Alpha-actinin localization in Zasp52 mutants during dorsal closure

Dorsal closure is a stage of Drosophila embryogenesis in which lateral epidermal cell sheets bordering an eye-shaped dorsal opening ”zip” together to form a seamed and then seamless epithelium. Understanding this process is important because it models cell sheet movements such as neural tube closure and palate formation as well as wound healing in vertebrates. The dorsal opening itself is filled by a layer of thin, flat amnioserosal cells. The amnioserosa is bordered by actomyosin-rich purse strings that provide tension and maintain the shape of the opening as it closes. The expression of Echinoid in the lateral epidermis and the lack of expression in the amnioserosa is known to be essential for actomyosin cable formation, but what proteins govern the formation and stability of the purse string are not fully understood. I used a GFP-tagged alpha-Actinin fusion protein to visualize alpha-Actinin localization in Zasp52 and Echinoid double mutants as well as in Echinoid mutants only. Fluorescent imaging revealed that in Zasp52 and Echinoid double mutants, alpha-Actinin does not appear to localize to the purse string, while in Echinoid mutants it does. This provides evidence that it is Zasp52 that recruits alpha-Actinin to the purse string.

DIFFERENCES IN GROUND REACTION FORCES BETWEEN A STIFF AND COMPLIANT GAIT

Orthopedic injuries often occur while the knee is extended, in a relatively stiff (limited change in angle) position that generates relatively high ground reaction forces (GRF, forces applied to the body from the ground). Modern humans walk with their knees in this relatively stiff position while apes, our closest relatives, utilize a compliant gait that features greater knee joint yield (decrease in knee angle). Compliant walking has been predicted to lead to lower GRFs, but there is limited data to test that presumption in walking. The Animal Locomotion Lab sought to compare the differences in GRFs, using 28 participants walking with a stiff and compliant gait. If  participants walking compliantly are shown to have lower GRFs  compared to when they walked stiffly,  implications span from orthopedics to evolutionary anthropology. The data was collected using two methods to determine the validity of an open-source and marker-less gait analysis technique (OpenCap). Traditional gait analysis requires force plates and markers to gather force and joint angle data. This method, though accurate, is resource intensive and hard to utilize in nontraditional settings. OpenCap performs the same data analysis with fewer resources and is applicable in various settings. Even though we found significant differences in the numeric values of minimums of the force traces, Open Cap still calculated accurate data for peak forces that was not statistically different, which can still provide insight into how athletes, non-athletes, healthy, and injured people move. 

Pseudouridine as a Key Player in Plant Immune Response – BSURF 2023, Week 6

Pseudouridine, an isomer of the uracil nucleotide, is a common RNA modification. However, its function is not fully understood, especially during plant immunity. In collaboration with Dr. Chuan He’s laboratory in University of Chicago, we applied a new technique, BID-Seq, to quantitatively measure pseudouridine modification in plant RNAs at base resolution. This revealed that this modification is widely present in plant mRNAs. After being used in COVID vaccines in previous studies, it was found that pseudouridine helped in suppressing immune responses in humans from the vaccines. Due to this, we hypothesize that mRNA pseudouridylation plays a role in regulating plant immune response as well. In order to understand the impact of pseudouridine on plant immunity, we have taken seedlings of Arabidopsis and subjected them to various pathogen treatments. By measuring the pseudouridine levels in mRNAs, we are looking to see dynamic changes of pseudouridine levels which could be an important trigger for the plant’s immune response. Furthermore, we seek to identify what proteins are involved in the pseudouridine synthetic pathway during immunity by using a tagger on known proteins involved in the pathway. If we can understand the importance and mechanisms of pseudouridine in plant immunity, we believe it would be a significant step towards strengthening agriculture as understanding plant immune response mechanisms to pathogens will help us benefit our crops which ultimately will benefit our communities. 

