Wednesday, February 24, 2010

The new and notable models and Giant Molecules -Cardiac muscle and the failing heart

The cardiac muscle session chaired by Dr. Solaro and brought to the limelight the new key players of muscle contraction. A key observation also included the animal model systems and whole heart studies in vivo, ex vivo and in vitro done to understand the cardiomyopthies in totality as studies traversed from whole heart to the single molecules step by step.  Hence, I enumerate contractile players who are in the focus and models of the same.

Beta -MHC - Myosin Heavy Chain  (220 Kda) occurs in two isoforms in the heart - the alpha MHC and the beta MHC. As the transition from a non-failing to failing heart progresses, the beta MHC isoform takes over and is predominant in the heart. This isoform is slower than the alpha MHC and thus more energetically economical for the heart. Given the energy starved status of the heart, the switch takes over. The human isoform to begin with is predominantly beta. However, in small rodents like mice alpha isoform is predominant. Dr. Susan Lowey highlighted how a single mutation in MHC can severely affect the kinetics of cross bridge cycling in the herat and lead to cardiomyopathies. It is important to keep in mind here that even after PTU treatment of mouse hearts which would convert alpha to beta MHC - the kinetics of contraction are not the same as that found in higher mammals where the beta isoform is always predominant. Hence the need to study higher models like Guinea pigs, rabbits, pigs is very important. My own work in Dr. De Tombe's lab is targeting this issue as I have developed a Guinea pig model of Heart failure.

MyBP-C - Myosin Binding Protein C is a 140 Kda protein found in the contractile apparatus. MyBP-C puts a constraint on the mysoin heads as they bind of actin and regulates the contraction process. In KO models of MyBP-C cardiomyoptahies are known to develop. Dr. Richard Moss gave a very comprehensive talk on the phosphorylation states of the molecule and how they affect the kinetics of cross-bridge cycling. Dr. Sakthivel Saddyappan has worked on various KO mouse models of this protein and shown how important it is in maintaining the physiological function of the heart. Dr. Moss elegantly showed how phosphorylation of MyBP-C alters the kinetics of contraction in the cardiac muscle.

Titin -  The next molecule brought into focus was Titin - a 400 KDa protein. Titin provides a 3 spring system in the contractile machinery to modulate the stiffness of the molecule. Dr. Henk Granzier talked about the how various isoforms of titin alter the passive stiffness of muscle. Dr. Wolfgang Linkes work on the phosphorylation of isoforms of titin by PKG complements the studies shown by Dr. Henk Granzier. In the failing heart a switch takes place from the N2B isoform to the fetal isform N2BA and as this artio alters so does the passive stiffness of the cardiac muscle, leading to impaired relaxation and diastolic failure. Also was shown how PKA and PKC induced phosphorylation of titin alters the passive stiffness.

Also in limelight is Nebulin - the measurement ruler in the muscle, the SERCA pump and Phospholamban system (Dr. Beata Wolska gave a very elegant talk on the workings of the system) which regulate the calcium concentration within the muscle and the Troponin complex - which regulates contraction.

Cheeses, Wines, Fungi, Chocolates, Salami -Ferry Building

Today is the last day of BPS. Lot of us are packing our bags and checking out from the hotels. A must do before you leave the city of SFO, is a visit to the Ferry building. Just walk down the market street towards the Bay bridge and you will reach the Ferry building. From amazing cheeses you can taste and pick at the cowgirl creamery to mouth watering truffle oils to Napa valley wines to delectable chocolates - you got it all at the Ferry building!

So add a small item on your itinerary before you board that plane! You will not regret the visit at all. Not to mention, the view of the Bay bridge is awesome from here and you can have Peet's coffee as you admire the Bridge! The city's romance is unforgettable!

Tuesday, February 23, 2010

Spice up your palate at BPS -Dosas

Spicy, aromatic and delightful fusions of South Indian cuisine tantalized our taste buds at this restaurant tonight. The food is exotic and very tastefully prepared by the chef. The serving style is a delight to the senses. Without being overly priced and offering a most modern ambiance with excellent service, this place is not to be missed out in while you are in San Francisco.

Enjoy a hearty meal before you leave SFO!

