Friday, December 25, 2009

ABLE India Competition - Worth it

Time encompasses so fast that, till we are about to sort out memories of any incident or any event in our every day routine, some or other incident re brushes memory lane to wake it up to same experiences to show up.

I am on verge of letting our Bangalore trip's incidents to flow down here, in meanwhile its so exciting that I am jolting these down in midst of my preparation again to depart for same destination although with different mission in hand this time.

We (includes Me, Roshan -Team leader, Asmit, Moiz & Shreyash) had been to Bangalore for ABLE India competition with our team being selected one among top-20 at all India level for sending novel methodology to diagnose and treat Primary Glioblastoma (Brain tumor), with every team with their own concepts or technologies with some particular goal in mind. We became extremely proud when we read that we were selected among more than 200 applicants and selected one's being from different premier institutes like IIT's, etc. It's always great feeling to be with to compete with people of such grades.

We had booked our tickets for nice cool 2nd AC in Udhyan Express, with feelings reaching greater height when being informed that we would be getting reimbursement for all our traveling expenses and also that we have been provided accommodation in nice expensive suit of 5 star hotel. The journey in train was worth memorable from events like roshan starting installations in laptop as soon as we entered train from Kalyan, trying to get laptop charged, with Asmit sorting issues when he boarded train from Pune, watching movies like 'Andaz Apna Apna','Hangover', was really fun to get going.

We arrived in Bangalore on 24th morning and as we were given accommodation from 25th morning, we had to manage one day's stay, which we got it done with Shreyash's help, by getting flagship to stay at one of his friend's relative place. Typical South Indian welcome we got at their place, with warm coffee and a much needed breakfast just kept ready for us. We can hardly return back any favors from them for way we were made to feel being at home.

The day went quickly as lunch went heavy with most guys and made Roshan, Shreyash and Asmit sleep with wonderful dreams running around.Well as every team had to give power point presentation and restrictions like allowing only 5 slides maximum was itching us to rack our brains more for some innovation. With cheese serving as perfect trigger for moiz and with minor input from me of taking snapshots and adding them to slides. Moiz climbing perfectly on idea by not only taking snap shots but enlarging them on their occurrence during presentation. The idea worked perfectly fine.

Well as the lunch got digested and evening followed we had minor breaks in between and all 5 of us getting charged up. The dinner was again refreshing and was followed by small walk with moiz still searching for perfect food to satisfy his tummy. The night brought in more movies specially for moiz who loves hanging around not much far from any gadget when he is awake. The next morning was certainly more merrier as it was our time to get in to hotel.

The event was organized at Ramee Guest line Hotel and also place of our accommodation. We were split up during our on way to destination as Maruti 800 of the uncle (who took pains and time to drop us to venue) quite small in loading all of us heavy weights. So me and Shreyash had to board Auto, which dropped us in midway to catch bus as being instructed. The experience in that bus, I can never forget as our entry in bus was marked by large voice, which later on we realized was some south Indian hero giving war cry on villain from a movie running on television sets attached in bus. We very interestingly followed what went in the Kannada movie, trying to interpret with suddenly realizing that bus got in to last stop and we were suppose to catch some other vehicle to carry on, which allowed us to get experience of Volvo bus system, was truly memorable.

As we came across the ABLE India representatives at the venue, it brought us more cheers with we getting our part of the reimbursement. The rooms were extremely cool place to keep us attached to bed for long enough but as time schedule for the 3 day event was organized it needed us to be ready in short time interval and attend lecture series for next 2 days which were arranged to help us with our presentations on the 3rd day with topics ranging from Entrepreneurship, finance, new scientific innovations, Government initiatives for Biotechnology sector in India, etc with speakers like Kiran Mazumdar Shaw from Biocon, many leading personalities from Department of Biotechnology (DBT)-India. We were provided with wi-fi facility which was very much relief for all internet addicted generation staying there which helped also for building up blocks for the presentation along with inputs from different seminars.

The day of presentation was most thrilled as organizers had arranged Industrial Visits to Biocon but we choose of settling ourselves in our rooms and making presentation which was like brought in something like presentable form on 2nd day's night by moiz and shreyash. The morning sailed on with all of us forcing ourselves on laptops and trying give inputs for the presentation. The most memorable scene was that we had our presentation timed at 3 and we were still settling scores to prepare final copy and although we went for lunch it brought us sooner to room with tensions creeping to certain extent. At 2:30 few of us were almost ready and one guy which is moiz was very calmly making final touches to presentation and finally we did receive call twice from Nandita Mam (Chief coordinator) on our dear group leader Roshan's cell phone. As all of us reached to the seminar hall which was occupied only by the panel of judges waiting for each team to individually present their project proposals.