Investigating Tools to Study Calcium Dynamics in Arabidopsis CPK mutant Immune Response

Pattern-triggered immunity (PTI) is an ancient, conserved branch of the plant immune system activated by pathogen-associated molecular patterns. Calcium signaling is an important part of the immune pathway and plant responses to stress, yet the spatiotemporal dynamics of calcium during PTI are not well understood. Previous research has demonstrated the role of calcium-dependent protein kinases (CPKs) in downstream cytoskeletal regulation and actin reorganization, but the role of calcium in these pathways is unknown. Here, we validate tools to study calcium dynamics using the model plant system Arabidopsis thaliana, and mutations in CPK3-2 and CPK6, which have previously shown cytoskeletal phenotypes during PTI. T-DNA insertional knockout lines cpk3-2 and cpk6 were used to explore the connection between CPK pathways, calcium signaling, and the cytoskeletal PTI response induced by the bacterial flagellum-derived protein flg22. The knockouts were crossed with the genetically encoded calcium biosensor RGECO1/mTurquoise. Seed lines were bulked and genotyped to select homozygote lines for the TDNA insertional mutant, as well as screened via confocal microscopy to check the fluorescence and functionality of the RGECO1/mTurquoise cassette. Time-lapse images were taken of selected plants with the confocal microscope after inoculation with flg22, and their initial calcium responses were compared with the wildtype RGECO1 cross. The preliminary results presented here give further insight into the roles CPK and calcium play in the PTI response, and how best to continue studying calcium and cytoskeletal dynamics in plant cells.  

DETERMINING THE ROLE OF K63 UBIQUITINATION IN ER TRANSLATION DURING OXIDATIVE STRESS

Oxidative stress is a pervasive environmental challenge linked to the development and progression of several diseases, including cardiovascular disease, cancer, and immunodeficiency. Understanding various cellular responses to oxidative stress is essential in developing therapeutic interventions. The Ubiquitin Proteasome System (UPS) regulates several cellular processes, including protein degradation, synthesis, and gene expression. The Silva lab investigates protein translation by K63 ubiquitination during oxidative stress. Recent literature indicates significant translational regulation occurs in the endoplasmic reticulum (ER), and our lab highlighted preferential K63 ubiquitination of ER-bound ribosomes under oxidative stress in mammalian cells. Despite this, numerous functions of ER-localized translation remain unsolved. This study will determine how stress-induced K63 ubiquitination plays a role in ER-localized translation regulation using saccharomyces cerevisiae (yeast) as a model. To investigate this process, we will utilize several techniques, including cellular fractionation using differential centrifugation. We will further validate differences in accumulation between cellular compartments by performing western blot analysis targeting ubiquitin conjugates. These studies will allow us to better understand whether ER-localized K63 ubiquitination is conserved across species and provide avenues for understanding its essentiality for cellular responses to oxidative stress. Eventually, this will enable us to develop targeted therapies that leverage the power of this cellular modification to better control and alleviate the detrimental effects of oxidative stress and pave the way for future treatments and interventions.

