The Science Behind the Image Contest Winners: Group II Intron Ribozyme

The BPS Art of Science Image Contest took place again this year, during the 61st Annual Meeting in New Orleans. The winning image was submitted by Giulia Palermo, a postdoctoral fellow in the group of J. Andrew McCammon at the University of California, San Diego. A team of three scientists composed the image:  Giulia Palermo created the original design, Amelia Palermo (ETH, Zurich) made the handmade painting, and Lorenzo Casalino (SISSA, Trieste) performed digital manipulation on the picture. Giulia Palermo took some time to provide information about the image and the science it represents.

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With this picture we would like to send as the main message that Physics and Art try to interpret the beauty of Nature in different ways but there is a natural overlap between these disciplines, which could lead to wonderful discoveries and amazing beauty.

Group II intron ribozyme perform self-splicing reactions. In the picture, two scissors are used to represent this mechanism. What we like about this image is how a handmade painting could capture the fundamental aspects of the mechanistic action of the system. Besides the beauty of handmade painting, we enjoyed our teamwork and, fostered by the passion for this research, we have been motivated to submit this image to the Art of Science Image Contest.

This image has been inspired by the work we have done in the group of Prof. Alessandra Magistrato (SISSA, Trieste), in collaboration with Prof. Ursula Rothlisberger (EPFL), which resulted in the publication of our research in the Journal of American Chemical Society and in the Journal of Chemical Theory and Computation, while other equally exciting results are in preparation for publication. Below, we report details of our publications:

  1. Casalino, G. Palermo, U. Rothlisberger and A. Magistrato. Who Activates the Nucleophile in Ribozyme Catalysis? An Answer from the Splicing Mechanism of Group II Introns. J. Am. Chem. Soc. 2016, 138, 1034.
  1. Casalino, G. Palermo, N. Abdurakhmonova, U. Rothlisberger and A. Magistrato. Development of Site-specific Mg-RNA Force Field Parameters: A Dream or Reality? Guidelines from Combined Molecular Dynamics and Quantum Mechanics Simulations. J. Chem. Theory Comput. 2017, 13, 340–352.

My research exploits advanced computational methods – based on classical and quantum molecular dynamics (MD), novel cryo-electron microscopy (cryo-EM) refinement – and their integration with experiments to unravel the function and improve biological applications of key protein/nucleic acids complexes directly responsible for gene regulation, with important therapeutic applications for cancer treatment and genetic diseases. As a next-generation computational biophysicist, I aim at going beyond the current limits of time scale and system size of biomolecular simulations, unraveling the function of increasingly realistic biological systems of extreme biological importance, contributing in their applications for effective translational research.

The World Health Organization reported that ~8.2 million citizens die each year for cancer, while genetic diseases affect millions of people. As such, the clarification of the fundamental mechanisms responsible of gene expression and of their therapeutic implications is of key urgency to society.  By using advanced computational methods and by their integration with experiments, I seek to unravel the function and improve applications of biological systems of extreme importance. My current interest – as a post-doc in McCammon’s lab at UCSD – is in the clarification of the mechanistic function of the CRISPR-Cas9 system via computational methods. Additionally, I am interested in long non-coding RNA, which regulates gene expression, and in intriguing protein/DNA systems, whose mechanistic function is at the basis of genetic inheritance.

How to Write a Biophysics Article Worthy of Publication: Part 1- From Lab Notebook to First Draft

William O. Hancock
Pennsylvania State University
Member, Biophysical Society Publications Committee
wohbio@engr.psu.edu

This is the first part of a three-part series on How to Write a Biophysics Article.  Although the suggestions herein are geared toward a Biophysical Journal paper and are targeted for graduate students and postdocs, they apply generally to all scientific writing and all levels of scientists and engineers.  In this first paper, I will discuss the hardest part of writing a manuscript — writing the first full draft.  The important tasks of polishing your writing and figures to achieve publication quality will be tackled in the second paper, and the third paper will cover navigating peer review and getting your manuscript published.

