The Purdue Lecture Hall Series

Earlier this semester, I was invited to speak about my research at The Purdue Lecture Hall Series (TPLHS). TPLHS is an evening seminar series for high school students, teachers, and parents from the Greater Lafayette community organized by the Purdue Institute of Inflammation, Immunology and Infectious Disease (PI4D) in partnership with Mr. Joseph (Joe) Ruhl from Lafayette Jefferson High School. The series celebrates the work of a wide variety of the life scientists that represent the different areas of research within PI4D and it is meant to inspire high school students to consider life sciences as a career choice.

I began my talk by delving into my background in STEM — from high school and undergraduation to my Master’s research experience and starting my doctoral research at Purdue. I spoke about the brain and drug discovery for neurological disorders. I introduced the students to brain immunity and the role of microglial cells in phagocytosis and inflammation during Alzheimer’s disease. During the course of the talk, I also introduced the two undergraduate students who work closely with me in the lab since I was informed that many high schoolers in the audience were interested in pursuing undergraduate research while in college. This was a great opportunity to talk about how different labs on campus function and how one should go about getting involved in research early on during their undergraduate studies. Overall, this was a great experience since it made me step out of my comfort zone (which involves very little interaction with the general public) and also hopefully inspired a few students to be curious about science and research!

 

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The three tiers of science communication

Perhaps one of the most important yet widely ignored skills that us scientists need to cultivate is to communicate our science better. By “communicate science”, I am not simply referring to publishing research articles and reviews in journals and publications. This is about conveying ideas, research, theories, and facts to a wide audience. This is harder than it seems. On a day-to-day basis, we are so engrossed in our little scientific bubble that we hardly engage with people from outside our labs, departments, and universities. For example, I can prepare to present my work during our weekly group meetings with an accurate understanding of how to introduce my research project to my fellow lab members and what data to present during my talks. Most individuals in my program or department have an understanding of the common scientific language and the several jargons that are overused during the talks and seminars.

I would like to think that being in my research group has given me a better understanding of communicating my work to my fellow peers.  My lab is a “hybrid” wet and dry lab i.e., it is comprised of computer scientists, computational chemists, synthetic chemists, and biologists. Our group meetings are extremely interdisciplinary covering a multitude of topics ranging from machine learning and molecular dynamics to immunology and cancer biology. At this point in my career, I am certain and confident with my ability to convey the scope of my project and the several particular aspects of my current research.

The most challenging audience are individuals who are completely outside the realm of our scientific bubble. These individuals serve critical roles in our society but are overlooked by us all the time. I have interacted with my friends and family from different professions and they’re always intrigued by my work and more specifically about *what* we do in the lab and *how* we do science. These are important questions that not only establishes confidence in the scientific community but also bridges the gap between our worlds. Questions that may seem simple or even silly to us may be important in the large scheme of things. For example, the other day, my friend asked me “How do the lab mice get Alzheimer’s disease?” To answer this, I could have just said that there are several transgenic models of mice with genetic mutants that spontaneously develop Alzheimer’s over time. This is an answer that I would have had for someone in the scientific community. But for my friend who happens to be a business associate, I candidly described genetics of the disease, how mice are bred in laboratories, and how they develop plaques that can be viewed in their brain tissue sections. In order for the public to trust us, first and foremost, they need to be aware and educated on the basic scientific methods and principles. This includes communication about the bases of experimental design, process of gathering significant data, peer reviewing, reproducibility, etcetera.

This brings me to what I consider are “the three tiers of science communication” that scientists should cultivate. We need to learn how to communicate our science to:

  1. Our fellow peers in the field i.e., individuals from our specific area of research
  2. Our scientific colleagues from different areas of research
  3. The general public including individuals from other professions

Tier #1 is a no-brainer. Individuals from this tier read and review our work. They are critical of every aspect of our research and question the scientific methods used. They make signifiant contributions to our work and provide guidance for the growth of our research. Tier #2 is tricky. Why would I, a neurobiologist want to communicate my work to a computer scientist or a meteorologist even? A major aspect of creating new solutions to old problems is to collaborate with scientists from outside our specific focus areas. Drug discovery is not possible without computer scientists teaming up with chemists and biologists. Many of the problems in the areas of neuroscience such as understanding of neural circuits and systems, cognitive and behavioral neuroscience, etcetera would not be solvable without the help of electrical and mechanical engineers.

