Author Archives: Annika Sharma

As we wrap up the program and I mentally prepare myself for the 30-hour journey back home to India, I’ve been thinking a lot about how this summer has gone. BUSRF definitely didn’t go as expected—I had to change labs halfway through (for reasons I won’t go into here) and ended up working in Dr. Brian Hare’s Duke Canine Cognition Centre. Although having to start over was frustrating, the opportunity to experience two very different lab environments in two very different departments made the experience all the more valuable. Never underestimate the importance of good mentors (and a good mentor-mentee fit) in navigating the research world! Navigating science is just about as messy as the science itself.

Halfway through last semester, I had pretty much decided I was going to study neuroscience. Now, I’m not so sure. Dr. Hare insists that there is a way to merge my passion for animals and studying animal behavior with my interest in understanding the workings of the brain, a field he has termed evolutionary neuroscience (a behavioral/non-invasive approach to understanding the human brain by studying species such as chimpanzees, bonobos, wolves & dogs). As I leave with more questions than answers, I’ve promised myself that I will take the time to explore before settling on a field of study.

Next year should be exciting! But first, a much needed break awaits…

I can’t wait to see my dog Cinnamon (and relax like she is)!

7:50am… 8:00… 8:10… 8:20… 8:30… 8:35… These are all the alarms my roommate had to suffer through on a daily basis as I tried to convince myself to wake up for our morning faculty seminars. Although dragging myself out of bed and then rushing to get ready because I was running late (again) is not my idea of fun, the speakers made it worth it. Apart from learning about interesting research, it was reassuring to hear about the zigzagging career paths that many of the speakers had taken.

Two talks stood out in particular. The first was Provost Sally Kornbluth’s talk on research misconduct. I’ve grown up loving shows like CSI, Law & Order and White Collar, and the case study of Anil Potti, a former Duke medical researcher, sure felt like the academia version! Her talk also struck a chord because I’ve been thinking a lot about the failings of biomedical research after reading the book Rigor Mortis by Richard Harris (shout-out to Dr. Brian Hare for the recommendation). I’m not saying that basic science research is unnecessary—it has resulted in many life-changing (literally) discoveries. However, it isn’t perfect. The lack of severe repercussions for Anil Potti is a case in point (he is still a practicing doctor and will be allowed to conduct research again after 2020).

The second talk that caught my attention was that of Dean Stephen Nowicki who spoke about learning and mate selection in song birds. Having taken his class last semester, I knew some of the information but listened with newfound appreciation for the evolutionary perspective. It was especially fascinating to hear about his research on geographically comparing song repertoires between groups of male swamp sparrows. Song types varied between groups with groups farther away from each other being more distinct. Females tended to respond preferentially to males singing songs typical of their group with their responsiveness diminishing as songs from groups further away were played. This indicates that females’ notions of a ‘good song’ are learned through experience and exposure. Who would have guessed that songbirds are such an ideal species to study language and learning!

*This abstract is the first draft of what will ultimately be used for the poster presentation at the end of this program*

The gut microbiome & depression: what is the best method for DNA extraction?

Our gut microbiomes play an important role in immune functioning, nutrient processing, and regulating many aspects of brain functioning. This gut-brain axis has been implicated in major depressive disorder, and inducing a depressive phenotype in mice using social defeat (SD) is one commonly used model to study the shifts in microbial diversity that occur. To do so, an effective protocol to extract and sequence the bacterial DNA from the fecal matter of subject mice is required. As it stands, methods and results in the field vary widely. This study compared two DNA extraction kits—the Maxwell RSC PureFood GMO & Authentication Kit and the MoBio PowerSoil DNA Isolation Kit. The Promega kit was hypothesized to be superior due to its high degree of automation. In order to assess the quality of the extracted DNA, spectrophotometer, fluorometer and electrophoresis were used. Results from 16S rRNA gene sequencing are awaited for phylogenetic classification of the samples. Analyses indicate that the Promega kit is more efficient, requiring less sample, but may have co-purified contaminants. Sequencing results will indicate effects of this, if any. Further work must explore microbial changes after SD and treatment, and the roles of specific bacterial species on the CNS.

Since choosing a chalk talk to write about was so hard (everyone’s projects are so cool), I decided to go with the topic I found most intellectually intriguing. Martin Acosta’s presentation on ‘Rapid Tryptophan Depletion: Sex-mediated Differences in Rats’ stuck with me because of the parallels between our projects.

