BPS Members Making a Difference Beyond the Lab: Karen Fleming

Society members make a difference in their communities in many ways.  BPS member Karen Fleming a faculty member and undergraduate program director in biophysics at Johns Hopkins University, has taken on the barriers facing women in science, and decided to do something about it on her campus. 

Know a member making a difference in their community that should be featured here?  Let us know. We would like this to be the first of a series!

womenofhopkins

Pictured, (Left to Right), all of Johns Hopkins University: Dominic Scalise, graduate student in chemical and biomolecular engineering and Women’s of Hopkins Exhibit team member; Erin Gleeson,  Project and Events Specialist, Office of Instituional Equity; Gail Kelly, one of the Women of Hopkins; Ron Daniels, President; Karen Fleming; and Jeannine Heynes, Director, Office of Gender Equity.*  Photo Credit: Will Kirk, homewoodphoto.jhu.edu

Role Models.

We hear it over and over again—the need for diverse role models so that diverse students can see themselves succeeding in science. This includes gender.  Women often are underrepresented on panels, at conferences, and as recipients of prestigious awards.  BPS member Karen Fleming decided to do something about it.

Fleming, along with a handful of other JHU staff and graduate students, formed a committee, successfully sought institutional funding and support, and put together an exhibit entitled “Women of Hopkins.” The purpose of the exhibit was to highlight the many successful women that have graduated from the university and been pioneers in their fields, especially for current students.   Nominations were accepted and vetted by the committee, resulting in a photographic exhibit of 23 Hopkins graduates, displayed on the walls of Hopkins buildings. The women highlighted represent many different fields-not just science—and include Bonnie Basler, Bernadine Healy, Carol Greider, Mary Guinan, Nitza Margarita Cintrón, and Florence Sabin.  The project also includes a Women of Hopkins website, which has a biography for each woman included in the exhibition, as well as a presence on social media platforms Facebook and Twitter, giving the project greater visibility.

*Women of Hopkins team members not pictured are:  Anna Coughlin, graduate student in chemical and biomolecular Engineering; Jeff Gray, Professor of chemical and biomolecular engineering; and Valerie Hartman, instructional designer at JHMI.

Probing Water and DMSO near Lipid Membrane Surfaces

BPJ_111_11.c1.inddDimethyl sulfoxide (DMSO) is a powerful anti-freezing agent and has been used in biology as a cryoprotectant of cells. Thanks to a series of experiments and computer simulations  bulk properties of DMSO solution are reasonably well understood, yet the effects of DMSO on water molecules near lipid membrane surfaces, which are more relevant for elucidating the underlying physical chemistry of DMSO as a cryoprotectant, still remain elusive.

The consensus from a number of different experiments is that DMSO dehydrates phospholipid bilayer surfaces, which our study confirms. However, the DMSO-enhanced water diffusivity at solvent-bilayer interfaces, was not confirmed in our simulations. In order to resolve this discrepancy, we explicitly modeled Tempo-PC by appending Tempos to a few choline groups and conducted simulations and analyses.

Our cover image for the December 6th Issue of the Biophysical Journal depicts a snapshot from the molecular dynamics simulation of POPC phospholipid bilayer in 7.5 mol% DMSO solution. The lipid tails are rendered in grey, and the regions corresponding to phosphatidylcholine head groups are depicted in pale blue. Four Tempo-PCs, in the upper and lower leaflets are highlighted with the tail domain in yellow and the Tempo appended to the choline group in blue. Of particular note is that in contrast to the original intent of Overhauser Dynamic Nuclear Polarization (ODNP) measurements using Tempo-PC lipids to probe the surface water dynamics, the Tempo moieties are predominantly equilibrated at 8 − 10 Å below the solvent-bilayer interface, probing the water dynamics in the interior of bilayer. The water and DMSO molecules around the Tempo moieties are depicted in stick and surface representations, respectively. The inset magnifies the snapshot of water and DMSO molecules around Tempo. The image was produced using the molecular visualization system, PyMOL.