It was one of the most admiring moment for me as moiz who was suppose to present our proposal did not even go once through the final soft copy of presentation although time was also against it and it was indeed really interesting and pleasurable to watch roshan with such a big smile on his face with him getting free of giving presentation by getting nervous during our practice sessions. It is fun to watch how stress can change our expression status.

As we were called in for the presentation we all teamed up looking truly professional with everyone matching each other through their perfect formals and tie to go around. I definitely marked that we stood best among rest of the teams in being perfectly dressed up.

Introduction was given by me for our team leader and all of our team members followed by getting nod from panel for moiz to present and those 10 minutes he spoke like warrior trying to battling for his bride. After presentation time limit, we were put up with few questions with biggest hurdle which we understood was that we dealt with 2 things at one go the diagnosis and treatment. Choosing either of them would have helped us to give enough justice to push our idea in those most skilled brains present in the panel of judges. It was enough justice that moiz was still considered as person to give best presentation among all teams there ,which was award in itself.

As the presentation got over it was one of the most relief moments to follow with all of us heading for a much needed break by chilling out through photo session, interacting with other teams and relaxing by pool side although wait for results was building up among all.

The results were announced in evening session with 3 teams from different institutes getting awarded for ideas like 'Vein locator', 'Bio diesel project', etc. It left lump in the throat for not reaching final 3 but it did allow oxygen to enter with a feeling that we were left enriched with higher values, higher skills, loads of inputs at different aspects, giving us opportunity to truly be one among the best.

I would like to emphasize on the facts that when we come in terms on competition with people from institutes like IIT's or in our case the team which won 1st prize was from Stanford Biodesign is that to focus on resolving needs which seem very simple but are very effective in terms of their applicability like a 'vein detector'. Also point of focusing or addressing single aim at one time was principle lesson learn during our participation. It allowed us to self introspect at every aspect when we came in terms to other participants. It allowed giving us inputs which highlights term of 'acceptance' quality along with 'adaptability' like we were suppose to put forward budget for our proposal which needed us run our brains in finance.

The next day (28th July) was little shy in terms of activities as we were suppose to leave for our return journey back to Mumbai in evening through Udhyan Express, with we all choosing to relax in hotel room until our check out time. We got a Volvo bus back to station with ride giving Bangalore darshan sort of. The lunch comprised of KFC on the menu for non vegi's and me and asmit feasting on nearby mithai shop.

The return journey in train was again pleasurable with movies to recounting all the events to cheese feast by moiz next morning in train till Pune saw asmit waving off. As we got down at kalyan it did gave a feeling of satisfaction which was biggest gain for me from the journey and experiences throughout not only during journey but through our formulation of the project where moiz came up with idea, roshan working with high end animations to come up with some high end animated diagram, me getting chance to show skills by my writing by building requisites of proposal and getting laptops arranged, shreyash managing inputs from setting up meeting with pharma company owner to finding accommodation before our stay at hotel, asmit charging up with his knowing of scientist to allow us getting their inputs.

I can just summarize that it was made worth the part of life through loads of efforts, ideas, few months to put in from everyone of us each one contributing as much as possible, which has made richer in terms of experiences we all shared together, lessons we learned at each stage. Just wanna say 'Thank you Guys' to all my team members.

I would simply request people following this blog to keep a note of such encouraging events held regularly to actively participate which will help in scaling greater heights not only with individual self but also for finding applicability of one's own self to society.
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Saturday, October 3, 2009