PRENATAL ENVIRONMENTAL TOXIN EXPOSURE AS A RISK FACTOR FOR NEURODEVELOPMENTAL DISORDER PATHOLOGY IN ADOLESCENCE

The prevalence of neurodevelopmental disorders (NDDs) has increased rapidly in recent decades. Genetic mechanisms of these disorders have been studied extensively; however, environmental factors contributing to NDD development remain less understood. Current evidence suggests that alterations in synaptogenesis and pruning underlie NDD pathology. Sleep is essential to this synaptic remodeling process. Disturbed sleep is a highly conserved trait affecting nearly 86% of NDD patients. Additionally, sleep appears to be particularly sensitive to environmental perturbations, as negative sleep outcomes have been strongly linked to exposure to air pollutants. The Diesel Exhaust Particle and Maternal Stress (DEP/MS) paradigm was used to co-expose pregnant mice to DEP and a maternal stressor, modeling epidemiological data suggesting that combined prenatal exposure to DEP is strongly linked to socioeconomic stressors. Previous studies have revealed that DEP/MS offspring show male-specific social and behavioral traits consistent with NDD pathology. Based on preliminary data demonstrating sleep deficits in DEP/MS adult offspring, this study aims to characterize NDD phenotype of DEP/MS offspring during adolescence, focusing on possible female-specific sleep and behavioral alterations. To analyze sleep patterns in DEP/MS and control offspring, we performed electroencephalography and electromyography (EEG/EMG) recordings from P37 to P40. Additionally, we isolated astrocyte and microglia populations from parietal and frontal cortex tissue. Lastly, we conducted the forced-swim test to analyze depressive-like behavior, a common comorbidity in female NDD patients. We hypothesize that DEP/MS offspring will exhibit sex-specific depressive-like behavior and sleep deficits driven by changes in astrocyte gene expression.

The Contribution of Nrg1/ErbB4 Signaling to the Homeostatic Effects of Seizures in Epilepsy Models

​​Temporal lobe epilepsy (TLE) is known to be induced by seizures themselves, although there is much to learn about the mechanisms by which TLE develops and progresses (“epileptogenesis”). Briefly inhibiting BDNF/TrkB signaling induces regression of epileptogenesis, yet this only occurs when introduced following a seizure. Thus, we propose that seizures also activate signaling pathways that inhibit epileptogenesis. The receptor tyrosine kinase ErbB4 is expressed exclusively in the interneurons of the hippocampus where the activation and inhibition of ErbB4 signaling exerts anti- and pro- convulsant effects respectively in diverse seizure models. We hypothesize Nrg1 activated ErbB4 to be involved in this seizure-activated, anti-epileptogenic signaling pathway. Kainic-acid status epilepticus mice cohorts and PBS control mice cohorts were studied using in situ hybridization techniques such as RNAscope to label targeted mRNA sequences with fluorescent tags. Charting the spatial profile of KA-SE induced Nrg1/ErbB4 signaling was achieved using ImageJ software that produced image based, semi-quantitative analyses of the RNAscope data. We expect to be able to quantify and localize the increases in Nrg1 mRNA expression that has been seen in our preliminary data. Considering that ErbB4 receptors are expressed in hippocampal interneurons together with the requirement of parvalbumin (PV) interneurons for the anticonvulsant effects of Nrg1 suggested that seizures may enhance PV neuron mediated synaptic inhibition. Understanding the circuits and cells in which KA-SE activates Nrg1/ErbB4 signaling would facilitate elucidating its functional consequences.

Fabricating Microfluidic Devices for Modeling Microvasculature

Microfluidic devices are tools used to study cell behaviors and interactions with their microenvironments. Typical in vitro models, like 2D-culture dishes, insufficiently capture the complex structure of the extracellular matrix, whereas in vivo models like animal testing make it difficult to control experimental conditions. A microfluidic device is a small chip that permits 3D cell cultures while still allowing control over desired variables, such as flow rate and growth factor concentrations in the cell media. Although there are commercially available devices, these options normally lack design variability and are costly. Therefore, there is a need in the lab for a method to design and fabricate personalized chips for our specific applications. My project is to investigate existing protocols and formulate a procedure for producing microfluidic devices in our lab. The main fabrication method is lithography, which involves printing a design onto a silicon wafer and using it to mold several chips. Most current protocols leave the photoresist, or the “ink,” on the wafers when molding (Scott & Ali, 2021); instead, we physically etched our designs into the wafer to more economically achieve the feature depths needed for our lab’s application. By testing different elements, such as etching and sterilization, we hope to find the most effective method for promoting blood vessel formation in these chips. If perfected, our lab can readily fabricate its own chips for experimental use to advance our current in vitro models and improve the quality of our experiments.

