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From Bangalore to Boston-IISc Igem 2016

in SciWorld by

Many articles have already been written about research and the intricacies of a life in it. Most of them, though, are from the perspective of graduate students; written by graduate students themselves, or people reminiscent of their time in graduate school. This is probably because that is when a person generally evolves from a student into a researcher, and it is during this transition that the properties of life in research become more stark. The entire point of the IISc undergraduates participating in the iGEM competition was to shift this frame slightly to the left in the timeline. All of us were under the impression that the researcher in us, though far from being developed, has already started developing. iGEM, to us, was a path to verify this rather ambitious speculation of ours.

The IISC IGEM TEAM 2016 in BOSTON lead by Arunavo

iGEM stands for International Genetically Engineered Machines (I never really understood why the ‘i’ is in lowercase, but in defense of the ‘i’, ‘I’ never really ventured to determine the reason). As the name suggests, it is an international synthetic biology competition, mostly for undergraduates, though some high-school and over-graduate teams also participate in their respective categories. It started out as an effort from MIT to standardize plasmid backbones. Most people who do wet-lab work regularly in biology, would have faced this problem — “I have my gene of interest in plasmid A, and I want to put it downstream of some other gene in plasmid B. I need restriction enzymes m, n, o, and p for this relatively simple exercise, but there is no enzyme ‘q’ in the lab! Now all I have to do is get a quotation for it from the vendors, wait for about 6729 years for it to arrive while I finish a few more seasons of my favorite TV series, and lament after 20867 years how excruciatingly long a PhD in Experimental Biology takes to complete.” The next time a similar experiment needs to be performed, suddenly enzyme z is out of stock and the same sequence of events repeats. Instead, if most plasmids come with cut sites of a specified (and very small) set of restriction enzymes, having those in stock will be sufficient to do most research work. iGEM started an attempt to make a library of plasmids with specific cut sites (which, certainly, should not be present in your gene insert), and develop/validate this library either by using or improving the pre-existing plasmids (known as BioBricks), or making a new plasmid that fits the library criteria. Possibly the reason for the target participants being undergraduates, was because a failure for them is much less costly (with no PhD thesis at stake!), and because of this, they often tend to explore improbable regions, with hidden answers.

ABHIJEET KRISHNA in one of his moods

Like most student initiatives, the idea of forming an iGEM team came to us one day, over junk food, late at night, in our first semester at IISc. Arunavo, being a YouTube-phile, had come across a video of the formation of the iGEM team of King’s College, London, and suddenly, we thought, “Why can’t we have an iGEM Team?”. The next day, we went and talked to our instructors, who were pretty interested at the prospect, and took an active interest in discovering what the competition was and why it is worth participating in. But, like most sudden peaks of academic excitement and interest that originate in one’s hostel room, it died away soon. Thankfully, our instructor (Dr. Narmada Khare) had not forgotten about the conversation we had, and one day, while conversing with a PhD student at NCBS (National Centre for Biological Sciences, Bangalore), she came to know that this PhD student, known to her colleagues as Chaitra Prabhakara, was a member of the IIT-Madras iGEM team in her undergraduate days. Both Narmada and Chaitra considered the formation of an IISc iGEM Team a possibility, and Chaitra came and gave us a talk about her experience with the competition. That was the activation energy we needed, and a team suddenly formed out of thin air, comprising of 21 students (of course the number is not exact; all I can say is that it is of the same order of magnitude as the original number!). Anyone with any interest in biology was willing to help.

Aiswarya-You can tell she is the no nonsense person in the group

As many would have noticed in their personal life, or in the dynamics of academia around them, in the initial days after formation, the number of members in an undergraduate team follows a roughly exponential decay, until it hits a threshold number. Interestingly this threshold is often a number which is practically the minimum requirement of calling a collective a team. It finally settled down to the four of us — Abhijeet, Arunavo, Shreyas and I (Prabaha). Actually, at a point of time, the team was practically two people — Arunavo and Abhijeet, because Shreyas and I were busy discovering our startling incompetence at even starting to solve an assignment anytime before ‘the-night-before-the-deadline’. Now, since both of us were taking a lot of courses, once we solved an assignment at the last minute, the next day was always the last minute for some other assignment/test. Eventually, this oscillation in the number of members settled down, and the four of us consented in each other’s calling ourselves part of the IISc iGEM Team, which was still to be recognized by the authority – the Undergraduate Department.

Sreyas making the most of the Halloween

The next step was the rate determining step — convincing the authority of the existence and legitimacy of the first team from our college. No one had any problem with undergraduates trying out original research; after all, the entire objective of the UG program was to promote exactly this! But, the problem is, there exist factors like intellectual property rights, and many more words, whose exact definitions still elude me, which noobs like us had not exactly accounted for (to be truthful, we had, but iGEM was open source — a philosophy we wanted to be a part of, but the authority was still getting used to). Also, $!

The team and the mentorDr. Srinath T

After multiple discussions, Prof. Umesh Varshney, the UG Dean at that time told us one of the most encouraging things we had heard, “You people take care of the science, and we adults will handle the bureaucracy”. What more does an undergraduate need? We had spent almost one and a half years, discussing among ourselves, and getting our ideas validated (generally rejected) by Chaitra, Sachit (another PhD student from NCBS) and our biology instructors in finally coming up with an original idea that was interesting, and could be verified in the time window (roughly the summer time). But research needs funds, and though the UG department had funds, it was allocated for specific purposes. We discovered a competition called iBEC that was being organized by DBT (Department of Biotechnology) for the very first time that year, for Indian iGEM teams. The teams had to submit a grant proposal, and a select few teams would receive financial aid up to an amount of Rs. 10,00,000. We got the complete Rs. 10,00,000 that we had asked for. The UG department loaned us the registration fee (~Rs. 4,00,000), because the iBEC results were to be announced after the deadline to register for iGEM, and thus the IISc iGEM Team was officially established, lead by Prof. Deepak Saini from the MRDG department (Molecular Reproduction, Development, and Genetics) and Prof. Umesh Varshney, the contemporary UG Dean. We even took in 3 of our juniors — Aiswarya, Aneesh, and Ayan, so that next year’s team got a head start.

 

Then began a stream of failed experiments, with one or two successful ones in between, and the not-very-uncommon waiting periods for enzymes to get delivered (I know that the entire point of iGEM was to avoid this very thing, but one or two of the iGEM prescribed restriction enzymes were not the most commonly used ones in our labs). But that is all known to anyone who has done science; most people reading this article can teach advanced courses on failed experiments, and I am just a beginner. The challenging hurdles we faced, other than the scientific ones, were the non-scientific ones. I won’t be surprised if someone now shouts, “10 points from Ravenclaw for stating the obvious”, but what one needs to understand is that we had no idea about how to solve the non-scientific ones. The registration fee ensures the recognition of the existence of the team, and the shipment of a collection of pre-existing BioBricks; it does not cover for anything else! When the team goes to present at the Giant Jamboree (the event at which the teams present their work) another registration fee for each team member had to be paid, and that, again, covered nothing else. We had to arrange for our own lodging, food, and travel. We needed to raise more money for that, the amount promised by DBT was not even close to sufficient!

Thus, in addition to spending sleepless nights in the UG bio labs doing experiments, we started thinking about how to get more money. Our professors promised to manage the money, and even help us get it, but we had to come up with the sources. We jotted down the list of funding agencies that help startups, successful startups that might be interested in our work, people who might help us point towards a source of money, and started emailing any and everyone. One day I was talking to Kuldeep from MBU (Molecular Biophysics Unit), a PhD student I worked with in my first summer, lamenting about the soup we were in, and he told me about the existence of a Facebook group called Career Support Group (CSG), comprising largely of IISc students and alumni. I thought that this group might help stranded IISc students going abroad get a place to stay. It took me some time to discover that the pre-stated description of CSG was as complete as calling a university a place with classrooms.

