Category Archives: Week 2

Dr. Mike Tadross has always liked tinkering with things and looking to solve difficult problems. For his undergraduate years studied to become an electrical and computer engeerir at Rutgers. He liked electrical engeering because of viewing circuits as puzzles that he could solve.

After undergraduate he started his work on M.D. P.H.D in Biomedical engeering at Johns Hopkins. His P.H.D ended up taking three years longer than he expected because of a project that he could not get to work. This project has still yet to be solved. Following his P.H.D he started working on DART (Drugs Acutely Restricted by Tethering) before he had an established lab of his own. After the publication of paper with DART he was offered a faculty position at duke and to establish the Tadross lab. The Tadross lab focuses on making tools to study the brain whether that be chemical or electrical wiring. He believed that since the brain and computers are composed of circuits then one would be able to design computers to interact with each other. He came to the conclusion that one needs to be able to put all of pieces together to understand the brain. Dr. Tadross when putting together his lab embraced this. He brought in people that focused on the chemical, electrical and the biology of the brain.

His path has not been as linear as one would expect. At Johns Hopkins, he joined a lab that focused on a topic that was not particularly interesting to him. During his time in the lab, he would spend three years on a project that would never actually end up working out. Because of this he worked on a project that he was not particularly interested in, but this gave him the experiencing failure firsthand in the lab. After graduate school, he worked in a pre-faculty position where he was able to work on his own projects but did not have his own lab. This eventually led to him figuring out his greatest invention using halo tag technology to restrict binding for drugs nears the channels.

Talking with Dr. Tadross, I had learned that if you are flexible and are willing to work hard that you will eventually stumble upon success. That while diving deep into topics is important having a broader understanding of what is occurring at all levels is just as crucial. Overall, my talk with Dr. Tadross made me more comfortable and adjusted to his lab.

Every 40 seconds, someone has had a stroke in the United States [1]. This occurs when an obstruction in the blood vessels prevents the brain from getting ample oxygen and nutrients, causing a cavity of dead tissue in the brain. This brain tissue has a limited capacity to regrow, and the resulting cavity is lacking in blood vessels and neuronal connections.

The Segura lab is leading in this work, as they’ve come up with various hydrogel networks that carry growth factors (amongst other things) to promote the regrowth of this dead brain tissue. We use mice–model organisms–to study the impact of these gels, meaning I give a bunch of mice some strokes for the greater progress of science. I’ve been part of over 60 mice surgeries since I’ve been in the lab, and the general process includes anesthetizing the mice, injecting a substance to induce the formation of free radicals that can cause blood clots, projecting a laser onto specific portions of the open skull to induce stroke, and injecting the hydrogel into the stroke cavity a few days later. The mice are only partially impaired, and seem active immediately after inducing strokes, unlike human patients who need countless rehabilitation and medical attention. The mice will be sacrificed at predetermined time points and brain slices will be studied to determine the impact of the gels on the regeneration of blood vessels and neuronal growth.

I’m excited to be a part of this lab and collaborate with other researchers to study strokes. Blacks are twice as likely to have strokes than whites and have the highest death rate afterward [2]. I know of at least four people in my family who’ve had strokes, so this research hits close to home. I also hope to shadow some behavioral studies at the lab with the aims of studying the impact of strokes in general, as well as the use of hydrogel on the behavior of the mice. I’m just really happy to be a part of the Segura lab!

Before I was dropped off at college, I distinctly remember the ominous warning from my mother: be careful, don’t get meningitis! The disease is known for occurring in infants and college students, but I didn’t really know what it was…until this summer, now that I’m studying it. While there are several pathogens that are able to cause meningitis, Dr. Perfect’s lab studies Cryptococcus neoformans, or the fungal pathogen readily found in the environment causing cryptococcal meningitis. Though its environmental ubiquity seems alarming, Crypto. is actually an opportunistic infection, meaning it only causes disease when it is able to take advantage of weakened immune systems, such as those with HIV/AIDS, transplant recipients on immunosuppressants, or possibly even survivors of COVID-19. Even so, the prevalence of cryptococcal meningitis is significantly higher in poorer countries with less access to necessary treatments and supplies. Duke Hospital’s own survival rate for Crypto. infections is around 80%, while the global survival rate is only 50% (skewed largely by the populations lacking in adequate healthcare resources). Despite this frightening statistic, research on Crypto. is largely underfunded due to the fact that cryptococcal meningitis is a noncommunicable disease, or it cannot be spread person to person via a cough or a sneeze the way that the flu, COVID-19, or other common diseases can.

