Using Biophysics to Understand the Basis of Allosteric Inhibition in the Hepatitis C Virus Polymerase

July 28 is World Hepatitis Day, drawing attention to the 240 million people chronically infected with the hepatitis B virus and 150 million people with the hepatitis C virus. To recognize this worldwide awareness day, BPS asked member Ian Thorpe, Assistant Professor of Chemistry and Biochemistry at the University of Maryland Baltimore County, to answer a few questions about his research on hepatitis C.

HCV blog

What is the connection between your research and hepatitis?

We study the RNA polymerase responsible for replicating the Hepatitis C virus (HCV) genome. There is no vaccine for HCV infection and this enzyme has been extensively targeted with the goal of developing HCV therapeutics. One reason for this is the crucial role the enzyme plays in generating new viral particles. In addition, because it is an RNA-dependent RNA polymerase, there is no homologous human version of this enzyme. Thus, if this enzyme is targeted there should be a decreased likelihood of generating side effects from impacting human proteins. The search for therapeutics has yielded many small molecule inhibitors of the polymerase, including allosteric inhibitors that bind distal to the active site. We employ molecular modeling and simulation to understand the physical processes that underlie this allosteric regulation.

Why is your research important to those concerned about these diseases?

Understanding how allosteric inhibitors modulate the function of the HCV polymerase provides insight into what intrinsic properties of the enzyme allow it to effectively replicate viral RNA. In addition, understanding the underlying molecular mechanisms involved may allow for the development of novel and more effective inhibitors. While there are treatments currently available to treat this disease, these are generally expensive, can require significant time investments and often have serious side effects. In addition, the HCV RNA polymerase does not contain proofreading ability. The resulting high error rate during replication induces an elevated incidence of mutations in the virus and facilitates the development of viral resistance to treatment regimens. Thus, there is a continuing need to identify new molecules that could serve as HCV therapeutics.

How did you get into this area of research?

During my postdoctoral position at the University of Utah with Professor Gregory Voth (now at the University of Chicago), I began to consider research projects that I could pursue during my independent career. My wife worked in the field of liver transplantation at the time. She was familiar with HCV because it is one of the leading reasons for liver transplantation in the United States. She also recognized the need for additional studies of HCV due to the limited treatment options and extensive gaps in our knowledge of how the virus functions. HCV infection is a burgeoning health crisis because the virus was only conclusively identified within the last thirty years and many people were likely infected (for example via blood transfusions) before a test became available to screen for HCV infection. Those who are infected often go decades without displaying symptoms, only to be diagnosed later with serious complications including cirrhosis and liver cancer. Thus, a large fraction of the population who were infected in the past may be asymptomatic and are only now starting to display symptoms (or will do so in the near future).

How long have you been working on it?

I began working in this field after becoming a faculty member in the Department of Chemistry and Biochemistry at the University of Maryland, Baltimore County in 2009.

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

I do not currently receive federal funding for this work. However, I do receive federal support in the form of compute time on supercomputing resources supplied by the National Science Foundation via the Extreme Science and Engineering Discovery Environment (XSEDE).

Have you had any surprise findings thus far?

Yes, several! One of the key features of the HCV polymerase is that it likely undergoes transitions to distinct conformational states as it replicates RNA. These include a closed state required for the initiation of replication and an open state associated with elongation of the newly synthesized RNA strand. We have discovered that components of the polymerase may have a regulatory function by restricting the conformational sampling of the enzyme. In addition, we have seen that while diverse allosteric inhibitors can induce distinct effects on conformational sampling and enzyme dynamics, shared characteristics exist that may underlie the inhibitory action of these small molecules. Specifically, most inhibitors we have studied disrupt the conformational sampling of the enzyme in ways that can be related to their inhibitory capability. Some discourage conformational transitions by overly stabilizing one or the other conformational state, while others may destabilize both states to the extent that neither can be stably occupied. Finally, we have observed that free enzyme is able to explore both the open and closed states thought to have functional roles in RNA replication. This result is unanticipated given that the free enzyme lacks other components of the replication complex such as RNA template or nucleotides. This observation suggests that the presence of ligands does not engender new enzyme conformational states, but instead shifts the populations of preexisting enzyme conformations. Such a phenomenon is consistent with the conformational selection model of allostery.