DMSO deposited beneath the PC head group, where Tempo moieties are equilibrated, increases the area per lipid slightly, and hence water diffusion probed by Tempo is detected to increase with increasing DMSO. Our study suggests that the experimentally detected signal of water using Tempo, stems from the interior of lipid bilayers, not from the interface. The only viable tool for the direct probe of water dynamics on biological surfaces at present is ODNP measurements using a Tempo spin label. Given its significance, the equilibrated location of Tempo moiety in lipid bilayers revealed here calls for adequate interpretation of data and careful re-evaluation of the technique.

—Yuno Lee, Philip A. Pincus, Changbong Hyeon

Biophysics on World AIDS Day 2016

December 1 is World AIDS Day, a global awareness day to bring attention to the disease, the research being conducted in relation to it, and the many people living with HIV/AIDS. In honor of World AIDS Day this year, we spoke with Gildas Loussouarn, University of Nantes, about his research on cardiac channels dysfunction in Long QT Syndrome, a disorder seen much more frequently in HIV patients as compared to the general population.

What is the connection between your research and HIV/AIDS?

With the worldwide development of antiretroviral therapies, HIV patients now live longer. As a result, they encounter additional pathologies and these pathologies are over-represented as compared to the general population. Among these pathologies, the Long QT syndrome, a heart rhythm condition associated with arrhythmias. In the general population, this syndrome is a rare disorder, characterized by a delayed ventricular repolarization leading to tachycardia and/or sudden cardiac death. HIV patients present with a higher prevalence of Long QT syndrome, as compared to the general population suggesting a higher risk of sudden cardiac death. Here at the institute du thorax, the global objective of our team is to decipher molecular mechanisms of cardiac ion channels and their dysfunction in cardiac arrhythmias, in order to identify new therapeutic targets. We are thus interested in cardiac channels dysfunction in the context of HIV.

Why is your research important to those concerned about HIV/AIDS?

More than 10 studies lead to the conclusion HIV patients have a higher prevalence of Long QT syndrome, as compared to the general population. Despite that, it is still difficult to address whether the LQT syndrome is due to the virus itself or to drugs that are proposed to HIV patients, which are known to prolong cardiac repolarization. Sorting this out is essential, in order to limit arrhythmias and the potential sudden cardiac death in HIV patients. By directly looking at the effect of HIV proteins on cardiac repolarization, we aim at addressing if the virus itself participate to cardiac repolarization prolongation. Importantly, we have already observed that one of the viral protein, Tat, can delay repolarization in human cardiomyocytes generated from induced pluripotent stem cells.

figure-loussouarn

This figure shows that HIV-Tat is detected intracellularly in human cardiomyocytes but not simian fibroblasts (COS-7), after a 24h external application (200 ng/ml). Tat immunostaining is shown in red, plasma membrane is identified by hERG channel immunostaining (green), nucleus is in blue (DAPI). Tat remains in the extracellular compartment of COS-7 cells (red arrow) while in human cardiomyocytes, Tat is located inside the cytoplasm (asterisks) and colocalizes with hERG at the plasma membrane (yellow arrows).* (Adapted from Es-Salah-Lamoureux et al, 2016, JMCC 99:1-13, with permission.)

How did you get into this area of research and how long have you been working on it?

I was first contacted by a colleague, Dr. Bruno Beaumelle (CNRS Research Director, Montpellier). Dr. Beaumelle had previously observed that due to its high affinity to the membrane phospholipid PIP2 (phosphatitylinositol 4,5-bisphosphate), HIV-Tat could interfere with some PIP2-dependent mechanisms of neurosecretion. Bruno Beaumelle had read our previous works showing the impact of a decrease in available PIP2 on cardiac repolarization through specific cardiac channels KCNQ1 (Kv7.1) and hERG (KV11.1). Our previous works indeed suggested that a decrease in PIP2 or a decrease in KCNQ1 / hERG channels affinity for PIP2 leads to a decrease in the activity of the repolarizing channels and hence leads to Long QT syndrome. Bruno Beaumelle anticipated that we will be interested in looking at a potential effect of Tat on these channels. The hypothesis was that Tat may be a link between HIV and LQT syndrome. Our results seems to confirm this hypothesis since expression or extracellular application of Tat leads to a decrease in the activity of the repolarizing channels KCNQ1 and hERG. In human cardiomyocytes, HIV-Tat leads to a delayed repolarization and other Action Potential alterations, which are common triggers of cardiac arrhythmias.

