Controlled electroporation of the blood-brain barrier

BPJ_110_2_3C_v2Brain endothelial cells are the major constituent of the blood-brain barrier (BBB) and permeability is the key to successful drug delivery across the BBB. The BBB regulates the transport of different substances from blood to the brain by moving through the cell (transcellular transport) or between the cells (paracellular transport). However, it is considered an obstacle to efficient delivery of drugs that target the central nervous system.

Pulsed electric fields are one of several methods that have the potential to temporarily make the BBB permeable when substances are traveling through the transcellular or paracellular pathways.  The electroporation phenomenon is mainly responsible for opening the cell membrane and enhancing the transcellular pathway across the BBB. Therefore, in order to get a more accurate picture of this transport phenomenon, it is necessary to investigate the effect of electropermeabilization on the endothelial cell monolayer of the BBB.

For transcellular transport to occur, molecules are first absorbed through the apical side of the cell membrane into the cytoplasm, and then they pass through to the basolateral side. Therefore, monitoring cellular absorbtion provides insight into possible enhancement of transcellular transport for different substances and can help us determine whether or not a certain treatment is effective in transporting drugs across the BBB.

The cover image shows the permeabilization of the brain endothelial cell monolayer at different sections of the tapered microfluidic channel, which is implemented in this study as a platform for delivering the pulsed electric fields. The electric pulses were applied at the channel ends, making a gradient of electric field across the channel which is inversely proportional to the channel width. Therefore the electropermeabilization could be monitored for a range of electric field magnitudes in a single experiment. The cells were initially stained in green using calcein AM and then pulsed in the presence of propidium iodide (PI), which is naturally impermeable to the cells. Upon entering the cell, PI stains the cell nuclei red, making it a valid probe for monitoring absorption. The images are taken using an inverted fluorescent microscope with two filters which enable the visualization of red and green stains.

The results from varying pulse strengths and the number of applied pulses may provide information that can be used to find the proper parameters to enhance transcellular transport across the BBB using pulsed electric fields without causing any permanent damage.

– Mohammad Bonakdar, Elisa Wasson, Yong Woo Lee and Rafael Davalos

Biomolecular Dynamics, an Historical Account

Cover 110-1We are changing every second of every day. We travel around and between our offices and homes, and move our bodies to perform tasks. The proteins and other biomolecules that comprise us are no different. For us, our relevant timescales range from milliseconds to many years and we are conscious of experiences on these timescales. Biomolecules move around and perform their functions on timescales that are generally much faster, occurring between femtoseconds and seconds. To study motions of biomolecules on these timescales is a considerable challenge, but advances over the last 40 years have bought us to the point that we now can start to decipher the atomic goings on of biomolecules on their relevant timescales. In this issue we present an historical perspective on one of the early papers in our field by Lipari and Szabo, which was published in the Biophysical Journal in 1980. This work laid the foundation for the study of internal motions of biomolecules using the model-free approach that is now frequently used to analyze relaxation data measured by nuclear magnetic resonance spectroscopy (NMR) and fluorescence anisotropy. Lipari and Szabo demonstrated how to study the internal dynamics of molecules, where overall rigid body rotational diffusion is separated from the more local internal motions which are described by the order parameter S.

The cover image for this issue of Biophysical Journal includes one of the original figures from the 1980 Lipari and Szabo paper (center) and is complemented by representations of the protein myoglobin, with cones representing the amplitude of internal motion of methyl groups in the interior of the molecule, as inferred from the temperature factors obtained for a room-temperature crystal structure. In representing the molecule within the glassy sphere similar to a marble, we try to capture the concept of the rigid body rotation of the molecule, while with the pink cones we communicate the approximate degree of motion for the atoms. By determining the amplitude of motion in biomolecules on the timescales of ps-ns we discover the degree of flexibility of these entities and try to relate the degree of motion on these timescales to biological function.

The original paper from Lipari and Szabo was followed by two papers by the same authors that described the details of the model free approach. Since then, order parameters have been determined for many different backbone and side chain atoms of various proteins. These studies have illuminated the dynamic nature of biomolecules, which we continue to study at ever increasing spatial and temporal resolution.

– R. Bryn Fenwick and H. Jane Dyson

Get to Know: Paul Axelsen, BPS Treasurer

We recently spoke with Biophysical Society Treasurer Paul Axelsen, University of Pennsylvania, about who he admires, why he appreciates serving as treasurer, and what he loves about being a pilot.