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

Our studies highlight mechanisms of allosteric regulation that provide insight into how allosteric inhibitors decrease enzyme activity and suggest experiments that can be carried out to validate our hypotheses. Understanding the molecular mechanisms of allosteric inhibition is important in the development of HCV therapeutics because this knowledge may allow the discovery of new and more effective inhibitors or new ways of using existing inhibitors, such as in novel combination therapies. These studies also illuminate the fundamental processes involved in RNA replication by viral polymerases. HCV is related to several viruses that are serious human or agricultural pathogens, including the viruses that cause Dengue Fever, Yellow Fever, West Nile disease and Bovine viral diarrhea. Thus, knowledge gained from studying the HCV polymerase may be applicable to polymerases from these related viruses as well. More generally, our research helps to elucidate the many ways that allosteric regulation of enzyme function can occur. Allosteric regulation is a key way in which protein function can be modulated in biological systems. Thus, better comprehending the underpinnings of allosteric regulation may be applicable in diverse contexts such as in determining the molecular origin of disease states or in discovering drugs to treat other ailments.

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

While this work is fundamentally basic science research, the knowledge we obtain may ultimately be useful in identifying novel and more effective treatment options for infection by HCV and related viruses.

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Learning from Industrial Expertise: A Change in Perspective  

By Cecilia Read, Summer Research Program in Biophysics 2014 participant

When I think of industry, I think of cubicles and an endless amount of paperwork that consumes any livelihood you might have had at the beginning of the work day.  As an undergraduate in the field of biomedical engineering, industry is a very real possibility for me after college.  However, the mental image I’ve developed of industry since entering my field of interest has pushed me from even considering it as an option after I graduate; I would rather continue in academia.

My view on industry changed with the visit of Dr. Deborah Thompson and Dr. Drew Applefield, two employees of the Biotechnology Center in North Carolina who came as speakers for the Biophysical Society Summer Research Program I am participating in.  They provided great insight into what to expect if entering industry, the benefits of an academic background in industry (basically having a Ph.D.), and how to prepare an application for a job in industry.  They each talked about their personal backgrounds and finding their passion in industry.  While one was involved with organizing internships and industrial research, the other worked in advising new businesses and individuals looking for employment in industry. They spoke about finding a balance when choosing what field of industry to work in and the importance of recognizing “What I want” versus “What I bring” to the work place.  Each had their own perspective that made the presentation informative and useful.

When talking about what stood out from the presentation, my friend, Amanie Power, another student in our summer program, reminded me of the speakers’ most important advice: be aware of your own transferable skills.  What exactly did they mean by this? Each person has skills that they may not initially view as important for other fields of work.  In actuality, many skills are transferable; as Amanie describes, “skills that are good for anything.”  It’s hard to see one’s own skills as good for everything, but it’s important to recognize and highlight transferable skills, because they can be used for every setting.  My other friend and fellow student in the Biophysical Summer program, Olivia Dickens, described the whole experience in the most direct way possible:  “it was the best speaker presentation we’ve had all summer… they came, they presented, and they left.  They were well prepared and said exactly the right information that was useful for us.”

I still want to pursue a degree after my undergraduate experience, but I am no longer as opposed to industry as I was before.  Both Dr. Thompson and Dr. Applefield did a tremendous job in enlightening my misconceived notions about industry.   Industry is now no longer a negative alternative to academia in my mind; it’s just another option that can lead to future adventures, with its own kind of fulfillment.

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All-Atom Ensemble Modeling of Detergent Association to Membrane Proteins

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Small-angle scattering techniques are low-resolution techniques that can give information on the shape and size of complex systems in solution. When applied to membrane proteins, the contribution to the scattering signal of the detergent molecules present in the sample is significant and needs to be dealt with. This is the case for x-ray scattering but most of the times also for neutron scattering, since the amphiphilic nature of the detergent molecules results in residual scattering contributions even at the contrast variation match point (which, ideally, would mask the scattering contribution of the detergent).

The image on the cover of the July 1, 2014, issue of the Biophysical Journal shows a low-resolution surface representation of the bacterial outer membrane transporter FhaC, which is the first and prototypical structure of the Omp85 superfamily of protein transporters. The protein is surrounded by a belt of all-atom detergent molecules, covering the hydrophobic beta barrel of the protein. Here, only one configuration out of an ensemble of possible detergent arrangements is shown.