Imagination has no bounds

The first post I made on the Facebook page was requesting the members to allow the team members to stay at their apartment(s) for the duration of their stay. The response was overwhelming! Many people offered us a place at their apartment, but it was not limited to that. Dr. Selvaraj Nataraja from the group proposed that we try crowd funding, so that we could book an apartment for the entire team, and gave us a seeding amount of $300. Dr. Ananda Ghosh, the founder of the group contacted us and advised us to campaign in the group — regularly post about who we are, what we are trying to do, about the program we are a part of, about the Jamboree and the associated opportunities and exposure, etc. In addition to learning to do science, we started learning people skills, and the methods of pitching and funding one’s research endeavors. Finally, after campaigning, we asked the CSG members to help us in organizing the fundraising, and Dr. Kushagra Bansal (an IISc alumnus, and currently a Postdoc at Harvard) took up the responsibility of collecting the money for us. Once again CSG surprised us with its response and within a week, we had raised enough money to book an apartment for the duration of our stay.

In the meantime, we were trying other avenues to get funding for our travel and sustenance, and we managed to convince the IT, BT and S & T department of the Karnataka Government that our project was worth funding and got a grant worth Rs. 6,00,000 (toward consumables) from them. Also, we had applied for the Indian Alliance (Wellcome-DBT) travel grant, which is generally meant for PhD students, but surprisingly, we got that grant too! IISc-AANA (Alumni Association of North America) also agreed to make payments for the rest of the predicted amount of money required. Finally, we had all the money we needed. All that was left was the science.

If one tries to track the evolution of the answer to the question “What exactly are we trying to show?” in a project, often, the trajectory takes massive downward leaps along the axis of ambition. Our case was not much different. Our idea made sense, the genetic circuits made sense, the two modules we made also made sense, and so did the expected crosstalk between them. The only thing that did not make sense was the amount of time all the experiments we designed were taking to perform, and the astounding number of parameters that cause failures and delay work by days — reasons like “The liquid nitrogen we brought this morning has evaporated” or “The glycerol in the polymerase caused the entire PCR mix to precipitate (it took us a good part of a month to discover that that was what was going wrong with our PCRs; also, some wells in the PCR machine were not working!)”. Finally, when the week before our date of travel arrived, we had got both the modules to work independently, but did not have time to show the effects of combining both of them in a cell. We decided to present what we had done, and that in itself involved new scientific work, but were dissatisfied at the fact that we did not quite live up to our soaring ambition.

The Award

Finally, Abhijeet, Arunavo, Aiswarya, Srinath (instructor), Shreyas and I headed towards USA to present the work of IISc-UGs to the world. All of us were so relieved at the last minute completion of experimental work and documentation, it did not occur to us that we should be feeling excited about where we were going and what we represented.

None of us had ever visited Boston, but we did not feel any degree of alienation when we reached, on account of Dr. Kushagra Bansal and Dr. Gajendra Dwivedi (another CSG member in Boston) picking us up from the airport. That night, all of us enjoyed the placid, dreamless sleep brought on by exhaustion from the journey. The next day, all of team but I explored Jamaica Plain while I went to meet a professor I had mailed about the coming summer. The meeting was fruitful, the trip not only enabled us to present our work but also got me a funded project for the next summer at Harvard. On that same day, Dr. Selvaraja took us all to dinner; at this point, Boston was almost a home away from home. It was after the dinner that we realized that we had a significant portion of our presentation to complete, thanks to some experimental success right before our departure. In addition to the content, the design of the presentation was crucial; a brief look up of past years’ winning presentation revealed style was almost as important as content and delivery. And thus began a highly awkward yet effective process of outsourcing the design to India; a couple of our peers — Prokash and Sai had volunteered to draw the figures and design the layout of the presentation respectively. Another realization struck us on the night of the dinner – the poster needed significant revision; it has too much text and too few illustrations. We sat through most of that night annotating the pdf version of the poster with the changes and mailed it to Sai to implement. We needed the poster on the very next day!

We made the poster deadline (thanks to a nearby Kinko’s and the design team’s efficiency) and rehearsed the presentation in a room set up very similar to the way the presentation hall would be. We realized some visibility problems (some of the text was too small) and headed back home. Further changes were mailed to the exhausted design duo and we continued with a flurry of rehearsals during the next day and a half that finally culminated in our actual presentation. It went well, except for some unrehearsed time allotments to the different presenters. A wave of relief washed over all of us. Only 2 more days of poster presentations and we would know the result of our year and a half of blood, sweat and tears. We ended up winning a Bronze medal in the competition (not to be mistaken as the third prize; many gold, silver and bronze medals are given out, somewhat like the Olympiads). We had also contributed to something called the ‘InterLab Studies’ where volunteering iGEM teams across the world perform a particular prescribed experimental protocol, and produce robust statistically significant data. The theme for 2016 was standardizing fluorescence quantification. When we attended the InterLab committee’s presentation, we heard some ‘IISc iGEM Team’ had suggested a correction to the experimental protocol, that had reduced the error margins by 100x. “Must be a team of astoundingly brilliant students”, we thought.

We also spent a significant amount of our time watching other teams present. A trend we observed about the teams whose work was extremely elaborate (a large fraction of these teams eventually went on to win lots of awards) was the significant role of graduate students and Postdocs – some of these teams had up to 20 such researchers acting as mentors in addition to the PIs. And suddenly it hit us — a group of more experienced grad students, along with us charged-up UGs, could have hastened the lengthy process of troubleshooting our protocols, saving us precious time. Research requires both innovation and experience and the latter we lacked.

Overall, the experience was extremely valuable, not only in enabling IISc to put out better iGEM teams in the years to come but also in providing us undergrads a taste of all aspects of research, not limited to the science of it.

 

 

 

 

 

 

Prabaha Gangopadhyay is an undergraduate from Indian Institute of Science (IISc), Bangalore, India. Year: 3rd, Major: Biology This is what he has to say “I like learning about mechanisms underlying the existence of life. I find myself comfortable in the overlap of theoretical and experimental biology, because of the extreme interdisciplinary nature of the area, and being at IISc has allowed me to explore it. I am interested in doing my final year project, and eventually my PhD, in Neuroscience. Other than science, I love literature, classical music, and, like any other undergrad, food!”

Battle of Wisdom: CRISPR-CAS9

in Sci-IP/SciBiz/SciWorld by
Editor’s Note: Gene editing for a better (or worse) is coming to a store near you. Some of you may have followed the ongoing patent war on the ownership of CRISPR-Cas9 technology between University of California (Berkeley) and Broad Institute (MIT-Harvard). But there could be many who are wondering what is the fuss all about? At the Career Support Group (CSG) for STEM PhDs we might still continue the debate about CSG’s usefulness to biologists vs non-biologists, but as inventors we are always in unison about perfecting the art of claiming ownership. #ClubSciWri is always attempting to listen and respond to your expectations and we are pleased to present the “Battle of Wisdom:CRISPR-Cas9” from Dileep Vengasseri. Dileep has nicely deciphered the meshwork underlying this matrix of claims to the CRISPR invention. We hope this story helps make sure that the next big thing from your gray matter secures your rightful ownership to the intellectual property.- Abhinav Dey

My dear friend, this 60 minutes of my time and 1597 words are for you! As you rightly said, maybe we should discuss our opinion(s) in public at least for educating others on what we have learned during the due course of our time.

Disclaimer: All what is written/expressed here are my personal opinions, and are not to be construed in any manner as a reflection/opinion of the firm that I am associated with. My words are solely my words! I will try to be as generic as possible to ensure there is absolutely no conflicts of any interest. This is purely a personal blog, written within the constitutional freedom that my Country has offered me when I was born here.

Many great battles are won not in the battle fileds, but in the minds of the battle leaders. What we read, saw, and talked about were the after-effects of those battles won or lost inside those great minds. In the great epic Mahabharata, Arjuna was about to lose Kurukshetra battle even before it was fought. But, there was a Krishna to save him from that humiliation. Many may not be as lucky as Arjuna was.

Before I begin, with all due respect, let me remind all of us one trivia very clear. US is not the “World” … it is just one of the many countries [a privileged one, indeed] of this world.  A larger population residing outside that privileged country, do not play a “World Cup” between their states or clubs. They don’t re-spell a word to make it look like they have invented it. For them, the metal “Al” is still aluminium and not aluminum.

We, living at the periphery of the world of modern(?) science, have got enough fuel from CRISPR-Cas, the game-changing method of gene editing, to satisfy our ego of being a part of a ‘privileged community’ who understands (?) the words like ‘gene editing’ and ‘CRISPR-Cas’.  For all such ‘privileged souls’, the “IP Battle of CRISPR-Cas” is more than just another battle. Let me call it a “Battle of Wisdom”.

But, was this battle worth fighting?

