibitors of PKM zeta cause loss of memory, as demonstrated in rats, when their memories for tastes vanished after a single ‘shot’ in their taste cortex. So, LTP is not the only mechanism that fosters memory and neural plasticity. Long term memory also uses gene activation and expression, and protein synthesis. Any interruption in either of these mechanisms will have its own deleterious effect.

Genes have been identified which produce proteins that are necessary for memory formation and their maintenance. These genes act fast in the central nervous system and are known as IEGs (Immediate Early Genes). IEG Arc, one such gene codes for Arc protein, which is abundant in the hippocampal neurons. Obviously, the mRNA (messengerRNA can be thought of as ‘command’ from a gene, while proteins are like the results of this command) for that gene is formed in the nucleus by transcription, but once transcribed, it goes to the dendritic spines (especially those which are active) along the cytoskeletal rail road formed by microtubules. In the dendritic spines they (mRNA) translate themselves into proteins, Arc proteins, in this example.

Guzowski et al did an interesting experiment. They infused antisense oligodeoxynucleotide (for the inhibition of Arc protein synthesis) straight into the hippocampus of rats. Antisense oligodeoxynucleotides (antisense ODN) are short sequences of deoxynucleic acids which block the translation of mRNA into protein. The rats forgot the tasks they learned, while they could still form new memories. Their spatial memory was badly damaged. They concluded that antisense oligodeoxynucleotides interfered with the maintenance phase of LTP.

Rats can be challenged spatially using the Morris water maze. As we see in the picture, the water pool has two raised platforms, which rats can locate (and remember) spatially after some training. Even the rats already trained successfully, lost this spatial (co-ordinates in space) memory and had severe difficulty floating, when they were challenged with these chemicals. Looks like, You Have Been Erased!

Contradictory (against the motion) Link: Memories like diamonds may be forver

C. K. McIntyre (2005). Memory-influencing intra-basolateral amygdala drug infusions modulate expression of Arc protein in the hippocampus Proceedings of the National Academy of Sciences, 102 (30), 10718-10723 DOI: 10.1073/pnas.0504436102

LTP Ensures That Memories Are Forever

2008-10-19T02:37:00.006+05:30

LTP or Long term potentiation is a process that may explain how memory gets stored in the brain for long term use. When you stimulate the presynaptic neuron by giving a brief (of transient duration) but rapid train of stimulus, the post synaptic neuron adjusts its ‘weight of association’ with respects to the presynaptic one, in the form of a chemical reaction. Though LTP occurs throughout the brain, it has been studied mostly in the hippocampus. If we are to understand the underlying molecular mechanism of memory, we can not do without LTP.

Two different types of LTP are known: mossy fiber LTP and Schaffer collateral type LTP. While the basis of mossy fiber LTP is not clearly known; it involves modification of the presynaptic terminal, and is independent of NMDA. A schematic and functional diagram for Schaffer collateral LTP is presented here. But before that, allow me to digress a little bit.

Your computer has a DRAM (Dynamic Random Access Memory) memory chip: memory because it can store and retrieve information; Random access as it allows you to search anywhere within the memory at random (it does not have to reach D via A through B, and then through C, sequentially), and dynamic, since the memory needs to be refreshed from time to time. To store a bit of data in memory, your computer charges devices called ‘capacitors’ within the chip, which retain their charge; and all your computer have to do is to read the data in the form of those stored charges for later retrieval. But these capacitors lose their charge over time and hence dynamic refreshing is necessary to maintain their memory.

You’ll now understand why this digital analogy as we discuss LTP. The picture on the left portrays a presynaptic neuron which discharges glutamate, the main excitatory neurotransmitter of the brain and the spinal cord. Glutamate after being released upon the stimulation of the presynaptic terminal, binds with their ‘receptors’ in the postsynaptic neuron. The post synaptic neuron, downstream, has 2 types of Glutamate receptors: NMDA (N methyl D Aspartate) and AMPA (alpha Amino 3 hydroxy 5 Methyl isoxazole 4 Propionate). Glutamate binds with both NMDA and AMPA receptors. NMDA receptors have a Magnesium ion, guarding at its channel entry. So, for NMDA receptors to act, it needs to be partially depolarized first, so that this magnesium block is removed. This is achieved by the AMPA receptors, which upon binding with glutamate, allows the entry of Sodium ions inside, thereby raising the cell voltage. NMDA receptors now swing into action as it now allows huge amounts of Calcium ions (and Sodium ions) to enter inside.

