Author: Joshua Lepson

Reaching the summit: climbing with the burden of pain

My fellow science geeks, sadly, this will be my last World of Work blog post.  However, rather than focusing on the fleeting nature of summers, I wish to walk you through my achievements, insights, and trials and tribulations of working in a biomedical research lab with a severe chronic pain condition.  Since the age of twelve, I have endured an excruciating nerve pain syndrome known as Complex Regional Pain Syndrome (CRPS)[i].  Here, I will briefly mention how CRPS affects me, with the hopes of encouraging students living with disabilities and adversity to pursue their career passions and dreams.

One of the most common questions I am asked regarding my pain is “how?”: “how do you attend college?”  “How do you participate in a research lab?”  “How do you live with the pain?”  My response remains steadfast; human beings (and life in general) possess a remarkable ability for adaptation, even in the bleakest of circumstances.  I believe in challenging the notion that extreme adversity cannot be triumphed in some form.  As you read this blog post, I hope you will view my experiences as evidence for why your hardships should never preclude you from actualizing your dreams.

Shapiro Science Center (SSC) of Brandeis University. The SSC is the annual location of SciFest, an undergraduate poster session [ii].

A few weeks ago, I presented a poster of my summer research findings at Brandeis University’s SciFest VII [iii].  SciFest is an annual poster session showcasing undergraduate student research hosted in my favorite building on campus, the Shapiro Science Center.  In this very building, I learned a cursory understanding of journal style science writing in Dr. Kosinski Collins’s (Dr. K-C) Biology Laboratory course (thank you Dr. K-C!).  I only had a taste of journal diction, yet I relished the opportunity to learn the art behind science writing.  Generating a poster presentation of original research presented my next learning opportunity.  Thankfully, the post-doctoral fellow (“post-doc”) I worked alongside and my principal investigator (PI) were ecstatic to hear about Brandeis SciFest, and strongly encouraged me to create a poster of my summer research.  Thus, I began crafting selected “mini” sections of a journal style paper, beginning with an abstract, followed by a curtailed introduction and figure descriptions of my experimental evidence.  I was fortunate to receive invaluable advice from my co-workers; I passed my writing along to my supervising post-doc, asking her to tear my writing apart.  I wanted her to know “I mean business” when it comes to learning.  I circulated my writing amongst lab members, also gathering my PI’s sage advice.  This gave me a small taste of the manuscript writing process, an essential component of every research laboratory.  This process culminated in a poster, which, upon entering this summer, I knew little about.  My poster explored the role of cysteine restriction in energy homeostasis, focusing on a key metabolic pathway known as the trans-sulfuration pathway.

Pictured above is the intersection of the methionine cycle (from methionine to homocysteine), the folate cycle (far left), and the trans-sulfuration pathway (bolded in red). I focused on the enzyme CGL, or cystathionine-gamma-lyase. This figure was created with assistance of Yang et al. 2016  [iv].
I am immensely proud of my poster and presentation, given that my success represents triumph both over internal and external doubts regarding my capacity for achievement in the face of debilitating pain.  Given that my physical disability effects my left hand and arm, I was concerned regarding my ability to efficiently learn new experimental techniques.  However, with patience, I successfully completed methodologies such as Western Blotting [v], including the pain-inducing sonication step [vi].  Sonication involves “shooting” high energy sound waves into a sample containing proteins and nucleic acids.  The sound waves shear DNA into small chunks, thus liberating nuclear (nucleus-bound) transcription factors (proteins) for proteomic investigation.  I may have taken a few extra minutes to complete this step, but I obtained pure proteins, which I was able to immunoblot for [Western Blotting] analysis.  Another technique I am proud of learning is mouse dissection.  Although simpler than the microscopic Drosophila (fruit fly) dissections I have attempted at Brandeis, mouse dissection still requires significant dexterity and focus.  I was concerned I would lose control over my left hand, or that the pain would inhibit my precision.  However, I excelled, even learning how to excise “speck-like” structures such as the pituitary glands in the brain and the thyroid gland in the neck.  I also improved upon techniques such as RNA tissue extraction, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) [vii], study design, statistical analyses, and more.