The Proteomic Era of Structure and Post-translational modifications

As I attend more and more muscle and cradiac muscle talks at BPS, I realize how much the focus has moved from just studying protein function, to appreciating and studying the much more dynamic changes in proteins via structural studies and post-translational modifications like phosphorylation, methylation, oxidation and ubiquitination. Exchange studies wherein phosphomimetics prepared by single site directed mutagenesis are created and exchanged within the muscle preparations to study these effects. PTM (post-translational modifications) sites are identified using 2-D DIGE studies, mass spectrometry, IEF gels, ProQ diamond staining, phospho-tags. 

The mechanics and kinetics of cross-bridge cycling are then studied by langendorff hearts, intact fiber preparations, single cell studies, single myofibril technique, in-vitro motlity assays, 3- bead assay and exchange studies. Mechanical data are extensively being employed to study the nature of these PTM exchanges to understand how they regulate muscle function. Structural studies using X-ray diifraction, NMR, circular dichroism and mass spectrometry are key tools to really look at the molecular structure and protein folding to understand these phosphomimetics to see what changes within the molecule/s.

Muscle mechanics - the emerging techniques of note II

Many new techniques were brought to the forefront with the muscle mechanics sessions we have had thus far. Having detailed some in my previous article, I detail some more emergent techniques having a great impact in the field.

Single Myofibril Technique - It was developed in Dr. Poggesis lab in Italy and has been a focus of our lab (Dr. De Tombe) as well (check Ryan Matejas poster on Wednesday). My work heavily employs this technique as well. It enables us to measure kinetics of activation/relaxation in the muscle within milliseconds. The forces recorded are of the order of nano newtons and the muscle preparation itself is approx. 20-80 um long to 2-4 um in diameter. The technique uses phase contrast microscopy. The myofibrils (a single skinned string of sarcomeres ) are bound to the coverslip glass by electrostatic interactions. We pick them up between two glass needles. One needle is stiff and doe snot move while the second needle has an L-shaped tip of about 7um and is compliant. As the solutions are switched from relaxing to activating using a double barreled perfusion pipette, the muscle contracts, pulls on the compliant needle thereby making it move and the movement is then detected by video microscopy. The forces and kinetics are then calculated. 

Exchange Mass Spectroscopy - Deuterium water exchange is been exploited to study the structure of various molecules (myosin, troponins, tropomysoin) in the contractile apparatus in various conformations (calcium free, calcium bound, phosphorylated, methylated, binary complexes, ternary complexes). The idea behind the technique is simply to replace the hydrogen ions in the amino acid backbone by the deuterium labeled isoform of hydrogen and then capture the mass difference  in form of shifted m/z ratio of peptide/protein peaks by mass spectrometry. A qualitative as well as quantitative analysis ensues.

In Vitro Motility assay - This technique enables us to measure the kinetics of sliding velocity of myosins over the thin filament. The major advantage of this technique is that  one can  play around with a lot of the contractile proteins individually and study their effects very specifically. The  myosin heads are bound on the coverslip. As thin filament molecules (actin, actin with Tropomysoin, actin with tropomysoin and troponin) are injected onto the coverslips they start sliding under unloaded conditions. You could change the isotonic strength of the solutions, the ATP/ADP ratios, phosphomimetic protein modifications and study these changes very easily as the velocity of thin filamnet sliding varies. The movement is captured by video microscopy.

Flourescence Resonance Energy Transfer (FRET) - Intermolecular interactions are studied as two molecules in close proximity to each other are labeled with two different flourophores. The preparation is excited at two different wavelengths and the signal is captured. As the molecules interact based on the experimental conditions, conformational changes might take place which can be recorded using FRET by measuring the distance changes between the two flourescent signals under a confocal microscope. Currently FRET is being used in Dr. Seth Robias lab to take it to the next level wherein intramolecular changes can be recorded by measuring the distances between the signals.

STED - Developed in Dr. Lehnharts lab, STED is an emission microscopy based technique wherein a higher resolution of imaging is obtained in contrast to confocal microscopy. For molecules like t-tubules where sizes are less than 300nm, confocal does not capture all the nuances of the structure as the wavelength it uses is within the same range of 300nm. However with STED you get sharper images and better resolution. A doughnut shape around the area of excitation by laser is exploited in STED. A major advantage of STED is that it can capture structural dimensions in the z-axis as well and reveal in-depth details.