Axelsen, Paul - PHOTOWhat is your current position & area of research?

I am in the Department of Pharmacology at Penn with secondary appointments in the Department of Biochemistry and Biophysics, and in the Department of Medicine.

Everyone in the lab, in some way, studies the problem of amyloidogenesis in Alzheimer’s disease, which we suspect may result from protein-lipid interactions rendered pathological by oxidative stress.

What drew you to a career as a biophysicist?

The enthusiasm of my pre-doctoral and postdoctoral mentors for the field!  Before I was in any position to judge for myself, they held up the Biophysical Society, its Journal, and its Annual Meeting as a model for how science should be done at the highest levels.

What do you find unique or special about BPS?  What have you enjoyed about serving as treasurer?

Without question, the answer to both questions is: the people involved in leadership.  Becoming involved in BPS committees and Council greatly expanded the number of people I knew outside my field, and broadened my perspective on science.

Who do you admire and why?

That generation of scientists – now largely gone – who stayed “hands-on” in the lab throughout their careers, who made their own reagents and instruments, and who can be credited with creating the modern popular expectation that basic science can solve practical problems.

 

P1070213a

The Wright Brothers Memorial in Kitty Hawk, North Carolina is visible on a hill behind the airstrip where the Wright brothers first flew. 112 years later, Paul made it there in about 2 hours from Philadelphia.

 

I hear you are a pilot. What made you want to fly? What do you love about it?

Powered flight is one of the greatest human inventions of the past few hundred years, and many aspects of flight just cannot be experienced by watching videos or even IMAX films.  It is also an outstanding example of how government, private enterprise, and individuals can organize to create an extraordinarily safe transportation system.  As with the BPS, it is an extraordinary privilege to be a part of such an organization.

What is something BPS members would be surprised to learn about you?

I vaguely recall having worked as a professional musician throughout most of the ‘70s.

Science Fares Well in FY 2016

On December 18, Congress passed a $1.5 billion omnibus spending bill that funds the government through September 1, 2016. The bill increases funding for science at several federal agencies, which was made possible by the budget deal in late October that provided relief to sequestration for the discretionary parts of the federal budget (this includes all research programs).

With the budget settled, agencies can now move forward conducting business and making grant awards with the knowledge of how much money they have for the year. The chart below shows information for several programs and agencies of interest to the biophysics community.

Federal Funding for Science Agencies (in millions)

Agency

FY 2015 Enacted Level

FY 2016 Enacted Level

Difference between FY 15 and FY 16 Percent change between FY 15 and FY 16

National Institutes of Health

$30,073 $32,100  

$2,000

 

6.6%

National Science Foundation

$7,344 $7,460 $120 1.6%

Department of Energy Office of Science

$5,067 $5,350 $279

5.6%

NASA Science

$5,245

$5,589 $344

6.6%

NIST Science and Tech Laboratories

$676

$690 $755

2.1%

Department of Defense Basic Research $2,278 $2,309 $31.5

1.4%

Veteran’s Affairs Medical and Prosthetic Research $588.9 $631 $42.1

7.1%

And, here are a few notes:

  • The $2 billion increase for NIH is the largest increase for the agency since 2003. This is a huge win for the biomedical community! Within that amount $200 million is designated for the Precision Medicine Initiative (PMI); $936 million for Alzheimer’s disease research (which is a $350 million increase); $150 million for the BRAIN Initiative (an increase of $85 million); and $100 million to National Institute for Allergy and Infectious Diseases (NIAID) for antimicrobial resistance research.
  • Within the NSF budget, “Research and Related Activities”receives a $100 million increase over FY 2015; this is the account that from which funding for research grants comes.  The “Major Research Equipment and Facilities Construction” line decreased $45 million from FY 2015.
  • The language that appeared in a House appropriations bill for NSF earlier in the year and that would have decimated the Social and Behavioral Sciences (SBE) and the Geosciences Directorates was removed.  Instead, included language states that SBE should be funded at no more than the FY 2015 level. The House Science Commitee and NSF have been in an elongated battle over how NSF selects grants.  NSF must continue to certify that all awards are in “the national interest.”