Altogether, I am quite proud of my tireless work this summer, both experimentally and regarding my pain condition.  I see my work as another step towards achieving my career goals in medicine.  There is an expanding pile of evidence that my pain will not write my story; I will.  I wish to convey this simple fact to other students living with disabilities and adversity; you can achieve your greatest dreams and more.  Although I have yet to accomplish my goal of becoming a physician scientist, I know I will get there.  You will reach your goal too.

 

-Josh Lepson

 

References

[i] American RSDHope. 2017. CRPS OVERVIEW/DESCRIPTION. Accessed on August 17.

http://www.rsdhope.org/what-is-crps1.html

[ii]  Brandeis University. Integrated Media – CAMPUS BUILDINGS. Accessed on August 17.

https://www.brandeis.edu/communications/media/mediaresources/buildings.html

[iii] SciFest. 2017. SciFest VII Abstracts. Accessed on August 17.

https://kb.brandeis.edu/display/SCI/SciFest+VII+Abstracts

[iv] Yang, M., Vousden, K.H. 2016. Serine and one-carbon metabolism in cancer. Nat. Rev. Cancer. 16(10): 650-662.

[v] ThermoFisher Scientific. Overview of Western Blotting. Accessed on August 17.

https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-western-blotting.html

[vi] New England Biolabs. DNA Fragmentation – Application Overview. Accessed on August 16.

https://www.neb.com/applications/library-preparation-for-next-generation-sequencing/dna-fragmentation

[vii] ThermoFisher Scientific. Basic Principles of RT-qPCR: Introduction to RT-qPCR. Accessed on August 17.

https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/basic-principles-rt-qpcr.html

The Panacea for Obesity: Fat!

Hello fellow science lovers!  Since my last blog post[i], I have been quite busy and have generated exciting and perplexing data.  As a brief reminder, I am working within the Division of Endocrinology, Diabetes and Metabolism at Beth Israel Deaconess Medical Center and Harvard Medical School[ii], focusing on hydrogen sulfide signaling using genetic knockout mouse models.  In particular, I am focusing my research on a knockout (KO) mouse strain for the major hepatic (liver) endogenous hydrogen sulfide producing enzyme, cystathionine gamma lyase (CGL). When I wrote my last blog post, I was beginning to examine key gene expression and protein expression levels between wild type (WT) control mice and CGLKO mice by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR)[iii] and Western Blots[iv] respectively.  I continue to rely on these powerful molecular biology methods, where I attempt to connect the dots between differential gene and protein expression levels.  Recently, my data has lead me towards a nutritional framework, where I have been particularly interested in dietary-induced and dietary-resistant obesity.

Pictured on the right is the ob/ob mouse strain compared to a normal, wild type mouse strain on the left. Ob/ob mice are deficient in the feeding inhibiting hormone leptin, and thus are used in obesity and diabetes research [vii].
Given the pervasive rise in obesity and diabetes within the United States (US), therapeutic targets for dietary-resistance to obesity are a “hot” research topic within the field of Endocrinology and Metabolism.  In a special report published in 2005 within the New England Journal of Medicine (NEJM), the authors predict “that as a result of the substantial rise in the prevalence of obesity and its life-shortening complications such as diabetes, life expectancy at birth and at older ages could level off or even decline within the first half of this century.”[v] This stands in stark contrast to human trends, where human life expectancy has steadily increased over the past thousand years [v].  Thus, the need for breakthrough research discoveries regarding obesity, metabolic disease, and diabetes has never been more imperative.  A major research target in recent publications has been the heat-generating, master energy consuming mammalian brown fat, or brown adipose tissue (BAT) [vi].