tier three
Source: SMBC-comics.com

Individuals from tier #3 are probably one of the most significant yet overlooked in this regard. Science communication to the general public does not happen until there is a problem affecting people from the both worlds. Involving this tier should not be limited to the difficult times but should be an ongoing process. It should be a part and parcel of our work. Much has already been said about this. How do we make science outreach a regular part of our work? Should the burden of outreach not be imposed on scientists at all? We need more science communicators breaking out of our bubble and out into the real world. Furthermore, many grad students and researchers make contributions in their own way. For example, using social media (#scicomm on twitter and instagram) for science outreach is a great way to reach thousands of individuals from your fingertips while working in your lab. No fancy equipment, no travel money, no event organization necessary! Well established senior scientists with the means and resources should strive to connect with and impact a larger audience.

Grad school diaries: The preliminary exam

The past few weeks months have been terrifying, nerve-wracking, depressing, and scary. My friends and family have also been subjected to my constant irritable and grouchy behavior. I have been preparing for my preliminary examination and everything seems to be coming together (very) slowly. I have woken up to sweaty nightmares about missing deadlines, submitting a complete crap proposal to my committee, and being told that my “scientific caliber” is not up to the mark to pursue an academic career (gulp!)

The first week of November is officially my “prelim week” and I will continue to go through series of mini heart-attacks and one too many mood swings until then. What exactly is a preliminary examination, you ask? Well, also called as the “candidacy exam”, or “the OP” (short for the original proposal – mostly followed in life sciences), it is an examination that PhD students take (and pass) in order to officially become PhD candidates. Many schools and department do this differently, and I can only tell you what is done in my program. Here is a short excerpt about the exam from our handbook –

The purpose of the Preliminary Examination is to stimulate you to develop original research ideas and to assess your academic knowledge, preparation and ability to analyze and synthesize the literature on and surrounding your topic. In the written proposal, you are expected to provide the examination committee with adequate background and details to understand the current state of the chosen field of research and to evaluate your proposed experiments. The oral examination allows the committee the opportunity to test your knowledge of the chosen research project, your ability to formulate and address a few research questions to anticipate the types of results to be obtained, and to evaluate your understanding of its scientific foundation. The examination will not only assess the science involved in the proposal but also will evaluate the quality of the presentation and the writing.

Basically, we are required to come up with an original idea – a topic that is not our main thesis research, write a hypothesis-driven research proposal in the NIH Exploratory/Developmental Research Grant (NIH R21)-type format, and defend it in front of our prelim committee (which is different from our thesis committee and consists of new members). The proposal must be original and designed to advance the current state of knowledge in the chosen field. It cannot be based on our own (current or previous) research projects. Also, our advisor cannot critique the research proposal prior to submission of the proposal to the prelim committee. The whole process takes almost 8-9 months and I have briefly summarized the timeline of the process below –

March-April 2017: Brainstorming ideas for the topic; Reading, reading, and more reading. (My topic is about the role of myeloid-derived suppressor cells or MDSCs in mediating pancreatic beta-cell death in Type 1 Diabetes, which is an autoimmune disorder.)

May 2017: Topic approval by the program office.

June-August 2017: Literature review; Brainstorming ideas and key questions for experiments, techniques, aims, etc; Beginning to write… maybe…

August 2017: Prelim committee assigned; Serious writing and reviewing (rinse, repeat); More reading.

September 2017: First draft completion; Review by peers, friends, and colleagues; Schedule date and time for the oral defense with committee; MORE READING.

October 2017: Submission of written proposal to the program office and prelim committee (4 weeks prior to oral defense); Approval of proposal for oral defense (or, revise and resubmission of proposal aka “your proposal is indefensible at this stage and requires more work”); Practicing oral talk (aka “pre-prelim talk”).

November 2017: Defense! Drinking and crying (if pass); Drinking and crying (if fail); New sense of purpose in life.

