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Negotiation strategies for scientists: Preparing your counter offer

You finally did it! You got an interview for the job of your dreams. For years you have been working for this- first, persevering through five years of graduate school while your friends were beginning their careers and making money; then another five years of postdoctoral fellowships where you were not quite a student, but not faculty either.

The interview goes well, and you think this might be it. The chair of the department offers you the position, along with a salary amount, and asks if you think it is adequate. It’s much more than you made as a postdoc, but it’s difficult to know your own self-worth. You may be applying for a position outside of academia or in a slightly different field. In addition, you have been a trainee for such an extended period of your life that it is difficult to toot your own horn. Should you make a counter offer? And if so, how?

Negotiation skills are important for science careers, yet few scientists are trained to in this area. Further, women are less likely to negotiate than men, and receive lower salaries. According to a study at Carnegie Mellon University of graduating masters’ candidate salary offers, men were more likely to negotiate than women. The salary offers for men were 7.6% higher than for women.

At the FASEB Career Center on Saturday, Dr. Debra Behrens gave two sessions on Negotiation Strategies for Scientists. Dr. Behrens, a PhD counselor at the University of California, Berkeley, provided key strategies to determine the appropriate salary for your position and how to leverage your strengths to negotiate for your needs.

Learning to negotiate well has benefits beyond establishing a salary. Preparing to negotiate allows you to evaluate yourself: your financial needs, what you require for professional productivity, and the strengths and abilities that you can provide for the employer. This won’t be the only time you will need negotiation skills in life, so it pays to prepare for the first position. In addition, future salaries are often based on your previous pay.

Dr. Behrens outlined 5 strategies for effective negotiation.

  1. Preparation

Research the position and its typical salary range. For academic positions, you can visit The Chronicle for Higher Education and search by discipline and position for average salaries. AAUP, ACS, Association of American Medical Colleges, and The Scientist also provide salary information. At public institutions, it is a requirement that faculty salaries are made freely available, and can be found on the institution’s website. If you are currently affiliated with a university, you may be able to access their subscription to Vault, a career information website which contains employee surveys and career guides to individual industries.

Salary surveys, as well as federal research are available for private industry positions. Professional organizations are also a good resource for salary information in industry. When searching for salary information, make sure to factor in the region where the position is located, as cost of living will vary across the country.

It is often thought that negotiating your salary is not possible for jobs in the government and public sector. While the salaries for these positions are not as flexible, they may be more negotiable that you would think, as there are differences among agencies. Dr. Behrens shared a pro tip for career advancement in government: when considering your place in career grade and step, it is better to begin at the bottom of the pay grade. Even if the positions are similar in pay, there is more possibility for advancement by increasing your steps within a pay grade than to level up from on pay grade to the next.

During the interview, you may be asked what you would expect for a salary before you have actually been offered the job. It is in your best interest to wait to answer this type of question until you have an actual offer, when you have the most power. If you accept a salary offer during the interview, it locks you in early before you have a full understanding of what you can leverage during the negotiation process.

If they ask you during the interview what you would expect for salary before stating an offer, it is to your best advantage to wait until you have an actual offer. You could respond by saying, “At this point, I’m focused on learning more about the position and telling you about what I have to offer first before discussing salary.”

  1. Effective listening

During your interview, be observant for cues about the needs of the department or company. Consider how you can fill those gaps and use that knowledge as a leveraging tool during the negotiation period. In addition, pay attention to facial cues during your interview. If someone looks confused, it is better to stop and ask if you need to elaborate than run the risk that your future employers do not know your strengths and what you require from them for success.

  1. Big picture perspective

It is important to create a list of the must-haves and deal breakers when negotiating a job offer. However, it is also just as critical to keep the grand scheme of things in mind. What are the things you are willing to concede in order to achieve a higher goal? Dr. Behrens cited a concept called “firm flexibility” in which you remain firm about your goals, but flexible about the way you achieve them (Fisher & Ury, Getting to Yes 1991).