In this work, we show that explicit modeling of a protein-detergent complex, combined with small-angle neutron scattering and contrast variation can yield excellent results and provide valuable information on both the protein conformation as well as the organization of the detergent belt.

The flexibility and general applicability of the method make it an extremely powerful and significant tool for the study of membrane proteins, including their conformational span. For more information, do not hesitate to contact any of the authors.

This work was the result of a collaborative effort involving five research teams in France:

Center for Infection and Immunity of Lille:
Bacterial respiratory infections

Interdisciplinary Research Institute, Lille:
Computational molecular systems biology
Transcriptional regulation and mediator

Structural Biology Institute, Grenoble:
Membrane and pathogens

Structural Biology Institute and Institut Laue Langevin, Grenoble:
Extremophiles and large molecular assemblies

- Marc F. Lensink, Frank Gabel, Françoise Jacob-Dubuisson, Bernard Clantin, Vincent Villeret, Christine Ebel

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Using Biophysics to Uncover the Mechanisms of Neuromuscular Degeneration in ALS

The Biophysical Society is an an international society, with over 35% of its members residing outside the United States.  Thus, for this month’s feature on the relationship between biophysics research and disease, we are looking at ALS in honor of ALS Awareness Month in Canada.  Society member Jingsong Zhou, an Associate Professor in the Department of Molecular Biophysics and Physiology at Rush University Medical Center, is working hard in her lab to develop a deep understanding of the disease mechanism in hopes of that understanding contributing to new therapeutics and a better prognosis for those with ALS.

These are images of single muscle cells (“fibers”) derived from an ALS mouse model (G93A). The A panels show absence of electrically well-polarized mitochondria (marked by mitochondrial membrane potential probes, TMRE in green) in the vicinity of the neuromuscular junction (identified by alpha-BTX in red). The B panels show simultaneous imaging of mitochondrial function (marked by TMRE in red) and calcium release activity (indicated by a calcium dye, fluo-4 in blue) of G93A muscle fibers. Lack of TMRE staining identifies fiber segments with defective mitochondria (1). Note that calcium release activity is greater in the segments with defective mitochondria (2). (Zhou et al., JBC, 2010).

What is the connection between your research and ALS?
The goal of my laboratory is to explore the molecular mechanism underlying neuromuscular degeneration during ALS progression. Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease characterized by progressive motor neuron death and skeletal muscle atrophy and paralysis. Most patients die within 5 years of the disease onset. The lifetime risk of ALS is about 1 in 500. Since ALS is an age-dependent disease, as the U.S. population increases and ages, an increase in the prevalence of ALS can be anticipated. Despite intensive research efforts, there is no effective cure for ALS. My laboratory hopes to identify potential therapeutic means to alleviate ALS progression.

Why is your research important to those concerned about these diseases?
During ALS progression, the degeneration of motor neuron limits neuron-to-muscle signaling and leads to severe muscle atrophy, while the retrograde signaling from muscle-to-neuron, which is important for axonal growth and neuromuscular junction maintenance, is also lost in ALS progression. There are research groups that focus on muscle physiology, while others focus on understanding the mechanism of motor neuron degeneration. Taking advantage of working closely with both muscle and neuron groups, we are conducting translational research that bridges studies on the pathophysiology of muscle and motor neurons in ALS. My lab has been developing various genetic mouse models and molecular probes to examine the functional interplay between the neuron and muscle and between the intracellular organelles during the progression of muscle wasting in ALS. The signals reported by those molecular probes represent functional indices of ALS progression. In addition to understand the molecular mechanisms underlying the degeneration of motor neurons and skeletal muscle, our genetic model systems allow us to evaluate whether potential interventions have a beneficial effect on ALS.