EXPLORING THE PHENOTYPIC CHARACTERIZATION OF CRYPTOCOCCUS NEOFORMANS CLINICAL ISOLATES

Cryptococcus neoformans is a human fungal pathogen that can infect the lungs and later spread to the central nervous system. This disease is most associated with immunosuppressed individuals. C. neoformans is distinctive compared to other fungi because of its ability to produce a thick capsule comprised of polysaccharides, proteins, and other molecules, which shield this yeast from environmental stresses. The capsule can also interfere with the hosts’ immune response by dampening the response and preventing phagocytic cells from engulfing the yeast due to its increased size. We hypothesize that variation in capsule size among clinical isolates could result in different patient outcomes, as it could aid the cells in establishing disease and avoiding immune detection. For instance, smaller capsules may enable this yeast to survive better within macrophages through adaptation. These infected macrophages can then travel to other parts of the body and spread the infection, while yeast with large capsules pose a challenge for immune cells to engulf. To test this hypothesis, we carried out in vitro analyses to examine which conditions caused optimal capsule production in C. neoformans isolates from temperature, growth duration, media volume, and other growth conditions. In addition, 162 clinical isolates were evaluated for sensitivity to cell stressors, SDS detergent, and caffeine, that affect cell membrane and cell wall integrity. By identifying the conditions that promote optimal capsule growth–a major virulence factor– for this yeast cell, and those that hinder it, more comprehensive research can be conducted on C. neoformans and other fungi.

RNAi pathway components may contribute to starvation-induced gonad abnormalities in Caenorhabditis elegans

Caenorhabditis elegans that hatch into an environment without any nutrients are arrested in the first larval stage of development, L1. Larvae can remain in L1 arrest for weeks and resume normal development once they receive food. However, many C. elegans that experience extended L1 arrest develop abnormal gonads in adulthood, including germ-cell tumors and uterine masses. These growths suggest a misregulation of cell growth. Previous results indicate that genes involved in processing somatic RNA interference (RNAi) could be causing abnormality formation. RNAi refers to a cell’s response to exogenous or endogenous double-stranded RNA. It functions as an internal regulatory mechanism as well as an immune response, allowing sequence-specific suppression of corresponding genes. We hypothesize that endogenous RNAi pathways are involved in the occurrence of starvation-induced gonad abnormalities. It is possible that aberrant small RNAs produced by somatic RNAi machinery are transported to the germline, causing abnormalities to form. We examined rrf-1, ppw-1, rde-1, dcr-1, rde-4, sid-1, alg-1, and rrf-3, which correspond to components of various RNAi pathway branches. Knockout strains and RNAi were used to evaluate the impact of these genes on gonad abnormality frequency in adults following 8 days of L1 arrest. So far, results align with previous findings about somatic RNAi and our transportation hypothesis. We expect future results to further corroborate these findings and clarify the role of different RNAi pathway branches in gonad abnormality formation. Overall, these experiments provide greater insight into the little-known mechanisms regulating pathologies that result from early-life starvation.

Assessing C. elegans innexin location phenotypes in touch-sensitive neurons

Gap junctions are intercellular channels made up of hexameric units, connexins in vertebrates or innexins in invertebrates, that allow ions and small molecules to pass between adjacent cells. Although gap junctions are essential to the survival and function of organisms, relatively little is understood about them. To understand more about gap junctions, we used TurboID, a proximity biotinylation enzyme fused to a neuronal innexin, UNC-7, folowed by HPLC Mass Spectrometry to identify proteins of interest. We generated single gene deletion strains, guided by the TurboID data to gauge its effect, if any, on innexin localization. The effect the proteins of interest have on innexin regulation was assessed by introducing deletion alleles into a strain that labels an innexin closely associated with UNC-7, UNC-9, with GFP and expressed in a single pair of touch-sensitive neurons in the animal’s tail, the PLM neurons. PLM neurons form gap junctions with several other important neurons in C. elegans, including the PVC, PVR, and LUA neurons. These neurons should appear as bright GFP puncta on the PLM axon. Using this system, we found that the deletion of syg-1, unc-116, vab-1, and src-1 lead to phenotypes in the localization of the GFP labeled UNC-9 innexin. When looking at these genes, we sought to find out whether the deletions affected function – forming nonfunctional gap junctions, interrupting cell-cell communication, or not forming gap junctions – trafficking, or positioning at the correct synaptic face.