Although many students and postdocs put off writing until they absolutely have to, there are important reasons why you should tackle the first draft of your manuscript earlier rather than later.  The most important is that writing up your work in manuscript form is the best way to clarify which experiments are essential and which are less essential or even superfluous.  Although it may seem that you are losing productivity by stepping away from the bench to write, in the end you will save a lot of time by avoiding unnecessary experiments, and you will have an added focus for those experiments that you realize are needed to complete your story.  The second reason for starting early is the unavoidable truth that good writing requires extensive revising, and revising takes time.  So, do not wait, start writing now!

Telling your story

A good paper is one that addresses an important question and changes the way that the reader thinks about a problem.  When you write a manuscript, it is important that you remember that you are writing for an audience.  For this reason, it is often helpful to think of your paper as a story that you are telling the reader.  The story is broken down into four sections:  Introduction, Methods, Results, and Discussion. In writing your story you should aim to fulfill four goals:

  1. Explain why the question you have chosen to work on is important — guide your reader’s thinking and get them excited about your work;
  2. Explain how you did the experiments — help your reader evaluate whether the methods are appropriate for the problem at hand;
  3. Clearly describe the results you obtained and the control experiments you did to substantiate your conclusions;
  4. Discuss how these results change the way in which we should think about the question at hand — educate your readers and convince them of the impact of your findings.

No bones about it, writing is hard.  To minimize writers’ block and the intimidation of a blank page, I lay out a series of steps here to help you build a first draft.  It is assumed that you have a collection of data in your notebook, and you may even have an important breakthrough to report, which motivated you to write up your work.  But writing is a very different activity from carrying out experiments or doing theoretical work, so having a clear game plan is vital.

Step 1:  Define your story.

What is the point you are trying to get across to your reader?  This story is in the context of specific questions in your field, and you have a set of data that you want to present to try to tell this story.  Defining the story early on is important because it will help you decide how you want to organize the presentation of your results.  Defining the story is also important because it streamlines the Introduction and defines the specific background points you’ll need to get the reader up to speed.  Finally, the Discussion will hammer home the narrative of the story you presented in the Results — reiterating it, extending it, putting it in the context of what has been done before, and pointing to where the story will go in the future.  You should be able to summarize this story in a sentence or two and, in fact, it is a good idea to write these sentences at the top of your document that will grow into the first draft of your manuscript.

It is important to point out here that the “narrative” you present in your manuscript need not follow the historical sequence of your actual experiments.  In fact, because research often takes a circuitous path, the ordering of the results in the manuscript generally should not follow the timeline of your experiments (and no, this is not “cheating”).  Remember that you are writing a science “story” and not a science “diary”; hence, the trials and tribulations you encountered along the way (even though they took up a lot of your time) are not important to the reader.  A related point is that you should avoid the urge to include all of your experimental data in your paper.  The more threads you try to weave into the story, the more risk there is that you’ll detract from the main storyline.  To sum up:  Think about how to create the best narrative that presents the work in a logical and memorable manner.

Step 2:  Organize your figures.

Your figures are the most important part of your manuscript.  A good rule of thumb is that a reader should be able to look through your figures and the associated figure legends and get the gist of your story.  Hence, deciding how you organize the various plots, images, and diagrams into discrete multi-panel figures is a key task.  The Results section will be written around these figures, so a helpful approach is to “divide and conquer.”  Many journals (like the Biophysical Journal) allow the Results section to be broken into subsections, each with its own subhead, which makes your job much easier.  Just as you wrote down the main point of your story above, write down a series of active statements that describe the data you are presenting, and use these statements to organize your figures.  Then you can think of your Results as a series of chunks, each of which has an actively worded subhead that states a result (e.g., “Protein X activates complex Y”), has a figure and legend that present the data, and has one or more paragraphs that describe the data presented in the figure. These are the bullets that make up the key points of your paper.