Spirochete staining - Silver Impregnation Technique

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Thursday, September 3, 2009

Aurobindo Pharma acquires Cefepime

Bangalore, Aug 27, 2009: Hydebadad-India based Aurobindo Pharma has received Swiss drug regulator Swissmedic's approval for the license of Cefepime APL for Injection 1g and 2g.
A press release by Aurobindo said Cefepime APL for Injection 1g and 2g falls under the anti-beta-lactam segment and is indicated for Moderate to severe pneumonia, septicaemia, UTI, skin & skin structure infections, intra-abdominal infections.This is Aurobindo’s second product approval in Switzerland.
But without going much into it in this article I wish to focus on some basic information on Cefepime.
Cefepime (pronounced /ˈsɛfəpiːm/, /ˈkɛfəpiːm/) is a fourth-generation cephalosporin antibiotic developed in 1994. Cefepime has an extended spectrum of activity against Gram-positive and Gram-negative bacteria, with greater activity against both Gram-negative and Gram-positive organisms than third-generation agents. Cefepime hydrochloride was first marketed in 1994 and is currently marketed under various trade names including Maxipime, Maxcef, Cepimax, Cepimex, and Axepim. A 2007 meta-analysis suggested that when data of trials were combined, mortality was increased in patients treated with cefepime compared with other β-lactam antibiotics. In response, the U.S. Food and Drug Administration performed their own meta-analysis which found that there was no mortality difference.
Cefepime is usually reserved to treat severe nosocomial pneumonia, infections caused by multi-resistant microorganisms (e.g. Pseudomonas aeruginosa) and empirical treatment of febrile neutropenia. The use of cefepime might become less common, since it has been associated to an increase mortality when used for different types of infections.
Cefepime has good activity against important pathogens including Pseudomonas aeruginosa, Staphylococcus aureus, and multiple drug resistant Streptococcus pneumoniae. A particular strength is its activity against Enterobacteriaceae. Whereas other cephalosporins are degraded by many plasmid- and chromosome-mediated beta-lactamases, cefepime is stable and is a front line agent when infection with Enterobacteriaceae is known or suspected.

The combination of the syn-configuration of the methoxyimino moiety and the aminothiazolyl moiety confers extra stability to β-lactamase enzymes produced by many bacteria. The N-methylpyrrolidine moiety increases penetration into Gram-negative bacteria. These factors increases the activity of cefepime against otherwise resistant organisms including Pseudomonas aeruginosa and Staphylococcus aureus.
Click to read more about cefepime

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Tuesday, August 25, 2009

Glycogen Biosynthesis

Glyccogen structureBiosynthetic and degradative pathways rarely operate by precisely the same reactions in the forward and reverse directions. Glycogen metabolism provided the first known example of this important principle. Separate pathways afford much greater flexibility, both in energetics and in control. In 1957, Luis Leloir and his coworkers showed that glycogen is synthesized by a pathway that utilizes uridine diphosphate glucose (UDP-glucose) rather than glucose 1-phosphate as the activated glucose donor.
Step 1. UDP-Glucose Is an Activated Form of Glucose

UDP glucose structure 
UDP-glucose, the glucose donor in the biosynthesis of glycogen, is an activated form of glucose, just as ATP and acetyl CoA are activated forms of orthophosphate and acetate, respectively. The C-1 carbon atom of the glucosyl unit of UDPglucose is activated because its hydroxyl group is esterified to the diphosphate moiety of UDP. UDP-glucose is synthesized from glucose 1-phosphate and uridine triphosphate (UTP) in a reaction catalyzed by UDPglucose pyrophosphorylase.