Let me begin with disecting this IP battle to four main sections: (1) Technology (2) The Battle Field (3) The Win and (4) The Strategy. May be, in future, I can complete this article with “Lessons Learnt”.

  1. Technology: At least from what is publically available, we know that Doudna/Charpentier’s team made that beautiful gene editng system work in-vitro in prokaryotic cells, in a neater, simpler manner than what it was in the nature itself. Instead of using a 3-component system including tracrRNA, crRNA and Cas9, her team beautifully designed a 2-component system, including a key synthetic, single guided RNA (sgRNA), that effectively performed site specific genome editing along with Cas9 (It is interesting to note that in-vitro 3-component system is also IP protected!). What was the big deal? The big deal was its simplicity, efficiency, and marketability. It was not that gene editing methodologies never existed before… however, now the World has access to an elegant gene editing system that is much more easy to perform (no more protein engineering!) & predictable. We also know that Feng Zhang (don’t forget George Church’s back-to-back publication in Science along with Feng Zhang) made it work in the eukaryotic system.
  2. The Battle Field: No one (at least the majority of money makers) wants a gene editing system that works only in prokaryotic systems. So, the “Battle of Wisdom” eventually boiled down to the IP on gene editing in eukaryotic system with CRISPR-Cas. Duodna filed a US patent application (remember, US is not the World, more so when it comes to IP protection) first and Feng Zhang got the first granted patent in US (note that the USPTO could have  provoked an interference at that time itself, but they didn’t!). Feng Zhang’s patent ‘claims’ to ‘cover’ eukaryotic CRISPR/Cas gene editing system (no comments on its “claims” and/or its “coverage” as the battle is still on…at least let the battle be fought under the belief that the land that is going to be conquered is still fertile!).  Duodna had anyway made it easier for Feng Zhang to get his patent granted by ‘boasting about’ her team’s achievement in multiple forums and explaning ‘how difficult it is/was to make it work in a eukaryotic system’.   Alas! enough of such wisdom on eukaryotic system was passed on to that Patent Attorney who filed her provisional applications, at least before the one on 19th October 2012 that is prior to the Feng Zhang’s priority date of 12th December 2012. Now, the battle of wisdom (what we call as “Interference Proceedings”) is to establish who invented (i.e., conceived and/or reduced-to-practice) the “eukaryotic CRISPR-Cas” first. Duodna will be fighting to make a point that porting CRISPR to  eukaryotic system is just a non-inventive aspect. Feng Zhang is going to fight back at least on the ground that if it is that obvious why did it then take Doudna a good 6-9 months to achieve the same.   I refrain from making any comments on how long or short is 6-9 months in a field like Molecular Biology. I know that my dear friend, who forced me (as usual) to write this long article, has wandered in the wilderness of IISc campus behind an elusive protein for a good 6 years :-)). And, I must admit that I have made the entire story of this Battle of Wisdom to a deeply  abridged version as the facts of this case are much more than what this layman article can handle. But, I believe that this much background is good enough to make my “teaching moments” convincing.
  3. The Win: Does it matter who wins this battle? Of course, YES! All battles are known after the leader who has won it (Aravind Kejriwal and Hilary Clinton are no where near their counterparts, as of today). Generally, the winner get the privilege to write the history that we all can read and study. But, is this Battle of Wisdom the same as any other great battles fought, lost and won? No. What is required to win this battle? It is required to show that who has invented the “eukaryotic CRISPR-Cas” first; it is required to show what is inventive/not inventive in this field; and it is required to show what constitutes an adequate written description/enablement in this field so that the “public disclosure function” (spirit and letter of any patenting system in the world) of the patenting sytsem is intact.  But, as with any other battle, only one person can be the winner. But, what will they both win or lose? The loser will any way have a deep wound in ego that may take years to heal. But, will he/she lose everything? Need not be. It depends on what other IP portfolio or picket-fencing that he/she has done around this gene editing tool. For example, a good claim on the synthetic guide RNA, a good IP portoflio on a better Cas9 proteins,  a better method for transfecting the cell, or an alternative to Cas9 itself… all these can make or break a commercial deal.  Is the winner going to get everything? Need not be.  During this entire process, it might open a pandora box and a myriad of avenues to potentially invalidate the patent claims that the winner can take home, to limit its claim scope, to limit its application coverage etc.
  4. Strategy: Isn’t it important for everyone in the field of IP to realize that most often a “hand shake” may do more good than “a fight”.  Before taking the army to a battle, it is important to know if raisng a white flag will be more beneficial than a gruelling battle. It is important to understand for what one is fighting a battle.   Does anyone fight for satisfying an ego or to make a point?  It is imporant to  understand that in a patent battle field, a wiser does not fight from their heart, but from their mind!. It is  important for each of the fighting members to know “What will happen if we do not fight, but rather collaborate?”.  Both Doudna/Charpentier and Feng Zhang could have been still partners in Editas, and they could have ruled the field.  When you fight in public, you expose yourself…you expose more than what you wanted to. And, what you have exposed can kill you even if you win YOUR fight.

Three more points to ponder:

  1. IP protection of PCR technology made Roche the king of DNA amplificaiton for quite sometime. Why? It is true that PCR was a technology that literally transformed the world of Biotechnology. But, was the IP protection on PCR probes for important pathogens less important? Were Taqman probes for real time PCR less important? Were the chips that made thermal cycling easier less important? No. All of them “together” made PCR a “cult” technology. That’s what a strategy means.
  2. IP protection in the field of ESC took Thompson and Wisconsin Alumni Research Foundation (WARF) to the center of the scientific world. Many IP/Tech Transfer cells in the Universities across the world wanted to be like WARF. As far I know, WARF gave its rights for free to any academic instituties, but made any industry pay for the same. Great! What were the other things that were needed to sustain and progress that technology ? An environment that morally support ESC research, a completely synthetic media to grow ESC, a culture that is devoid of mouse fibroblasts … all these were essential for taking ESC to reach its maximum potential. In modern day science, it is unlikely that we will see a winner of a single battle emerging as the “real winner”. A real winner is going to be the one who knows the game and strategize accordingly.
  3. US is not the “World”, and IP rights are jurisdictional. So, make yourself open to strategize for the real world!

Another Disclaimer: While starting my blog in WordPress, I had promised that I will not proof-read what I have written. In the past, many times, I had become a victim of my perfectionism and my writings had never seen the light. So, please pardon any typographical, grammatical, or otherwise errors. I hope factual errors are not there. Please let me know if you find any errors so that I can correct the same.

Authored by

 

Dr Dileep Vangasseri, PhD (Indian Institute of Science, IISc); Post-Doctoral research, University of Pittsburgh; Senior IP Professional, John F. Welch Technology Center, GE India Technology Center Pvt. Ltd., GE Global Research, Bangalore, India). Dileep has over ten years of in-house IP experience in Life Sciences, Healthcare and Medical Diagnostics industry after eight years of academic research experience in Bio-Organic Chemistry, Gene Therapy and Cancer Immunotherapy. He is well versed in all facets of patent analytics, techno-competitive intelligence, technology forecasting and business development.

This blog was originally posted here on December 7 (2016).

Featured image source: Pixabay

Creative Commons License
This work by ClubSciWri is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

The question: Burning excess calories post exercise

in SciWorld/That Makes Sense by

Combining resistance and endurance exercises potentiates fat loss and muscle hypertrophy

You don’t burn calories while working out alone, body continues to burn calories even after the cessation of the workout. It was attributed to excess post-exercise oxygen consumption (EPOC), which remains high after aerobic exercise as well as anaerobic exercise. In addition, lactic acid produced, during strenuous exercise, in muscle cells has to be diverted/oxidized back to other metabolites, which might also contribute to the excess calorie consumption after the workout. These 2 hypotheses however could not completely explain burning of more calories after exercise.

pic-1

The science behind

Researchers at Harvard University detailed the science behind these hypotheses. They found that endurance induces a hormone which converts white adipose tissue (tissue which stores fat) into brown adipose tissue (tissue which burns fat). Irisin is the hormone produced upon endurance exercise in mice and human subjects which regulates this process. Irisin has been in the news ever since as an exercise hormone. In another study, by the same group, they found the scientific reason why resistance exercise induces muscle hypertrophy. When human subjects performed resistance exercises such as leg press, chest press etc., Insulin like growth factor (a hallmark protein for muscle hypertrophy) production was enhanced.