These Ca++ then bind with Calmodulin present within the cell to form a complex, which then activates calcium-calmodulin kinase 2 (Ca/Cam k2). This newly formed compound then activates (phosphorylates) AMPA receptors, resulting in: 1) increased activity conductance of the already existing AMPA receptors in the cell membrane 2) Recruitment of AMPA receptors from within the cell to the cell membrane. So we can see that the synaptic strength is increased with each firing by both AMPA recruitment and increased AMPA conductance. The synapse stops at not only this, the postsynaptic neuron also discharges a ‘diffusible’ messenger, nitric oxide (NO), which 'tells' the presynaptic neuron to discharge more quantal release of glutamate next time. The phenomenon epitomizes Hebbian learning: Cells that fire together, wire together.

But the memories so formed need to be stabilized as in the case of DRAM. In the central nervous system, dendritic spines are the main postsynaptic sites. These tiny protrusions form and change over a few hours. In hippocampal slice cultures it was shown, by De Roo and colleagues, that application of theta burst remodeled the dendritic spines; unused ones were shed (trimmed) while used ones were stabilized and new spines were formed. LTP was the chemical basis of all these modifications. They used GFP or green fluorescent protein for visualizing these changes of neural plasticity. However, they (physical units of memory) can also be seen by restorative deconvolution microscopy, in the form of flattened synapses (as if the ohmic resistance getting diminished in their electronic cousins) and hence more area for contact between the pre and postsynaptic neurons. So like DRAM chips, our memory chips too need to be constantly refreshed, even long term memories need maintenance.

Last modified: Jun 26, 2010
Reference:Dominique Muller, Morgan Sheng, Mathias De Roo, Paul Klauser, & Morgan Sheng (2008). LTP Promotes a Selective Long-Term Stabilization and Clustering of Dendritic Spines PLoS Biology

Neurons: Digital or Analog?

2008-10-05T23:15:00.005+05:30

Its very hard to stamp neurons solely as either an analog device or a digital device. Actually its both. I'll tell you why it is analog. When an impulse arrives at the cell body of neuron, it creates an EPSP or excitatory post synaptic potential. During an EPSP, the sensitivity of the post synaptic ending to another stimulus is increased, hence the name. At this phase, stimuli add up resulting in spatial or temporal summation. Thus individual stimulation may not elicit a propagating action potential, but together they can cause enough inflow of sodium ions to produce an action potential. So from this point of view, we can see that the neuron is not following an all or none law; and the actions may best be described as analog.

Once an action potential has occurred, it always spreads down the axon to the synaptic knob. Actually, when a neuron is stimulated at any point in its course, the nerve depolarizes in both the directions. Since the cell body of a neuron is devoid of the neurotransmitter machinery of a synapse, the impulse is not chemically forwarded to the adjacent neuron. Thus this part of the neuron is rather like a diode in electronics. On the whole, the axonal and synaptic part of a neuron resembles a Boolean machine, a ‘0 or 1’ response. So this part of the neuron is undoubtedly digital.

The story doesn’t stop here. There’s a quantum touch to a neuron too. Quantum mechanics can be foxy. You can imagine of a cat as either alive or dead. In quantum mechanics, the cat (called the Schrodinger's cat) may have another state: BOTH dead and alive at the same time. Quantum superposition is thought to exist in the brain too and even Bose-Einstein condensate may form among the neural proteins. But how an interaction like this forms in the warm (thermal noise) environment of the brain and how the brain avoids decoherence at this temperature, has been studied by Stuart Hameroff and Roger Penrose. Their proposed the Orch OR (orchestrated objective reduction) hypothesis addresses the issue of ‘binding’ of consciousness and the mechanisms of the thalamocortical circuitry by quantum interactions.

The neuron thus seems to behave in analog, Boolean and quantal ways, so far, until string theory comes up with another!
Last modified: never
Reference: hyper-links, unless specifically mentioned

Smelling The Intentions Of Olfaction

2008-10-04T22:03:00.004+05:30

One simple thing puzzles me as to why the olfactory pathway has to bypass thalamus while most pathways from sensory receptors and sensory organs say hello to the thalamus, as they pass to the cortex. In fact, the cortex and thalamus are so intricately connected to each other, in a bidirectional way, they are sometimes referred together as thalamocortical system. Thalamic excitation of the cortex is necessary is necessary for almost all cortical activity. Why is there an exception in the case of olfaction (smell)?