  1. Interactive exchange

Treat the job negotiation as an interactive, not a static exchange. In other words, it’s a lot of give and take. First, developing a rapport with your employer during the interview will build trust that you can utilize during the negotiation process. Second, it’s ok to ask questions such as, “What would it take to resolve X?” “Would it be feasible to do Y?” or “Under what circumstances might you consider Z?” By keeping the exchange interactive, you are able to gain key insight into the rationale behind your job offer and what accommodations are actually possible. Other valuable questions include, “What issues do you foresee with this request?” and, “What might be other ways we could go about this?”

  1. Win-win outcome

Finally, focus on a win-win outcome. Both sides of a job offer have the same objective of fair compensation. Indicate how fulfilling your needs will benefit the organization, making it easy for them to say yes. If you have received a job offer, the employer really does want you. As in the movie Jerry Maguire, your employer is really saying, “Help me help you,” though hopefully without all the melodrama and sweating.

When negotiating a job offer, you have more power than you likely ever will with the organization. Knowing that, remember that employers expect you to negotiate. Finally, know that there is more to negotiate than salary. You can negotiate things such as housing benefits, course release, the size and location of your lab space, and many more items listed below that contribute to your quality of life.

Money isn’t the only issue

  • availability of dual career programs (check out Stanford’s website on dual career programs).
  • housing benefits for new faculty
  • moving expenses
  • sabbaticals/vacation
  • tuition reimbursement
  • summer research stipends
  • intellectual property/patent rights
  • teaching load/ course buy out
  • use of teaching assistants/readers/graders
  • advising, theses and committee work
  • work schedule
  • telecommuting
  • on-site childcare
  • parking
  • start up package
  • size and location of office/lab space
  • lab equipment
  • computing/software
  • research assistants
  • conference and travel funds
  • intramural research funds
  • grant-writing support
  • journal subscriptions/books

In part two of Negotiation Strategies for Scientists, we will learn how to make the counter offer and how to navigate an impasse in the negotiation.

 

 

 

 

 

 

 

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Investigating the links between alcoholism, impulsivity, and neurogenesis

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A model of impulsivity in humans. http://xkcd.com/1466/ 

Impulsivity is defined as behavior performed with little or insufficient forethought. Another more technical description is disinhibition of reward-driven behavior that is not appropriate for the current demands. Impulsivity has strong correlations with substance abuse, including alcohol addiction. People with impulsive behavior may be more at risk for addiction, and degeneration due to chronic alcohol abuse can increase impulsivity, progressing severity of the disorder. Understanding the connection between impulsivity and addiction may lead to better treatments for substance abuse. However, their relationship is relatively unknown. In this post, we will follow the work of two scientists who demonstrate the persistence and curiosity required for biomedical discoveries.

Scientists can recreate alcohol addiction using transgenic mice. These mice lack a particular transporter known to function in alcohol consumption called ENT1, or equilibrative nucleoside transporter 1. After drinking alcohol, this transporter is inhibited. Mice without this transporter protein are less affected by alcohol and tend to consume more than normal mice, mimicking alcohol addiction in humans.

Normally, ENT1 transports adenosine, a neuromodulator that has an inhibitory effect on the central nervous system. When ENT1 can’t transport adenosine, it is unable to regulate excitation in the brain and tell it to “slow down.” In other words, alcohol keeps the foot off the brakes, possibly promoting impulsivity.

Alfredo Oliveros is a Mayo Graduate School PhD candidate in the lab of Dr. Doo-Sup Choi at the Mayo Clinic College of Medicine. He is using these transgenic ENT1 negative mice to discover exactly how alcohol addiction alters adenosine levels in order to cause impulsive behavior.

edited choi

So, how do you measure impulsivity? A famous example of impulsivity in humans is the marshmallow test. In this test, children are told they could have one marshmallow now, or wait and receive more marshmallows later. The more impulsive kids are unable to wait for more marshmallows, and instead eat the one and receive instant gratification.

Oliveros translated the marshmallow test for mice to test their impulsivity. Mice were trained to get food from a receptacle only when a special sound-cue is played. However, if they were impulsive (i.e. were unable to wait for the next sound-cue) and checked the receptacle, their next opportunity for the reward was delayed. Impulsive mice would check the food receptacle frequently, while less impulsive mice were able to wait. He also used a test, which differed in that the reward was only provided when a certain pattern of lights was illuminated, adding a cognitive aspect to the test.