Luckily, Dr. Perfect’s lab is doing the hard work, spending every day working towards understanding Crypto. and trying to find new ways to target it through drugs and other methods to help prevent or cure future cryptococcal meningitis infections. This is where I come in (with my bench mentor/graduate student Julia, of course). One of the most interesting things about Crypto. is where it causes infection: the brain/spinal cord. A vast majority of other pathogens struggle significantly with the lack of nutrients and overall harsh environment of cerebral spinal fluid in the central nervous system, which is why Crypto. is such an intriguing fungus; where others fail at mere survival, Crypto. seems to thrive in this nutrient-deficient environment, hence its deadly survival rates. One project I am helping work with Julia to look at this summer is one particular set of genes that may be influencing this growth. These genes regulate nitrogen catabolism, or the identifying and processing of available nitrogen sources for use. Nitrogen catabolite repression (NCR) is the name for the regulation of these genes via certain regulators based on the nitrogen sources available (normal, easily metabolized sources results in a negative regulation — turning the genes off — and sources that require several steps to break down cause a positive regulation response — activating the genes encoding for certain necessary enzymes to break the nitrogenous compounds down –). Our first steps so far have been looking at two potential NCR genes, tagging them with a fluorescent tag (we are using mCherry, but for comparison it’s like GFP), and looking for any interactions between the proteins based on localization (if the proteins are co-localizing, we will see a certain fluorescent pattern showing them near each other). Knowing if these genes work together to regulate nitrogen catabolism is critical for moving forward in the overall investigation of identifying these regulation pathways in Crypto.

These last two weeks, we have been assembling all the necessary pieces to create the tagged strains of Crypto. I first had to design primers that would work in a PCR reaction to amplify each of the 5 segments needed for the tagged strain (the 5’ UTR, for attaching to a plasmid backbone, the NAT antibiotic resistance cassette, for incentive to integrate the plasmid into Crypto., the promoter + gene itself, the mCherry fluorescent tag, and the 3’ UTR region to attach to the other end of the plasmid backbone). The PCR results so far have been mostly successful, with two stubborn fragments still left to successfully amplify (let’s just say I’ve run a LOT of electrophoresis gels this week). Once we know that the PCR worked, we cut out the bands and extracted the DNA from them to be saved until all the pieces are ready. This week, we are finally going to do a Gibson assembly to put all the puzzle pieces together.

These two weeks so far have been a whirlwind of reading papers and learning techniques and working towards an exciting goal; I feel like a supersaturated sponge, but I can’t wait to keep learning more!

My first (of many) electrophoresis gels showing the successful PCR reactions for gene 1: URE3. Our 2nd fragment had the best band, meaning it worked the best (NAT cassette), and was extracted, but most of the other four fragments had to be run again in a new PCR reaction.

This was the gel electrophoresis showing the PCR reactions for gene 2: TAR1. The brighter bands were the successful amplifications, and the ones that were very faint or didn’t show up at all in the column had to be run again. Here, we were able to gel extract from 1 and 2, and some from 3 and 4 (This was a vast improvement from the first URE3 gel, which was one of the first PCRs and gels I ran in the lab).

Genetics intrigues me because of its ability to explain the mysteries of biology. It helps us understand the biological programming behind all life forms, including ourselves. In the past 100 years we have discovered DNA, developed ways to read it, and now we are working on methods to write and edit this code. It is the growing understanding of the universal language of life that provides us the incredible power to shape the future of humanity. This is a scientific revolution that, with the right amount of careful consideration, will change the human condition for the best. Most obviously it will transform healthcare. 

A person’s healthcare treatment today is based on what works for the average human in a population of 7 billion. But with the decreasing cost of genome sequencing and a better understanding of the genome itself, a person’s treatment is becoming increasingly based on their biology. Healthcare is therefore beginning to shift from generalized to precise. 

One of these novel precise treatments is gene therapy and the researchers at Asokan Lab work to find novel ways of improving it. Gene therapy is a technique that targets the cause of the disease by finding genetic solutions for genetic disorders. The classic model of gene therapy is to use a viral vector to deliver a working copy of a defunct gene. The introduction or change of genetic material into the cells of a patient is all about changing how a protein or group of proteins is produced by the cell. For example, one of the genetic disorders targeted by the project I’m currently working on is Duchenne muscular dystrophy (DMD). Patients with DMD have severely reduced muscle strength as a result of alteration to a protein called dystrophin that helps keep muscle cells intact. The goal of the project I’m working on is to increase levels of functional dystrophin expression in DMD patients through RNA editing. 