Interestingly, both the endurance and resistance exercise benefits were under the control of a master protein called PGC1 α. This protein is differentially produced in the body according the nature of the exercise performed. If endurance exercise is performed it produces the beneficial effects of burning fat; if resistance exercise is done muscle hypertrophy results.

PGC1 α is very important protein, a person’s athletic performance is determined in part by it. Genetic mutations in this protein affect athletic performance of the individual.

Kill two birds with one stone: resistance and endurance exercise

It was also reported that PGC1-α is induced at a higher level when resistance (anaerobic) exercise is performed after endurance (aerobic) exercise, which is called concurrent training. Combining both exercises, thus, will have a synergistic effect on overall health.

The future

There has been no golden rule for how much workout has to be done for achieving desirable health benefits- either fat loss or muscle gain. It could be possible, in future, that amount of production might be used as readout for endurance or resistance exercise for each individual. Proper exercise regime and nutritious diet could help maintain general wellbeing and attain dream physique.

 

 

About the Author:

srinvas

 

Srinivas Aluri is postdoc at Albert Einstein College, NY. He is a fitness enthusiast, exercise and diet expert. He is also an international sports science association certified fitness trainer as well as American Heart Association’s CPR/AED certified professional.

P.S: This article was blogged at an untraceable place. It’s been edited and published here.

 

Photo source: builtlean.com and Pixabay

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This work by ClubSciWri is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

My story on how I got my H1B

in SciWorld by

Hello everyone, I would like to share my experience towards getting the H1B visa. I’m happy to let you know that I am joining the University of Michigan, Ann Arbor, as a postdoctoral fellow. I am very grateful to CSG, as early discussions on this forum encouraged me to begin applications even before thesis submission. My PI offered to do an expedited processing of the H1B visa, and I was also assigned an immigration lawyer for the same.

I chose Mumbai as the city of choice for my visa interview, but there were no dates for H1B available until Feb 2017!! (They have now increased availability, but from July-Oct it was tough to get dates within 3 months for this visa category). So I did the biometrics at the VAC in Mumbai, and had my consular interview at Kolkata,as that was the only place where dates were available. I’m sure many of you know this already, but just for the benefit of the minority (I was one of them) who don’t, it is perfectly fine to take visa appointments anywhere in India, as long as you mention the same city on your DS-160. In case you have already submitted the DS-160 and then see no dates available in your city of choice, or if you need to correct and/or update information, you can select the “Retrieve an Application” option on the site where you filled the form, enter your previous visa application ID, and then select “Create a New Application”. Your personal information will then populate into the new application. In the new application, update the details and proceed further. Applicants who have completed a new DS-160 after scheduling an appointment are required to carry both the old and new DS-160 confirmation pages to their Visa Application Center appointment for biometrics. 

At the Kolkata consulate, I was given a few minutes to explain my current research in IISc and future research in USA, and I was told that further administrative processing would be needed before they could grant me the visa. They kept my passport, and handed me a form which stated that my application was pending under section 221G, and I needed to email them a detailed word document describing my current research as well as proposed research in USA and its practical applications, following which I would get my visa in 2-4 weeks. Two other postdoctoral candidates at the consulate were also given the same form, and this is pretty common for researchers working in fields belonging to the ‘Technology Alert List’ that includes biomedical research, nuclear physics, chemical engineering among others. After a very tense and frustrating wait where I kept imagining the worst case scenario (that my visa would get rejected), I finally got my visa after 3 weeks 🙂

Take home message – Visa processing takes time, but it is rarely rejected.

 

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Awanti has done her PhD in Systems Biology at the Indian institute of Science,Bangalore, and will be joining the University of Michigan for postdoctoral research. She identifies as a compulsive chatterbox

Of clothes, clocks and lice

in SciWorld by

We humans are the only species who wear clothes. And, it is obvious that we are obsessed with clothes – about the designing, making and procuring of clothes and materials that are used to make clothes. As the ‘Page 3’ would testify, we are fascinated about who wears what, and also who did not wear what!!

But, how did it all come about? What were the first ‘baby’ steps? When did we start wearing clothes?

The trouble with this kind of investigation is the paucity of ‘hard’ evidence. Clothes, unlike bones, do not fossilize, and unlike stone and metal, they perish fast. Thus, except under special environmental conditions in which some paleo-humans (such as iceman Ӧtzi) have been unearthed, the direct evidence of prehistoric clothing is scanty and so the origins of clothes have been lost in the mists of Time.

But, as always, there is evidence – it is only about properly looking for it. In this case, the evidence lies in the well-known pest – the human louse.

THE PEST FAMILY

Almost all mammalian and avian species are host to various species of lice. But, humans are among the few species that are host to, not one, but 3 species (or subspecies) of lice! The human head louse (Pediculus humanus capitis), the body louse (P. humanus corporis, also considered P. humanus humanus) and the pubic louse (Pthirus pubis) are obligate ectoparasites (Figure 1) to the human body and cannot survive on other species, including pets. Head louse are slightly smaller in size than body louse and usually have a darker pigmentation. There are subtle differences in the lengths and widths of the antennae and the front legs. But, not surprisingly, the head louse and body louse have considerable morphological similarity. Their main difference lies in the choice of habitat.

fig-1

The head louse is a blood-sucking insect that lives only on the head scalp and lays eggs only on scalp hair. The body louse, in contrast, doesn’t venture towards the head. It feeds from the skin and notably, it lives and lays eggs in human clothing. The head louse and the body louse are fastidious about their habitats – neither encroaches into the other’s ‘territory’ (in fact, the head louse cannot live on clothes). And, neither species can survive away from a human host for long – the head louse perishes within 24 hours, while the body louse (which reside on clothing) can live without human contact for about a week. Noted biologist Mark Stoneking (Figure 2) – who had already made a name for himself by studies on the ‘mitochondrial Eve’ – got interested to study the migration of these obligate parasites across the globe hoping that would lead to insights about human migrations. Stoneking hypothesized that the head louse was the ancestral species and body louse have evolved from head louse only when a new ecological niche got available – in the folds and creases of human clothes. When did this happen? – the most likely answer is when humans started regular wearing of clothes. This leads to the intriguing possibility that finding out the time when the body louse evolved from head louse would (by inference) correspond to the beginning of extensive use of clothing by ancestral human populations. The answer, as was elegantly shown, lay in using a molecular clock approach to calculate the origin of body louse.

 

fig-2

BOX: What is a molecular clock?

The molecular clock is a method to determine when 2 species/ sequences diverged from a common ancestor. It is based on the principle that, as time passes, random errors/substitutions take place during DNA replication and get transmitted down the generations. More the time since the 2 sequences diverged greater the number of differences between them (as substitutions happen independently). Thus, if the ‘number of substitutions per million years’ is known, it is possible to estimate how many years have passed since the 2 sequences had a common ancestor. This calibration can be done by knowing the number of substitutions that have accumulated in 2 DNA sequences whose divergence-time is actually well-established from other lines of evidence, for eg, fossil record. Assuming the error rate stays constant across time and in different species, this allows to calculate the unknown time points (Figure 3).

fig-3

THE GENETIC TALES OF THE LICE…

The molecular clock for dating the evolution of lice was built by using 2 mitochondrial DNA and 2 nuclear DNA segments. To avoid any bias in their investigation, the scientists collected lice from 12 geographical regions – Ethiopia, Panama, Germany, Philippines, Iran, Ecuador, Laos, Papua New Guinea, Florida (USA), Taiwan, Nepal and the United Kingdom – and extracted nuclear and mitochondrial DNA. They also collected DNA from chimpanzee head louse. Since it is well known that hosts and their parasites often co-evolve, it was assumed that the chimpanzee louse (Pediculus schaeffi) and P. humanus must have co-speciated with their respective hosts, and this must have happened at around 5.5. MYA – the scientifically-established period when humans and chimps diverged. Thus, the differences between DNA sequences of chimp louse and human head louse must have accumulated over 5.5 million years. Using this specific time period as a calibration point for the clock, the time when head louse and body louse diverged could be estimated.

For the sequence analysis, Stoneking’s group first analysed segments of the genes ND4 and CYTB present in mitochondrial DNA of the louse. They followed it up with comparative sequence analysis of 2 bits of nuclear sequence – from the important genes of elongation factor EF-1α and RNA polymerase II subunit RPII. The size of fragments ranged between 400-600bp. The results obtained were fascinating to say the least.