How did you get into this area of research?
After graduating from Xiangya Medical College, I found my passion in medical research. Luckily I was accepted by Dr. Eduardo Rios as his first Ph.D. student at Rush University and trained as a muscle physiologist with a focus on understanding how calcium signaling is controlled during muscle contraction. As a core muscle physiologist I have been often asked how I found my career path in ALS research. The collaboration with Dr. Rios led us to examine the role of mitochondria in regulating fast calcium signaling during muscle contraction. My medical training also puts my interests in studying diseases. I wondered the role of mitochondria in regulation of calcium signaling in muscle pathophysiology. Dr. Han-Xiang Deng at Northwestern University is among the scientists who first identified ALS mutations and generated ALS mouse models. We graduated from the same medical school and often had interesting scientific discussions, which inspired me to begin my research in ALS. The mitochondrial defect is a pathologic hallmark in motor neurons during ALS progression. Skeletal muscle comprises around 40% of whole-body lean mass and is substantially affected in ALS. But little is known about the role of mitochondria in muscle degeneration during ALS progression. Using an ALS mouse model provided by Dr. Deng, I initiated my research in ALS by examining mitochondrial function and calcium signaling in skeletal muscle of ALS.

How long have you been working on it?
In 2006, we obtained the first ALS mouse from Dr. Deng. Since then, our efforts never end to understand the mechanisms of neuromuscular degeneration in ALS and to explore potential therapeutic interventions to treat ALS.

What organizations funded your work?
In the past 8 years, my research in ALS was first funded by the Muscular Dystrophy Association USA (MDA) and continuously funded by the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) at NIH.

Have you had any surprise findings thus far?
Most ALS cases are sporadic (SALS), with about 10% being familial (FALS). Both SALS and FALS manifest similar pathological and clinical phenotypes, suggesting that different initiating causes lead to a mechanistically similar neurodegenerative pathway. Mouse models expressing ALS-linked mutations effectively recapitulate many features of the human disease. By studying live skeletal muscle of an ALS mouse model, we have discovered that there are localized mitochondrial defects, which occur near the site of the neuromuscular junction and lead to uncontrolled intracellular calcium transients. This finding opens a new window to understand the role of mitochondria and calcium signaling in the crosstalk between muscle and neuron during ALS progression and to target the neuromuscular junction for developing potential therapeutic means to combat ALS. In addition, mitochondria are dynamic organelles that constantly undergo fusion and fission to maintain their normal functionality. We have found that ALS mutations directly slow down the dynamics of mitochondria in ALS skeletal muscle, suggesting skeletal muscle is also a primary target of ALS mutation. Restoring mitochondrial function in skeletal muscle could provide potentially beneficial effects for alleviating muscle wasting and slowing down disease progression.

What is particularly interesting about the work from the perspective of other researchers?
Our research defines the role of Ca2+ signaling and mitochondrial function in health and diseased skeletal muscle. I am particularly interested in the control of local intracellular Ca2+ signals mediated by the interaction between the SR (sarcoplasmic reticulum) and mitochondria. The mechanisms controlling this signaling represent critical points at which many cellular phenomena (e.g. contraction, secretion, transcription, etc) can be modulated. This work is thus clinically important because it defines potential sites for pathological failure and/or therapeutic intervention in many other diseases.

What is particularly interesting about the work from the perspective of the public?
ALS is an age-dependent fatal disease with no cure. The only FDA-approved treatment for ALS, Riluzole, only extends patients’ life for a few months and has limited efficacy on symptom relief. New treatments are needed for improving the life quality of ALS patients. Relying on deep understanding of the disease mechanism, our research may lead to advances in developing therapeutic interventions.

 

 

 

 

 

 

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Gram-Negative Bacterial Outer Membrane Molecular Complexity

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When many of us think of a biological membrane on the molecular scale we picture a textbook image of a symmetric phospholipid bilayer that looks somewhat uncomplicated. The outer membrane of gram-negative bacteria is anything but symmetric and simple. We wanted the cover image to convey the molecular complexity and asymmetry of the outer membrane that these bacteria have as their first line of defense from their immediate environment.

The cover image for this issue illustrates a typical E. coli outer membrane and the molecular system that we used to represent the complexity in molecular dynamics simulations. In the image the diffuse background molecules represent the bilayer composed of (from the top, external leaflet) glycosylated amphipathic molecules known as lipopolysaccharide (LPS) consisting of an O-antigen polysaccharide (small red and orange spheres), a core oligosaccharide (red and blue spheres), and lipid A (pink spheres) and (the bottom, periplasmic leaflet) various phospholipid molecules (green, orange, and magenta spheres).