With 2016 wrapped up, its time to start the process for funding the government for 2017. The Administration  have been working on their proposals since the summer. The process should be made a bit easier by the budget deal that was struck in October; it created a top line number for both 2016 and 2017.  The President will  lay out his vision for his last year in office during the State of the Union on January 12.  He typically sends his budget request to Congress the first week of February.

Two phases on two faces

109-11 Cover ImageThis image of domains on a lipid vesicle was captured by Matthew Blosser, who is now an NIH NRSA fellow at the University of Oxford. In the image, the lipids within the membrane of a ~70 micrometer giant unilamellar vesicle have demixed into coexisting liquid phases. The bright domains are the liquid-disordered phase, which are diffusing in a background of dark, liquid-ordered phase.  The image was obtained with an epifluorescence microscope using a 40x air objective. The spherical vesicle was imaged at several different focal planes, and these images were then projected onto one 2-dimensional image.

One striking aspect of our image is how cleanly the vesicle divides into regions of light and dark. All of the have nearly the same brightness (accounting for geometric effects), and each individual domain has a single, uniform brightness. This means that, although this vesicle is made of several different lipid components, only two phases are present within the membrane, each with well-defined physical properties. It also means that the domains on each face of the membrane are perfectly aligned with each other, at least on length scales observable by fluorescence microscopy. As a result, the two monolayer leaflets of the membrane must be coupled.  This result is somewhat non-intuitive because interactions between the two leaflets are at least partially mediated by the lipid acyl chains, which are floppy.

We were inspired by our collaborator Aurelia Honerkamp-Smith and by other researchers to measure this effect. Specifically, we measured the energy penalty a vesicle would have to pay in order to misalign the domains on each face of the membrane. Because we knew that domains did not misalign on micron length scales of their own accord, we decided to give a push to the domains on only one side of the membrane.  Because that push had to be a very strong one, we built a microfluidic chamber into which we flowed vesicles, which burst on the solid support. Flowing water through the chamber moved the upper monolayer of the membrane over the lower monolayer, which was firmly stuck to the substrate. By measuring how hard we needed to push an individual domain to make it move (and by collaborating with Mikko Haataja and Tao Han, who produced some beautiful theoretical work), we extracted a value for the interleaflet coupling. The value we found agrees with a previous theoretical prediction (there were no previous measurements to our knowledge), and it could help explain how changes in lipid organization are communicated across cell membranes. More information about our research is found in our article in the new issue of Biophysical Journal and at http://faculty.washington.edu/slkeller/.

-Matthew Blosser, Aurelia Honerkamp-Smith, Tao Han, Mikko Haataja and Sarah Keller

What’s Going on With the Federal Budget?

budget3It’s Thanksgiving Week and Congress is in recess.  Perhaps the last you heard about the federal budget for FY 2016 was that there was a bipartisan deal at the end of October.  Sounded like a good outcome.  That is true, but that deal didn’t actually provide funding for the coming year; it just increased the amount of money that could be spent.  Congress has until December 11 to figure out how it is going to divide up those additional dollars and pass a bill to fund the government for the coming year.  So why haven’t you heard much?

After the October budget deal, Congress began working behind closed doors on how to appropriate the additional dollars. The appropriations chairmen let their subcommittees know how much money they had to divide up among the programs for which they were responsible. These numbers were not made public. The subcommittees were supposed to send their proposals back to the Congressional leadership by November 20.  It is rumored that the conversations have not only focused on dollar amounts for each programs, but also on what policy riders will be included in the final bill.  Policy riders are directives that require certain actions or disallow certain actions by federal agencies.  The Democrats prefer a spending bill without riders; Republicans are pushing to include riders that reflect their priorities.  An example of a potential policy rider that affects scientists would be one that would require the National Science Foundation to certify that all funded grants represent research that is the national interest by making the U.S. more secure or improving the economy.   (This rider was in a spending bill approved by the House earlier this year, and could end up part of the ominbus bill currently being worked on.)

The rumors are that Congress will release an omnibus bill funding all federal agencies and programs on December 1, at which time we will be able to see how the agencies we care about have fared.  It is expected that the next ten days will be spent working out the riders and final numbers.

What has BPS been up to?

While the Hill has not been forthcoming with information during the past month, the Society has remained active in advocating for science funding in the final bill.  When the budget deal was reached, the Society sent a thank you letter to the White House and Congressional Leaders.  The Society has also sent communications to the Hill as a member of several coalitions in which it participates.  Many of these groups are also working on FY 2017 funding; a letter as sent by a coalition of coalitions, in support of raising science funding 5.2% across the board in 2017.