Oliveros found that the alcohol preferring ENT1-negative mice were more impulsive than normal mice and checked the food receptacle frequently. Since ENT1 transports adenosine, he then studied the involvement of adenosine as the next step between alcoholism and impulsivity. To promote impulsive behavior, adenosine binds to a particular receptor on neurons involved in inhibitory behavioral control. Oliveros used a drug to antagonize that receptor, blocking any downstream effects. Once again, he saw that impulsivity was increased, suggesting that adenosine is the brake that controls impulsivity.

If ENT1 regulated adenosine levels to promote addiction and impulsivity, what did adenosine regulate? You can see that scientists are never content with one answer. They are always asking, “why,” tunneling further and further toward the truth. Oliveros and Choi knew that the adenosine receptor activated a protein called ERK. Was ERK also responsible for enhanced impulsiveness in alcoholism? To test this question, the scientists used a drug to inactivate ERK and examined the mice for impulsive behavior. Sure enough, mice with inactive ERK demonstrated more impulsivity.

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ERK plays many roles in the brain, one of which is cell proliferation. Normally, brain cells do not divide. However, it has been discovered that neurogenesis, or the creation of new neurons in the brain, does occur. The function of neurogenesis is still unclear, though current studies implicate neurogenesis with learning and memory. Could modulation of impulsivity be a novel function of neurogenesis via ERK?

Interestingly, neurogenesis was reduced in the ENT1 negative mice that displayed alcoholic and impulsive behavior. It appears that alcohol addiction promotes impulsivity by inhibition of adenosine, which inactivates ERK. ERK caused a reduction in neurogenesis, suggesting a new role for neurogenesis in alcohol addiction and impulsivity. How do altered levels of neurogenesis cause these behaviors? The questions continue, and Oliveros and Choi will continue to follow the trail.

By mapping out the connections between addiction, impulsivity, and neurogenesis, Oliveros and Choi provide potential targets for therapeutics to treat alcohol addiction. Scientists such as these who persist, continuing to ask “why,” are the key to future drug discoveries and improved lives.

 

References

Choi, D.S., et al. (2004) The type 1 equilibrative nucleoside transporter regulates ethanol intoxication and preference. Nature Neuroscience. 7: 855.

Nam, H.W., et al. (2013) Adenosine Transporter ENT1 Regulates the Acquisition of Goal-Directed Behavior and Ethanol Drinking Through A2A Receptor in the Dorsomedial Striatum. Journal of Neuroscience. 33: 4329.

Verdejo-García, A., et al. (2008) Impulsivity as a vulnerability marker for substance-use disorders: Review of findings from high-risk research, problem gamblers and genetic association studies. Neuroscience and Biobehavioral Reviews. 32: 777.

 

Glycoprotein-130 and Chitosan-Coated Nanoparticles: Two Keys to Bladder Cancer Treatment

Studies show that glycoprotein-130 may hold the key to bladder cancer treatment. Inhibition of this IL-6 cytokine receptor can reduce bladder cancer cell proliferation and tumor volume in mice, as shown in a study performed by Dr. Darryl Martin, an Associate Research Scientist working with Dr. Robert Weiss at Yale University in the Department of Urology.

In the search for bladder cancer targets, the Weiss lab found that the cytokine IL-6 was overexpressed in several bladder cancer cell lines. Unfortunately, inhibition of IL-6 expression did not alter cancer cell proliferation–but the scientists were not discouraged.  The IL-6 receptor is located at a central point between 3 important pathways involved in cancer regulation: JAK/STAT, PI3K/AKT and ERK/MAPK. Perhaps greater success would be found by studying the IL-6 receptor. Glycoprotein-130, or GP-130, is a critical component of the IL-6 receptor, important for activation of downstream targets stimulated by IL-6 binding.

3101113_cmr-3-177f3

Courtesy of Tawara et al. (2011) Cancer Manag Res. E3: 177-89.

To begin, Martin evaluated GP-130 expression in human bladder cancer specimens by immunohistochemistry and found that levels of GP-130 protein were correlated to tumor grade. He also looked at GP-130 levels in cell lines derived from human bladder cancers. Cell lines from high grade tumors showed elevated GP-130, while a cell line from a low grade papillary tumor showed less protein as seen by Western blot.