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

Our work that was just published in the Journal of Molecular and Cellular Cardiology was partially funded by the Fédération Française de Cardiologie, the Fondation Genavie, the Marie Curie European Actions, the French Regional Council of Pays-de-la-Loire, the National Research Agency, the Fondation Lefoulon Delalande, the Fondation pour la Recherche Médicale, the Association of Scientific Orientation and Specialization and Campus France. We hope to get specific funding from AIDS focused agencies allowing us to study the effects of Tat in further details.

Have you had any surprise findings thus far?

Yes! Our first surprise was that external application of Tat, which is known to penetrate cells, had an effect on hERG channels depending only in specific cell type! Tat did not have any effect on hERG channels expressed in COS-7 cells. Paradoxically, the same application halved the activity of the same channels in cardiomyocytes! The absence of Tat effect in COS-7 cells is surprising since Tat is a promiscuous ligand that binds a plethora of receptors (such as Heparan Sulfate Proteoglycans), internalization of which supposedly allowing Tat entry. To identify the reason of this cell-specific effect, we looked for intracellular Tat in both models, after its extracellular application. We observed that intracellular Tat could be detected by immunofluorescence, in cardiomyocytes but not in COS-7 cells (cf. also figure). We suppose that Tat requires specific cellular components to be internalized in sufficient amount to have an effect on the potassium channels. It has been shown that Tat interacts tightly with some receptors such as LRP and CXCR4 receptors, which represent a cell-specific way for internalization. Such receptors may lack in COS-7 cells. In addition, these observations illustrate the great value of induced pluripotent stem (iPS) cells derived cardiomyocytes, a model closer to mature human cardiomyocytes as compared to COS-7 cells. Human iPS cells are quite easy to get: they were obtained by reprogramming renal cells contained in the urine of a patient. This is clearly a non-invasive (but long!) way of getting human cardiomyocytes.

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

Tat is present in the patient’s serum and seems to target different organs and interfere with various PIP2-dependent processes in these organs: Bruno Beaumelle showed that Tat can interfere with the secretory activity of neuroendocrine cells, by sequestering PIP2. We then showed that Tat can interfere with cardiac repolarization, also by sequestering PIP2. Now, we could test if Tat is also a link between HIV and epilepsy, which is also overrepresented in HIV patients.  This hypothesis is founded on the observation that PIP2 activates neuronal channels (KCNQ2/KCNQ3), alteration of which leads to epilepsy.  We can hence speculate that AIDS may be seen as a “PIP2-pathie” with multiple organs targeted by Tat.

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

I think it is important for the public to bear in mind that despite huge progresses made in treatments against HIV, living with AIDS is associated to many other severe pathologies, including cardiac diseases. It is thus important to remain aware of risk- behaviors. Regarding potentially new therapies, an anti-Tat vaccine may represent a new therapeutic strategy, as tested currently by another French laboratory (ETRAV laboratory, Aix Marseille University/CNRS).

 

*Image scale= 5μm. 

Post-Election: BPS President’s Message

Dear Biophysical Society Members,

sfscarlataNo matter what side of the aisle we are on or what part of the world we live in, the recent US election and the campaigns leading up to it have left us with a feeling of divisiveness and unease. This is only the latest world event to bring to the forefront fissures that exist among people.  It has made many want to shut themselves off from the world around them.  As scientists and as citizens, we cannot do that.

The amazing strength of biophysics is its inherent diversity, and it is this diversity that has impacted fields far beyond biophysics.  Now more than ever, we as biophysicists need to work together to demonstrate through our actions, our work, and our outreach, the power of inclusivity, diversity, civil discourse, and collaboration.  The Biophysical Society has always been an international, global organization, representing incredible breadth and diversity of scientific areas, as well as diversity in every demographic aspect possible. I cannot stress enough that the Society has always been committed to inclusivity, respect of others and selves, and collaboration among disparate groups.  This will never, never, never change. We appreciate our members as scientists and human beings, and we appreciate what every member brings to the Society.