The first result (Table 1) showed that genetic diversity of the African louse (although collected only from Ethiopia) is significantly greater than the global samples of non-African louse. The finding mirrored the greater genetic diversity of humans seen in Africa compared to that in other continents. Since greater genetic diversity almost invariably occurs at the source, the results indicate, as in the case of humans, the African origin of the human louse.

Table 1: Comparing genetic diversity of African versus Non-African lice (adapted from data present in Kittler et al (2003))

Genetic Diversity

African louse Non-African louse
mtDNA 3.31 1.76
EF-1α 0.29 0.10
RPII 0.94 0.56

 

The next set of results (Table 2) similarly showed that human head louse was far more genetically diverse compared to its cousin, the body louse – proving that the head louse was the ancestral species.

Table 2: Comparing genetic diversity of Head louse and Body louse (adapted from data present in Kittler et al (2003))

Genetic diversity

Head louse Body louse
mtDNA 3.42 0.19
EF-1α 0.23 0.18
RPII 0.93 0.61

 

(BOX: A few definitions: Phylogenetic tree: a tree-like diagrammatic representation that describes the evolutionary relationships between the organisms/sequences being studied (Figure 4).

fig-4

Monophyletic sequences: Two or more DNA sequences that have evolved from a common ancestral DNA sequence.

Clade: A group of monophyletic sequences that consists of all the sequences included in the analysis that are descended from the ancestral sequence at the root of the clade.

Outgroup: a homologous sequence that has originated from a common ancestor as the sequences under investigation, but is not as closely related to the being-studied sequences as they are to each other. In this case, the DNA from chimp louse serves as an outgroup and helps to locate the root of the tree)

 

THE FINGERPRINTS ON THE TREE…

Next, a phylogenetic tree was constructed using all these mitochondrial sequences of human lice (Figure 5). The tree showed presence of number of clades. The deepest clades contained only head louse sequences, confirming that body louse had originated from head louse. Notably, one particular clade contained all body louse and 16 head louse sequences and included samples from all over the world. The molecular clock, calculated using the sequences from the chimp louse as an outgroup, showed that this clade is 72000 +/- 42000 years old. Since it contained all body louse sequences, the estimated age of this clade has to be the upper limit for the time since body louse originated. And, since body louse exclusively inhabits human clothing, this must be the time period when modern humans started regular use of clothes.

fig-5

Very similar results were obtained from studying the nuclear sequences, in spite of the fact that DNA recombination can make such analysis difficult compared to that for mitochondrial DNA. As a final piece of evidence, Stoneking’s team also analysed parts of the Cytochrome oxidase (COX) gene, also present in mitochondrial DNA and showed that the results were in agreement with that obtained from ND4-CYTB i.e. anatomically modern humans, residents of Africa, started wearing clothes around 70,000 years ago.

In a latter study, David Reed’s group from the University of Florida carried out a more robust analysis using a Multilocus Bayesian isolation-with-migration coalescent method and concluded that body louse had diverged around 170,000 years ago, and certainly not after 83,000 years ago. The difference between the two sets of data is not surprising given the different methodologies used (moreover, Reed et al also used 18S ribosomal RNA from louse nuclear genome for their analysis), but they are in broad agreement. Importantly, both conclude that the body louse evolved in presence of anatomically modern humans in Africa because of the availability of a new ecological niche – clothes.

But, what if clothing originated much earlier, and louse colonized this ecological niche later? This is an intriguing possibility and cannot be discounted entirely. Stoneking et al believe that, since a new ecological niche is colonized fairly rapidly, it is unlikely that clothing could have existed for thousands of years before body louse occupied it. Indeed, the molecular data also corresponds well with the archaeological finding that the earliest eyed needles – the only prehistoric tools that can be definitely associated with clothing – are ~ 40000 years old, and they have been found only in settlements of modern humans and not archaic humans like Neanderthals.

TO SUMMARIZE,

The genetic and archaeological data converge on the conclusion that the chimp louse and the human head louse are close cousins who must have originated from a common ancestor. The human head louse got confined to one relatively small habitat (i. e. scalp) when ancestral humans lost significant amount of body hair/fur ~1.2 million years ago. However, sometime between 70,000-170,000 years ago, anatomically modern humans started stitching and wearing clothes and the lice could now colonize a new niche. Indeed, it is quite possible that clothing protected the modern humans against the vagaries of environment and allowed them to explore the world out of Africa ~50000-80000 years ago – a time period that has been validated by the latest studies. And, along with humans and their clothes, the human lice have spread across the globe.

 

POSTSCRIPT:

Not surprisingly, this is not the end of ‘louse research’. Lice found in 1000 year-old Peruvian mummies have subsequently given insights into how and when humans migrated to the New World…..just imagine the unearthed treasury if we could genetically score for lice present in the various populations of the Indian subcontinent.

 

ACKNOWLEDGMENTS:

Prof. Mark Stoneking; Eugene Dubois Foundation.

 

REFERENCES:

  1. Kittler, R., Kayser, M. and Stoneking, M. (2003) Molecular Evolution of Pediculus humanus and the Origin of Clothing. Biol. 13, 1414-1417.
  2. CARTA: Unique Features of Human Skin – Mark Stoneking: The Molecular Evolution of Human Lice https://www.youtube.com/watch?v=S9rl8o8cGZ8
  3. https://en.wikipedia.org/wiki/Louse
  4. https://en.wikipedia.org/wiki/Head_louse
  5. https://en.wikipedia.org/wiki/Body_louse
  6. Whitfield, J. (2003) Lice date first human clothes. http://www.nature.com/news/2003/030818/full/news030818-7.html
  7. Toups, MA. et al (2011) Origin of Clothing Lice Indicates Early Clothing Use by

Anatomically Modern Humans in Africa. Mol. Biol. Evol. 28, 29-32.

  1. Choi, CQ (2011) Humans Got Lice When We Clothed Our Naked, Hairless Bodies. http://www.livescience.com/9225-humans-lice-clothed-naked-hairless-bodies.html
  2. Choi, CQ (2008) Lice Shed Light on Ancient History of Americas. http://www.livescience.com/2291-lice-shed-light-ancient-history-americas.html
  3. https://en.wikipedia.org/wiki/Ӧtzi#Clothes_and_shoes
  4. Venkateswaran, TV (2011) Clothing is distinctly human. But how old is it? http://beta.bodhicommons.org/article/clothing-is-distinctly-human-but-how-old-is-it
  5. Eugene Dubois Foundation, Eijsden, The Netherlands http://www.eugenedubois.org/en/lecture-by-mark-stoneking-archaic-genomes-and-insights-into-human-evolution/
  6. Zimmer, C. A Single Migration From Africa Populated the World, Studies Find. New York Times, Sept 21, 2016. http://www.nytimes.com/2016/09/22/science/ancient-dna-human-history.html?_r=1

Featured Image source: http://imgur.com/gallery/2ELegP1

Author Profile:

for sciwri

Anirban Mitra, Ph.D

Anirban Mitra did his PhD from the Department of Microbiology and Cell Biology, Indian Institute of Science (IISc), Bengaluru and is now a teacher of biology, based in Kolkata. His interests range from biological evolution to history of science and facets of India’s past.

 

 

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From writing to reviewing…. my story, my experience

in SciWorld/That Makes Sense by

bh_doskcmaeh_bm

 

Image source:“Piled Higher and Deeper” by Jorge Cham www.phdcomics.com

How to write a scientific article for a journal? Does writing require training? Does the training come with experience or does one take courses for that? How does one write it so that lucidity is maintained and the scientific message is still conveyed straight across? How much does writing PhD thesis help beforehand?