Martin then began experiments knocking out GP-130 in vitro. siRNA for GP-130 caused a 60% decrease in cell viability and 50% reduced migration in the scratch assay. Crystal violet staining of cells showed a reduced number of cells. This showed promise for GP-130 as a target for bladder cancer treatment.

To explore the mechanism by which GP-130 regulates cell proliferation and viability, the effects of GP-130 knockdown on the PI3/STAT pathway were investigated. Western blots showed reduction of AKT by 20% and a 50% decrease in mTOR, suggesting that GP-130 indeed utilizes this pathway, but that others may be involved, as well.  ERK1/2 and other pathways are currently being explored.

In his final set of experiments, Martin evaluated the effectiveness of GP-130 inhibition in vivo. Heterotransplant tumors were formed by subcutaneous injection of UMUC3 bladder cancer cells in nude mice. Upon tumor formation, GP-130 siRNA encapsulated in chitosan-coated nanoparticles was injected into tumors. Martin and Weiss developed this delivery system in collaboration with Drs. W. Mark Saltzman and Jill Steinbach in the Department of Biomedical Engineering at Yale in an attempt to enhance the amount of siRNA delivered to the cancer site. By delivering siRNA within nanoparticles, its stability is increased, and lower doses are required. One issue with cancer treatments instilled in the bladder is that the drugs do not always penetrate the urothelial wall. The coating of chitosan, a mucoadhesive polysaccharide, allows greater penetration of this bladder permeability barrier.

When the tumors were observed following treatment, tumor volume was significantly decreased in the siRNA-treated mice compared to the control mice.  In addition, levels of the GP-130 protein were reduced, as well as those of the CK20 protein, a marker correlated with advanced tumor stage. The success of this experiment was a critical step for the advancement o f GP-130 as a bladder cancer target, as tests in animals are required before clinical trials may begin.

Future experiments will include injection of cancer cells into the bladder to create a model more similar to bladder cancer in humans. This would also allow Martin to evaluate the effectiveness of the chitosan nanoparticle to penetrate the urothelial wall, inhibit GP-130 expression, and tumor growth.

The anti-obesity drug Belviq® may be repurposed for treatment of cocaine abuse.

belviq

Image courtesy of stockimages and David Castillo Dominici at FreeDigitalPhotos.net.

Belviq®, an anti-obesity drug that acts on serotonin receptors in the brain, shows signs of dampening the rewarding and reinforcing effects of cocaine in rhesus macaques. Dr. Gregory Collins of the University of Texas Science Center at San Antonio and the South Texas Veterans Health Care System San Antonio is working with Dr. Charles France to determine if this drug could be safely repurposed for the treatment of cocaine addiction.

Belviq®, also known as lorcaserin, is an anti-obesity drug developed by Arena Pharmaceuticals that was approved by the FDA in 2013. It works by activating the serotonin receptor 5-HT2c in the brain hypothalamus to induce satiety. However, activation of the 5-HT2c receptor can also dampen dopaminergic neurotransmission, which has important implications for cocaine abuse. Cocaine leads to the increased release of dopamine in the brain, a neurotransmitter which activates the reward pathways of the brain. Drs. Collins, Lisa Gerak, and France are studying whether the activation of 5-HT2c receptors could counteract the increase in dopamine caused by cocaine, reducing the rewarding and reinforcing effects of the drug. “There have been a lot of preclinical studies looking at 2c receptor agonists and 2a receptor antagonists as phamacotherapies for cocaine abuse but since this one is actually in the clinic it should be pretty easy to repurpose it for a different indication if we can find effects in the lab,” stated Collins.

Drug repurposing, or the study of a drug currently used for one disease or condition for its use in other diseases, is a strategy which can speed the time it takes for a drug to travel from the lab to the clinic. By beginning with a drug which has already been proven safe for humans with one disease, the success rate is greatly increased for its use in another. The NIH National Center for Advancing Translational Sciences developed a program in 2012 to support drug repurposing, which can be read about here.

In order to repurpose lorcaserin, Collins needed to determine the effectiveness of the drug to reduce cocaine self-administration in animals, to confirm that the dose was still safe, and to determine that tolerance to lorcaserin didn’t develop. For his experiments, Collins used the rhesus macaque, a species of monkey with behavioral responses to cocaine very similar to that of humans.