How do we smell things? It is believed that volatile odorant molecules first mix with the mucus in the nose. They then interact with receptors in the olfactory cilia, called G-Protein coupled receptors. An enzyme called adenyl cyclase is produced as a result, which opens up a sodium channel. Sodium ions (Na+) pour into the olfactory cells, which then fire and smell is thus created. Marshall Stoneham and colleagues, at University College London, propose that olfaction may involve quantum mechanics. The odorant molecules, in addition to having a shape, have another property called vibration. They argue that this (vibration) property may allow electrons to tunnel through to the receptors within the nose to evoke the sense smell. They found phonon assisted electron tunneling, from a donor to an acceptor, mediated by the odorant activated a receptor, in perfect harmony with present physics provided the smell receptors fulfilled some criteria.

We all know that rats fear cats and avoid them so that they are not harmed. They can smell cat’s urine and don’t tread their paths. However, when cats are infected with the parasite Toxoplasma gondii, the rats no longer fear the cats’ urine. The parasite possibly alters the rats’ perception of smell in such a way, so that the intrinsic avoidance becomes passionate attraction. Manipulation of smell sensing thus ensures that the parasite is transmitted from their definitive host (cat) to their intermediate host (rat), thus completing its life cycle. In a similar way it has been conclusively found that dogs can smell some human cancers like lung cancer, breast cancer and skin cancer.

Scientists now can measure minute amount of acetaldehyde, a biomarker of lung cancer. Using tunable diode laser absorption spectroscopy, they could ‘smell’ the telltale signature of those odorant biomarkers in the exhaled breath of the patients. May be they would be able to devise practical explosive smelling devices as well. Sniffing with reliable accuracy and precision would herald a new era in diagnostic oncology.

Apart from its bypassing the thalamus, olfaction puzzles at another point: its stem cell reserve. Why out of the whole brain has it been selected to hold an adult neural stem cell reserve? May be it’s just a coincidence. These cells may come in very handy in the treatment of a variety of diseases.

While a newer pathway of olfaction has been discovered in the monkeys which pass through the thalamus, olfaction on the whole continues to elude me.

Last modified: never
Reference: Diagnostic Accuracy of Canine Scent Detection in Early- and Late-Stage Lung and Breast Cancers; DOI: 10.1177/1534735405285096
Perception without a Thalamus How Does Olfaction Do It? doi:10.1016/j.neuron.2005.03.012
Jennifer C. Brookes, Filio Hartoutsiou, A. P. Horsfield, A. M. Stoneham (2006). Could humans recognize odor by phonon assisted tunneling? arXiv:physics/0611205v1

Neural Networking, Alzheimer's Disease and Memory Share a Few Things

2008-10-03T00:26:00.006+05:30

The hippocampi, as has already been discussed, are endowed with the task of binding elements of memory into a coherent trace. There are extensive interconnections within the hippocampi in the form of a local area network (LAN) or intranet. This LAN like architecture resembles the “Hopfield Network” of artificial neural networks. It is especially abundant in the CA3 neurones of the hippocampi. CA stands for Cornu Ammonis; meaning the horn of Ammon (After the ancient Egyptian God Amun).

In a Hopfield network, neurodes (neuronal equivalent of neurons) are connected to each other in the form of a bidirectional interconnection. However, a Hopfield network assumes that each neurode can have either of the two states: on (1) or off (0). But human neurons, we know, can also have additional states when it is in the summation or subtraction mode (spatial summation, temporal summation etc, whereby one neuron is incapable of eliciting a binary 1 or no action potential; but when suitably combined in a time [temporal scale] or a space scale [spatial], it can ). Reverberatory Hopfield networks reinforce each other, thereby strengthening the associations among themselves.