In mammals, BAT is a major tissue site for chemical production of heat (thermogenesis) from fats, which has made BAT a promising target to induce weight loss[vi].  Traditionally, when exposed to cold temperatures, humans generate heat by shivering [vi].  However, mammals such as mice and human infants possess vast BAT depots, allowing thermogenesis during cold exposure to be driven by the chemical uncoupling of cellular energy production, oxidative phosphorylation [vi].  This chemical uncoupling of oxidative phosphorylation is achieved in part through expression of uncoupling protein-1 (Ucp1) [vi]. Additionally, white fat or white adipose tissue (WAT), the classic form of stomach fat we all attempt to minimize, can be induced into a BAT like state, known as “beige” or “brite” fat [vi].  This beige fat has thermogenic capacity, and because thermogenesis relies on the breakdown of fat depots in order to generate heat, beige fat has the ability to burn excess fat depots and promote a healthier metabolic system [vi].  Countless studies have demonstrated that “expanding the activity of brown fat, beige fat or both in mice through genetic manipulation, drugs or transplantation suppresses metabolic disease.”[vi] One such stimulus for expanding beiging of WAT is dietary control.  Thus, because of the vast therapeutic potential of beige fat and BAT, I have been particularly fascinated by diets that can induce beige fat and or increase BAT activity.  Such a diet could have broad reaching implications for metabolic disease, and could help reduce the estimated 300,000 deaths per year related to obesity [v].

Here, major anatomical depots of brown adipose tissue (BAT), white adipose tissue (WAT) and beige adipocytes are depicted. This figure portrays differences between fat locations in (a) mice and (b) humans.   Genetic markers are given for each adipocyte type in the lower right hand corner  [viii].
Compared to my classroom studies at Brandeis, working in a biomedical research lab allows me to explore complex physiological topics that I would never confront in an undergraduate class, such as BAT and beige fat thermogenesis.  After running experiments on RNA, DNA, and proteins extracted from both control (WT) and CGLKO mice, the results almost always spur me to read a slew of research papers and reviews, which guide me towards a holistic understanding of what is occurring inside my mice. For example, I have examined Ucp1 expression levels in my mice, leading me towards reviews regarding thermogenesis. This ability to read beyond only what is assigned to me is a wonderful aspect of research which is mostly absent as an undergraduate at Brandeis.  I find this freedom allows me to become more excited about the material, and often causes me to gleefully share theories of mine with my co-workers, most of whom are post-doctoral fellows.

Similar to last summer, I am loving the environment of working in a basic science research lab.  I am continually refining my molecular techniques, learning new assays weekly, such as the protein concentration quantification bicinchoninic acid (BCA) assay[ix].  With each data result or conversation with the post-doctoral fellow I work alongside, I learn new complex signaling pathways within mammalian physiology.  After each biweekly lab meeting, I learn new elements of modern thyroid research, continually building upon my knowledge base of intricate thyroid endocrine regulation.  These molecular biology techniques combined with novel biology concepts will serve me well both in my future Biology coursework at Brandeis and in my future pursuits in and after medical school. Who knows, I may even end up a practicing Endocrinologist and participating in BAT thermogenesis research!  Only time will tell.

– Josh Lepson

[i] Brandeis University Hiatt Career Center. 2017. World of Work (WOW) Summer Internship Blog: Harnessing Science for the Common Good. Accessed on July 2.

Harnessing Science for the Common Good

[ii] Beth Israel Deaconess Medical Center. 2017. Endocrinology, Diabetes and Metabolism. Accessed on July 2.

http://www.bidmc.org/Centers-and-Departments/Departments/Medicine/Divisions/Endocrinology.aspx

[iii] ThermoFisher Scientific. Basic Principles of RT-qPCR: Introduction to RT-qPCR. Accessed on July 2.

https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/basic-principles-rt-qpcr.html

[iv] ThermoFisher Scientific. Overview of Western Blotting. Accessed on July 2.

https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-western-blotting.html

[v] Olshansky, S.J., Passaro, D.J., Hershow, R.C., Layden, J., Carnes, B.A., Brody, J., Hayflick, L., Butler, R.N., Allison, D.B., Ludwig, D.S. 2005. A potential decline in life expectancy in the United States in the 21st century. N. Engl. J. Med. 352(11): 1138-1145.

[vi] Harms, M., Seale, P. 2013. Brown and beige fat: development, function and therapeutic potential. Nat. Med. 19(10): 1252-1263.

[vii] The Jackson Laboratory. B6.Cg-Lepob/J. Accessed on July 2. https://www.jax.org/strain/000632

[viii] Bartelt, A., Heeren, J. 2014. Adipose tissue browning and metabolic health. Nat. Rev. Endocrinol. 10(1): 24-36.