A few weeks into this process (around May), the horror stories start – stories about seniors failing their defense and “Mastering out” (which is seen in a really bad light), stories about committee member issues, stories about inadequate writing, etc. I have heard one too many stories about people dealing with depression and constant stress during the period of writing and oral defense. There are tons of useful advice about what to do and what not to do during the process. Of course, the experience is unique and different for every student but it would certainly be easy if I could get on with it without constantly being traumatized by every little detail (like feeling guilty every minute that I’m not thinking about my OP or working on it).

However, a few things have indeed helped me so far:

  • Finding a studying/writing spot outside of work and my apartment. I have been working at WALC until wee hours of night these days. (WALC is the active learning center on campus and is always hustling and bustling with students.) Just being among other students and the white noise in the background seems to be a great environment to focus and get stuff done.
  • Biking to and from work every day (around 6.5 miles). My friend recently convinced me to buy a bike and I must say that it has helped me get around the campus faster and save a ton of time. Not to forget the kick of endorphins in the morning that helps me focus on my experiments in the lab and plan things more effectively through the day. I spend most of the mornings doing cell culture work (I get done with this the first thing in the morning in order to make time for meetings and other experiments through the day) and afternoons on tissue processing and protein work. This gives me sufficient time from evening until late night to work on my OP.
  • Eating regularly, but not fussing over cooking. Most of the time spent on cooking and cleaning can be replaced by quickly grabbing something to eat on the go. (I can hear my sister squeaming at this already!)
  • Talking Ranting to friends, especially colleagues about the OP, work, life, and everything in general to relieve all the stress. I am fortunate to be on the same boat as many folks who can relate to my situation and listen to my rambling.
  • Reading something completely un-related to my research or the OP over the weekends. I have read three books in the past few months (check out my reading list!).

Alright, I should probably get back to work now (this was some major procrastination and I am feeling guilty already). Perhaps I should talk about my topic in detail on the next post. Until then, I will try to keep calm and carry on.

Religion, science, and believing.

I don’t usually talk about my personal views on this blog. However, this topic is something that I have contemplated for a while now and think is fair to be open about. I am still learning and evaluating my outlook on approaching this subject. Below are some bits revolving around the themes of religion and personal belief systems that were hidden away in my drafts folder for a long time. I have decided to publish all of them together. I’m sure I’ll have more to say about this topic in the future, but here’s a start.

***

Recently, I had a conversation with a fellow grad student about religion and his personal beliefs. Most academics shy away from this discussion in a professional (and sometimes even in a personal) setting. It is considered uncommon or rude to talk about it and people keep it to themselves. It is often acknowledged that as scientists, “we do science for science’s sake”, or that “a person’s religious beliefs has no place in his/her scientific pursuits.” This is something that has always boggled my mind. As a biologist and an atheist, I have confidence in my work/study because the underlying laws of biological systems are established and follow a set of proven scientific principles. For example, when we design an antibacterial drug against a particular strain of resistent bacteria, we know for a fact that the bacteria has mutated (or evolved) and therefore the old drug doesn’t work anymore. Similarly, we use mouse, worm, and other animal models for testing compounds in vivo because we have evidence to prove that humans are genetically related to other animals through a common evolutionary ancestor. Therefore, we can study the effects of the drugs in other animals before testing them to humans. The empirical evidence that exists as the basis of our research is inherently acknowledged to be the underlying force that drives scientific research. Now, how can someone who does similar work in a laboratory setting have a completely contradictory viewpoint in his/her private life? How can someone believe in a book (or many books) that preaches blatant falsehoods about our understanding of the universe and at the same time come to work every day and do science with a conscious mind? For me, science is deeply woven into our personal lives. No, I cannot pretend that science does not affect my personal views about the world. Similarly, my conscious will never let me pretend like my personal views have no affect on my scientific work.

***

One of the most common arguments that I have come across during such discussion is that people often say “I don’t believe in *everything* that this book says. I only believe in a few things that are important for my moral framework.” This is complete BS and hypocritical. One cannot disregard a particular theory written in a book (for example, “the earth is 6000 years old”, or, “when humans die we come back as another life form on earth”), and at the same time believe in another theory written in the same book. One can’t pick and choose what you want to accept and reject from a book, and then claim the book to guide one’s moral framework.