Tryptophan is an important amino acid which we obtain through our diets and which is metabolized into serotonin (an important neurotransmitter) in the brain after passing through the blood-brain barrier (BBB). Martin’s project offers an alternative to the social defeat (SD) paradigm to induce depression in rodents since SD is not effective in females and hence cannot be used to study sex-differences in depression. In their protocol, the Kuhn lab feeds rats a mixture of several large neutral amino acids (LNAAs) or a mixture of LNAAs & tryptophan. LNAAs compete with tryptophan molecules to pass through the transport proteins in the BBB. Therefore, rats fed only LNAAs have a higher proportion of them which prevent tryptophan from passing into the brain and being converted into serotonin (serotonin deficiency is associated with depression).

Although preliminary results from Martin’s study (which measures the levels of various metabolites in the rats’ blood and brains) have shown no differences in how male and female rats are affected, this is not the aspect interests me. Tryptophan plays an important role in the gut-brain axis—some bacteria are known to use up tryptophan leaving less to be metabolized by our bodies, while others produce it. Additionally, the kynurenine pathway is another important pathway for tryptophan metabolism. Kynurenine is produced from tryptophan and can be further metabolized in two ways with opposite effects—the end product of one pathway is neuroprotective while the other is neurotoxic. Altogether, tryptophan and its metabolites seem to play an important role in depression and are an important signaling pathway between the gut and brain. This is where my project comes in: I can’t wait to see the bacterial DNA sequencing results from the feces of the SD mice in my project to determine whether the bacterial species which are more/less abundant are implicated in the tryptophan pathway.

P.S. Tryptophan is one of my new favorite sounding words—try saying it, it’s addicting!

Although my lab is in the division of translational neurosciences, most of our day-to-day work involves cellular, molecular and microbiology. We’re currently comparing two methods for bacterial DNA extraction from mouse feces to study shifts in their microbiomes during depression, analyzing the levels of four proteins in plasma samples from our longitudinal alcohol dependence study, and searching for biomarkers for PTSD susceptibility in soldiers.

There is no such thing as a ‘normal’ day in my work. Sometimes I spend hours labelling slides which will be used for the PTSD brain sections—they have to be labelled in pencil or else the staining process will dissolve the label/ink. It’s really interesting to watch Dianne cutting the sections and see the tissue adhere itself to the special, positively-charged slide.  Other days I weigh 2 ml tubes to ensure that we have a healthy stock of pre-weighed tubes since they save tons of time during experiments. I also help pull samples for the protein assays we’re currently running—as we’ve learnt the hard way, finding and randomizing hundreds of tiny PCR strip tubes with IDs handwritten on the sides is NOT easy. (An aside: I’m totally in awe of dry ice).

We’re still waiting on the sequencing results from the mouse poop extractions (which is my primary project) and so, in the meantime, I’ve been learning about and analyzing data from the quality tests we’ve run—260/280 & 260/230 ratios from the spectrophotometer which measure purity, DNA concentration from the fluorometer which measures yield, and the DNA Integrity Number or DIN from the TapeStation which measures degradation & quality.

On some days work can seem dreary or slow, and I have realized that research is not at all like it’s romanticized to be. However, learning from my awesome mentors, Michelle and Tulay, makes everything worth it! Although I’ve mostly been handling experiment prep, I’m looking forward to getting my hands dirtier in the next few weeks!

Our gut microbiomes have long been known to be critical for immunity, nutrient processing, etc. More recently, research has suggested that gut bacteria play an important bidirectional role in brain development & function, and the modulation of stress response. Major Depressive Disorder (MDD) patients have altered microbial compositions and many metabolites which play a role in depression are byproducts of gut microbiota.

My lab is focusing on the effects of chronic social defeat on the microbiomes of mice (as a pre-clinical model). In short, the social defeat (SD) paradigm involves placing the subject mice into the same cages as aggressive mice, allowing them to fight, and then separating the mice using a divider while but keeping them in close proximity to each other for a day. After the process is repeated 10 times with different aggressive mice, the subjects present with anxiety and depressive symptoms.

The aim is to study changes in microbial richness and diversity, and differences in the relative abundances of gut bacteria at the family and phylum levels between depressed and healthy subjects. This is measured through fecal samples which are collected from the subjects’ cages before the paradigm (a baseline) and after SD. The mice are then treated with electroconvulsive shocks (ECS), and post-treatment samples are taken to examine whether reductions in symptoms are also accompanied by a stabilizing microbiome. Many people don’t realize that ECS is still used today to treat severe depression in humans!

The aggregate sequencing results from a pilot study by Kara McGaughey. Each color represents a bacterial phylum, and the shifts in the microbiomes of the depressed group are easily visible.