In the first set of experiments, the possibility of negative behavioral effects was tested in monkeys. This allowed them to determine a safe and pharmacologically active range of lorcaserin doses. Importantly, they observed that lorcaserin treatment induced yawning in monkeys, a behavioral response that is characteristic of 5-HT2c activation, indicating that the scientists were maintaining receptor specificity with their treatment method.  They also evaluated the effects of lorcaserin on food-maintained responding, a test that was used to confirm that the changes in animal response to cocaine were actually due to the lorcaserin treatment, and not just a general disruption of the animals’ behavior. This test showed only modest changes in food-maintained responding, allowing Collins to proceed to testing the effects of lorcaserin against cocaine.

In the next phase of research, Collins and colleagues measured blood levels of lorcaserin in monkeys to determine the point at which lorcaserin is highest in the body following treatment. This information is important for identifying the appropriate pretreatment time to study the effects of lorcaserin against cocaine. In behavioral treatments, Collins was then able to treat the monkeys with cocaine at a point in which lorcaserin levels were highest.

Finally, the key experiment for lorcaserin was performed. Would treatment with the drug decrease the monkeys’ interest in cocaine? To determine this, Collins and colleagues used a cocaine self-administration assay in which the monkeys were trained to press a lever to self-inject cocaine. Normally, monkeys will respond at high rates to earn injections of cocaine. However, if the rewarding and reinforcing effects of the drug are reduced by lorcaserin, the monkeys should start to reduce this cocaine-taking behavior.

Amazingly, when the monkeys were pre-treated with lorcaserin, their preference for the lever delivering cocaine was significantly reduced. In fact, this effect was consistent over 14 days, suggesting that the monkeys did not develop a tolerance to lorcaserin. In addition, the effects of lorcaserin treatment were also observed with larger doses of cocaine, suggesting that the monkeys were unable to surmount the effects of lorcaserin by taking more cocaine.

This is excellent news for Dr. Collins and the France lab, suggesting great promise for lorcaserin in the treatment of cocaine abuse. As investigations of the anti-cocaine effects of lorcaserin move into human cocaine-abusers, Drs. Collins, Gerak, and France are now beginning to assess the effectiveness of lorcaserin against other stimulants of abuse such as methamphetamine.

To learn more about the France Lab, click here.

Reynold Spector Award in Clinical Pharmacology Lecture by Dr. Scott A. Waldman

Dr. Scott A. Waldman received the Reynold Spector Award in Clinical Pharmacology at this year’s national ASPET meeting at Experimental Biology. Dr. Waldman gave a presentation regarding his award-winning scientific achievements titled, “Bench-to-Bedside Translation in Clinical Pharmacology: From Knowledge Generation to Healthcare Delivery.” Dr. Waldman outlined the history of his research on the gastrointestinal receptor guanylyl cyclase C, or GCC, from the initial characterization of its overexpression in colorectal cancer to its use as a biomarker and therapeutic target for the disease.

Colorectal cancer is the 4th leading cause of cancer in the United States and the 2nd cause of cancer-related death. It is typically caused by the sequential accumulation of mutations that cause normal intestinal epithelial cells to transition to a hyperproliferative state, followed by the formation of adenoma and final progression to carcinoma.

Guanylyl cyclase C is a receptor localized to the intestine. The molecules which bind the GCC receptor include the hormones guanylin and uroguanylin. Interestingly, this receptor also mediates the symptoms of Traveler’s Diarrhea by binding to the enterotoxin released by harmful bacteria.

One theory behind the initiation of colorectal cancer is that of paracrine hormone insufficiency. The hormones guanylin and uroguanylin are the most commonly lost gene products in colorectal cancer. This reduction occurs during the early phases of cancer. When these hormones are lost, the receptor GCC is silenced. Studies have shown that loss of GCC results in an increased incidence of colorectal cancer in rodents, leading to its identification as a tumor suppressor. Therefore, Waldman hypothesized that hormonal replacement therapy could prevent the occurrence of colorectal cancer recurrence. By maintaining homeostatic levels of guanylin and uroguanylin, perhaps the silencing of the receptor could be avoided and carcinogenesis prevented. Fortuitously, since the GCC receptor is exposed to the lumen of the small intestine, hormone therapy can be delivered orally, making it amenable to clinical use. When tested in mice, hormone replacement therapy eliminated tumorigenesis. Currently, Dr. Waldman is collaborating with the NCI Division of Chemoprevention, Ironwood Pharmaceuticals, and the Mayo Clinic to test paracrine hormone replacement therapy in humans.

