Transient receptor potential vanilloid-1 (TRPV1) ion channels are polymodal signal integrators of noxious stimuli including heat, vanilloids such as capsaicin, peptide toxins, acid, and inflammatory mediators. It is unknown whether activation of TRPV1 by different stimuli is achieved through the same structural mechanism or if different stimuli activate the channel through different structural mechanisms. Clinical trials using TRPV1 antagonists resulted in patients exhibiting hyperthermia, suggesting that TRPV1 plays a role in maintaining body temperature, and highlighting the need to ensure that therapeutics targeting the channel do not disrupt thermal homeostasis. Hence, knowledge of different structural mechanisms for channel activation would aid in the design of therapeutic agents targeting TRPV1. To address this, we have expressed a series of functional single-cysteine rat TRPV1 channels for spectroscopic analysis, with techniques such as electron paramagnetic resonance, double electron-electron resonance, and Förster resonance energy transfer spectroscopy. By probing several structural regions within TRPV1 we can determine which regions of the channels move during activation and whether those are the same for different noxious stimuli.

Membrane trafficking in the eukaryotic cell is a highly controlled and significant process that is related to various inherited disorders and cancers. Among numerous regulatory proteins, Uso1 protein – an essential, long, coiled-coil protein – plays a key role in the tethering process, capturing and pulling the vesicle toward the target membrane. Despite many years of work, the tethering mechanism has not been fully understood. Although several tethering models have been proposed, none of them were tested with Uso1 protein. Hence, to test these models, we engineered a mechanically deficient version of Uso1 protein, which lacks the critical tethering region for functioning. After the protein characterization, in cell survival tests and in test tube tests in chemically defined fusion system have been performed. The failure of the trafficking event, which would support our hypothesis, along with other functional test data, would provide a promising demonstration for the tethering mechanism of the long coiled-coil tether and the role of Uso1 protein.

Cortical plasticity is the substrate for learning and memory. It is the basis of an organism’s ability to adapt in response to a changing environment and is central to functional recovery after an injury involving the nervous system. Vagus nerve stimulation (VNS) has already been shown to be effective in altering neuroplasticity. Most VNS research has been conducted on epileptic animals and thus offers little information on how VNS may affect a healthy human brain. The final goal of this project is to establish a minimally invasive VNS protocol aiming to augment neuroplasticity and ultimately affect behavioral performance in a cognitive task. Because this study is conducted using non-human primates, and because of the limited invasiveness of the procedure, the outcomes can be directly translated to applications for human subjects. Non-human primates are implanted with cortical electrodes allowing both recording and stimulation of the cortex at select locations. Closed-loop cortical stimulation triggered by the troughs of beta (15-25Hz) oscillations leads to phase-dependent changes in excitability which outlast the oscillatory episodes for a couple of seconds. By delivering current through a clip electrode attached to the right or left cymba concha, we paired the cortical stimulation with auricular branch VNS (abVNS). This stimulation protocol is used to investigate the effects of cycle-triggered cortical stimulation with abVNS. Preliminary results show a suppression of excitability when the abVNS is delivered in phase with cortical stimulation which lasted for minutes after the cycle-triggered stimulation has ended. However, more control experiments need to be run before conclusions can be drawn. These findings of lasting change to cortical plasticity are novel and suggest that vagus nerve stimulation can affect medium-term neural plasticity.

Chromosome segregation during mitosis and meiosis ensures accurate division of a cell's nucleus to produce daughter cells that each contain a proper set of chromosomes. An important hallmark of mitotic and meiotic cell division is the attachment of microtubules to protein structures called kinetochores. The phenomenon is susceptible to error, causing mis-segregation of chromosomes, which can result in aneuploidy, miscarriage or cancer. During the 1960s, Dr. Bruce Nicklas established the present model for how meiotic error correction transpires. By directly manipulating individual chromosomes in grasshopper spermatocytes, he misaligned individual chromosomes from the metaphase plate and then observed as the chromosomes spontaneously realigned. Dr. Nicklas’ work demonstrated that mechanical tension is required for stabilizing proper kinetochore-microtubule attachments, and that the lack thereof results in detachments that allow the chromosome to reorient correctly. The Asbury Lab aims to further his experiments by using small molecular inhibitors in combination with micromanipulation to determine the role of enzymes such as Aurora B kinase in chromosomal error correction. Here, we tested the impact of Aurora B kinase, using a small molecular inhibitor that has been reported in human cancer models, AZD. Cricket spermatocytes were dissected and soaked in a solution of insect medium and AZD. After treatment, the spermatocytes were spread on a glass slide and filmed under a phase-contrast microscope for three hours. Metaphase cells were filmed because it is expected that Aurora B kinase releases erroneous attachments during this phase of cell division. Our results show that AZD treatment of cricket spermatocytes delays the transition from metaphase to anaphase, confirming that Aurora B kinase plays an important role in chromosome segregation in these cells. Our latest work focuses on fluorescent tagging of chromosomes to better distinguish misaligned chromosomes and to further understand the mechanisms of error correction.

The transient receptor potential vanilloid 1 (TRPV1) ion channel is well known for its role in sensing numerous “noxious” stimuli. It responds to noxious heat, acid, capsaicin (the active compound that makes chili peppers “spicy”), and inflammatory signals, which can be perceived by the brain to be painful. When the TRPV1 channel senses a signal such as capsaicin, it goes from a closed, non-conducting state, to an open state that allows the passage of ions into the cell, generating an electrical signal. Since TRPV1 is activated by a variety of different stimuli one of our aims is to determine if the structural mechanisms that open the channel is the same or different for each stimulus, which could have important clinical implications. For example, failed clinical trials of some TRPV1 antagonists resulted in elevated body temperatures in patients, indicating that the body’s ability to regulate body temperature is impaired when TRPV1 is completely inhibited. Hence, if TRPV1 can be inhibited in most cases, but still respond to temperature, it will be a better target. We aim to combine cysteine-scanning mutagenesis with spectroscopic techniques to map the structural changes of TRPV1 activation in response to different stimuli. Since introduction of mutations can result in impaired channel function, each mutant needs to be functionally verified. My research will be to generate a broad library of single-cysteine TRPV1 mutants and verify their function using calcium imaging. Mutants that preserve TRPV1 function can then be used for further spectroscopic studies.