I’m involved with comparing two DNA extraction kits using the pre-SD fecal samples to choose the more suitable one for the rest of the experiment. I was initially surprised to learn that there is no ‘gold standard’ in DNA extraction and that the various kits in the market all detect varying species & proportions of bacteria. Although my project has ended up being more about microbiology than neuroscience, learning about the different bacterial DNA extraction and sequencing protocols is definitely cool. I’ve also learnt a ton about the measures used to assess the purity (microplate spectrophotometer), concentration (fluorometer) & quality (TapeStation) of extracted DNA. I’m really looking forward to seeing & comparing the sequencing results!

Dr. Williamson hails from a small, working-class family in Pittsburgh, Pennsylvania and was the first of his family to go to college. During our discussion, he recalled having no idea what he wanted to do when he was 19 except for his drive to receive an education and obtain a Ph.D. He did just that after his bachelor’s in Psychology, receiving a Ph.D. in psychiatric epidemiology (the study of the distribution of psychiatric diseases in a population and the risk factors that impact the distribution).

Over time he has migrated towards integrating preclinical models, brain imaging, biomarkers and genetics into his study of stress-related disorders. He defined himself as a translational epidemiologist and really struck a chord with me when he said, “in life you can call yourself whatever you like and become it”. Given a chance to go back in time, he said he would have enhanced his biology training as an undergraduate, however he advised not worrying “about fitting everything into an end-product that you imagine for yourself because in 10 years I don’t think you’ll find yourself in the position you’re imagining now”.

I love how Dr. Williamson’s current projects are very much a product of happenstance and seized opportunities. The project I’m involved with is sequencing the gut bacteria of mice pre & post a chronic social defeat paradigm (which induces a depression-like phenotype) to measure shifts in their microbiome. What began as Kara’s (an undergrad who now works in the lab) senior thesis and has now grown into one of the lab’s main focuses—a beautiful example of Dr. Williamson’s teaching style in which he likes to work with students one-on-one and “to learn together, teaching and being taught”.

Their second project is within a national PTSD consortium (partially funded by Veteran Affairs). Before moving to Duke, Dr. Williamson worked in San Antonio where a colleague approached him about taking on a genetic study of soldiers pre & post deployment to find biomarkers for PTSD susceptibility. The happy accident which started out as helping a friend has grown exponentially, and Dr. Williamson is now the director of the ‘Genomic and Biomarkers Core’ within the consortium. Although Dr. Williamson has been lucky both professionally and financially to be able to branch into new fields, he mentioned that allowing investigators to access money not tied to project-specific grants would help science move forward. Additionally, he would like to see more incentives for scientists to be unselfish and collaborative so that one’s life experiences can become the whole group’s knowledge.

The lab’s final project is a longitudinal teen alcohol and depression study for which Dr. Williamson’s epidemiological background has been critical. “Depression is not depression is not depression”, each individual’s depression is characterized by their family history, presence early-life anxiety, age of first onset, etc. and one must look at the disease in that framework. In the pursuit of better understanding these factors, he is following children with high-risk backgrounds during the peak onset period of 12-15 years old.

As a final piece of advice for aspiring scientists, he emphasized the importance of being generous, gracious and dependable as a collaborator and of giving more than one takes. “No one will help you if you’re a pain in the ass,” he said jokingly, but also with the wisdom of one who has dealt with such a colleague before.

I started in my lab only midway through the week, and by then, the imposter syndrome had set in. Hearing about all the things that my fellow BSURFers were doing and learning, I felt incredibly incompetent. However, when I finally met Dr. Williamson (my primary mentor) on Wednesday afternoon, he assured me that mistakes and misunderstandings were to be expected and that his motto was always to “trust, but verify”. I still feel like I’ve been thrown into the deep end, but at least I have a life vest! I’ve realized that being a budding scientist involves living in almost constant and see-sawing states of confusion and exhilaration.

The Williamson lab studies the bidirectional role of gut microbiota on depression in preclinical mouse models (among other projects). In my first few days during my first real lab experience, a couple of things have quickly become apparent: 1) science involves a lot more waiting around than one would think – for example, the Illumina HiSeq DNA sequencer that we use takes 65+ hours to run! This leads to 2) how incredible the equipment and technology that exists today is, and 3) how single sentences in published papers often represent extremely elaborate and laborious protocols. Science requires patience and perseverance, and it demands knowledge and experience.

This summer I expect to get a glimpse of life as a researcher, I anticipate failing often while learning how to ask for help, I wish to feel the rush of an unexpected discovery, and, most importantly, I hope to foster relationships with my lab that I can build on in the future. I know that research often involves time-consuming lit review and data analysis, but I’m really looking forward to getting my hands dirty!

At our weekly lab meeting!

Shoutout to Tulay for giving me the coolest pen!!