This step is key, so here are a few pointers:  (1) A good way to build your manuscript over time is to assemble your notes and data into a PowerPoint presentation that you can present at lab meetings and easily modify and reorganize.  (2) For the first draft, don’t worry too much about finalizing formatting of the panels in your figures, you can do this later; if some data are missing at this point, that’s okay, put in a mock figure and keep pushing forward.  (3)  For journals that don’t allow section headings, this type of organization is still helpful; just delete the headings.

Step 3.  Write the Results section.

Now that you have your figures together and have divided your Results into subsections, it is time to write.  Each subsection should describe: (1) the specific question being addressed, (2) the methods employed, and (3) the results obtained.  Each section should logically connect to and set up the next section.  One good way to achieve a logical flow and a compelling narrative is to organize the sections of the Results as a series of questions.  Another useful approach is to organize each section around a specific hypothesis that is being tested.

For the methods, be brief because full details are in the Materials and Methods section, but give sufficient information for a reader to understand the essentials of what you did.  And for the results, as you proceed logically from one figure panel to the next, you should describe the key result contained in each panel, perhaps provide additional details that are not in the plot or legend, and summarize the “take-home point” before moving on to the next result.  For your initial draft, include all details (err on the side of verbosity) and distill down to essentials in later drafts.

Writing the Methods in parallel with the Results makes sense because you can progress through the same sequence (for each Results section you write, write the corresponding Methods section).

A note on verb tense. It is generally accepted that your narrative should be in the past tense when you are discussing what you did and what you found. In contrast, when discussing data that are in the literature, we typically use the present tense — which may seem surprising. But most importantly try not to mix past and present tense in your manuscript.

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Step 4. Write the Discussion.

For writing the Discussion you need to step back a bit.  Whereas the Results section is very specific and detailed, the Discussion needs to put your work into a larger context.  It is good to start the Discussion with a paragraph that reiterates the question set up in the Introduction and then reiterates the key results in a concise way.  An added benefit of summing things up here is that it provides a running start for your Discussion.  You then need to relate your work to previous work that has been done and put it in the context of the field overall.  You should also critically evaluate your methods and results — what are the strengths and limitations of your approach, and how do they compare to previous or related work?  You should extract as much meaning from your results as possible (without going overboard).  What results amplify and confirm others?  What subtleties in the data suggest other phenomena beyond what you’re looking at specifically?

Step 5.  Write the Introduction.

Now that you’ve written most of the manuscript, it’s time to write the Introduction.  Return to the story you defined at the start (maybe you need to revise it somewhat after laying out all of the results?), and think about the points you’ve made in the Discussion.  In the Introduction you want to lay out the basic logic and motivation for your study — build a framework that makes the reader excited and hungry to see your results.  To achieve this, you need to provide the key background material that enables the reader to understand the state of knowledge in the field.  Avoid a comprehensive review of the field, and instead focus on the important open questions and why they are important. Build a convincing argument for why you did what you did.

In setting up the background, you should write with the literature that you reference close at hand, and be checking that what you think is in the papers is actually written in the papers. Beware of boldly stating what you assume to be true — provide evidence and references when stating any “fact.” Also, avoid referencing review articles whenever possible, and instead reference the original papers where key observations were made — if you make an important discovery wouldn’t you rather have people reference your hard work rather than a review article written by someone else?

The last paragraph of the Introduction is key.  It should briefly describe what you did and what you found, and it should set up the Results section.  In this way, the Introduction creates tension and intrigue, and this last paragraph gives a sneak preview of what is to come.  Ideally this last paragraph of the Introduction should also link to the first paragraph of the Discussion, providing two bookends of the Results.

Step 6.  Write the Abstract, Title, and Reference List.

Now that you have your complete text, you should write the Abstract.  Be brief and to the point (check word limit for the journal).  Minimize background, clearly state your results and include any methodological details you need.  Finish with the implications of the work.  You will hone your abstract later.

If you haven’t settled on your title yet, this is the time.  Be specific and be precise.  Also, finishing your first complete draft means that you have a complete reference list with proper formatting.  Bibliographic software is essential.  Suitable packages include EndNote, Mendeley, and Zotero; use whatever works best for you.  One consideration in choosing software is that editing subsequent drafts is much easier if you and your coauthors use the same package.

