Life Sciences – Page 5 – UROP Spring Symposium 2021

Life Sciences

RNA Sequencing Analysis of the Early Response to Retinal Ishemia-Reperfusion Injury

Ischemia-reperfusion (IR) injury causes damage is caused when a tissue is deprived of blood flow blood (ischemia) followed by a sudden reflux of blood into the tissue (reperfusion). This injury can occur in the retina and contribute to several different issues in the eye, such as diabetic retinopathy and glaucoma. In order to get a better understanding of this injury, we used a retinal IR model in C57BL/6 mice where ischemia is caused by injecting saline to raise pressure in the eye, thus shutting off blood flow. Reperfusion naturally occurs when the injection is halted. The design of this study was to use RNA sequencing to examine retinal transcriptome at different points in time following the injury. This report focuses on deferentially expressed genes (DEG) identified by comparing sham treated (control) and IR injured retinas at two early time points, specifically 6 hours and 1 day after reperfusion. iPathwayGuide was used to perform bioinformatics analyses, including KEGG pathway identification, for the sets of DEG identified. The RNA sequencing and analysis identified 4358 DEG at 6 hours and 6664 DEG at 1 day. Cytokine-cytokine receptor interactions were identified as the most significant KEGG pathway for DEG sets at both 6 hours and 1 day. Direct comparison of 6-hour and 1-day transcriptomes showed that cytokine mRNAs were particularly upregulated at 6 hours, while mRNAs for their receptors were more upregulated after 1 day. This suggests a rapid and robust chemoattraction of leukocytes from blood into the retina in response to IR injury. DEG corresponding to additional markers for leukocyte subsets were examined to indicate if these cells had actually entered the retina by 1 day after injury. The results provide an insight into the inflammatory response to retinal IR injury, including the chemokines that may be responsible for the attraction and retinal infiltration of immune cells into the injured retina.

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Analysis of brain c-fos expression in response to the presentation of two temporally separated stimuli by Protein Immunofluorescence Combined with Intronic Fluorescent In situ Hybridization (PICIFISH)

This project aims to develop an understanding regarding the neurobiology of vulnerability to addiction and post traumatic stress disorder (PTSD). Oftentimes when different environmental cues that have been associated with something emotional are triggered, it can lead to an emotional or motivational state that influences the way one behaves and can lead to undesirable consequences. For example, drug-associated cues including seeing or smelling drugs can induce craving which can lead to drug use or relapse. We are particularly interested in the neuronal basis on incentive salience – the cognitive process that attributes a “desire” or “want”, which includes a motivational component, to a rewarding stimulus. To investigate this phenomenon, our lab experiments with animals, particularly rats, and introduces a lever cue followed by a food reward in the cage. A distinction between rats is made: Rats who consistently approach the lever are known as sign-trackers (STs), rats who mainly ignore the lever and go straight to the location of the food are known as goal-trackers (GTs), and then there are unassigned rats who do not particularly show interest in the lever or the place where the food will come down, until the food is given. It is evident that STs attribute incentive value to lever while GT rats attribute predictive value to the lever as they go to the location where the food will eventually come after seeing the lever. Throughout this process, neuronal activity is measured through immunohistochemistry and protein immunofluorescence combined with intronic fluorescence in situ hybridization (PICIFISH). These are methods of staining and amplifying the c-fos MRNA signal through the application of multiple antibodies and blocking serums, which allows us to see the varying levels of c-fos mRNA, a marker for neuronal activity, in various rat brain slices. After these processes are complete, the brain slices are viewed under the microscope, helping us develop an understanding of how much c-fos mRNA was created. By understanding and inspecting the levels of c-fos mRNA expression and neuronal structure of the rats, we hope to develop an understanding of the neurobiology that correlates with addiction and PTSD based on the varying levels of c-fos expression in the distinct types of rats.

Analysis of brain c-fos expression in response to the presentation of two temporally separated stimuli by Protein Immunofluorescence Combined with Intronic Fluorescent In situ Hybridization (PICIFISH) Read More ยป

Analysis of brain c-fos expression in response to the presentation of two temporally separated stimuli by Protein Immunofluorescence Combined with Intronic Fluorescent In Situ Hybridization (PICIFISH)

This project aims to develop an understanding regarding the neurobiology of vulnerability to addiction and post traumatic stress disorder (PTSD). Oftentimes when different environmental cues that have been associated with something emotional are triggered, it can lead to an emotional or motivational state that influences the way one behaves and can lead to undesirable consequences. For example, drug-associated cues including seeing or smelling drugs can induce craving which can lead to drug use or relapse. We are particularly interested in the neuronal basis on incentive salience – the cognitive process that attributes a “desire” or “want”, which includes a motivational component, to a rewarding stimulus. To investigate this phenomenon, our lab experiments with animals, particularly rats, and introduces a lever cue followed by a food reward in the cage.

Analysis of brain c-fos expression in response to the presentation of two temporally separated stimuli by Protein Immunofluorescence Combined with Intronic Fluorescent In Situ Hybridization (PICIFISH) Read More ยป

Schwann cell-derived signals on skeletal muscle growth

Schwann cells play a pivotal role in the maintenance and regeneration of axons in the peripheral nervous system by directly communicating with motor neurons. Under conditions of acute oxidative stress, Schwann cells produce cytokines and neurotrophins to communicate with immune and nerve cells to orchestrate the nerve repair process. However, less is known about the direct communication that occurs between schwann cells and myofibers. In an in vitro setting, the communication between schwann cells and myofibers, as well as the effects of oxidative stress on myogenesis were investigated. Schwann cells were activated with hydrogen peroxide to induce growth factor and cytokine expression. Conditioned media from both activated and non-activated Schwann cells were then added to skeletal muscle progenitors cells (C2C12) to assess the effects of treatment on myogenesis. We hypothesized that the C2C12 cells receiving the conditioned media derived from activated Schwann cells would display larger myotubes than C2C12 cells that were treated with non-activated conditioned media and non-treated controls. Images were taken with a fluorescent microscope six days after treatment and the results showed that myotube diameter was not significantly different between the three groups. More experimentation and analyses are needed to fully understand the communication between Schwann cells and myofibers, especially in conditions of elevated oxidative stress such as aging. Further analysis of such data and gaining a thorough understanding of the relationship between these cells can be vital in the discovery and implementation of new treatment methods for neuromuscular diseases.