And then there is an argument that science is not perfect and that not everything published in all of the scientific literature is true. This is absolutely correct. This is why science is constantly changing – because our understanding of the world is constantly changing. This is why scientific literature constantly undergoes modifications and updates to accommodate our latest understanding of the world and the universe.

This is not the same with religious texts. These texts were written hundreds and thousands of years ago and are obsolete in this day and age. These texts were written to accommodate the worldview of an ancient time period. They are not relevant to the 21st century and we certainly do not have to submit to these texts in order to live within a moral framework of society. As of 2017, we have discovered around 8.7 million species on earth and can estimate a hundred billion galaxies in the observable universe. We have achieved things that were once considered unfathomable by humankind. Why do we have to be stuck in the ancient past and live by some 12th century law in order to be considered as “good humans”? Of course, religious texts provide interesting insight into various philosophical questions that one can ponder over. However, they do very little to the understanding and practice of science in this day and age.

It is also often argued that we need religion to understand morality and differentiate between good and evil. Religion does not equal morality. One does not have to be a good human just to please an invisible supreme being or to go to heaven. Altruism and kindness can exist on their own.

***

Talking about scientists with personal religious beliefs, I remember a wonderful conversation between Richard Dawkins and Lawrence Krauss many years ago. I can’t help but bring up a part of their conversation while thinking about this topic –

Krauss: I’ve had people write to me and say “I’m a medical doctor and I don’t believe in evolution.”

Dawkins: That’s a disgrace. I’m not supposed to say that, especially in this country (referring to the US) because one’s private beliefs are supposed to be irrelevant. But I would walk out of a doctor’s office and not consult him anymore if I heard that he said that. Because what that doctor is saying is that he’s a scientific ignoramus and a fool.

Krauss: In fact, in that regard, it is interesting to me at the same time how people can hold beliefs which are incompatible with other beliefs they have. And in some sense, everyone is a scientist and they just don’t realize they are, and yet in the time of crisis, that’s when.. (breaks). The example I gave is when George Bush was president, he said intelligent design must be taught alongside evolution so the kids will know what the debate is all about. And it wasn’t a stupid statement at priori, it was ignorant because he didn’t realize that there’s no debate. And that’s fine. I don’t mean ignorant in a pejorative sense, I just mean he wasn’t aware.

Dawkins: Ignorance is no crime.. you just don’t want to consult a doctor who’s ignorant.

Krauss: What amazed me is that in the same administration, when the avian flu was going to be a problem and mutating to humans, president Bush said “We’ve got to find how long it takes before the avian flu will mutate into humans.” And what amazed me is that no one in the administration – not a single person said “It’s been designed to kill us, forget about it.”

Dawkins: That’s a very good point. This kind of split-brain business which you’ve been referring to, the most glaring example I know, is more in your field (referring to Theoretical Physics and Astrophysics) than mine. I was told by a professor of Astronomy at Oxford, about a colleague of his who’s an astronomer and an astrophysicist, who writes learned papers – mathematical papers, published in astronomical journals, assuming that the universe is 13.7 billion years old. But he privately believes that the universe is only 6000 years old. How can a man like that hold down a job in a university as an astrophysicist? And yet, we are told “Well, it’s his private beliefs, you mustn’t interfere with this man’s private beliefs as long as he writes competent papers in astronomical journals”.

Krauss: Well, I mean, as long as he doesn’t teach his private beliefs.

Dawkins: Well, let’s hypothetically suppose that he teaches absolutely correctly – that the universe is 13.7 billion years old. How could you want to take a class from a man who teaches one thing and believes in something that is so many orders of magnitude different?

***

About “believing” in science.

My advisor once pointed out not to use the word ‘believe’ when someone said “I believe that..” during a lab meeting presentation. Back then, I didn’t understand what was wrong in saying we “believed” in something. I now understand. As scientists, we evaluate something on the basis of observation, experiment, and evidence. The evidence is dependent on the observations made and experiments performed. Therefore, something is either likely or unlikely to occur. It is either more probable or less probable. We don’t have to believe in evolution or the big bang theory. We accept the evidence that supports them. Believing in evolution or not doesn’t make it true. The evidence for evolution suggests that it is true. Belief is not a part of rational enquiry. Belief relies on faith and not on evidence.