Earlier, mathematician cum scientist von Neumann tried to find an analogy between the memory architecture of a computer with that of a human brain. In a von Neumann architecture based computer, data can be fetched from their ‘address locations’ within the database when queried. Retrieval of data in a von Neumann model is sequential, that is the information flow is one by one. Human brain, on the other hand, handles data parallely, and there is no ‘bottleneck’ (which is inherent in the von Neumann model due to different locations of CPU and memory). No bottleneck means higher data transfer rate in the human brain. Human brains differ in another aspect. Secondly, human brain can think in terms of abstract terms, while a fixed program computer like von Neumann’s one, can not do so easily. In fact, human brain is far too superior in ‘fuzzy logic’ or abstract thinking compared to their silicon cousins.

The reverberatory circuit in the hippocampi is continuously synchronized with each input stimulus. This is akin to the synchronization of our circadian rhythms with that of daylight, via the retino-hypothalamic tract. The hippocampal anatomic correlate is somewhat like this: entorhinal cortex---> dentate gyrus---> CA1 & CA3 pyramidal neurons---> subiculum--> back to entorrhinal cortex. This circuit is heavily damaged in Alzheimer’s dementia resulting in loss of episodic memories and preventing acquisition of new memories. In artificial neural network model simulations, Traub and colleagues showed that the synchronization was done in the gamma frequency range. They showed that with the arrival of each theta cycle, the attractor-based autoassociative memory process got stronger and the attractor got stable after a few theta cycles. An attractor is somewhat like a binder, which indexes and binds information.

The hippocampi also get input from the basal ganglia and other portions of the brain. These regions have a diverse range of neurotransmitter chemicals. They include acetyl choline, GABA, NMDA, Dopamine, AMPA and many others.

In acetyl choline deprived state, as occurs in Alzheimer’s disease, the frequency of gamma discharge diminished, leading to diminution of theta frequency thus delaying learning (and promoting unlearning or unbinding too).

Its time to disentangle the intracytoplasmic neurofibrillary tangles; and clear the mystery of the amyloid plaques which are so characteristic of AD. The stage, it seems, has been set.

Last modified: never

Reference: Attractor neural network models of spatial maps in hippocampus

Misha Tsodyks

Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel

E . Menschik (2003). Neuromodulatory control of hippocampal function: towards a model of Alzheimer''s disease . Artificial Intelligence in Medicine, Volume 13, 99-121

The Organization of Memory And Internet Analogy

2008-10-01T21:02:00.005+05:30

You are reading this particular article now on my website (in your computer and browser, of course!). There are billions of such websites and you picked mine. Perhaps you put your queries in the search box, which then showed you a list of results. You clicked my link there and lo, you are here!

The search engines (Google, Yahoo!, MSN and others) parse or read the webpages, index them and rank them. Each search engine has its own search robot, called bot, which crawls the pages. Now, this particular webpage that you are seeing is a part of my website. If you think of a website as a tall building, webpages may be considered as its floors. The search spiders not only crawl the website vertically (that is vertically up and down its individual floors), but they also move horizontally (clicking hyperlinks will lead you to arrive at some floor of another building) to be navigated to another webpage of another website.

Longitudinal (vertical) scanning picks up individual elements of the content, which are indexed (tagged) and remembered with respect to their locations, much like the human episodic memory in the hippocampus.

Now, its time for the bot to leave. It follows a hyperlink, if you have one, to a page of another website. So the search engine spider is directed to some floor in another building. Thus one might think of this search engine analogy to “neurodes and synapses” in artificial neural network. Here neurodes are the floors of the buildings and synapses are the hyperlinks (connections between them).

Next the search engine (Google) looks around in the floor to find similarities and dissimilarities from the floor it came. That is, it compares the webpages for relevance, in much the same way the anterior cingulated cortex (ACC) calculates “error related negativity” (ERN), and learns from it. It then calculates the ‘weight’ of association. It too learns from time to time by constant error related feedback, as happens in the ACC. Thus it assigns its hallowed “pagerank”.

PageRank is the relative importance of a page in Google’s eyes. ‘Page’ could come from Larry Page, co-founder of Google or more likely after ‘webpage’. One can see them in the Google toolbar in IE or Firefox, as a small green bar (Google’s own browser ‘chrome’ is yet to provide with a pagerank though). The lowest rank a page can have is 0, and the highest 10. Though the algorithm of pageranking is kept a secret like that of CocaCola, some insights may be had from “The Anatomy of a Large-Scale Hypertextual Web Search Engine” by Sergey Brin and Lawrence Page, founders of Google. I found a lot of resemblance of this paper with the architecture of learning and memory.