And now more than ever we as biophysicists need to engage in civil discourse to show how working together creates a better future for all.  We need to reach out to non-scientists and speak to them as fellow citizens in understandable terms so that they can appreciate the implications of the science policy decisions they make through their votes.  We need to work at the grassroots level within our own communities to make everyone understand that scientific research leads to better jobs, to a better environment, to healthier lives, to more prosperity.

This is not the time to isolate ourselves from one another, but quite the opposite.  It is a time to work together to use what we know best to help educate those around the world about the hope that science research brings to all. It is a time to come together and continue our tradition of respect and inclusivity.

Suzanne Scarlata
Biophysical Society President

Toward Modeling More Realistic Cell Geometries

BPJ_COVER-1Nowadays, computer-driven numerical simulation is becoming increasingly popular as an integral part of all areas of research and engineering. Simulations can often be performed faster, cheaper, and in a potentially safer manner than benchtop experiments. Describing the real-world with systems of equations also helps us to verify the existence of different phenomena, predict future behavior, and discover new idiosyncrasies in complex systems.

In our work, we study how biological cells respond to externally-applied electric fields using numerical simulations based on the finite element method (FEM) for the specific purpose of cancer treatment. FEMs are based on the concept of approximating the geometry of a real-world problem using smaller two-dimensional shapes (often triangles or quadrilaterals) or three-dimensional solids (often tetrahedra). A complex global problem may be reduced to a number of local problems on each of these smaller geometries—called elements—and each of these elements is related to those surrounding it, forming a mesh of elements representing the global geometry and a large system of coupled equations. For the specific application reported here, the geometries of biological cells are described in two dimensions using triangles that are refined around high-aspect ratio features, such as at the cell and nuclear membranes. The cells in our simulation need to be described mathematically as accurately as possible, to render appropriate spatial, temporal, and physical characteristics. Typically, many simplifications are made to a cell’s morphology in order to more easily represent it computationally, which include simple shapes in the form of circles/spheres and ellipses/ellipsoids and a limited amount of physical phenomena. However, biological cells are complex structures with ever-changing geometry and physics that span many length and time scales. To truly capture and explore physical phenomena in such a system requires accounting for more of the complexities of a biological cell, such as its irregular geometry.

Our cover image for the November 15 issue of Biophysical Journal was created during a project where we showed the importance of using realistic cell shapes when studying electric field exposure. We used fluorescence microscopy images to extract realistic cell shapes and convert them into a two-dimensional numerical model. The image shows a triangular FEM mesh, fit to the cell boundaries, where each system of equations is generated on each triangular element that describes their relation to other neighboring elements. Each element is also assigned material parameters, such as electrical conductivity and permittivity. The cell and nuclear membrane boundaries are discretized using numerous boundary nodes to resolve the irregular geometry and minimize the numerical error introduced into the calculation, while retaining sufficient solution efficiency. Conversely, fewer nodes are needed in the middle of the nuclear regions for example, where material boundaries are not present. The above mesh consists of almost one million triangles in a rectangular computational domain that measures around 200 microns across. The image is a zoom of a large model system with realistic cell geometries, containing 91 tightly packed cells and nuclei. The nuclei were highlighted in blue during the assignment of parameters to the nuclear regions in the software used. A glowing effect was added during post-processing for a bit of a dramatic flare.

These types of simulations are not just for research purposes though. Historically, we have utilized robust FEM models to predict how electric currents will propagate in a tissue. Such models are also utilized during treatment planning for tumor ablation procedures in which a series of electrical pulses are delivered to the tumor via two or more electrodes to maximally destroy malignant cells while preserving critical stromal components, such as vasculature. More realistic models of biological cells allows treatments to be delivered to the appropriate malignant region and further limit the damage to the surrounding healthy tissue.