Muscle Mechanics - the emerging techniques of note - I

The Monday morning muscle sessions started with two awesome talks on the relay loop of the myosin and its function in stabilizing the myofibril structure assembly and force generation. While the morning session talks were super interesting, of note were the new emerging techniques that were talked about.

Gravitational Force Spectroscopy (GFS) - a new technique developed in the Root lab, essentially exploits a two microsphere (small glass beads) system wherein the larger of the two micro-spheres is anchored and the smaller one is free floating. The distance between the centers of these two microspheres is at its minimum. As a myosin molecule tethers itself between the microspheres, gravitational force is exploited such that it is either parallel or perpendicular to the orientation of the molecule. As the free floating microsphere rotates based off the gravity, one can calculate the min. and max. distance between the centers of the two microspheres and thereby calculate the flexibility of the myosin tethered. It is a novel and neat technique, with lots of potential for experiments contrasting diseased and control states of various molecules and post translational modifications altering flexibility.

3- Optical bead Assay - Highlighted in the last two talks of the session was the 3-optical bead assay. The gist of the technique - mount two anchored polystyrene glass beads on two opposite ends on a coverslip. Motion sensors are attached to each of these beads. Actin filaments attach these two beads together. A third bead is positioned in between these two beads and a myosin head is tethered to it. As the myosin head forms a bond with the actin filament, the anchored beads move, the distance is sensed by the motion sensors and the step size of force generation by one head is revealed. Now if you attach small loads to each of the two beads attached to the actin filament, you get the working stroke  of the muscle under loaded conditions!

The other notable techniques that are emerging are the single myofibril technique, FRET, mass spectroscopy and cryo- EM. which i would cover in part II of this article later in the day. Have to rush to career workshops now!

Monday, February 22, 2010

Students go Hungry, BPS Grad Student Breakfast runs out

Bright and early students wake up and jaunt up to Room 301 at 7:30 am to get to know fellow graduate students and have a hearty breakfast hosted by BPS for graduate students. Lo and behold they run out of food in less than 10 minutes. BPS calculations went off by far! Students were not happy about the situation.
I am headed to the muscle mechanics sessions now!

Until later updates - have a good day!

Sunday, February 21, 2010

The Spooks in SFO on a dark rainy night!

Its a dark night, I can hear the wind howling, the owls hooting and squelching of wet sneakers as I walk down the Octavia street. Its the Old Victorian district of San Francisco. Beautiful mansions line up the street on both the sides with the scent of eucalyptus trees and rosemary bushes stimulating the neurons. Me and my labmates are getting ready to get the spooks in the city - we are headed to a Ghost tour! After a long day of science at BPS it seemed like a perfect way to end the day walking in the old neighborhoods of the city.

We enter the Queen Anne hotel where we are designated to meet the ghost hunter. The hotel is deserted with antiques lined up in the lobby, piano music playing and a warm fire crackling up. We start warming up to the place, after all we were just out in the damp weather on a bus. Of course, there are sudden footsteps and a man with a long beard, an overcoat, black boots, a hat and a lantern walks in the room. The ghost hunter has arrived! The tour starts as he animatedly talks about the ghost of the hotel - the friendly ghost Mary Anne! She supposedly wraps you in blankets as you sleep in room #410! We start walking out in the neighborhood and a jolly evening of part architecture, part theatrics and part eerie stories, begins!

There were the romances of Gertrude and clashes of the sisters, ghosts who dont like kids and of course the Voodoo queen of SFO - Marry Ellen who is a historical figure in the times of Gold Rush! While we didnt really see any ghosts, a haunted key turned by 180 degrees on the palm of my hand because I willed it so - as claimed by the ghost hunter! It was an interesting night - just the brisk walk and the fun of the theatrical ghost hunter was worth it. Do visit the district on your BPS spook night.

Until the awesome science tomorrow - good night folks!

People denied access, Overcrowding of the platform session rooms!

As the enthusiastic scientists from across the globe have come together for the talks, platforms sessions have become over-crowded and people were denied access to rooms. This left many people upset and complaining. Once the chairs filled up, everyone started sitting at the floors and the rooms were jam packed for some of the sessions. Given the expansion in BPS meeting members this year, the rooms need to be bigger to accommodate everyone interested in attending the talk. This was observed in Room 304 and then 308 today morning where the cardiac muscle session and then the calcium signaling session took place.