Dr. Waldman’s lab then investigated the use of GCC as a biomarker for colorectal cancer. GCC is only found in the small intestine and colorectum normally. However, they found that it can also be detected in colorectal cancer cells. Could GCC be used as a marker to test for colorectal cancer metastasis? Indeed, upon further research his lab proved that RT-PCR analysis of extraintestinal tissues can indicate the presence of metastatic cancer cells, as well as the harder to detect micrometastases. Dr. Waldman is also investigating the use of GCC as a biomarker to differentiate tumor stage and risk of recurrence. Currently, clinical trials are underway to determine whether chemotherapy, a treatment typically reserved for metastatic cancer, will be effective for patients who are not categorized with metastatic cancer by current staging guidelines, yet have extraintestinal sites of GCC-positive cells, indicating micrometastases.

The third area of research which Dr. Waldman has pursued is the development of GCC-based vaccines to prevent colorectal cancer recurrence. He hopes to direct the immune system to metastatic cells expressing GCC.  The mucosal and systemic systems have different lymphoid organs and separate effectors, providing a dual immune system with minimal cross talk. Waldman is taking advantage of this to immunize the systemic compartment against GCC-expressing cancer cells, without inducing a response in the mucosal compartment where GCC is normally localized. In fact, a vaccine containing GCC elicited antibody and killer T cell responses in mice, preventing metastatic tumor formation without creating or exacerbating inflammation. Currently, Dr. Waldman is testing a GCC-based vaccine in a Phase 1 trial of stage I and II colorectal cancer patients. So far, positive antibody and killer T-cell responses have been observed.

The advances in cancer research that Dr. Waldman has accomplished is the dream of many scientists. To discover the basic mechanisms of a disease and use that information to not only diagnose, but also treat the disease is a feat that few scientists ever accomplish, and Dr. Waldman is indeed worthy of the Reynold Spector Award in Clinical Pharmacology for his research. Throughout his presentation, Dr. Waldman also took the time to highlight scientists who have acted as mentors and friends, supporting him throughout his career. “I think it’s a rare opportunity that we get to thank our mentors in public,” Waldman said. “These are the people that set our feet on the path for our careers and shaped us… For me these are the people on whose shoulders I’m standing, trying to catch a brief glimpse of the horizon in the distance.”

During his doctoral training at Thomas Jefferson University, Dr. Waldman worked in the lab of Dr. Ken Chepenik.  “He is the one that instilled in me a love of science and research,” said Waldman. He also expressed appreciation for the friendship he has maintained with Dr. Chepenik through the years. “While he taught me many things scientifically, the more important gift that he gave me was his enduring friendship.”

In 1979, Dr. Waldman began a postdoctoral fellowship in the lab of Ferid Murad, co-winner of the 1998 Nobel Prize in Physiology or Medicine. “He taught me how to do big science,” stated Waldman. “The most important thing that he taught me was actually a passion for translational research. He is the quintessential physician-investigator. Every day it was, ‘What are we going to discover today that we can translate to better manage a patient tomorrow?’” In addition to the science he taught Waldman, their families have remained close friends for 35 years.

Dr. Waldman also thanked his colleague Dr. Andre Terzic, whom he met as a junior faculty member at Thomas Jefferson University. Waldman expressed gratitude for several lessons in personal and professional development. “There’s no ‘I’ in team,” said Waldman of advice gained from Dr. Terzic. “When you are working on something, you are doing it for the betterment of the institution and organization, not for the betterment of the individual. In every interaction, always take the high road, and I’ve tried to do this throughout my career.” Waldman and Terzic have produced a textbook together in basic and clinical pharmacology, and have been the co-editors of Clinical Pharmacology and Therapeutics for ten years. “While scientifically and professionally he’s given me a lot, more importantly he’s given me his friendship, which is of paramount importance,” said Waldman.