Last modified: never
Reference: hyper-links, unless specifically mentioned

The Binding Of Memory And Hebbian Learning

2008-09-30T22:11:00.002+05:30

Imagine this. Your physics professor is taking his regular class, and a boy sneezes. The professor is startled and the chalk falls from his hand. The whole class breaks into laughter. The memory of this particular incident gets stored in your head.

The whole process is somewhat like this. The visual scenes (your teacher’s attire, the chalk and the blackboard etc) are analyzed and processed in the visual association areas; the sounds (sneeze, laughter) get processed in the sound processed in their respective association areas and so on. Thus memory seems to be broken down to its individual elements; and these elements confine themselves in the areas in the neocortex where they were first processed.

Hippocampus (means sea horse in Greek) is a small banana shaped structure inside the brain. It gets its input from these association areas via another structure called the parahippocampal cortex. Hippocampus, as if, queries those areas: ‘what happened’, ‘when did it happen’ and ‘where did it happen’? Then the hippocampus binds all those information in the form of an event. For this particular episodic memory (classroom drama); the hippocampus wires together the respective areas so that the whole event is now bound together into an ‘engram’, the proposed neuro-anatomical representative of a particular memory.

While all these are happening in the medial temporal lobe (MTL), more specifically the hippocampus, the actual memory elements are still in the neocortex. With each recapitulation, voluntary (by thinking about the incident) or involuntary (someone else’s sneezing reminds you of that event), the association gets stronger. Thinking of sneezing (memory stored in neuron A) reminds you of chalk falling (neuron B). This way as A becomes active (fires), B is associated too, and this leads to wiring them. This is known as 'Hebb’s rule', after Donald Hebb, a Canadian scientist. Simply put, it says, ‘cells that fire together, wire together’.

The strengthening of synapses as a basis of learning were later found to have been mediated in part to LTP or long term potentiation, a chemical process. While the memories are being strengthened this way, the neocortical areas become more and more inter-connected. This releases the MTL connection, as the memory finally gets settled in the neocortex. This 'plasticity' is important. The neocortex stores memories effectively, has a large storage space, but it learns slowly. The MTL learns quickly, but has little storage space. One can think of neocortex as the ‘hard disk’ and MTL as the ‘RAM’ of a computer. Freeing the MTL would enable it to acquire memory more efficiently.

No wonder my teacher said, ‘Read once, write twice, think thrice’. Its time we consolidated our memories by recap.

Last modified: never
Reference: hyper-links, unless specifically mentioned

Of Diabetes, MAPK and Memory

2008-09-27T22:59:00.008+05:30

In the previous article we saw how MAPK translocated from the cytoplasm to inside the nucleus. In the nucleus, it forms cascade of responses, which end up in transcription of some portions of DNA and forms proteins that strengthens the synapse. This is one example of chemical basis of memory.

There are some diseases where MAPK levels rise. For example, in type 2 diabetes mellitus, a form of diabetes, where the end organ sensitivity of tissues to the effect of insulin decreases. Insulin has many diverse functions, one important function of which being the lowering of blood glucose. Insulin helps glucose to enter into the cells; this is one of many ways it lowers plasma glucose.

In type 2 DM (constituting 90-95% of all diabetics), glucose lowering effect of insulin is not there, as if the cells are refusing to listen to the instructions of this hormone. The cells are now deprived of their regular dose of sugar, and blood sugar level rises. The body senses danger and is driven to the ‘wrong’ conclusion that it is due to the ‘inadequate’ insulin level the glucose is not entering. So, it revs up the production of insulin. Thus, in type 2 DM blood insulin level is raised.

While it is true that insulin’s instructions for glucose entry are no longer obeyed, not all commands of insulin are null and void. Insulin’s command on MAPK is still valid! Insulin plays an important role in mitogenesis, i.e. growth of cells. It does it through MAPK or mitogen activated protein kinase pathway. We just learned that insulin level is increased in type 2 DM, and so does MAPK activity. One nasty effect of enhanced MAPK activity is atherogenesis, which is a fallout of enhanced mitogenesis.