Does arginase mediate immune suppression in the brain?

This week I want to talk about an interesting enzyme: Arginase-I (Arg1) and its metabolic pathway in an immune microenvironment. Arg1 is a cytosolic protein that is involved in the urea cycle. Specifically, it catalyzes the hydrolysis of L-arginine to L-ornithine and urea. It has been shown that the expression of Arg1 by macrophages has an important role in tumor growth. Macrophages that are recruited into the tumor microenvironment (tumor associated macrophages) express high levels of Arg1 resulting in the depletion of arginine – an essential nutrient required for T cell metabolism. Cytotoxic T cells, therefore, can no longer function and inhibit the tumor cells from proliferating. Arg1 is implicated in several inflammatory diseases as well as in autoimmunity. Arg1 plays a significant role in mediating immune suppression and blocking its metabolism is a novel strategy in preventing tumor growth and other inflammation-related conditions.

T cell suppression by MDSC
Myeloid-Derived Suppressor Cell suppresses cytotoxic T cell function through Arg1 metabolism. (IFNγ, IL-4, and IL-13 are cytokines that induced MDSC activation)

One hypothesis is that the immune suppressive cells in other immune microenvironments in the body must be similar to the Myeloid-Derived Suppressor Cells (MDSCs) that induce T cell suppression in the cancer microenvironment. The question is, do such immunosuppressive cells express increased levels of Arg1 and act through the Arg1 metabolic pathway? Since I am interested in the brain and neurodegeneration, this hypothesis can be extended to the brain immune microenvironment. Microglial cells in the brain also upregulate Arg1 and are neuroprotective in nature (in a healthy brain). These cells are the resident macrophages of the central nervous system and function by phagocytosing cell debris and toxic misfolded proteins (that eventually form aggregates and lead to neuronal death as seen in Alzheimer’s disease) out of the brain environment. The question now is – do the microglial cells exhibit immunosuppressive behavior by altering their Arg1 metabolism?

Kan et al., 2015, recently showed that CD11c positive microglial cells are immunosuppressive in the CVN-AD mouse model and that immune suppression is caused due to the deprivation of arginine (increased levels of extracellular Arg1 causing decreased levels of total brain arginine). What isn’t explicitly mentioned in this study is that arginine is also the substrate for nitric oxide synthase (NOS) that makes nitric oxide (NO) in an alternate L-arginine metabolic pathway. L-arginine is a substrate for both Arg1 and NOS. The Arg1 pathway polarizes the macrophages to M2 phenotype and the NOS pathway polarizes the macrophages to the M1 phenotype (Rath et al., 2014). The current model of microglial activation in the CNS is limited to these two polarized states, where, the M1 microglia are neurotoxic and the M2 microglia are neuroprotective. Arg1 is upregulated in microglia in the healthy brain and aids in phagocytosis of misfolded proteins and other cell debris. The classical microglial activation is through the M2 phenotype wherein the induced nitric oxide synthase (iNOS) is upregulated thereby accelerating inflammation in the brain (neuroinflammation is one of the hallmark characteristics of several neurological diseases such as Alzheimer’s Parkinson’s, Multiple Sclerosis, Traumatic Brain Injury, etc).

M1 and M2 microglia
The current model of microglial activation is limited to the Arg1-mediated M1 and iNOS-mediated M2 polarized states.

So, if an immunosuppressive cell exists in the brain, is it possible that the immune suppression is regulated through the M2 activation and that M1 activation is absent? In other words, L-arginine is metabolized through the Arg1 pathway and not through the NOS pathway. Other questions to consider: MDSCs upregulate both Arg1 and iNOS – so how does that fit into the two-state polarization model? How does iNOS modulate MDSC activity? We know that increased NO expression by MDSCs increases T cell suppression in the tumor microenvironment. Currently, both Arg1 and iNOS inhibitors are being developed to block the immune suppressive activity of MDSCs in the cancer microenvironment. However, understanding immunosuppression in the brain is still a long way to go and the idea is not widely accepted within the neurobiology community (my understanding from the currently available literature or published studies). Investigating this mechanism in the brain will be useful in developing potential therapeutic strategies for treating neuroinflammation and neurodegeneration.