The Internet Kiosk, a lost umbrella, a request!

The line waits at the internet kiosks peaked at around lunch time at 12:45pm!  A beautiful black and white umbrella with red venitian gondolas was left in the drawer of the internet kiosk!

A request to all - The umbrella is missing and has not been returned to the LOST AND FOUND section. If you have found it then please turn it in at the BPS desk by the internet kiosk or at the lost and found desk at BPS.

Riveting Cardiac Muscle sessions ensue

In contrast to the outside dull and gray weather conditions, BPS meeting is buzzing with activity today. Even as you walk the streets of the city, you see people with yellow string tags crowding the bars and cafes all along. As you step in the Moscone center, you see people walking briskly towards the rooms where they are about to attend the talks of their interest or people sitting scanning the green colored BPS meeting book sorting their itineraries.

My morning starting crisp and sharp as I headed towards the cardiac muscle symposium in Room 304. The very first talk contrasted STED (a new imaging technique) with confocal microscopy, showing beautiful pictures of imaged t-tubules, showing the sharper images you get with STED and the better resolution you get being able to capture the z-plane as well. A lot more people have trickled in the room by this time and it is beginning to getting cramped. The second talk was from Dr. Poggessi's lab in Italy by Cecilia highlighting novel experiments looking at the impact of loss of t-tubules on cardiac muscle function. It was a very interesting talk. Following this was a FT-ICR mass spectrometry talk by Dr. Alan Marshalls group showing deuterated water exchange studies on the troponin complex in calcium free and calcium bound state. By now the room was super crowded and people were sitting on the floors. 

The next talk kind of zoomed by really fast as the multiple structural conformations of troponin complex simulations were talked about. The simulations were picoseconds to nanoseconds long. Cool electrostatic mapping of the structures revealed positive charged areas which allow repositioning of molecular lobes within Tn complex. The next few talks focused on the specific mutations in either tropomyosin, troponinC, which altered the cross-bridge cycling kinetics and showed altered calcium sensitivity. The platform session concluded with a very interesting talk showing the Drosophila wing heart m-mode echocardiography videos studying the an UNC-45 knockdown mutation and its effects.

It was an intense session marred only by the fact that the room was very crowded and some people were denied entry because of over crowding!

Saturday, February 20, 2010

Imminent Arrivals!

A 4:30 hr long flight took us from wintery cold of the Midwest to the warm city of San Francisco! Ahoy BPS!

So here we are at the convention center, just collected our badges and scanning through the humdrum of the Moscone center. Travel awards night tonight, and some good sea food at Fishermans Wharf! Tomorrow morning sessions start bright and early at 8:15 - watch out for updates!

Friday, February 19, 2010

The BPS network and me!

This year the BPS will  be bringing together 6000 researchers, 4000 poster presentations, 200 exhibits and over 20 symposia to the table. Not to  mention the fact that there are the career workshops, the institution fair, National lecture and the society reception and dance!

Just going by sheer numbers, one can calculate the number of probabilistic connections, collaborations, networks and outreach such a nexus provides one with. Given the vast diversity of talks it is an awesome platform to launch yourself and to learn about the latest innovations in research and technology. Sure it will leave you dumbfounded sometimes when you sit through some of these talks but then again there are the talks that are so riveting that you cant wait to get back to your  lab and experiment!

As for myself, I am a Ph.D. student working on developing the Guinea pig Heart failure model and studying the post-translational changes in the cardiac regulatory proteins using the single myofibril technique, mass spectrometry and proteomics.

During my time at Biophysics, I would be focusing a lot on the symposia, sessions, posters and talks on single molecule techniques, mass spectroscopy, muscle mechanics and regulation, cardiomyopathies, stem cells, systems biology and heart failure! Sunday and Monday are packed with sessions and talks on these topics. Tuesday is relatively light but there are some very interesting career workshops and Wednesday has a whole session on muscle again! 

You will be reading constant updates on my blog about all the science and much more aka things to do in SFO, the visit to the Fisherman's Wharf, the Ghost tour, Dosa restaurant, Chinatown, the visit to the California Science Academy and the bike ride on the Golden gate bridge!