 

As any other beginner I had the same questions with my first-ever scientific article as a 2 years old PhD student at the Max Planck Institute for Biology of Aging, Cologne, Germany. I was still figuring out best ways to interpret results, optimize protocols, and busy learning new things, when I was invited by my boss to participate in writing a review. On the one hand, I was overwhelmed with joy at the opportunity to face this new challenge. On the other hand, I was confronting the pressure to prove my writing skills not only to my ambitious and detail-oriented supervisor but also to my ambitious self. The task was made easy by two facts. First, it was an invited review from the journal, and was supposed to be short. Second, my highly organized supervisor divided the review into two equal parts and allowed me the option to choose mine. I chose the introduction and the first topic. She was responsible for the last two topics. Since I was naïve I decided to follow directions. I was first directed to read up past and current literature regarding my parts and draft a very rough plan on what I was going to write and whom I was going to cite (with proper justifications regarding the latter). I had strict time limits and needed to fulfill this task within two days. This strict deadline helped me to be structured and to streamline my reading. After getting a green signal on the rough draft, I was given a week to hand over the final draft. I had to make sure I read most of the articles I cited, followed a scientific diction of that from leading journals, and that it made a smooth reading. It felt like a herculean task as a beginner and required utilizing all my scientific fervor and burning my night oils. First of all, I read up reviews written by my supervisor, just to have an idea of her writing so that the review doesn’t read completely foreign when both the parts are combined as a final article. I made sure that I stayed close to the topic and included past and current knowledge as well as discussed contradictions wherever applicable. Of course flair for writing and a bit of talent was necessary to fulfill the task in that short time. Her honest feedback and constructive criticisms were very beneficial, and it was a huge relief to hear that it was well written. After combining both the parts, we still went through several editing sessions, which is part of the ball game anyway. Finally, it was accepted and published, without a single correction or suggestion from the editor. That was a big exercise!! Looking back at that article I always feel a sense of accomplishment, as it smells the sweet fragrance of my hard work and learning.

 

The next challenge was to write my first manuscript ever during the first few months of my Post Doc. This was now a different lab than my PhD lab. Two things helped again. First, by this time I had finished my PhD, hence by now I was a pro in literature survey, and second, I already went through the review writing and thesis writing. As anyone else would do, I started first with writing the results part and prepared the figures. Preparing figures with the adobe illustrator was a part of my PhD learning, hence it was easy now. Next, I wrote the easiest part, the materials and methods. The introduction was a bit challenging because I was pretty new to the topic and needed to read up past and current literature, but having written the review on a strict time schedule helped. The challenge was the discussion because I needed to discuss all past and present contradictions, conundrums and speculate possible mechanisms. Another point was the format. In certain journals, the discussion is written like one long story, putting the results in a bigger perspective. In some others, each result is discussed under a separate heading. Hence, I needed to first decide which journal we were going to communicate this manuscript to, in order to be able to choose one format over the other. Since ours was a specific topic, hence we narrowed down to a few journals dealing with this topic. Next, we chose the one that was closest to our interest in terms of peer review, audience spectrum and impact. I then wrote the discussion according to the norms of our chosen journal. In all, I took a good one month in preparing the entire manuscript for this journal. The manuscript writing was a fun learning, more so because my Post Doc supervisor gave me complete freedom to explore my writing skills. She edited and improved certain parts but thankfully the overall story was narrated in my style and therefore I feel a strong ownership to this one. The story has just been accepted!

 

The most recent learning however has been the review of another manuscript. Many PIs request their senior PhD students or post docs to review a few articles to relieve some workload. I look at it as a symbiotic act because the post doc gets trained to be objective, improve his/her analytical skills and becomes even more critical without being biased and the PI saves his/her time. Since I had no experience or opportunity in reviewing during my PhD, I had once mentioned it as one of my interests to my Post Doc supervisor. One fine day, she gave me an article to review and I took this task happily to my stride. I was naïve, therefore, my boss advised me to judge the following points in the manuscript:

  1. If the abstract is making justice to the title.
  2. Is the introduction justified, too long or too short according to the journal norms?
  3. Are the experiments performed with proper controls, if statistics are sound, if the conclusions are drawn well or are there over interpretations?
  4. Are the techniques sufficient to answer the question or other ones could have been used?
  5. Have they discussed adequately.

I took into account all the above points. Since the manuscript was a small story and was quiet close to our field, it was easy to judge as a beginner. Ideally speaking, a Post Doc is not a beginner in reviewing anymore. By this time he/she has critically evaluated his/her own data, has discussed the data of peers during lab/departmental seminars and has attended numerous scientific talks from speakers around the world and hopefully participated in stimulating scientific discussions. It is only that one has not penned down the critical points in a structured manner. Hence, I began. It was not too difficult to find the strong points of the story as well as the deficiencies. First of all I wrote down what kind of controls or experiments were missing. Next I checked if the techniques they used were justified, if not, what else could have answered their question better. I spotted the over conclusions/under statements in their data as compared to the numbers on their graphs. And finally I assessed the contribution of their work in the field by doing a little bit of background check on the exact topic. I wrote down all the above points in a well-structured way and discussed with my boss, who also came up with her own judgment. Finally, we came to a unified conclusion and she gave the verdict.

Overall, it was a very unique experience to be on the other side of the coin, to be able to be unbiased and critical and to be able to judge as a peer.

 

So this was my small journey with my baby steps from writing a review to writing my first manuscript and to reviewing a small article. With these steps I not only came to realize my interests in scientific writing, but also that we all can learn new things and venture into the unknown if we are passionate, determined, and focused. And yes, every single small step counts! I hope that, through my tiny story, I could somehow inspire the beginners and the skeptical. So….what are you waiting for? Grab the opportunity or create one!

sushmita-headshot

About the author: Sushmita Ghatak completed her PhD from Max Planck Institute for Biology of Ageing (Cologne, Germany) in 2015. As a graduate student, she worked on the role of the ageing niche in regulating skin stem cell homeostasis. She is currently a postdoctoral fellow at Uniklinik (Cologne). She is exploring the domain of skin wound healing and fibrosis by studying the role of collagen binding integrin receptors in skin homeostasis.

 

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Ten years of sailing aboard a cockroach

in SciWorld by

The story of the cockroach milk protein started about a decade ago with the ‘curiosity of discovering’ and continues with the ‘curiosity of understanding and developing’. While observing these minute crystals inside the embryos of pregnant cockroach females, little did Nathan or Prof. Ramaswamy know that one day it will be popularly known for being the future protein supplement. During these years, while all the authors of the paper collaborated towards the understanding of the protein, we all marveled at the Biology of these cockroaches and the fascinating crystallographic features that the milk proteins exhibited.

cockroach-birth

Image: Diploptera punctata cockroach (source: livescience.com)

Generally, As an advantage for the organisms, the proteins are under negative natural selection pressure for crystallizing inside their cells. However, the knowledge of several proteins crystallizing inside an organism (in vivo, either in cellulo or ex cellulo) is also known. These proteins are proposed to be under positive selection pressure for crystallizing in vivo with functional importance. The crystals observed by us in the Pacific Beetle cockroaches, usually found near Hawaiian regions, are one of the examples of in vivo crystals. Cockroaches are known to be very sturdy organisms having survived for over 300 million years. During this period, one of the features that it has evolved for higher survival chances is its nature of reproduction. There are three types of reproduction found in the cockroaches; oviparous (eggs-laying), ovo-viviparous (fertilized eggs laid in maternal brood sac without her nourishment) and viviparous (fertilized eggs with maternal brood sac protection and nourishment). The pacific beetle cockroach, scientifically known as Diploptera punctata, is the only known viviparous cockroach till date which gives birth to the young ones like mammals and provides nourishment to the developing embryos. In-keeping with the nomenclature of “milk” used for the maternal nutrition in new-born of mammals, the nourishment provided to the embryos in these cockroaches is also termed the same. The brood sac of the pregnant females secretes this ‘cockroach milk’, which is taken up by the embryos. As the embryos continue to drink the milk, there is a surplus of the protein in their gut. This excess amount is stored inside the embryos’ gut in the form of crystals, which maintain equilibrium with the liquid milk in solution that is readily available to be ingested. Storage of food in the form of crystals allows for a high concentration of food to be stored as well as controlled release of nutrients as needed by the embryos.

Cockroach crystals

Image: From the research article jt5013, showing in vivo-grown Lili-Mip crystals from D. punctata. Polarized microscopy reveals birefringent protein crystals enclosed inside the embryo midgut and an enlarged view of the extracted crystals (inset).