Optimizing assembly of asymmetric peptide nanofiber vaccines for delivery through mucosal membranes

Mucosal vaccines are gaining increased attention due to their potential to tackle distinct bodily mucosae and induce immune responses to prevent and treat infectious diseases and autoimmune conditions such as inflammatory bowel disease (IBD). Some benefits over traditional vaccination methods include needle-free delivery and localized action through the production of IgA and tissue resident T cells. Firstly, materials need to cross a dense layer of mucus before reaching the epithelium and achieving the desired therapeutic goal. Moreover, each mucosal membrane is distinct and presents its own properties, for instance, pH, immune cell type, turnover rate, etc, that call for specific biomaterial properties for successful delivery. Inspired by infectious organisms that naturally cross human mucus to impair cellular function (e.g., Influenza A virus), this project aims to develop a safe and effective carrier platform that can both attach to and cross mucosae in time before being carried away by natural mucus turnover in the host organism. The asymmetry of the fibers is essential so that they can perform a Brownian ratchet-like motion, where one subunit of the fiber is constantly cleaving mucus whereas the other provides support by attaching to mucus. After conjugating each subunit with a fluorophore, we observed through confocal microscopy that they combine most effectively at 95°C for 10 minutes. We also tested the binding activity of two mucin-binding peptides identified from the literature as well as epitopes of interest through a mucoadhesion assay. C3a1 showed the highest binding activity. Knowing this, we aim to conjugate this binding subunit with a mucus cleaving enzyme and assess their overall dynamics in mucus. 

ELASTIN-LIKE POLYPEPTIDES WITH MODIFIED CDC-19 AMYLOID INSERTS 

J Savage, M Ney, A Chilkoti

Biomedical Engineering; Duke University; Durham, NC 27708 

Elastin-like polypeptides (ELPs) have the ability to phase separate in liquid which provides many applications (like protein purification), and with modifications they can have pH dependent properties to form functional materials. ELPs reversibly change solubility based on temperature, becoming insoluble after reaching a critical solution temperature. CDC-19 amyloids are found in yeast and sequester proteins during stress. When added to ELPs, the ELPs become irreversible below a certain pH threshold. However, this threshold is below physiological conditions and pH changes alter ELP transition temperatures, limiting use and predictability. It was hypothesized that 1) substituting the aromatic amino acids will increase the threshold pH and 2) adding positively charged amino acids to neutralize the amyloid’s negative charge will stabilize the phase transition temperature across pHs. Polypeptides were placed into a UV-Vis spectrophotometer at various pHs, then heated and cooled to record transition behavior and measure phase reversibility. Polypeptides were also imaged under a microscope to show phase behavior assemblies (such as droplets and solids) at different pHs. It was found that the substitution of aromatic amino acids increases the pH threshold of polypeptides. In contrast, the addition of positively charged amino acids, arginine and lysine, abolished pH sensitivity. A material that is pH sensitive potentially has many applications, such as researching cancer as cancerous cells have distinct pHs. Future research should seek to increase the pH at which the ELPs change to a physiological one and stabilize the temperature-dependent behavior at various pHs.



What is the Genetic Cargo Necessary for Ferroptosis Sensitization of MCF7 Cells?