[ix] ThermoFisher Scientific. Pierce™ BCA Protein Assay Kit. Accessed on July 2.

https://www.thermofisher.com/order/catalog/product/23225

Harnessing Science for the Common Good

Division of Endocrinology, Diabetes & Metabolism, Beth Israel Deaconess Medical Center, Harvard Medical School

Working in a basic science biomedical research laboratory within the Division of Endocrinology, Diabetes and Metabolism at Beth Israel Deaconess Medical Center and Harvard Medical School[i] has been an incredibly exciting experience.  I started to work in the Lab two weeks ago, located within the Center for Life Science in the heart of the Longwood Medical area.  Since I worked in this same Laboratory during the Summer of 2016, I was welcomed into the research environment, and was able to pick up where I left off last summer.  After recently completing animal research facility training, I began working with laboratory mice, focusing on a knockout (KO) mouse strain of the major hepatic (liver) endogenous hydrogen sulfide producing enzyme, cystathionine gamma lyase (CGL).  The Lab I work in is interested in the regulation of human metabolism by master endocrine regulator, thyroid hormone.  Thus, I have been investigating the relationship between thyroid hormone and endogenous hydrogen sulfide production capacity, with an emphasis on extension of longevity using mouse models.

Inside the laboratory, much of my work consists of analyzing key gene expression and protein expression levels between wildtype (WT) control mice and CGLKO mice through various physiological states.  My research consists of dissecting mouse tissue ex vivo, performing an RNA extraction from that tissue type (i.e., liver tissue, brown adipose tissue, etc.), running a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR)[ii] using several key gene markers, and performing statistical tests on differences in gene expression levels between WT and CGLKO mice.  For proteomic analysis, I perform Western Blots[iii] and statistical tests to establish potential differential protein expression in CGLKO mice.  Once I have gathered meaningful data, I present the results informally to the post-doctoral fellow I work alongside and to my Principal Investigator (PI).  However, living systems are complex, and bewilderment can punctuate results.  At these times, I turn to scientific journals for answers.

Pipettors and laboratory reagents (Sigma-Aldrich, Fisherbrand by ThermoFisher Scientific): friends of the biomedical researcher.

Biomedical literature publications, such as Brent et al. 2014[iv], have guided me through the complex physiology of thyroid endocrine regulation.  As an incoming third year undergraduate student, dissecting complex signaling pathways with my current learning foundation is a daunting task, especially considering the wealth of knowledge and graduate degrees that my co-workers possess.  However, my co-workers and PI have been and continue to be excellent learning resources.  Bouncing theories back and forth with the post-doctoral research fellow I work alongside is a daily occurrence.  This collaborative environment is characterized by persistent questioning of results and interpretations, which has filled my scientific soul with joy.  This stands in stark contrast to undergraduate classes, where the measure of performance is reflective of the individual, rather than a research team.

Looking forward, the skills I am learning, both in molecular methods and thinking as an experimentalist, will bolster my ability to succeed as a Biology major at Brandeis, and as physician scientist in the future.  I wish to exit this summer with the framework to think as a biomedical researcher, with the ultimate goal of generating meaningful research that can mitigate human suffering.  This can be easy to lose track of in the busyness of a lab, but I hope this goal remains tethered to my being; science for the common good.

[i] Beth Israel Deaconess Medical Center. 2017. Endocrinology, Diabetes and Metabolism. Accessed on June 4.

http://www.bidmc.org/Centers-and-Departments/Departments/Medicine/Divisions/Endocrinology.aspx

[ii] ThermoFisher Scientific. Basic Principles of RT-qPCR: Introduction to RT-qPCR. Accessed on June 4.

https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/basic-principles-rt-qpcr.html

[iii] ThermoFisher Scientific. Overview of Western Blotting. Accessed on June 4.

https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-western-blotting.html

[iv] Mullur, R., Liu, Y.Y., Brent, G.A. 2014. Thyroid hormone regulation of metabolism. Physiol. Rev. 94(2): 355-382.

Josh Lepson