Now that the MAPK activity is raised in type 2 diabetics, can we expect more efficient memory in this disease? Other factors that are relevant here are glucose metabolism and blood flow to the brain. Atherogenesis more commonly affects the lower extremities (in the form of peripheral arterial/vascular disease: PAD or PVD), while upper extremity involvement is uncommon. Again, the entry of glucose into the brain is, largely, independent of insulin action. So it seems enticing to assume that patients with this disease will have better memories. Is it the case actually?

We need to develop better and preferably objective tests to quantify memories, if we are to find the significance of such a finding. Any statistical correlation of memory in patients of Alzheimer’s dementia (or other dementias) or diabetes mellitus along with disease duration and plasma glucose, insulin level, HbA1c and so on with those of normal control subjects need to be searched for. As a matter of fact, insulin enhances cognition not only in normal individuals but also in patients with mild AD, as was seen by Watson and Craft. At the same time, chronic hyperglycemia in diabetes has been associated with impaired cognition. But even today the role of insulin and memory is unsettled and debated.

Last modified: Jan 8, 2009
Reference:

Watson GS, Craft S. (2004). Modulation of memory by insulin and glucose: neuropsychological observations in Alzheimer's disease. Eur J Pharmacol., 97-113

Learning Memory Lessons From Aplysia

2008-09-08T01:02:00.000+05:30

While you are reading this on your computer, there may be many distractions in the background. Your mom may be shouting at someone or your daughter may be receiving her piano lessons. But after you finished reading this, no memory trace of this background remains. They are not registered with your memory. We seem to ignore this 'negative memory' by a process called 'habituation'. We remember by consolidating memories by another process known as 'sensitization'. But what is this that we call memory? Memory helps us to store, retain and retrieve information. To start it all, learning is needed.

Now you might ask why Aplysia out of all animals?

Aplysia californica, called 'sea hare' by the ancient Greeks, is a marine snail which has some resemblance to a rabbit. A hermaphrodite by sexual orientation, Aplysia feeds on marine algae, and often takes the color of the algae that it eats. When threatened, it liberates a colored, irritant compound to blind the attacker, as seen in the left. It was Eric Kandel who used this phenomenon to study how neural transmission occurred through synapses. He was awarded the Nobel Prize in Physiology or Medicine in 2000 for his pioneering work on Aplysia. Aplysia offers a distinct advantage by: 1. eliciting a visible (measurable) response (siphon-mediated gill withdrawal reflex) to a stimulus, that can be studied directly; 2. the response is triggered by several electrical synapses firing simultaneously, exemplifying several output in response to a single input; 3. Smaller number of neurons, about 20,000; 4. BIG neurons. Hence this animal is considered a role model in neurobiology.

The organism can be stimulated by the application of a minor electric shock to one side of the siphon, and the magnitude of response can be measured by a force transducer, a device, usually piezoelectric, which converts mechanical pull in terms of electricity. Applying a more intense shock to the tail, or administration of chemicals into its abdominal ganglion are other ways of stimulating the mollusk. After careful observations, scientists postulated that habituation, sensitization and classical conditioning were responsible for the learning that occurred in Aplysia.

In the picture on the left, you can see a presynaptic neuron on the left side, a postsynaptic neuron on the right and another neuron (facilitator terminal) on top left stimulating the presynaptic neuron. The flow of impulse propagation is from left to right, that is, from presynaptic to postsynaptic neuron. When the presynaptic neuron was stimulated alone, the post-synaptic neuron responded in a ‘what is it’ response. When the stimulus was given repeatedly, then the response of the post-synaptic neuron became less and less. This implies that the post synaptic neuron became as if ‘habituated’. On the other hand, when a noxious stimulus was applied at the ‘facilitator terminal’ at the same time the presynaptic neuron was stimulated, the response became stronger and stronger. They called this ‘sensitization’.

In the case of habituation, it was found that the release of neurotransmitter diminished in the presynaptic neuron, possibly due to progressive inactivation of calcium ion channels. It is the calcium ion channel which allows degranulation and release of neurotransmitters. Thus inactivation means blockage of impulse. Structurally, both the presynaptic vesicle number and size are seen to decrease in habituation.