In vivo crystallography is one of the new facets of Structural Biology that deals with structure determination of in vivo protein crystals. Apart from naturally occurring crystals, scientists have also engineered a baculovirus based system to induce in vivo crystal formation. Recent advancements in X-ray free electron lasers (XFEL) and serial femtosecond crystallography (SFX) have resulted in the increase of structures from in vivo crystals. One of the major challenges of in vivo crystallography has been the size of the in vivo-grown crystals that are limited by the volume of the cells that are usually of the micro-nanometer range. However, since these crystals were formed in the gut of the embryos, their sizes were not limited by cellular volumes and were comparatively larger. Therefore, single crystal X-ray diffraction was possible but obtaining the phases of the atoms was tricky. The structure was finally solved using sulfur single wavelength anomalous dispersion method. The protein is a lipocalin with β-barrel forming the lipid binding pocket and a single α-helix. Mass spectrometric and crystallographic studies revealed that each crystal was a heterogeneous mixture of not just multiple protein sequences, but also the sugars bound to these proteins in the form of glycosylation and the fatty acids bound at the binding pocket. More than three sequences of similar proteins (85-95% sequence identity) were found in these crystals. Additionally, multiple N-linked glycosylation sites (3-4 sites) with pauci-mannose and high-mannose structures and variable branching were observed. Further, the fatty acid bound at the pocket was found to be either an oleic acid or linoleic acid. With such an extent of heterogeneity, we were amazed to see that the crystals diffracted X-rays to atomic resolutions of 1.2-1.8 Å.

 

Generally, in macromolecular X-ray crystallography the rate-limiting step is the crystallization of proteins and obtaining good quality crystals. Usually, when we purify and crystallize recombinant proteins in vitro, we make several modifications such that the protein solutions are homogenous and monodisperse. This usually drives the protein away from its native state in which it naturally occurs. Further, proteins with post-translational modifications have been observed to be heterogeneous and mostly polydisperse in physiological conditions. Hence, when we make these proteins in vitro, we tend to remove all possible glycosylation to enable crystallization of the protein. The high-resolution diffraction of the milk protein crystals and its successful structure determination is till date the first structure reported with this amount of heterogeneity. The precise role for heterogeneity optimized for crystallization in a single lattice is currently unclear. Understanding the molecular structure of these in vivo grown milk protein crystals enables us to appreciate the evolution of viviparity in these cockroaches.

 

Analysis of the calorific value of these crystals shows that it has three-four times more the energy provided by the equivalent masses of cow, buffalo and other mammalian milks. This cockroach milk protein with proteins, sugars and lipids is a complete food for the embryos. The heterogeneity in the protein sequences provides all the essential amino acids to the embryos. The knowledge of the high energy values gave us an idea that if we produce these milk proteins in vitro in yeast, these recombinant proteins could be used for human consumption as a protein supplement. The curiosity still continues and we hope that this wonderful system can be used for multiple innovations in the future.

Below is the table showing comparison of milk calorific values:

Species

kcal (per 100 g)

Lili-Mip

230-300

Cow

66

Goat

60

Sheep

95

Water Buffalo

110

Human

72

AAEAAQAAAAAAAAinAAAAJGVlNTBmOGVkLTc0YjQtNDY5ZC1hNjQ2LWZhNGQ4YWQ1NTk1Ng
About the author: After completing her Ph.D. from Molecular Biophysics Unit, Indian institute of Science in 2014, Sanchari worked in Syngene International Limited for a very short time. Then she joined the Institute of Stem Cell Biology and Regenerative Medicine (Bangalore, India) as a postdoc. Since the time of joining she has been involved with the cockroach milk protein project. The other areas that interest her are in teaching and education. Her hobbies include dancing and cooking.
To know more, read her paper here: jt5013

Maria Sibyllia Merian, who rendered science pretty

in SciWorld/Theory of Creativity by

Maria Sibyllia Merian was an illustrator and entomologist (1647-1717). At a time when education was scant for women, she learnt miniature painting from her step father. She used this skill to depict her observations on insect metamorphosis across a variety of specimens. Her work contributed to the shift in belief from theory of spontaneous generation prevalent at the time. She travelled to the forests of Surinam where she spent six years studying insects and plants. She worked at a time, when illustrations were the only ‘photographs’ available. For financial support, she sold her work as art and published books. Linneaus later used her work to classify insects. Here are recreation of four of her plates.

maria betonien rose 1

Bentonein rose

 

maria chocolate tree

Chocolate pod

maria lime tree with butterfly

Lime tree with insect metamorphosis

maria insect of surinam

Insect metamorphosis

 

While Maria used copperplate etching for her illustrations, here Adobe illustrator software has been used to revisit those.

 

IMG_20151008_111034_1444282874501

About the illustrator: Ipsa is pursuing a Ph.D. at Indian Institute of Science. She loves to draw and paint. Biologist by training. Wants to gather and spread interestingness.

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The Earliest Englishman who never was

in Sci-Pourri/SciWorld by

 

Piltdown_gang_(dark)

    • 1912 – the meeting of the Geological Society, London. Charles Dawson, an amateur anthropologist, stuns the world by presenting bone fragments which, he claims, are that of an extinct species of ancestral humans!
    • The find – from a gravel pit at Piltdown village of Sussex – includes parts of a skull and lower jawbone, a canine and prehistoric tools made from bones. They are estimated to be about half a million years old.
    • Arthur Woodward, head of the department of Geology of the Natural History Museum, reconstructs the skull and announces that it had belonged to a ‘missing link’ between apes and humans – it is named Eoanthropus dawsoni, after the discoverer.

piltdown-image-1160x435

    • The crux of the discovery is that the skull indicates that the brain size would have been about two-thirds that of a modern human. It is quite similar to a human skull, except for the occiput (the part of the skull that sits on the vertebral column). BUT, apart from two human-like molar teeth, the jaw bone is identical to that of a modern, young chimpanzee.
    • ‘Piltdown Man’ stuns the world. And the British rejoice. Many of them had been sad that no fossils of ancestral humans had been unearthed in the British isles, while Neanderthals and Cro-Magnon fossils had been found in Germany and France respectively. Now, here is THE EARLIEST ENGLISHMAN AND THE EARLIEST EUROPEAN.

PSM_V82_D210_Reconstruction_of_Eoanthropus_dawsoni

    • And of course, Piltdown Man is an EURASIAN and not of African origin. [in the days of colonial imperialism and racism, Darwin’s 1871-hypothesis that human origins lay in Africa had caused much controversy; here was proof that Darwin had been wrong].
    • And the evidence nicely fits into the predominant hypothesis that the cranium had evolved first followed by jaws i.e. the large brain preceded the omnivorous diet . That also implies that the British were the ‘first to be smart’ and the first to start eating like humans, not beasts.
    • And Piltdown Man loved sports too – a sculpted elephant bone, discovered alongside the skull and jaw, is even interpreted as being the prehistoric cricket bat!
    • But, not all is rosy – many scientists, including the famous anthropologist Sir Arthur Keith, are skeptical (some of them in continental Europe and the US had ‘extra-academic reasons’ to be so too). And even The Royal College of Surgeons demonstrates that the bone fragments can be reconstructed differently such that it’d be identical to a modern human skull.
    • It is even suggested that the skull bones and the jawbones belonged to two different species and had accidentally come in close juxtaposition in the pit!
    • Besides, could an ape-like canine snugly fit into a jawbone that had human-like molars?   Scientists doubt – heated debates rage.
    • But, in 1915, Dawson discovers 3 more skull fragments from a site 2 miles away from Piltdown. And they look convincing. Now, even skeptics have to accept the data…grudgingly.
    • Dawson dies in 1916. Woodward digs more, but the Piltdown pit has nothing more to unearth.
    • Over the decades, more fossils come up – predominantly in Africa…and the line-of-evidence they present is rather different from the Piltdown Man. ‘He’ seems to be a strange, ill-explained aberration – almost an outlier…odd…funny…but, 40 years go by…
    • November, 1953. TIME magazine publishes the findings of Kenneth Oakley, Sir Wilfrid Le Gros Clark and Joseph Weiner. The article is titled ‘End as a Man’. Using the latest techniques, including Fluorine absorption dating, the trio proves that the Piltdown Man is a forgery.
    • The paleoanthropological hoax-‘fossil’ is a composite of bones from 3 species – a medieval-era human skull, a medieval-era orangutan’s lower jaw and fossilized teeth of a chimpanzee!!!
    • They were made to look prehistoric by staining the bones in a solution of Iron and Chromic acid.
    • Microscopic examination of the teeth shows file-marks – they had been modified as if they used to chew a human diet – Eoanthropus dawsoni had never existed. It was a scientific fraud, crafted with sinister care and then deliberately thrown at the world.
    • WHODUNIT?? – more than a century later, it is still unclear. Dawson was certainly involved (later investigations showed that many of the antiques and artifacts he had collected were hoaxes – a serial bluffmaster ? ), but probably there were others too.
    • WHY? – no one knows. Perhaps Dawson, an amateur, yearned for international recognition – maybe fellowship of the Royal Society….we will never be sure of the motive…
    • What had been achieved? People – researchers and commoners – had wasted a LOT of time and money and enthusiasm had been funneled into a ‘blind lane of knowledge’ for years. An estimated 250+ papers had been written on the topic!
    • No use at all? – well, it shows science can never compromise on its stringency – especially when cultural, natural or ideological emotions and the pursuit-of-glory may tend to cloud judgment. A successful hoax is one that ‘presents what one expects to see’ – and the Piltdown Man was just that.
    • And, of course, the great thing is that it was rigorous science that finally unearthed the hoax.