More simulations like this are sure to follow that include detailed geometries, extend these and similar models to three dimensions, and account for additional relevant biophysical phenomena. Though simplifications must be made to any simulation, the trend of increasing computer power and performance enables many of these inhibitions to be overcome by simulations that achieve a greater predictive capacity and come ever closer to simulating precise experimental and clinical conditions.

—Tomo Murovec and Daniel C. Sweeney

Using Biophysics to Understand Diabetes

November is National Diabetes Month in the United States. Twenty-nine million people in the US live with diabetes. To recognize this awareness month, we spoke with BPS member Roger Cooke, University of California, San Francisco, about his biophysics research related to the disease.

figure-cooke

This cartoon shows the 3 states of myosin. In the active state the myosin head is attached to the actin filament producing force and motility. In the super relaxed state, shown above, myosin heads are bound to the core of the thick filament, where they have a very low ATPase activity. In the disordered relaxed state myosin heads extend away from the core of the thick filament where they have a much higher ATPase activity and are available for binding to actin.

What is the connection between your research and diabetes?

Our laboratory has studied the physiology and biophysics of skeletal muscle for many decades. Recently we have concentrated on the metabolic rate of resting skeletal muscle. Skeletal muscle plays a major role in diabetes as it is the organ response for metabolizing a large fraction of the carbohydrate that we consume. Recently we have discovered a mechanism, which we believe can be manipulated to up regulate the metabolic rate of resting muscle, thus metabolizing more carbohydrate. This would be particularly helpful in Type 2 diabetes.

Why is your research important to those concerned about diabetes?

Type 2 diabetes is thought to be caused by or an overconsumption of carbohydrate coupled with a sedentary lifestyle that does not need the carbohydrate as fuel.  The excess carbohydrate leads to high levels of serum glucose. Our laboratory has focused on the motor protein myosin, which is responsible for producing the force of active muscle and also responsible for using much of the energy ingested in the form of lipids and carbohydrates. We have shown that myosin in resting muscle has 2 states with vastly different functions and metabolic rates. In one of these, the super relaxed state, the myosin is bound to the core of the thick filament where its metabolic rate is inhibited, See Figure.  In the other, the disordered relaxed state, the myosin is free to move about and its metabolic rate is more than10 fold higher.  By analogy with another motor, myosin in active muscles is akin to a car racing down the road. Myosin in the disordered relaxed state is similar to a car stopped at a traffic light with the motor idling, and the counterpart of the super relaxed state is a car parked beside the road with the motor off.

For energy economy in resting muscle most of our myosins are in the super relaxed state. If all of these myosins were transferred out of the super relaxed state into the disordered relaxed state they would consume an additional 1000 kilo calories a day. This is a large fraction of the standard daily consumption, which is approximately 2000 kilo calories a day.  Thus a pharmaceutical that destabilized the super relaxed state would lead to the metabolism of a greater amount of carbohydrate providing an effective therapy for Type 2 diabetes. Such a pharmaceutical would address one of the fundamental problems in Type 2 diabetes the consumption of more carbohydrates than are required as fuel.

How did you get into this area of research?

In 1978 a group in England showed that purified myosin in a test tube had a much greater activity than it has in living fibers. This observation showed that myosin in vivo spent much of its time in a state that had a very low metabolic rate. I felt that this inhibited state of myosin could have important consequences for resting muscle and whole body metabolic rates. Although we studied this problem for a number of years, we were not able to find an in vitro system that replicates the in vivo activity. In 2009 we started using quantitative epi-fluorescence spectroscopy to measure single nucleotide turnovers in relaxed skinned muscle fibers, and finally we were able to observe the elusive inhibited state of myosin, the super relaxed state.  This ability allowed us to now study the properties of this state.

How long have you been working on it?

I have been interested in this problem since the original observation in 1978, described above. However it was not until 2009, and the discovery of the in vitro assays, which allowed us to observe the super relaxed state, that this project became the central focus of our laboratory.

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

Our work has been funded by the National Institutes of Health.