Ferroptosis is a form of programmed cell death characterized by the accumulation of reactive oxygen species, which lead to lipid peroxidation and membrane rupture. To search for novel ferroptosis inducers and inhibitors, we combined two ferroptosis inducers: erastin and RSL3 with different drug-like compounds from Pandemic Response Box, a compound library for possible Malaria treatment. We identified tipifarnib as a candidate, which was shown to be particularly effective in sensitizing MCF7 cells to ferroptosis. Aside from the known target of farnesyltransferase we believe that tipifarnib has other targets that lead to the observed phenotype. We performed a one-pot assay and identified exportin/importins as the potential targets of tipifarnib to trigger ferroptosis. We hypothesize that tipifarnib disrupts certain import/exportation of “cargos” resulting in the sensitization to ferroptosis. We treated MCF7 cells with compound inhibitors of exportin, importin, and tipifarnib. Also we shRNA to knockdown exportin, importin, to look for potential pathways that results in the phenotypic change. Through the usage of RT-PCR assays we have data that suggests possible targets and non-targets that make MCF7 cells sensitive to ferroptosis treatment. With further investigation, this provides an avenue for an alternative strategy to treating tumors. 

Optimizing Cyanobacteria Detection for Comparing Lichenized and Environmental Cyanobacterial Communities

Cyanolichens are symbiotic associations between fungi and cyanobacteria. When lichenized fungi reproduce sexually, the fungal spores are dispersed without the cyanobacteria, and they must find a new partner to form a cyanolichen thallus. However, how common these potential cyanobacterial partners are in the substrates where the cyanolichens grow is unknown. A robust sequencing procedure is necessary to compare the communities of lichenized and environmental cyanobacteria. This study aims to optimize the procedure for sequencing 1800 environmental samples from Alberta, Canada. We tested the sensitivity of the 16S rRNA and rbcLX genetic markers for cyanobacteria detection and the role of substrate depth on cyanobacterial community composition. The rbcLX genetic marker is more variable but is single-copy, while the 16S rRNA gene is less variable but exists in multiple copies. Sequencing the 16S rRNA gene may be better for detecting trace amounts of cyanobacteria in environmental samples. Additionally, cyanobacteria are expected to be more diverse in top substrate layers because they need light for photosynthesis, so sampling from deeper depths may not be necessary. We sequenced the two markers from 112 environmental samples, including 12 with known taxonomic compositions. The remaining 100 samples have unknown compositions and include both top and bottom substrate layers. We will use the sequence data to compare the detection sensitivity of the two genetic markers. Preliminary results favor the 16S rRNA gene. The results will inform the procedure for large-scale characterization of environmental cyanobacteria to understand the formation of the cyanolichen symbiosis.

Abstract: Localization and function of ARC6 in cell division

Chloroplast division of photosynthetic eukaryotes is a highly regulated process with divisionary machinery involving a division ring and other essential components. To gain insights into this process, our lab uses the green alga Chlamydomonas reinhardtii, which possesses a single chloroplast that coordinates with the rest of the cell during division. We investigate the role of the ARC6 protein, a potential linker between the inner chloroplast membrane and the FtsZ division ring. We aim to investigate the dynamics of ARC6 localization and its potential role in the context of various division components and the FtsZ ring. While previous studies have indicated that ARC6 localizes at the division site, the underlying mechanisms and its relationship with FtsZ and other divisionary machinery are not fully understood. In our study, by employing fluorescent microscopy, we characterized the localization patterns of ARC6 during cell division. Our findings reveal a dynamic assembly and disassembly behavior of ARC6 at the division site, enabling movement along the chloroplast membrane to subsequent division sites. Previous research has suggested that cytoskeletal actin may be important for the formation of the inner division ring. Surprisingly, we found an apparent loss of ARC6 at the division site in the absence of actin in our experiments. This result suggests that the cell uses the cytoplasmic F-actin to regulate the localization of ARC6 in the chloroplast inner envelope. Possible mechanisms for this regulation and the consequences of loss of ARC6 from the division site are being examined.