The central dogma of biology is that the pattern of information flow in our cells goes like this: From DNA to new DNA (DNA replication), from DNA to new RNA (transcription), from RNA to new proteins (translation). RNA gene therapy targets disease-causing mutations at the translation step. In our project, we aim to edit RNA via trans-splicing by manipulating the splicing pathway by which pre-mRNA turns into mRNA. In doing so, we can replace a mutant exon in the DMD mRNA transcript, which is known to abolish dystrophin production, with a functional version of that exon. When this correction is made, therapeutic levels of dystrophin restoration can occur. DMD is only one of the multiple genetic disorders we will be targeting with this RNA editing mechanism. I’m very excited to be working on a project with this level of therapeutic novelty and medical relevance.

What do you think creates a habit? Habitual behaviors are defined by learning  an automatic response to a certain stimulus. For example, you could have breakfast in the morning, even if you are not feeling particularly hungry at the time. Here, the action(having breakfast) is executed regardless of the goal(consume calories to feel full), but instead follows a learnt stimulus(time). Thus, habitual behaviors are not conducted for the aim of fulfilling a goal, but is dependent on the stimulus.

Calakos lab investigates the cellular and circuit changes in the brain(with a focus on striatum) as a behavior changes from goal-directed to habitual. This can be done by overtraining, where an initial goal directed response to press a lever to obtain food pellets becomes habitual/automatic over repeated trials. In this case, the point when the researcher knows the behavior is no longer goal-directed is when the mouse still presses the lever with same frequency, regardless of whether it was full or hungry before the behavioral test. This is called the devaluation test as you devalue the utility of the outcome.

Striatum is a brain area known to be involved in not only initiating or inhibiting movement, but also changing from goal-directed to habitual behavior. My research project is to image the afferent connections into the DMS and DLS-dorsomedial striatum and dorsolateral striatum- using retrograde tracer AAV. Active brain circuits during goal-directed and habitual behavior will be selectively labeled, which would be used to compare how the inputs into the DMS and DLS changes as lever press behavior turns from goal to habit.

The project that I am working on in the Volkan Lab fits neatly into one of the lab’s broader questions: how chromatin regulation around master genes affects behavior. In regards to the fruit flies I will be studying, the behavior in question is male courtship and the master gene I will be looking at is called Fruitless, or fru. This fru gene transcribes a transcription factor that has been found to be the primary effector in the development of appropriate courtship rituals in male flies.

My specific project is to try and find the mechanism through which peripheral sensory systems affect courtship behavior. The sensory system I will be studying is a particular receptor in the olfactory system called IR84a. This receptor is responsible for sensing (and be activated by) phenylacetic acid and phenylacetyl aldehyde, two chemicals that are found on the fruits and plants that the flies use as oviposition sites and food sources. Upon exposure to these chemicals, and thus activation of the receptors, male fruit flies experience increased courtship behavior. In addition, when mutants are made that lack the IR84a receptor, the male flies not only lose their ability to sense these aphrodisiacs but they also display a lowered base courtship rate compared to wild type male flies. These two phenotypes clearly demonstrate IR84a’s link to courtship.

Studying IR84a ties into the overall question of the lab because we hypothesize that the mechanism through which IR84a affects courtship behavior is by regulating the chromatin state around the master gene fruitless. A process called ChIP, Chromatin Immunoprecipitation, will be performed to assess the chromatin state around fruitless in both the peripheral and central nervous systems of both IR84a mutant and wild-type male flies. If there is a difference between the two chromatin states, then this will provide evidence in favor of our hypothesized mechanism.

First full week at the Mooney Lab: complete. What can I say, this week has assuredly presented some steep learning curves. From accidentally sacking my bird (an affectionate euphemism I suppose) during a brain injection surgery to being unable to find the left ventricle during a perfusion (the process of draining the blood from a sacked bird), this week has posed many challenges. Nevertheless, learning all of these techniques and being thrown into the deep end of neurobiology research is quite exhilarating. I constantly find myself thinking, “Woah, this is sooo cool! (for lack of better words haha).” 