What can you do?

BPS has also been encouraging members to get involved.  A call for members to write to their Senators and Representative to thank them for the budget deal and advocate for science in FY 2016 went out to all U.S. members in early November. Thus far, 54 advocates have sent 166 letters.  If you haven’t written yet you can do so here.

Enjoy the quiet of Thanksgiving Week and stay tuned for more budget news in early December!

 

Dear Molly Cule: How do I staff my lab?

Professor Molly Cule is delighted to receive comments on her answers and (anonymized) questions at mollycule@biophysics.org, or visit her on the BPS Blog.molly_cule_blog1

I’m a new PI. How do I go about staffing my lab?

First, congratulations on becoming a principle investigator! Now how do you make your laboratory successful and productive?  Many resources exist to help get you started, one of which is a guide to scientific management called Making the Right Moves.  This guide was developed by the Burroughs Wellcome Fund and the Howard Hughes Medical Institute, and can be downloaded as a PDF from the HHMI website that provides resources to early career scientists: http://www.hhmi.org/programs/resources-early-career-scientist-development/making-right-moves. A full chapter of the guide focuses on staffing the laboratory, as well as managing a laboratory and developing a vision for your laboratory. Take advantage of this helpful resource.

An important step towards staffing the laboratory is considering what type of laboratory you want to run, which may be highly dependent upon your institution and startup package. As an example, there are big differences between the type of laboratory and laboratory personnel at a liberal arts college, a mid-sized research university, and a large medical school. This is where your vison for your laboratory comes in to play.  A helpful exercise to establish this vision is to look around your department and institution and observe the types of laboratories that are successful, but also to recognize that it takes time to build a successful laboratory. In generating the vision for your laboratory, you must weigh the costs and benefits of hiring a technician vs. recruiting a postdoc or recruiting undergraduate vs. graduate students to your laboratory. These costs and benefits do include monetary costs and benefit packages, but they also include differences in scientific acumen, capacity to work independently, and expected productivity. It is also important to recognize that technicians and postdocs are employees, but students are not. There are some subtle details that you will have to learn about related to these differences, but your departmental business manager or chair is usually a good resource for understanding these differences at your institution.

When I started my own laboratory, I thought the best place to start hiring was with a postdoc or lab technician. I wanted to hire a person with some knowledge of research, who would need minimal training, and ultimately be able to help get my lab up and running as quickly as possible. Next, I chose to proceed by acquiring students, who require more training. Do not be afraid to be picky about who joins your laboratory, it is okay to tell a student that he/she cannot join the lab. Although saying “no” can be difficult, it is necessary. Focus on quality, not quantity, in your hiring, particularly when you are just starting out.

Now that you’ve established where you want the laboratory to go and what types of people you want to have in the laboratory, you need to go out and get them.  You will need to create a job description that you can distribute on the human resources site at your institution, on the website for your laboratory, on  email list serves, and on job boards hosted by scientific societies to which you belong. It is very important to write a job description that attracts the specific skill set that you need regarding techniques that will be required, areas of research that you study, any minimum requirements that will be required for the level of the position, etc.

Once you have a set of applications, you will need to select candidates to interview  The interview is an important part of the hiring process, because you will want to determine the quality and ‘fit’ of an individual with your particular laboratory. Spend time generating a list of questions to ask during the interview. Think about why you are asking these questions, and be able to articulate (in your head or out loud) how and why the candidates’ answers to these  questions are important to the future success of your laboratory. Be aware of any red flags that suggest a person may not be a good fit for the position. For me, personality and ease of engagement between a perspective member of my laboratory and me are critical components of the interview process. You may have the most qualified candidate on earth, but if you and that person cannot easily communicate or get along, the working relationship will suffer. Remember, it is your laboratory and you need to assemble the best, most productive team possible to achieve the scientific vision that you’ve set out for your laboratory.

Once you’ve determined who would be the best person to hire, you will have to make an offer.  Many of the details related to these offers are less flexible that you may think, particularly when starting up a new laboratory. The pay scale may be dictated by the institution or tied to an offer letter related to your startup package. Hopefully these details won’t get in the way of you hiring the best person for the job, but you may want to investigate these details at the start of your hiring process, when you are drafting the job description

Good luck in staffing your laboratory,

Molly Cule