In sensitization, both the presynaptic vesicle number and size increase. Here the ‘noxious stimulus’ delivered alongside, causes release of serotonin from the facilitator terminal. This chemical, also called 5HT, binds with 5HT receptors in the presynaptic terminal. This is followed by activation of an enzyme known as adenylyl cyclase, which in turn produces cyclic AMP from ATP. cAMP then activates another enzyme, protein kinase A (PKA), which phosphorylates potassium ion (K+) channels. As a result K+ channels get blocked. When an imulsee arrives at the synapse, the membrane gets depolarized, i.e., the inside of the cell becomes positive with respect to the outside. For the cell voltage to return to its normal polarized state, the potassium ions must diffuse out, through K+ channels. But the exit route is blocked now. The action potential remains longer; more Ca++ enters into the presynaptic terminal.

Recent evidence points at the role of post synaptic neurons in habituation, sensitization and classical conditioning. Glanzman et al have proposed that activation of postsynaptic glutamate receptors might play a critical role in mediating long-lasting habituation of gill withdrawal reflex in Aplysia. A whole new range of activity starting at the synaptic knob to the nucleus and thence back to the knob again has been proposed for memory storage.

Short-term memory, which usually lasts for a few minutes, involves covalent bonding of pre-existing proteins leading to alterations in the strength of already existing connections. By contrast, long-term memory requires mitogen activated protein kinase (MAPK), CREB and new mRNA and protein synthesis; in addition to PKA.

MAPK migrates into the nucleus where it phosphorylates cAMP-responsive element binding protein (CREB). This then regulates gene expression, resulting in transcription and translation. Moreover, long-term memory is associated with the growth of new synaptic connections, phosphorylation of post synaptic densities (PSD is like a scaffold on which proteins are assembled) and many other processes. Thus it seems post synaptic mechanisms might have a bigger role than imagined.

So far, we discussed about only one form of memory: implicit or non-associative memory. Declarative or explicit memory consists of semantic (book) and episodic (related with places, memory of events) memory, which are consciously stored. Humans are perhaps, the best or only known subject in this regard. We certainly can't expect Aplysia to speak out.

Last modified: never
References:
Prolonged Habituation of the Gill-Withdrawal Reflex inAplysia Depends on Protein Synthesis, Protein Phosphatase Activity, and Postsynaptic Glutamate Receptors
Youssef Ezzeddine, David L. Glanzman

Molecular Mechanisms of Memory Storage in Aplysia
Robert D. Hawkins, Eric R. Kandel and Craig H. Bailey

C. Bailey, M Chen (1983). Morphological basis of long-term habituation and sensitization in Aplysia Science, 220 (4592), 91-93 DOI: 10.1126/science.6828885

Math Flaw Allows The Proverbial Tortoise Beat The Hare

2008-08-24T10:26:00.005+05:30

How fast can a man run? More importantly, what is the speed limit for us human beings? The mere answer of 'the speed of light' (for bodies having a rest mass) isn't enough. You might also suggest that going at sound's speed ain't a good idea too, due to the 'sonic boom' that happens at 1 Mach. Recently,Usain Bolt took 9.69 seconds in 100 meter dash in Beijing 2008 Olympics. He broke his own previous record. What if someone bettered him or even someone surpassed this 'bettering man'?

Speed will certainly depend on various biomechanical and metabolic factors, and will be limited by some constraints. The height and weight of the individual, the efficiency of anaerobic glycolysis, ATP and phosphoryl creatine stores, the training, which induces certain (fast twitch fibers, for sprint) are important. The strength of bones and the Young's modiolus, governing the stress-strain relationship (also of cartilages) are important factors of biomechanics of sports.

Nerve conduction velocities, refractory period (the time during which an excitable tissue like the nerve or muscle is NOT responsive to stimuli) of nerves and muscles are determining factors too, as is the pumping of calcium ions from muscle fibers back into the sarcoplasmic reticulum. Here I am disregarding external factors like friction, wind drag; and other physiological parameters like metabolic build-up like lactates or temperature, for the sake of lucidity.

So how much is the maximum possible speed? We are yet to decide using the physical and physiological attributes. But how about 'running' slowly and claiming a trophy? The bottom line is: you can even beat Bold, boldly! Just run the 100 meter dash in 100 seconds. A flaw in the mathematical calculation system allows you to do the trick. Read about it at 'cosmic variance'.

Last modified: never
Reference: hyper-links, unless specifically mentioned