The sharply focused atoms in the foreground represent the molecular system we used in our simulations. Here the LPS contains only the core oligosaccharide (white sticks) and lipid A (pink spheres). The cyan atoms interspersed with the core oligosaccharides are calcium atoms, which immobilized the membrane by mediating the cross-linking electrostatic interaction network. The ÿ-barrel protein shown as a yellow ribbon diagram represents outer membrane phospholipase A (OmpLA), a typical integral outer membrane protein. We chose this protein for our simulations because it has been extensively studied experimentally in synthetic phospholipid membranes.

We wanted to investigate the microscopic environment of OmpLA in a native E. coli asymmetric membrane, especially the thickness of the hydrophobic region and the interaction of core oligosaccharides with external loops of the protein. Currently it is not possible to create an experimental asymmetric membrane with the complexity found in nature and in silico studies are the only way to study this system.

Please visit http://im.bioinformatics.ku.edu (Wonpil Im) and http://pages.jh.edu/~fleming/frontpage.html (Karen G. Fleming) for more information on our research interests.

–Emilia L. Wu, Patrick J. Fleming, Min Sun Yeom, Göran Widmalm, Jeffery B. Klauda, Karen G. Fleming, and Wonpil Im

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Biophysics and a Better Understanding of Osteoarthritis

With May being designated Arthritis Awareness Month by the Arthritis Foundation, BPS went searching for a member conducting research related to this debilitating condition.  We were fortunate to find Alan Grodzinsky, Director of the Center for Biomedical Engineering at MIT, whose research group studies problems motivated by diseases of the musculoskeletal system including arthritis, connective tissue pathologies and, more generally, the molecular biology and biophysics of the extracellular matrix.  Grodzinsky  offers us a glimpse of the exciting work going on in his lab related to osteoarthritis.

What is the connection between your research and Arthritis?
We work on biophysical, biological and biochemical aspects of osteoarthritis (OA), a disease that has been estimated to affect over 150 million individuals worldwide. OA is a complex disease caused by a combination of mechanical and biological factors. It is not a single disease, but rather constitutes a heterogeneous combination of subtypes associated with risk factors for initiation and progression that include age, gender, genetic factors, obesity, improper mechanical joint articulation and congenital deformities of joints. Importantly, it is now recognized that OA is not just a disease of old people; traumatic joint injury in young active individuals involving knee ligament and meniscal tears, for example, can lead to OA at a young age. These injuries are especially common in young women of high
school and college age and are known to progress to OA within 10‐15 years. Finally, OA is much more common than rheumatoid arthritis (RA), the latter involving autoimmune pathways distinct from the pathology of OA.

Why is your research important to those concerned about these diseases?
In our lab, we use in vitro models of joint injury involving living cartilage specimens from animal or human donor sources. Cartilage subjected to mechanical injury is co‐cultured with inflammatory cytokines known to be present in human joint synovial fluid during the weeks following a traumatic joint injury. We use these in vitro systems to try to quantify the cell biological/biochemical pathways of cartilage degradation that is a hallmark of progression to OA, pathways that are additionally initiated by mechanical damage to cartilage that can be imposed in a quantitative fashion using incubator‐housed
loading instruments. These instruments also enable us to measure the biophysical changes in cartilage material properties to can occur in vivo. Additionally, these in vitro systems provide invaluable means to study the efficacy of potential drugs that can halt cartilage degradation and progression to OA. We have an active program for the design of nanoparticles that can be functionalized with small molecule or biologic drugs; the nanoparticles are designed to home directly into cartilage (and other adjacent soft
tissues) for drug release inside cartilage after intra‐articular injection, thereby avoiding problematic systemic side effects that may be associated with sustained release of many otherwise suitable drug candidates. Finally, we also have a research program in our group aimed at the repair of cartilage defects which, if not otherwise treated, could rapidly lead to OA and joint failure. These studies involve the use of a functionalized self‐assembling peptide hydrogel scaffold that will soon be incorporated into animal studies in vivo.