Lastly, but most importantly, Dr. Waldman credited his success to his loving family. “All the mentors and all the science is great, but at the end of the day there’s got to be something that inspires you. My inspiration, the thing that gets me up every day, is my family,” said Waldman. “Of all the things that I’ve ever done, of all the cool science that I’ve done and the projects that I’ve been a part of and the people who I’ve met, it’s all wonderful, but truly my family is probably the most important thing I’ll ever do in my life.”

The Reynold Spector Award in Clinical Pharmacology was established in 2014 by ASPET in recognition of Dr. Spector’s dedication and contributions to clinical pharmacology. The award recognizes excellence in research and/or teaching in clinical pharmacology. This award is made possible by an endowment to ASPET from Dr. Reynold and Mrs. Michiko Spector.

Is Optogenetics Too Good to Be True?

Optogenetic Light Alters Gene Expression in Wild-type Microglia

New research indicates that the use of optogenetic light for cell-selective control may have effects on surrounding cells, an important consideration for the use of this recently popularized neuroscience technique. Kevin Cheng, a graduate student in the lab of Dr. Jyoti Watters at the University of Wisconsin-Madison discovered this phenomenon while performing research with microglia.

For those unfamiliar with optogenetics, this technique utilizes the ability of light-responsive proteins to control cells. By targeting these transporters to certain cells, scientists can activate cells of their choosing by exposure to blue and other wavelengths of light. This also allows cells to be turned on and off at the researcher’s choosing. This technique was originally used to excite or inhibit neurons, though it has now expanded to various cell types and can also be used to control other functions of the cell such as GPCR activity.

To learn more about the basics of optogenetics and why it is so cool, check out this clip by Nature Video

Originally, Cheng was trying to transduce microglia cells with the construct for the light-responsive protein. They expected that optogenetic light would alter gene expression in the optogenetic protein-containing cells without effects on the wild-type cells lacking the light-responsive transporter. However, what they found was very different. When exposed to the light, control microglia lacking the light-responsive protein showed changes in gene expression. “The first time that I brought this to our weekly lab meeting, they laughed. Do you think it is possible that this is happening?” asked Cheng. “And they said no, it’s not.” After rigorous experimentation, Cheng was able to prove to his labmates that indeed, there were side effects of optogenetic exposure in normal cells.

To prove his hypothesis, Cheng utilized a black-walled 96-well plate aligned with an LED array. This setup allows for control of light duration and intensity in each well. Microglia were grown on the plate, then exposed to blue light at 450 nm, a common wavelength used for optogenetic experiments. Following exposure, pro-inflammatory gene expression was determined by quantitative RT-PCR.

Since the inflammatory response within the central nervous system is mediated by activated microglia, Cheng compared gene expression in both basal and LPS-activated microglia. He also evaluated the difference between a single bolus of light and a light pattern commonly used in optogenetics.

Of the pro-inflammatory genes measured, COX-2 expression was induced in basal cells exposed to both bolus and optogenetic light patterns. In LPS-activated cells, the genes IL-1b, iNOS, COX-2, IL-10, IL-6, and VEGF were decreased and IGF-1 was increased after bolus light exposure. Further, IL-1b, iNOS, COX-2, and IL-10 also exhibited decreases in gene expression following light with LPS activation. To make sure the energy dose delivered was not harmful to the cells Cheng also measured the potential for blue light to induce cell death and DNA damage in microglia, and found no effect. This suggests that wild-type microglia respond to blue light and that it may exert a surprisingly anti-inflammatory effect.It also demonstrates that optogenetic patterened light exposure can have effects on wild-type cells near the cells of interest.

To date, this is the first report of non-specific effects of optogenetic light. This study has important implications on the field of optogenetics, a technique which has named “Method of the Year” in 2010 by Nature Methods. Cheng plans to expand his experiments to include other cell types and to explore the mechanism behind this unexpected phenomenon.

For the abstract of this work, visit here.

To learn more about research in the Watters Lab, click here.

New Tamoxifen Analog Reduces Amphetamine Neurochemical and Behavioral Effects

Colleen Carpenter

Colleen Carpenter

Amphetamines are the second most abused drug worldwide, but no effective treatments are available for amphetamine abuse. At this year’s Experimental Biology meeting, graduate student Colleen Carpenter from the University of Michigan presented data revealing that the chemotherapy agent tamoxifen can serve as a platform for the creation of a pharmacological treatment against amphetamine abuse.