Author Profile:

for sciwri

Anirban Mitra, Ph.D

Anirban Mitra did his PhD from the Department of Microbiology and Cell Biology, Indian Institute of Science (IISc), Bengaluru and is now a teacher of biology, based in Kolkata. His interests range from biological evolution to history of science and facets of India’s past.

*This blog summarizes the findings from the research articles that can be found in this link. http://www.clarku.edu/~piltdown/map_prim_suspects/ABBOTT/Abbot_defense/piltman_englishmystery.html

*The overall conclusion derived from these studies have been voiced at the website of Natural History Museum http://www.nhm.ac.uk/our-science/departments-and-staff/library-and-archives/collections/piltdown-man.html

One man’s Junk is another man’s treasure or the secret art of cellular talking

in SciWorld by

Like begets like. And so bags of membranes shed other bags of membranes. A cell during its course of its short (or long) tortuous life is expected to shed membranous structures. After all it is the primary container in which all life processes are carried. Does this process have a higher purpose is a question that had been pondered over and decided that it must be how the cells get rid of unwanted stuff. A few years ago, If I were offered to investigate these cellular garbage bags, my answer would have been an enthusiastic “Thanks but No thanks”.

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(Image courtesy Johan Skog and Casey Maguire, Massachusetts General Hospital. I have met Johan, who is a wonderful person and is the CSO of exosome diagnostics.)

 

And hence I focused on disparate variety of things with no relations to the above, specifically on how neutrophils co-ordinate movement in complex tissue spaces. Neutrophils are the SWAT teams of the body. They are the first emergency responders that reach the site of a pathogen attack or injury to initiate a wide variety of inflammatory responses. Their modus operandi can be described right out of a scene of CSI. A police officer patrolling the highway smells a faint smell of smoke. He turns his head to detect the direction of the smoke and determines to be coming out of the wood. He decides to go into the wood to investigate, and in the meantime calls for a backup. He fears his backup wouldn’t know the direction he has taken into the woods and decides he should light up flares as he goes. As he approaches the scene of crime, shots ring out and he is fatally wounded. Before he dies, he shoots his flare gun into the air and calls for help from the backup that has arrived in wood’s edge. Seeing their comrade dead, the crack police team come out with guns blazing shooting down any one in their path.

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Now replace the officer with neutrophil, smoke with bacterial peptides released by a pathogen, highway as the blood vessel, wood with interstitial tissue space, guns with the deadly neutrophil proteases. What was not known till a few years ago, is the nature of the flares or simply put how does neutrophil coordinate during subsequent neutrophil swarming during an inflammatory response. The answer turned out to be a rather unintuitive molecule called leukotriene B4 or LTB4. I call the molecule unintuitive not because it is not a potent molecule, it is one of prime targets in asthma medication, but because it is a lipid. And that is a problem. Let me illustrate.

In a perfect world, neutrophils sense a gradient of chemoattractant (e.g. Bacterial peptide) and move towards a region of higher concentration, a process that is called chemotaxis. In situation where the gradient is too shallow for the following cells to detect, it would release a second molecule which now can diffuse far and wide to recruit other neutrophil, hence increasing the range of response. This phenomenon was dubbed as signal relay. This second molecule was found to be LTB4. But being lipid it doesn’t diffuse well. It does not play very well with aqueous environment of the tissue and of course, it would not create a defined gradient (remember neutrophil can find its way by comparing differences or gradients of attractants in space). Unless of course there is something that facilitates in doing its job.

 

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(from New paradigms in the establishment and maintenance of gradients during directed cell migration Current Opinion in Cell Biology, Volume 30, Pages 33-40 Ritankar Majumdar, Michael Sixt, Carole A Parent)

We started to find the facilitator by trying to locate where LTB4 was in the cell. We traced the location of the site of its active synthesis by mapping the proteins that were responsible for making this molecule. We found that these proteins were localized on atypical vesicular structures not found in a neutrophil’s natural repertoire. We took a very close look and found these were special bags-in-a-bag arrangement called multivesicular bodies. These are big bag that contains smaller bags which under certain conditions fuse with the cell membrane and release their content of smaller bags. These released smaller bags (or vesicles if you may) are called as exosomes. After painstaking ultrastructure sectioning of neutrophils migrating towards a bacterial tripeptide, we deciphered a series of events showing the genesis of the bag at the nuclear envelope to the fusion with the plasma membrane releasing smaller vesicles.

The rest of the story comprises of relatively boring monotonous sequence of validating the hypothesis by purifying exosomes, quantitating their content, knocking down the production of exosomes and their effects in chemotaxis etc. But one of the key insights came from a form of mixed cell experiments where we found cells talked to each other while they migrated. They talked through LTB4 by releasing them in exosomes. Without them the cells lose their sense of directionality. Without exosome, the dying police officers would have shot a very short feeble flare that the other police officers would have never seen. The exosomes acted as molecular beacons to the guardians of our body.

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Very few engaged in biological research would believe exosomes to be the garbage bag of cells. They have been shown to be carrier of extracellular miRNA, they can transfer genetic material between cells, and even active proteins. They have been heralded as the best candidate for liquid biomarkers in cancer especially in glioblastomas where getting biopsies mean boring through your skull or spinal cord. Researchers who work with exosomes have an immensely difficult task of unravelling these microcosm of things, especially when one encounters the very fundamental question of “Did I just purify exosomes or some other stuff that the cells throws out?” The lack of standardization of markers and the tedious pain-in-the-butt purification process pose additional challenges. (It took ~ 500 ml of blood to purify ~ half a billion neutrophils to yield ~ 50-100 ug of pure exosomes)

All said, working on exosomes gives a feeling that a genome researcher would have felt in the last decade of the last millennia or the geneticist of the last century. The path to research is full of promise and is littered with red herrings, where paradigms change in matter of days and the rule of the game is that there is no rule. For me the moral of story is that One man’s junk is other man’s treasure. Literally.

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(From Exosomes: secreted vesicles and intercellular communications, an excellent article by Clotilde Thery)

A few good reads and see.

One of my favorite video made by David Rogers in 1950s. This gives an idea of the persistence of a neutrophil chasing a bacterium. https://www.youtube.com/watch?v=I_xh-bkiv_c

An excellent review on leukocyte migration. For advanced readers I am afraid http://www.nature.com/nri/journal/v14/n4/full/nri3641.html

The finding of evidence of signal relay in neutrophils and how LTB4 plays a critical role. http://www.cell.com/action/showRelatedArticles?pii=S1534-5807%2812%2900084-6

The reason why I am writing this piece. “Exosomes Mediate LTB4 Release during Neutrophil Chemotaxis.” http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002336

Richard Robinson has done a wonderful job of explaining the findings in the above paper in layman terms. “To Attract Others, Immune Cells Release a Packet Which Releases a Signal” http://journals.plos.org/plosbiology/article?id=info:doi/10.1371/journal.pbio.1002337.

An excellent review on the current standing and status of exosome research by Clotilde Thery. http://f1000.com/prime/reports/b/3/15

About the author: Ritankar Majumdar is a Post-Doctoral fellow at the National Cancer Institute, NIH. He has been trained as a biochemical engineer, although after completing his thesis on  structure-function relationship of GPCRs from IISc, he refuses to identify himself as an one. He enjoys working on complex chemotactic systems, good music, decent food and living the fresh breathe of science.

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