Final notes

The key task to remember here is to get all of your results and all of your thoughts down on paper — the honing and polishing will come later.  Remember: it is better to start writing earlier rather than later.  Your next step is to refine your writing.  It has been said that the last 10 percent of the work takes 90 percent of the time, which is a bit extreme but not too far from the truth where writing is concerned.

Revising your draft will be the subject of Part 2, published Wednesday, March 8.

Interested in learning more and having a chance to ask questions?  Dr. Hancock will present a webinar on this subject Friday, March 10, at 1:00 PM ET.  Register at http://www.biophysics.org/Education/Webinars

Mechanical Interplay in Clot Contraction

BPJ_112_4.c1.inddBlood clotting, thrombosis, and blood cells all have great biological and clinical significance. Clotting is necessary to stop bleeding yet thrombi can obstruct blood flow, which can cause heart attacks, strokes, venous thrombosis, and pulmonary embolism. Although much is known about various aspects of clotting, much less is known about clot contraction or retraction. Clot contraction is thought to play a role in hemostasis, wound healing and the restoration of flow past otherwise obstructive thrombi.

The cover image for the February 28 issue of the Biophysical Journal shows a colorized scanning electron microscope image of a coronary artery thrombus extracted from a heart attack patient. We chose this image because contraction occurs in such thrombi and all of the elements described in our paper are visualized here: platelets (gray), fibrin (brown) and red blood cells (red). Thus, this image represents a real-world example of the practical significance of our research. Furthermore, we have found that clot contraction is altered in patients with certain thrombotic disorders, such as acute ischemic stroke. Our model provides the fundamental mechanical basis for understanding the contraction of blood clots.

The contraction of blood clots and thrombi is an interdisciplinary problem related to fundamental aspects of cell biology, including cell motility and interaction of cells with an extracellular matrix. The biophysical mechanisms of clot contraction have been poorly understood, although it has been shown that it results from the interaction of actively contracting platelets with the fibrin network, the structural matrix of the clot that has unique mechanical properties. Though many of the same basic principles of motility of other cells are employed in this system, the specialized mechanisms of cellular contractility represent a novel biological application. The consequences of cell-matrix interactions in blood clots are unique and result in massive compaction of the network, rather than motility or alignment of fibers that occur in other cellular contractile environments.

Blood clot contraction is driven by platelet-generated contractile forces that are propagated by the fibrin network and result in clot shrinkage and deformation of red blood cells. We developed a model that combines an active contractile motor element with passive viscoelastic elements consisting of fibrin and red blood cells. This model predicts how clot contraction occurs due to active contractile platelets interacting with a viscoelastic material, and explains the observed dynamics of clot size, ultrastructure, and measured forces.

– Andre E.X. Brown, Chandrasekaran Nagaswami, Valerie Tutwiler, Hailong Wang, Rustem Litvinov, Vivek Shenoy, and John Weisel

Note: This image originally appeared in a different form in Science 325:651, 2009.

Highlighting Biophysics Research During American Heart Month

February is American Heart Month. Heart disease is the leading cause of death for men and women in the United States, causing 1 in 4 deaths each year. In recognition of this awareness month, we spoke with BPS member Anna Grosberg, University of California, Irvine, about her research on cardiomyocytes, which play an essential role in contracting the heart and pumping blood.

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What is the connection between your research and heart disease?

My laboratory studies the structure dynamics, and function of cells. We are mostly focused on cells that make-up the heart muscle – cardiomyocytes, which play the essential role in contracting the heart and pumping the blood. Recently, we have made discoveries elucidating how the organization on multiple length-scales affects the ability of engineered heart tissues to generate force. This is especially important for the future progress in developing therapies following heart attacks and for understanding heart diseases, such as dilated and hypertrophic cardiomyopathies.

Why is your research important to those concerned about heart disease?