Currently, the mainstay of amphetamine abuse treatment is psychotherapy. “The problem with behavioral treatment methods is that they are not very effective,” Carpenter said. “We’re looking at a pharmacological way to block amphetamine abuse.” In the lab of Dr. Margaret Gnegy at the University of Michigan Department of Pharmacology, Carpenter is looking for pharmacological treatments to inhibit the rewarding and reinforcing effects of amphetamine.

The effects of amphetamine are caused by the release of the neurotransmitter dopamine into the synapse via the dopamine transporter in the brain. “People have actually tried to target the dopamine transporter directly,” says Carpenter. “The problem is, many ligands that bind to the dopamine transporter act as dopamine uptake blockers. Blocking uptake of dopamine is bad, because that’s how cocaine works. We still have the increase in extracellular dopamine at the synapse which has its own reward functions.” The Gnegy lab is taking an alternative approach. Rather than targeting the dopamine transporter, they are now looking at the upstream regulator of the dopamine transporter, protein kinase C.

It is known that amphetamine activates protein kinase C, or PKC, which ultimately enhances the release of dopamine through the dopamine transporter. By modulating the activity of PKC, perhaps dopamine release could be reduced, decreasing the rewarding and reinforcing effects of amphetamine.

There are numerous available inhibitors of PKC but the selective estrogen receptor modulator tamoxifen is the only PKC inhibitor known to cross the blood-brain barrier.   Tamoxifen acts at a regulatory subunit of PKC, so it causes less problems than one that acts at the active site.

At this year’s ASPET meeting, Carpenter reveals recent data suggesting that a compound modeled after tamoxifen called C091, designed and synthesized by the Vahlteich Medicinal Chemistry Core at the University of Michigan, is able to reduce PKC activity and dopamine release without affecting the estrogen receptor at relevant concentrations. In addition, behavioral studies with the new compound showed significant decreases in amphetamine-related behavior in mice.

First, Carpenter determined that C091 is a better inhibitor of PKC activity than tamoxifen, as indicated by changes in the ability of PKC to phosphorylate specific protein targets when treated with C091 versus tamoxifen.

She then investigated the potential for C091 to bind estrogen receptors, as this nonspecific binding could cause unwanted side effects if C091 is purposed as a therapeutic against amphetamine abuse. To determine this, she performed an estrogen receptor competitive binding assay. When compared with estradiol and tamoxifen, C091 did not bind to the estrogen receptor at concentrations where it effectively inhibited PKC.

To determine changes in actual dopamine release at the synapse, Carpenter measured the levels of dopamine release induced by amphetamine with and without treatment with various doses of C091. In order to measure dopamine release in vitro, the ends of dopamine transporter-expressing nerve terminals from the striatum are pinched off to form small spheres called synaptosomes. These small spheres are able to respond to amphetamine exposure and secrete dopamine via the dopamine transporter. A suprafusion system is used to collect the dopamine released from the synaptosomes. In short, this system allows the controlled perfusion of drug solutions over the synaptosomes and the dopamine-containing eluent is collected in fractions for quantification. Treatment with amphetamine stimulated dopamine release from striatal synaptosomes. However, dopamine efflux was reduced with increasing doses of C091.

After proving that C091 inhibits PKC activity and reduces dopamine release, Carpenter moved on to a critical experiment: would C091 have actual effects on amphetamine- induced behavior in an animal? For this experiment, mice were pretreated with C091 followed by amphetamine treatment, and changes in locomotion were observed. Normally, amphetamine exposure significantly increases mouse locomotion due to its action in the brain. When mice were treated with C091 systemically prior to amphetamine, their locomotion was greatly reduced.

Future studies with C091 will include a model in which the effects of C091 on amphetamine self-administration can be determined, as well as microdialysis for measurement of dopamine levels in vivo.

To read more about the research performed in the Gnegy lab, click here.

For more information about the creation of tamoxifen analogs, check out

de Medina, P., Favre, G. & Poirot, M. Multiple targeting by the antitumor drug tamoxifen: a structure-activity study. Current medicinal chemistry. Anti-cancer agents 4, 491-508 (2004).