UDP glucose formation

 The pyrophosphate liberated in this reaction comes from the outer two phosphoryl residues of UTP. This reaction is readily reversible. However, pyrophosphate is rapidly hydrolyzed in vivo to orthophosphate by an inorganic pyrophosphatase. The essentially irreversible hydrolysis of pyrophosphate drives the synthesis of UDP-glucose. The synthesis of UDP-glucose exemplifies another recurring theme in biochemistry: many biosynthetic reactions are driven by the hydrolysis of pyrophosphate.
overall reaction of UDP glucose formation
Step 2. Glycogen Synthase Catalyzes the Transfer of Glucose from UDP-Glucose to a Growing Chain
New glucosyl units are added to the nonreducing terminal residues of glycogen. The activated glucosyl unit of UDPglucose is transferred to the hydroxyl group at a C-4 terminus of glycogen to form an a-1,4-glycosidic linkage. In elongation, UDP is displaced by the terminal hydroxyl group of the growing glycogen molecule. This reaction is catalyzed by glycogen synthase, the key regulatory enzyme in glycogen synthesis. Glycogen synthase can add glucosyl residues only if the polysaccharide chain already contains more than four residues.Thus, glycogen synthesis requires a primer. This priming function is carried out by glycogenin, a protein composed of two identical 37-kd subunits, each bearing an oligosaccharide of a-1,4-glucose units. Carbon 1 of the first unit of this chain, the reducing end, is covalently attached to the phenolic hydroxyl group of a specific tyrosine in each glycogenin subunit. How is this chain formed? Each subunit of glycogenin catalyzes the addition of eight glucose units to its partner in the glycogenin dimer. UDP-glucose is the donor in this autoglycosylation. At this point, glycogen synthase takes over to extend the glycogen molecule.
overview Step 3. A Branching Enzyme Forms a-1,6 Linkages
PhotobucketGlycogen synthase catalyzes only the synthesis of a-1,4 linkages. Another enzyme is required to form the a-1,6 linkages that make glycogen a branched polymer. Branching occurs after a number of glucosyl residues are joined in a-1,4 linkage by glycogen synthase. A branch is created by the breaking of an a-1,4 link and the formation of an a-1,6 link: this reaction is different from debranching. A block of residues, typically 7 in number, is transferred to a more interior site. The branching enzyme that catalyzes this reaction is quite exacting. The block of 7 or so residues must include the nonreducing terminus and come from a chain at least 11 residues long. In addition, the new branch point must be at least 4 residues away from a preexisting one. Branching is important because it increases the solubility of glycogen. Furthermore, branching creates a large number of terminal residues, the sites of action of glycogen phosphorylase and synthase. Thus, branching increases the rate of glycogen synthesis and degradation.Glycogen branching requires a single transferase activity. Glycogen debranching requires two enzyme activities: a transferase and an a-1,6 glucosidase. Sequence analysis suggests that the two transferases and, perhaps, the a-1,6 glucosidase are members of the same enzyme family, termed the a -amylase family. Such an enzyme catalyzes a reaction by forming a covalent intermediate attached to a conserved aspartate residue . Thus, the branching enzyme appears to function through the transfer of a chain of glucose molecules from an a-1,4 linkage to an aspartate residue on the enzyme and then from this site to a more interior location on the glycogen molecule to form an a-1,6 linkage.
Glycogen Synthase Is the Key Regulatory Enzyme in Glycogen Synthesis
The activity of glycogen synthase, like that of phosphorylase, is regulated by covalent modification. Glycogen synthase is phosphorylated at multiple sites by protein kinase A and several other kinases. The resulting alteration of the charges in the protein lead to its inactivation . Phosphorylation has opposite effects on the enzymatic activities of glycogen synthase and phosphorylase. Phosphorylation converts the active a form of the synthase into a usually inactive b form. The phosphorylated b form requires a high level of the allosteric activator glucose 6-phosphate for activity, whereas the a form is active whether or not glucose 6-phosphate is present. 
Glycogen Is an Efficient Storage Form of Glucose
What is the cost of converting glucose 6-phosphate into glycogen and back into glucose 6-phosphate? The pertinent reactions have already been described, except for reaction 5, which is the regeneration of UTP. ATP phosphorylates UDP in a reaction catalyzed by nucleoside diphosphokinase. Thus, one ATP is hydrolyed incorporating glucose 6-phosphate into glycogen. The energy yield from the breakdown of glycogen is highly efficient. About 90% of the residues are phosphorolytically cleaved to glucose 1-phosphate, which is converted at no cost into glucose 6-phosphate. The other 10% are branch residues, which are hydrolytically cleaved. One molecule of ATP is then used to phosphorylate each of these glucose molecules to glucose 6-phosphate. The complete oxidation of glucose 6-phosphate yields about 31 molecules of ATP, and storage consumes slightly more than one molecule of ATP per molecule of glucose 6-phosphate; so the overall efficiency of storage is nearly 97%
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Electrophoresis is the study of the movement of charged molecules in an electric field. The generally usedsupport medium is cellulose or thin gels made up of either polyacrylamide or agarose. Cellulose is used as support medium for low molecular weight biochemicals such as amino acid and carbohydrates whereas agarose and polyacrylamide gels are widely used for larger molecules like proteins. The general electrophoresis techniques cannot be used to measure the molecular weight of the biologicalmolecules because the mobility of a substance in the gel is influenced by both charge and size. In order toovercome this, if the biological samples are treated so that they have a uniform charge, electrophoretic mobilitythen depends primarily on size. The molecular weight of protein maybe estimated if they are subjected to electrophoresis in the presence of a detergent sodium dodecyl sulfate (SDS) and a reducing agent