Have you had any surprise findings thus far?

The Holy Grail in this area of research is to find pharmaceuticals that will destabilize the super relaxed state. Recently we were able to devise new methods of measuring the population of the super relaxed state, methods that were amenable to use in high throughput screens. We screened over 2000 compounds looking for ones that destabilized the super relaxed state.   We found only one compound that did so, a compound named piperine, which provides the pungent taste in black pepper. After working for over a year developing assays and running the screen, to our surprise the one molecule we discovered was already known to mitigate Type 2 diabetes in rodents. Although piperine lowered serum glucose, no one knew how it did this. We propose that piperine acts by destabilizing the super relaxed state, thus up-regulating the metabolic rate of resting skeletal muscles. We showed that piperine had no effect on active muscle and no affect in cardiac muscle, both desirable qualities to have in a pharmaceutical targeting resting skeletal muscle to treat Type 2 diabetes. Although piperine is effective in lowering blood glucose in rodents, it only does so at very high doses, too high to be useful as a therapeutic in humans.  We now need to find molecules whose action is similar to piperine, but which bind with greater affinity.

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

Our results provide the proof of concept that pharmaceuticals targeting resting muscle metabolic rate, can be found, using the high throughput screens we developed.  These new pharmaceuticals have the potential of more effectively treating Type 2 diabetes.

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

Type 2 diabetes is a growing problem worldwide. Almost 10% of the US public has Type 2 diabetes.  Our research holds out the hope that new pharmaceuticals will be found to treat this disorder more effectively than those available today.

Our studies have also shown that the super relaxed state is destabilized when muscles are activated, and that it will remain destabilized for a number of minutes afterwards, due to phosphorylation of myosin. This extended period of destabilization adds to the metabolic cost of activity, particularly during light and intermittent activities. In fact a number of studies have shown that even modest and intermittent activity will improve serum glucose, help prevent weight gain and lead to better health. The worst thing that people can do is to sit for extended periods of in front of a computer or TV screen. For example when working at a computer I get up and walk around the room every 10 minutes or so, and I avoid elevators, taking the stairs to my laboratory on the 4th floor.

Be a Voice for Science in Washington, Apply to be the Next BPS Congressional Fellow

 

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Randy Wadkins, BPS’s 2016-17 Congressional Fellow, in the Halls of Congress.

With the election two weeks away in the United States, it is almost impossible to miss talk of politics.  Even if it is missing from the public discussion at the moment, what these politicians are tasked with doing when they are in office is set policy.  While the public and the mainstream media aren’t focused on science policy, it is something that elected leaders have to consider.  So, where will these elected Senators and House members, most who don’t have a science background, get their information?

From their staff.

And the Biophysical Society want to make sure that scientists are among those staff members.

 The Society is currently accepting applications for the 2017-2018 BPS Congressional fellowship.  The individual selected for the Fellowship will spend a year working in a Capitol Hill office advising the senator or congressman for whom they are working on science-related issues.

The BPS fellow will be one of 30+ AAAS Congressional Science and Technology Policy Fellows.  The AAAS Fellowship program has been bringing scientists to Washington DC to work both on Capitol Hill and in federal agencies for 43 years.  The purpose of the program is two-fold:  1) provide scientific expertise to policymakers and 2) have scientists understand the policy making process.

Worried you wouldn’t have a clue what to do in a congressional office? The AAAS has that covered. The program kicks off each September with two weeks of intense training on how the government operates, who the players are, and what your roll will be.  The program also guides you through the process of finding a placement for your fellowship.  The training continues throughout the year.  In addition, each cohort of fellows usually form a pretty tight bond.

This is a very unique opportunity, open to BPS members that hold a terminal degree (PhD, MD). Fellows could have just graduated, or have 20 years in the lab under their belt.  Individuals that have completed the AAAS fellowship have found the experience to be professionally rewarding—whether they have chosen to return to bench science or use their science knowledge in other fields.

Have we peaked your interest?  Learn more about the BPS fellowship and the AAAS Science and Technology Policy Fellows.  Applications are due December 15!