References:

  • Kan MJ, Lee JE, Wilson JG, et al. Arginine Deprivation and Immune Suppression in a Mouse Model of Alzheimer’s Disease. The Journal of Neuroscience. 2015;35(15):5969-5982. DOI:10.1523/JNEUROSCI.4668-14.2015.
  • Rath M, Müller I, et al. Metabolism via arginase or nitric oxide synthase: two competing arginine pathways in macrophages. Front. Immunol., 27 October 2014. DOI: https://doi.org/10.3389/fimmu.2014.00532

Blots, cultures and assays concludes rotation two

This week officially concludes my second laboratory rotation in the neuropharmacology lab with research focussed on  G protein-coupled receptors and their application in several neurological disorders such as depression and anxiety. In the eight week duration of my rotation, a few things were achieved with respect to validating the activity of the newly developed M4R-DREADD (a designer M4 muscarinic receptor exclusively activated by a designer drug). Designer receptors are engineered such that they are solely activated by a synthetic ligand. This opens new avenues in the activation and control of G protein-coupled receptors’ function in vivo.

After a long break from my Master’s research, I got back to maintaining two cell lines – CHO (Chinese Hamster Ovary) and HEK293 (Human Embryonic Kidney) cells, in which the opioid receptors were expressed for all my experiments. These cells were used to characterize the receptor signaling by western blot analysis of the downstream MAPK/ERK signaling  upon stimulation by a few agonists/drugs of interest. Luckily, the lab acquired a new fluorescence microscope during this period which helped us observe the recruitment of the β-arretin2 protein by δ-opioid receptors in HEK293 cells stimulated with clozapine-n-oxide, a synthetic ligand.

mrrd-gfp barrest-cherr cno 0 min 20x_Overlay copy
HEK293 with M4R-dreadd 20x
mrrd-gfp barrest-cherr cno 10 min 20x_Overlay copy
HEK293 with M4R-dreadd 20x

This week, I had a lot of difficulty in handling the mice. Being my first experience with animal work, watching the mice anxious and struggle while we held them down was hard. I am still pretty unsure about how I feel about animal work (if I HAVE to do it to save my research in the future, I will) but I definitely need more exposure and practice with them.

Overall, this lab taught me a lot, even if some days were stressful and  tiring. I feel like I learned and enhanced many skills in the process (primer design, restriction analysis, cell culture, cloning, western blot, cAMP assay), and got a feel for the lab at the same time. Through the course of these past two rotations, I have met some really smart and dedicated people. In the end, I am grateful to have had this opportunity.

Two months in: Last day in cancer lab

Today marks the last day of my first laboratory rotation. I want to pen down a few things that I learned and experienced during my time in the cancer lab:

  • Starting fresh in a new field of research was challenging at first, but got interesting once the different pieces of the puzzle were pieced together along the way.
  • Understanding the nitty-gritty of the investigation entails failures, failures, failures, followed by lots of optimizations and practice. Patience and perseverance is the key.
  • Staying positive and motivated throughout the journey can go a long way. My mentor/ senior grad student in the lab is one of the most optimistic people I’ve met in recent times.
  • Almost always, grad students manage multiple projects at the same time. It is essential to have a main project (or two) and a few side projects to keep the lab active, and research moving forward.
  • Taking a computing course along with the Biochemistry course has kept my study diverse and helped me broaden my thought bubble. At the same time, it has contributed to an additional pressure of having to take exams and submit assignments frequently (which I don’t want to be doing a lot of at grad school).
  • I nearly broke my arm while working with the french press for cell lysis (it really is a workout in itself!)
  • Having flexible working hours in the lab was good, but there were days when I took this for granted and ended up being awfully lazy. I am still learning to implement a fixed schedule for the weekdays to increase my productivity through the week.
  • Caffeine addiction is a real thing. I now even have a brew preference to satisfy my taste buds and more importantly, my brain.