The label “heart disease” encompasses a wide variety of patient symptoms and pathologies. Yet in many presentations of the disease, the structure of the heart is changed over a wide variety of length-scales. Indeed, the normal heart consists of sheets of elongated cardiomyocytes arranged to generate maximal force in parallel with each other. Conversely after an infarct, a scar forms that disturbs this organization and prompts the muscle to remodel. Often such remodeling causes the cells to become less organized within the tissue, and it can also occur without an initiating scar. Understanding how these progressive architectural changes affect function is important to identifying the dominant mechanism to target in modulating heart disease. Through the type of research my lab does, it was discovered that changes to the organization also cause downstream variations to expression levels of a variety of genes some of which are linked to both electrophysiological and contractile function. This makes it problematic to determine how much of the force reduction of diseased muscle is caused by disorganization, and to what extent it is influence by the downstream biological effects. We have recently found that providing muscle cells with local organizational cues, while maintaining a global organization results in engineered cardiac muscle with predictable organization-force generation relationship based on simple physical understanding.

In general, remodeling of the heart muscle and, even, the enlargement of the heart are part of normal physiology. For example, it is known that for both competitive athletes and pregnant women, the heart can enlarge for the duration of the higher load, but it will remodel to normal size once the excessive exercises are over or the baby is born, respectively. Thus, there is hope that by understanding the functional aspects of remodeled heart tissue, it will be possible to reverse some of the pathological remodeling observed in heart disease. Additionally, we also work with cells from patients with genetic mutations that cause heart disease. A deeper understanding of the mechanisms that make these cells abnormal can provide more clues for how genetically normal patients can still develop heart disease.

How did you get into this area of research?

In the past decade, advances in tissue engineering have allowed researchers to guide the architecture of engineered heart tissues. It has also been possible to image the detailed structures within the tissues – thus visualizing the organization of the sarcomeres (the force producing units within muscle cells). However, it was also found that the reduction in force generated by badly organized tissues was two times higher than predicted from basic physics principles. Unfortunately, it was not possible to change the organization of the tissue without also triggering biological downstream effects that affected force generation in complex, poorly understood ways. When I started my lab, one of our big goals was to create a system to both quantify organization of cardiac muscle over multiple length-scales and to build an experimental system where the organization could be changed without triggering any downstream effects. These tools now allow us to greatly deepen our understanding of the relationship between structure and function in both normal and diseased heart tissues.

How long have you been working on it?

I have been working on the relationship between structure and function in the heart for the past 15 years. However, only in the past few years we have built-up the tools to study multi-scale organization and its connection to force generation.

Do you receive public funding for this work? If so, from what agency?

Yes, our work has been funded by both the National Science Foundation and the National Institutes of Health.

Have you had any surprise findings thus far?

We have found that in the absence of guidance cardiac tissue forms spontaneous patches of organization. Yet, the force generated by such a tissue is almost two times weaker than if the patches of organization are introduced with guided parquet patterns. The first surprising finding was that it is possible through very basic physical principles (i.e. force vector addition) to predict the amount of force generated tissues if their organization was guided in parquet tiles. Second, we found that the spontaneously organized patches are only 3 times smaller than the engineered parquet tiles. This implies that either the cells are very sensitive to a narrow range of local signals, or the temporal change in self-assembly provided by guidance cues plays an important role in resultant force generation. Our current aim is to solve this mystery.

What is particularly interesting about the work from the perspective of other researchers?

Our results have shown that in the case of eliminating biological downstream effects, simple physical principles are predictive for cardiac tissue force generation. This provides a way to interpret the reduction in force observed in stem-cell derived cardiomyocytes that are incapable of achieving sarcomeric structures that fully match the adult or even neonatal hearts.

What is particularly interesting about the work from the perspective of the public?

Heart disease is still the number one cause of death in the developed world. Yet, there are no cures – only therapies that help manage the disease. By exploring the basic physical principles behind heart muscle structure and function, it might be possible to provide better targets for therapies or surgical intervention. Additionally, it might become realistic to use the principles we discover to build better engineered cardiac tissues, which would provide better predictability for testing pharmaceuticals for cardiac toxicity.