A word of thanks to BPS for giving me this opportunity to blog and to all you  my new readers - so stay tuned :)

Tuesday, February 16, 2010

Morning caffeine, the nanotech geeks and Science at Biophysics

A bright and sunny morning has dawned on Chi town as we all  get set to go to the Biophysical Society  meeting in four days! The excitement is brewing up as everyone around is getting their posters ready, putting their itineraries in place, zeroing in on the talks to attend and juggling with places to dine in San Francisco!

With morning symposiums starting as early as 8:15 am, the demand for caffeine is high. As I have noticed in the earlier Biophysics meetings, getting to the conference center a good 30 minutes earlier than the talk you are attending is always a great idea so as to get your morning coffee well in time. The lines are long and the rooms are spaced out. Not to mention the fact you never know who you will bump into and start conversing with.  For all you know it might be the person reviewing your latest grant application or publication! Those 5 mins of conversation are like a double espresso shot in itself - you might be talking about the sea lions in the bay area one second and the next you would  be playing quid pro quo about the latest innovations in nanotechnology and drug delivery systems! To top it all such times are like time warps when scientists from all around the globe exchange ideas. 

So a key point to keep in mind as you plan your itineraries and get ready for Biophysics - slot in those few extra minutes when you stand in queues for caffeine, meet the coolest people in science whose papers you read and ensue those storming ideas which might be the next project you work on! Its the beehive and we are heading in it! See you all soon!

Wednesday, January 13, 2010

The Last Decade of Nobel in Physiology or Medicine

The Nobel Prize - an annual tradition which started in Sweden in the year 1901 by Sir Alfred Nobel to award the most notable achievements in Physics, Chemistry, Medicine, Literature, Peace and Economics. It is an award coveted by scientists across the board. 100 Nobels have been awarded in Physiology since 1901 to 195 individuals of which 10 are women. No Nobel in Physiology was awarded during World war I and World war II. Here I enumerate the advances in Physiology or Medicine in the last decade which have captured the Nobel.

2009 - awarded to E.H. Blackburn, C.W. Greider and J.W. Szostak "for the discovery of how chromosomes are protected by telomeres and the enzyme telomerase".

2008 - awarded to H. zur Hausen "for his discovery of human papilloma viruses causing cervical cancer", to F.B. Sinnoussi and L. Montagnier "for their discovery of human immunodeficiency virus".

2007 - awarded to M. R. Capecchi, Sir M. J. Evans and O. Smithies "for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells".

2006 - awarded to A. Z. Fire and C. C. Mello   "for their discovery of RNA interference - gene silencing by double-stranded RNA".

2005 - awarded to B. J. Marshall and J. R. Warren "for their discovery of the bacterium Helicobacter pylori and its role in gastritis and peptic ulcer disease".

2004 - awarded to R. Axel and L.B. Buck "for their discoveries of odorant receptors and the organization of the olfactory system".
2003 - awarded to P. C. Lauterbur and Sir P. Mansfield "for their discoveries concerning magnetic resonance imaging".

2002 - awarded to S. Brenner, H.R. Horvitz and J.E. Sulston "for their discoveries concerning 'genetic regulation of organ development and programmed cell death'".

2001 - awarded to L. H. Hartwell, T. Hunt and Sir P. M. Nurse "for their discoveries of key regulators of the cell cycle".

2000 - awarded to A. Carlsson, E. R. Kandell and P. Greengard "for their discoveries concerning signal transduction in the nervous system".

1999 - awarded to G. Blobel "for the discovery that proteins have intrinsic signals that govern their transport and localization in the cell".

Monday, January 11, 2010

From Organs to Molecules

A selection of images I captured as part of my research. The images show the transition from organs to molecules. The right most image is an echo-cardiogram of the Guinea Pig heart, middle image shows a single cardiomyocyte held by two metal needles and left most image is that of a single myofibril suspended between two 7-10 micron thin glass needles.

TED - A Beehive of Ideas

TED talks - Technology, Entertainment, Design - Ideas Worth Spreading

I stumbled upon this website and ever since my iTunes podcast has been channeled to the talks and videos on TED. TED is a unique platform where you hear the news from new frontiers of science. The talks are inspiring and educational. It is a window to new ideas and innovation. TED is a convergence of many aspects of science. The talks are wide ranging from Jane Goodalls rendezvous with the Apes to Bill Gates new ventures! While the talks give us invaluable insights into varied worlds, they leave a lot of room for exploration of these worlds and deeper understanding of these concepts. TED piques your curiosity to learn. Get those 20 minutes of inspiration today!