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Suppressing heterotopic ossification and fibrodysplasia ossificans progressiva in mouse models through targeting BMP type 1 receptor kinases

Fibrodysplasia Ossificans Progressiva (FOP) is a rare genetic condition distinguished by the development of bone in soft tissue, known as heterotopic ossification (HO). FOP is a gain-of-function mutation, causing substitution of an arginine by histidine (R206H) within BMP type 1 receptor ACVR1 protein. This disorder severely impacts an individual’s life, as it leads to body malformations and disabilities. While the exact mechanism of HO progression is still unclear, studies have found that HO development is accompanied with an increase of BMP-SMAD signaling and inflammation. In this research project, we aim to discover methods to decrease HO in FOP mouse models, by attempting to decrease BMP signaling. ACVR1, BMPR1a, and BMPR1b are BMP type 1 receptors. Our previous studies showed that BMP-SMAD dependent signaling is decreased by the knockout of BMPR1a or ACVR1 in specific tissues. By deleting one and/or multiple of these receptors, we predict that it will decrease BMP signaling, ultimately suppressing HO. We use the constitutively active ACVR1 model (caACVR1-Q207D), which develops massive HO within two weeks after activation of BMP signaling with tissue injury. But our study showed our anticipation was wrong. We found that the HO was not inhibited in ACVR1-Q207D mouse model by knocking out BMP type 1 receptors. Additionally, the BMP-SMAD dependent signaling pathway was not inhibited by knocking out BMP type 1 receptors in the HO mouse models. Due to the fact that the outcomes of this project did not support our initial hypothesis, we need to analyze other ways to decrease BMP signaling to suppress HO formation. This research hopes to bring us closer to finding the first effective treatment for FOP/HO, helping the individuals whose lives are permanently impacted by this genetic disease.

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Modeling cardiac aging with stem cell-derived cardiomyocytes carrying a mutation for advanced aging

Using various images taken through a confocal microscope, the area of the nuclei in the heart cells of mice were measured in an effort to compare two varieties of cells. Those that were extracted from mice suffering from Hutchinson Gilford Progeria Syndrome (HGPS), a severe and rare cardiovascular disease. Comparing these two types of cells provided a better idea of the meaningful ways in which cell morphology is different in regards to the presence of HGPS. Differences reflecting statistical significance were identified between the heart cells of “healthy” mice and those afflicted with HGPS. This data collection helps build upon the foundation and overarching goal of the lab which is to model cardiac aging through the use of stem cell derived cardiomyocytes.

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Gene Editing for Combating Disease: Biology

The use of non-viral vectors to deliver the CRISPR/Cas9 complex to target cells is beneficial as they can potentially negate the immunological response that a host may have to the viral vector used in the present. In order to validate this, one must first identify the optimal guide RNA for the Cas9 complex to the target DNA sequence, which is done through PCR amplification and gel electrophoresis. The next phase is to image the cells by immunofluorescence. The transfected cells were then compared to the baseline cells to visualize the effects that the CRISPR/Cas9 complex on the cells.

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Gene Editing for Combating Disease: Biology

The use of non-viral vectors to deliver the CRISPR/Cas9 complex to target cells is beneficial as they can potentially negate the immunological response that a host may have to the viral vector used in the present. In order to validate this, one must first identify the optimal guide RNA for the Cas9 complex to the target DNA sequence, which is done through PCR amplification and gel electrophoresis. The next phase is to image the cells by immunofluorescence. The transfected cells were then compared to the baseline cells to visualize the effects that the CRISPR/Cas9 complex on the cells.

Gene Editing for Combating Disease: Biology Read More ยป

Gene Editing for Combating Disease: Biology

The use of non-viral vectors to deliver the CRISPR/Cas9 complex to target cells is beneficial as they can potentially negate the immunological response that a host may have to the viral vector used in the present. In order to validate this, one must first identify the optimal guide RNA for the Cas9 complex to the target DNA sequence, which is done through PCR amplification and gel electrophoresis. The next phase is to image the cells by immunofluorescence. The transfected cells were then compared to the baseline cells to visualize the effects that the CRISPR/Cas9 complex on the cells.

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Design of a PPI Inhibitor to Treat AAV

Many diseases, such as ANCA-associated vasculitis (AAV), are caused by mutated proteins involved in protein-protein interactions (PPIs) that can be inhibited by designer proteins that would serve as small molecule inhibitors. In the case of AAV, the targeted interaction is between TNF-alpha and its associated TNFR1 receptor, which induces the activation of neutrophils in endothelial cells, causing inflammation in AAV patients. By designing a protein which will bind to TNF-alpha, its interaction with TNFR1 can be decreased, thus decreasing patient inflammation. UniDesign from the Zhang Lab was used to design de novo protein sequences from a PDB model of the TNF-alpha and TNFR1 interaction. The results were then analyzed by comparing the binding energy of the designer protein against the native sequence. These results are very promising in the development of protein design as a viable treatment method for rare diseases. This new protein could provide a novel type of treatment to AAV patients that would mitigate the use of glucocorticoids, a common anti-inflammatory agent with long-term side-effects such as increased risk for infections, heart disease, etc.

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