Back to the grind

Didn’t get a chance to post on the last day of the BPS. We had to check-out at noon while many of the posters I wanted to see were scheduled that morning, which made the morning quite hectic.

My poster session on Tuesday went well. Met with fellow blogger Christopher Lockhart who had his poster at the same time. I received a lot of very good suggestions from people who spoke to me, and I am extremely grateful to everyone who did. This is what I look forward to the most at BPS meetings: the opportunity to present my work and getting feedback from the top scientists. Presenting the work in an organized manner is an extremely important component of what we do, and the feedbacks are invaluable for improving current research and planning future work.

Had dinner at the Acme Oyster House on Tuesday. I love to try out new food, and this time it was oyster. I wish I had had oyster before, because it was really good! Another nice discovery for me in this trip was Po-boy. This Louisiana sandwich had been my steady meal for a major part of the trip.

Back in the snow and chilling weather of West Virginia, and already missing the New Orleans weather. Like everyone else, I am eagerly waiting for San Francisco 2018. Hope to see everyone there.

From Cellular Footprints to Atomic Force Fields: Reflecting on BPS 2017

After BPS every year I take at least a day to review my notes from the meeting, lest I let the new and exciting biophysics slip out of my mind. After reviewing my notes I realized something extraordinary. Every single talk that I went to used or discussed an array of techniques; from experimental to computational, from long timescale dynamics to crystal structures, from cellular footprinting to atomic force fields. This variety of techniques is quite the tribute to the diversity and “do what it takes” attitude that astounds and inspires me about biophysical researchers.

Two of the most influential talks for me came at the beginning and the end of the meeting.

At the beginning of the BPS 2017 meeting, I was fortunate enough to attend Isaac Li’s talk “Mapping Cell Surface Adhesion by Rotation Tracking and Adhesion Footprinting,” … the very first talk on Saturday. The methods and results amazed me. Li was able to demonstrate the role of colocalization of proteins at the cell surface in conferring variability to cell adhesion footprints. Before this talk, I was unaware that such methods were even possible, and now I’m fascinated with how these methods could reveal molecular level details that vary from cell to cell. Additionally, I could not help but imagine how this sort of technology could be applied to understanding the surface chemistry of aerosol particles, as I have just jumped ship from a biophysics to atmospheric chemistry for my postdoc. To learn more about techniques from this laboratory, click here.

One of the last talks I went to at the BPS 2017 meeting, was Maxwell Zimmerman’s talk “Fast Forward Protein Folding.” Unlike the first talk, I was familiar with this work and have applied the described FAST algorithm to sampling conformational space in molecular dynamic simulations in my own research. Yet, in spite of the fact that I was already familiar with this work, again, I was amazed. Not because of the methods or the results, but because of how this work was communicated. Zimmerman did a remarkable job of creating visualization tools and choosing his words carefully to reach all members of his audience; a clear reminder that how scientists communicate matters. To learn more about the FAST algorithm, click here.

As with all BPS meetings, I came away inspired as well as regretful. Reading through the program this morning, I found so many talks that I wish I could travel back in time to attend. This got me thinking: What if the BPS recorded these meetings in the future? These recordings would allow those who attended to revisit the lectures and talks that inspired them and catch the sessions that they had conflicts with and unfortunately missed. Perhaps 2017 is not the time, the place, nor the political climate for such measures.  But I hope one day this will be possible!

JMS

Biophysics, by its nature is interdisciplinary

Having always worked in an interdisciplinary field, even taking courses from various departments, I have perpetually felt I do not fit into a certain category. With my computer science friends I do not seem to belong, as I do biology, and among the science ones, I am the computer science geek.

However, here at my first BPS meeting, I was at ease, attending talks and symposiums one after the other variedly different from each other. I realize it is indeed only a matter of doing good science, and adding to the knowledge of the working of things around us. I was glad to see the optimism and so many motivated individuals around me.

This was very appropriately summed up when I heard on the Biophysical Society TV outside Great Hall A while charging my phone, ‘Biophysics, by its nature is interdisciplinary’.