In the short time I have spent with the lab, I am by no means an expert on the project I am about to conduct; however, I will try my best to explain it here. Yet, before I do that, I think it will be helpful to define a few terms (learning these definitely helped me in understanding my project):

dopaminergic neuron cells: a class of neurons that synthesize the molecule dopamine

tyrosine hydroxylase (TH): enzyme that is essential in the synthesis of dopamine; all dopaminergic cells will have the TH enzyme

VTA/SNc: areas located in the midbrain of the zebra finch that are noted by a high concentration of dopaminergic cells

green fluorescent protein (GFP): protein that fluoresces green when exposed to certain wavelengths of light; commonly used as a reporter of gene expression

For my project, I will be testing whether a new cell editing technology is functional and expressed in the dopaminergic neuron cells of the zebra finch. For proprietary reasons, I can’t say the name or describe exactly how this new technology works. However, at a surface level, the technology is able to recognize and bind to a chosen RNA sequence within a cell (in my specific case, it is the sequence that encodes the TH enzyme for dopaminergic neurons). The technology also contains the sequence for GFP. If the technology successfully binds to the TH sequence, then GFP should be expressed. To test this, I will surgically inject the technology into the VTA/SNc region of the zebra finch and “let it sit” for around a month. After this waiting period, I will sack the bird and analyze the dopaminergic neurons within the VTA/SNc and determine if the technology was successful. 

So, in a nutshell, that’s my project! Right now, I am familiarizing myself and learning how to conduct all of the necessary techniques. So, I still have a little time before I actually start the experiment. For now, it’s just learning surgical procedures and how to do bird haircuts (yes, it is exactly what it sounds like).

In 2006, a Japanese researcher named Yamanaka discovered a chemical concoction which would allow the dedifferentiation of fated cells to once again become induced pluripotent stem cells (iPSCs). This discovery had significant impact in the world of regenerative tissue engineering, as it allowed for the indefinite creation and proliferation of complex cell types cultured from the patient’s own cells. In particular, the Bursac lab under which I am currently researching studies how iPSCs can be used to specify functional synthetic myocytes, both skeletal and cardiac. However, the large roadblock in tissue engineering research currently is that tissue fated from iPSCS are not able to generate as much force and muscular volume as primary myocytes.

The projects I am working on seek to better understand the roles that vascularization and innervation play in the development of functional skeletal muscle tissue, and more importantly, how co-culturing endothelial cells or neurons can allow muscles to exhibit greater forces. As one may expect, working with cell cultures are very demanding and requires a comprehensive set of procedures to ensure the growth and differentiation of each tissue type, as well as the necessary processing in order to understand the capabilities or functionality of the cells.

Although I’m only one week in, I’ve definitely picked up a plethora of laboratory techniques which were necessary in order to cover all aspects of an experiment. I learned how to create 2D and 3D growth media which facilitates the growth of cells within the first week of culture. After this, the cells will spend 2-3 weeks in a differentiation media which causes them to fuse and interact with each other and form tissue. It is during this step where endothelial/neuron cells will interact closely with the formed myofibers and form supporting vasculature or neuromuscular junctions. To understand the state of the muscle fibers at different points of the experiment, the tissue can be used in many ways. One of the most important of course is force testing, in which we rig bundles to a sensitive force guage and electrically stimulate them to understand their impulse responses or tetanus response. Another common way to understand the bundles is to do cross section stains, which requires the use of the cryostat machine I described last week, or whole bundle stains under which the entire skeletal muscle tissue will be imaged. Another procedure would be to use whole bundle RNA isolation which allows for the analysis of RNA expression at some week to better understand the genetic effects the presence of supporting cell types may have on muscle development. Additionally, we may use qPCR to quantify the relative amounts of certain genes and which ones are expressed more than others.

Although it sounds simple, there are inherent limitations to such research. For one, cells are fickle and it is difficult to understand why an experiment may go awry or why cells may behave differently, and these fluctuations can only be mitigated through strict sterilization practices. Additionally, the growth cycles for engineered tissue take weeks to months to produce tissue, which is currently ineffective for clinical application. However, I am optimistic that the research I am doing will allow for us to take one step closer to the goal of quick tissue regeneration and integration to save victims suffering from dire wounds.

My research project involves studying a particular type of pitcher plant called Sarracenia purpurea. These carnivorous plants have leaves that form pitcher-like structures that are able to collect water. This water, however, hosts a community of microorganisms that digests any prey that falls into these pitchers. The waste excreted by these organisms can then be taken up by the plant. 

This research project involves taking trips to various locations where these plants are found. We will be taking samples of the fluid in the pitchers as well as taking clippings of the leaves. The fluid will be studied to see what types of prey the plants are catching, what organisms live in the water, and what enzymes are present. Furthermore, nitrogen analysis will be conducted on the plant’s leaves. So far, we have visited and surveyed about half of the sites we will be sampling from, and we will soon begin sampling and studying these complex plants.

Although these plants may seem simple and uninteresting, there is much more to them than initially meets the eye. Within these plants lives an entire food web, and, even though the interactions within the food web will be studied on a small scale, learning about these interactions can help us understand those that occur at a much larger scale.