How did you get into this area of research?
As an electrical engineer by training, I spent a year early on in my career on sabbatical at Children’s Hospital in Boston, working with Dr. Mel Glimcher, who was then Chief of Orthopaedic Surgery and a leading basic scientist in bone and cartilage research. Since cartilage is the mostly highly electrically charged tissue in the body (due to the presence of negatively charged proteoglycans (aggrecans) in the tissue matrix), I found that my deep interest in this subject could fit right into an academic career in research and tissue at the interface between engineering and biology. Continuous ongoing collaborations with cell biologists, extracellular matrix biochemists and clinicians have been extraordinarily helpful and exciting.

How long have you been working on it?
There is still no “cure” for OA, and there are currently no disease modifying drugs available (unlike the situation with RA, where new biologic drugs have emerged during the pasts 15 years that help ~65% of the individuals afflicted with RA). As a result, we and many other groups around the world are still working actively in many aspects of this field to try to achieve a better understanding of ways of halting disease progression, regenerating injured cartilage tissue and eventually identifying drugs that can halt
the progression of OA disease. My research group has been involved in various evolving aspects of this research since the late 1970s.

Do you receive federal funding for this work? 
Most of our funding related to cartilage biophysics, OA disease, and cartilage repair has comes from NIH.  We’ve also received important funding over the years from programs within NSF, especially targeted to  the discovery and use of biophysical tools for studies of cartilage and matrix molecular nanomechanics.

Have you had any surprise findings thus far?
Researchers in our group have recently discovered pathways and potential therapeutics that may help to preserve the collagen network of cartilage even after initial loss of other essential matrix components (such as aggrecan). However, delivery of such therapeutics to involved tissues, like cartilage, has not been solved. But students in our group have discovered that highly positively charged nanoparticles may provide an ideal means to home into cartilage with attached drug molecules in tow. So we’re pursuing those studies and also using the same potential therapeutics functionalized to hydrogel scaffolds for
cartilage repair. In addition, Atomic Force Microscopy‐based imaging of cartilage tissue molecules and nanomechanical properties of cells and matrix have opened a window to the study of cartilage repair that we did not anticipate at all.

What is particularly interesting about the work from the perspective of other researchers?
The interdisciplinary nature of this kind research has been one of the most important and exciting features that have been of great interest to a wide variety of researchers in the field.

What is particularly interesting about the work from the perspective of the public?
Of course the hope of this kind of research is that it may lead to advances in diagnostics and patient treatment for OA disease, which has been a major concern of the public for many decades. While OA is not necessarily life‐threatening, it is the major concern for our aging population in terms of quality of life and freedom from pain and disability that is so common as a result of OA.

Do you have a cool image you want to share with the blog post related to this research?

may blog
These are “aggrecan” macromolecules (tapping mode AFM imaging, Laurel Ng et al., J Structural Biology, 2003) which are critically important for the ability of cartilage in our joints to resist static and dynamic loading in our daily activities. These molecules are extremely densely packed inside cartilage and are the first molecules to be degraded and lost from cartilage at the very earliest stages of osteoarthritis. These molecules also regulate the transport of drugs to cells inside dense cartilage tissue, and they are currently the subject of nanomechanical and biophysical studies within our lab as a means to understand the progression of OA and attempts to stop OA.

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Continuing to Bring Together Biophysicists in the Bluegrass State

The third BPS-supported Kentucky Networking Event took place on May 12 at the University of Kentucky. The event, organized by Trevor Creamer; University of Kentucky, focused on molecular biophysics and had over 80 registrants from numerous institutions including the University of Kentucky, University of Louisville, University of Cincinnati, University of Tennessee, IUPUI, Miami University of Ohio, Ohio State University, Northeastern University of Illinois, Transylvania University, Georgetown College, and Wright State University.

Trevor wraps up the event for us:

The number of registrants, the distances many were willing to travel for a one day event and the quality of the talks and posters, indicates that there is a lot of very good molecular biophysics being done on the area. I am surprised every year that people are willing to drive 4-6 hours each way for a one day event like this. I am also pleasantly surprised at the number of people who have attended all three years we have held this symposium. Although many of the attendees have participated in one or both of our previous events, we also draw new people each year. This means that there are plenty of opportunities for people to make new contacts/colleagues/friends. This certainly seemed to be occurring this year. We hope to continue this symposium annually.

Were you at the Kentucky Networking Event? Share your experience in the comments below!

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