mercaptoethanol (b ME). SDS disrupts the secondary, tertiary and quaternary structure of the protein to produce a linear polypeptidechain coated with negatively charged SDS molecules. 1.4grams of SDS binds per gram of protein. Mercaptoethanol assists the protein denaturation by reducing all disulfide bonds.
SDS-Polyacrylamide Gel Electrophoresis (PAGE)
Polyacrylamide gels are prepared by the free radical polymerization of acrylamide and the cross linking agent N N’ methylene bis acrylamide
Acrylamide + N N’ methylene bis acrylamide
Add Chemical Ammonium persulfate (catalyst)
Polymerisation ↓ TEMED (N,N N’ N’ tetramethylethylene diamine
1. Assembling the glass plate
( Gloves should be worn at all times while performing SDS-PAGE. To insure proper alignment and casting, the glass plates, spacers, combs and casting stand gaskets must be clean and dry. The glass plates should be cleaned with 70% ethanol.)
1. Assemble the glass plate on a clean surface. Lay the longer glass plate down first, then place two spacers of equal thickness along the rectangular plate. Next place the shorter glass plate on top of the spacers so that the bottom ends of the spacers and glass plates are aligned
2. Loosen the 4 screws on the clamp assembly and stand it up so that the screws are facing away from you. Firmly grasp the glass plate sandwich with the longer plate facing away from you, and gently slide it into the clamp assembly. Tighten the top 2 screws of the clamp assembly.
3. Place the clamp assembly into the alignment slot of the casting stand so that the clamp screws faceaway from you. Loosen the top 2 screws to allow the plates and spacers to sit firmly against the casting stand base. Gently tighten all the screws
4. Pull the completed sandwich from the alignment slot. Check that the plates and spacers are aligned. If not, realign the sandwich as in steps 1-3. Before transferring the clamp assembly to the casting slot,recheck the alignment of the spacers. Do this by inverting the gel sandwich and looking at the surface of the 2 glass plates and the spacer. Make sure that they are aligned.
5. Transfer the clamp assembly to one of the casting slots in the casting stand. If 2 gels are to be prepared, place the clamp assembly on the other side of the alignment slot.
6. Press the acrylic pressure plate bottom, so that the glass plates rest on the rubber gasket. Snap the acrylic plate underneath the overhang of the casting slot. Do not push the glass plates or spacers because this could break the glass plate.
2. Casting the gels (Demonstration)
Prepare 10% resolving/separating gel and 4.5% stacking gel.
1. Prepare the separating gel monomer solution by combining all reagents except ammonium persulfate(APS) and TEMED. Deaerate and mix the solution after adding each reagent by swirling the container
2. Place a comb completely into the assembled gel sandwich. With a marker pen, place a mark on the Glass plate 1 cm below the teeth of the comb. This will be the level to which the separating gel is poured. Remove the comb.
3. Add APS and TEMED to the monomer solution and mix well by swirling gently. Pipette the solution to the mark.
4. Immediately overlay the monomer solution with 1 ml. of water. Use a steady, even rate of delivery to prevent mixing with the gel.
5. Allow the gel to polymerize for 45 minutes to 1 hour. Pour the water overlaying the gel and drain the excess water with strips of filter paper.
6. Prepare the stacking gel monomer solution. Combine all reagents except APS and TEMED. Deaerate and mix the solution by swirling gently.
7. Place a comb in the gel sandwich.
8. Add APS and TEMED to the solution and pipette the solution down one of the spacer until the sandwich is filled completely
9. Allow the gel to polymerize for 15 minutes.
10. Remove the comb.
11. Gel is placed in the buffer chamber and running gel buffer is added into the chamber
3. Preparation of samples. ( here a cloned protein is put under analysis)
From the recombinant clone VC-25, the recombinant protein is produced as follows:
The clone was grown for 4hours and induced using IPTG for next four hours. The culture was pelleted and resuspended in PBS
4. Loading the samples
1. Rinse the syringe to be used for loading samples a few times with distilled water. Demonstrators will load the first well with LMW (7 ml of LMW). Insert the syringe to about 1-2 mm from the well bottom before delivery. Rinse the syringe a few times with distilled water after loading.
2. Load the second and other well with 20 ml of VC-25 protein as described above. Do not pipette the pellet at the bottom of the microfuge tube. Rinse the syringe a few times with distilled water after loading.
5. Running the gel
1. Check that the buffer in the upper buffer chamber are full because leakage of the buffer may occur.
2. Place the lid on top of the lower buffer chamber. Make sure that the connection is correct, ie. black to black and red to red.
3. Attach the electrical leads to a suitable power pack with the proper polarity (black to black and red tored). Run the gel at a constant current of 30 mA.
4. Stop the electrophoresis when the tracker dye is ~ 1 cm above the end of the glass plates.
6. Removing and staining the gel.
1. Remove the gel from the buffer chamber
2. Loosen all four screws of the clamp assembly and remove the glass plate sandwich from it.
3. Push one of the spacers out to the side of the plates without removing it.
4. Gently twist the spacer so that the upper glass plate pulls away from the gel.
5. Cut the gel on one side (to orientate the gel).
6. Remove the gel by gently grasping two corners of the gel and place it in the container containing the Coomassie blue stain. Make sure that the gel is fully submerged in the staining solution.
7. Stain the gel for 1 hour, agitate it slowly on a shaker.
8. Destain the gel in a destaining solution a few times until protein bands are visualised.
9. Approximately determine the molecular weight of the visualised protein bands by comparing them with the molecular weight markers.

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