Ph.D. Comics - 2 min Hiatus with fellow Grads

Ever so often, all graduate students have those few minutes in the day when we wonder "Is it just me!" and that is when a comic series dedicated to our lot comes to the rescue. Back in the days of Aristotle and Plato, such luxuries were not available, but today with the prime of technology and internet connections we can glimpse into each others lives and feel consoled that "There are others like me out there. I am not alone. Phew!".  Cheers to Ph.D. comic series Piled Higher and Deeper!


A glimpse into the story of muscle

Every time we see a phenomena/object we are not conversant with, we begin by giving it a name to start conversing about it. This rule applies across the board with a very strong emphasis in sciences. Each time there is a potentially new specimen still unrecognized, a scientific name is quickly alloted to it.

Folklore has it that long time ago, in Greece, a similar story began when the budding scientists of that era noticed a mouse moving up and down the upper arm of a disc thrower every time he exhibited his skills at the sport. To begin a simple conversation they named it - muscle, a simple diminutive version of musculus meaning mouse in Latin. Thus, was launched a whole new world of muscle studies.

Thereafter, it was recognized that the organ of motion is muscle. The understanding of muscle we have now, required long nights spent in graveyards digging bodies and studying science against the beliefs of the society then. Studying the anatomy of the human body and laying the foundations of this science of contraction has traversed from graveyards to the modern day science lab with the pipette tips and the microscope. It was in 1700s that Galvani and Volta recognized that movement of charges aka biological electric currents underlie contraction and cause movement of limbs.

It was only in 1864 that Kuhne isolated a viscous protein and named it mysoin. Amidst war years and complete scientific isolation, Albert Szent Gyorgyi and his colleagues established that "myosin" consisted of two molecules of varying viscosity. In parallel, Needham et al. discovered the effect of ATP on Kuhne's myosin independently. The two groups were never able to communicate. But a molecular motor driving movement had been discovered. A century later Actin molecules were discovered by Straub in 1942. The molecular foundation of the muscle had now been laid.

In 1953, the milestone of muscle research was established by Huxley and Huxley when they proposed the Sliding Filament Theory of muscle. It was based on the observation of constancy of the length of the A-band and the shortening of the I-band during contraction. Using interference microscopy and phase contrast miscroscopy it was shown that molecular motors do not change in length while contracting but thin and thick filaments slide over one another causing the change in muscle length thereby resulting in contraction. Thus heralded a new era in muscle science.

The race to discover the regulators of contraction, the structure of these threads, the ATP utilization of this phenomena had just begun. Lymn and Taylor proposed the cross-bridge cycle in 1971, breaking down contraction in steps of ATP utilization. Tropomyosin was discovered by Bailey in 1946. That calcium removal induced relaxation was discovered by Marsh and Bendell. And to complete the jigsaw Ebashi et al. unraveled Troponins.

I embarked on this journey when I did my rotation in Dr. De Tombes lab and saw a string of sarcomeres beautifully suspended between two 7-10 micron thin glass needles. The string is called a myofibril.  The shadow of a darkly coated glass needle fell exactly in between a pair of identical photodiodes when this string did not contract. As soon as calcium hit this string, it contracted, pulled the dark glass needle and its shadow biased towards one photodiode, thereby enabling us to record the force! It exerts a force of about 50nN/mm2. To understand this beautiful wonder, I delved deep into the past origins of this story.

Justice cannot be done to this discovery of the muscle and its molecules in a short one page entry. However, one can fathom the process of scientific discovery and the eons it entails as each piece of the big puzzle falls in place. What I have written here is just a peek into this wonderful world. The work on understanding muscle is still far from complete. A new brick has to be baked everyday and molded to perfection to be laid into the wall.

Follow up Reads:

1. Prime mover by Steven Vogel
2. The Early History of the Biochemistry of Muscle Contraction - Andrew G. Szent-Gyorgyi, J. Gen Physiol. Vol 123 (2004) 631-641.