Rescue of glycogen storage disease Ia in mice by liver-directed therapies – Research Abstract

Hannah Baetge

Mentors: Mercedes Barzi, PhD, Karl-Dimiter Bissig, MD, PhD

Department of Pediatrics | Division of Genetics

Rescue of glycogen storage disease Ia in mice by liver-directed therapies

Glycogen Storage Disease Ia (GSD Ia) is a rare inherited metabolic disease caused by a deficiency in the G6PC1 gene, which codes for an important protein called glucose-6-phosphatase-alpha (G6Pase-alpha), primarily active in the liver. Without G6Pase-alpha, a key enzyme in gluconeogenesis, the patient is unable to break down glycogen into glucose. Consequently, GSD Ia patients experience low glucose levels, glycogen accumulation in the liver, and toxic metabolite build-up in other organs of the body including the kidney, intestines and in some cases the brain. Long-term implications may include liver cancer and end-stage kidney disease. The primary treatment option for GSD Ia involves a dietary approach; however, patients’ quality of life remains considerably compromised. Additionally, the dietary regimen does not provide a preventive measure against long-term complications. A viable gene therapy would significantly increase the quality of life of these patients, but it has yet to be found. One key obstacle in delivering gene therapy lies in hindrance caused by glycogen accumulation in the liver. To address this issue, we are focused on developing a solution comprised of a drug and gene-editing therapy using liver-specific G6pc-knockout mice. Through this approach, we hope to enhance gene editing efficiency for GSD Ia and come closer to a promising therapeutic solution for the victims of this debilitating disease.

References

¹Cho JH, Lee YM, Starost MF, Mansfield BC, Chou JY. Gene therapy prevents hepatic tumor initiation in murine glycogen storage disease type Ia at the tumor-developing stage. J Inherit Metab Dis. 2019 May;42(3):459-469. doi: 10.1002/jimd.12056. Epub 2019 Mar 6. PMID: 30637773; PMCID: PMC6483852.

²Gjorgjieva M, Calderaro J, Monteillet L, Silva M, Raffin M, Brevet M, Romestaing C, Roussel D, Zucman-Rossi J, Mithieux G, Rajas F. Dietary exacerbation of metabolic stress leads to accelerated hepatic carcinogenesis in glycogen storage disease type Ia. J Hepatol. 2018 Nov;69(5):1074-1087. doi: 10.1016/j.jhep.2018.07.017. Epub 2018 Sep 5. PMID: 30193922.

³Pankowicz FP, Jarrett KE, Lagor WR, Bissig KD. CRISPR/Cas9: at the cutting edge of hepatology. Gut. 2017 Jul;66(7):1329-1340. doi: 10.1136/gutjnl-2016-313565. Epub 2017 May 9. PMID: 28487442; PMCID: PMC5878048.

Lisa’s Chalk Talk

This week in BSURF, we were tasked to present our projects in the form of a chalk talk. We had 8 minutes and a whiteboard, and had to communicate the central ideas and questions underlying our projects. Each talk was engaging and informative, and I enjoyed this dedicated time to further my understanding of the other fellows’ projects. Now, when I ask my BSURF friends about their days in the lab, I’ll have some background on the techniques they use!

For this week’s blog, I’m asked to identify a fellow’s chalk talk to reflect on. Lisa’s talk was particularly intriguing to me. Her research focuses on CRISPR-Cas9—more specifically, the guide RNAs used to direct the Cas9 protein to a DNA sequence. 

Lisa first provided background on the broader implications of her project. She explained how currently, the most pressing risk associated with the use of CRISPR is off-target DNA cuts. I learned about how Lisa’s project uses dCas-9, a deactivated form of Cas9 that finds, but doesn’t cut, a specific sequence of DNA. From my understanding, the overarching goal of her project is to gain a better understanding of different variations of Cas9 proteins. This, she explained, could have implications for clinical CRISPR use, particularly for patients who are resistant to commonly used forms of the Cas-9 protein.

The down-to-earth tone of Lisa’s presentation was what made her talk stand out. I admired her presentation skills–she presents with a confident voice, and isn’t afraid to incorporate humor into her talk. She effectively reduced complex topics into easily digestible terms, which kept the audience engaged and intrigued.

I found her topic very compelling, and I hope to be able to learn more about the CRISPR-Cas9 system in the future.