The inner ear is crucial for both hearing and balance and undergoes rapid changes during embryonic, fetal, and postnatal stages. Its complex structure poses challenges in studying auditory systems, especially the organ of Corti responsible for sound perception. Hair cells (HC’s) within the cochlea are essential for hearing, and their loss in mammals results in permanent deafness due to their inability to regenerate. We recently established Early B cell factor 1 (Ebf1) as an important factor for the development of the cochlea. Our lab’s work with conditional knockout (cKO) mouse models revealed that Ebf1 restricts sensory development within the cochlear duct. The Slc26a9-Cre Ebf1cKO model deletes on embryonic day (E) 9.5. We have seen over 2-fold increases in HC’s and their associated support cells (SCs). We have developed a tamoxifen-inducible Sox2Cre recombinase mice model for precise timing of genetic manipulation within the cochlea. Specific temporal activation of tamoxifen-inducible Sox2Cre recombinase in the cochlea will uncover critical regulatory time periods for the establishment of the sensory domain. Activation of tamoxifen-inducible Sox2Cre at embryonic day 11/12 only shows an increase in inner hair cells. These findings lead us to ask, what’s the critical window for Ebf1’s regulatory role in cochlear development? To determine the optimal window for Ebf1's regulatory role, I will activate the Cre expressed in Sox2 Ebf1-cKO mice with tamoxifen at different embryonic days (9-14) via oral gavage. Immunostaining experiments utilizing HC markers (Myo7a), inner HC markers (Vglut3), and SC markers (Sox2) will be conducted. I will quantify HC densities and cochlear length of Sox2 Ebf1-cKO and littermate control mice. Due to tamoxifen toxicity, samples will consist of embryonic day E18 specimens. Anticipated results include varying HC numbers, innervation and the presence of ectopic sensory patches. This study will offer valuable insight into the temporal dynamics of Ebf1's regulatory role.

The emergence and spread of antimicrobial resistance (AMR) in bacterial populations pose a significant threat to global health. A key factor in this process is the horizontal gene transfer (HGT) of resistance genes carried by mobile genetic elements (MGEs). This study focuses on understanding how mutations in plasmids influence the rate of HGT and its implications for the spread of AMR. Plasmids are small, extrachromosomal DNA elements that can transfer between different bacterial cells, a process crucial in spreading antibiotic resistance genes. These plasmids often use a mechanism known as conjugation for transfer, a process encoded by approximately 30 genes in the transfer (tra) operon. This research aims to understand how changes in the tra operon affect the plasmid's ability to move between bacteria. This movement can be either horizontal (between different cells) or vertical (from parent to offspring during cell division). Our project is divided into two main aims. The first is creating a library of mutated plasmids. I modified a large, well-studied conjugative plasmid, the F plasmid, and introduced specific mutations into the tra gene. This step is achieved using a method called Lambda Red recombination, a technique suitable for large plasmids where standard cloning methods fall short. The second aim is to measure the rates of vertical and horizontal gene transfer (VGT and HGT) in these mutated plasmids. We employed the Luria-Delbrück method for precise estimation of these transfer rates. Understanding the trade-off between VGT and HGT is crucial in predicting and controlling the spread of AMR. By mapping the relationship between specific mutations in the tra operon and their impact on gene transfer rates, our study contributes to a broader understanding of how antibiotic resistance spreads through bacterial populations. This knowledge is vital for developing strategies to combat the increasing threat of antibiotic-resistant bacteria.

Perinatal asphyxia or hypoxia, where the infant brain does not receive enough oxygen or blood flow, commonly occurs in premature newborns and is one of the leading causes of neonatal mortality worldwide. Survivors often have altered white matter with cognitive impairments, motor deficits, and increased rates of cerebral palsy. There is currently no standard treatment for preterm brain injury, so there is a critical need to research neuroprotective strategies as well as ways to assess their impact. The ferret is a promising model species for studying preterm brain injury due to its gyrified brain and white-to-gray matter ratio, which are similar to that of the human brain. The gyrification index (GI) can be used to assess cortical development and is calculated using magnetic resonance imaging (MRI) images. These are analyzed using ImageJ software to perform hemispheric tracing by dividing an internal trace, including the gyri and sulci, by an external trace that excludes them. A higher GI is indicative of a larger cortical surface area. This project seeks to evaluate the effects of postnatal (P) age on post-hypoxic-ischemic (HI) gyrification in two ferret models. In both models, HI ferrets underwent bilateral carotid artery ligation and exposure to hypoxia, differing by date of surgery, with randomly assigned control animals not undergoing surgery. Model One ferrets underwent surgery at P10 (extremely preterm equivalent) and tissue collection at P70, and Model Two ferrets underwent surgery at P17 (late preterm equivalent) and tissue collection at P42. I hypothesize that GI will be affected by HI injury, with both age of injury and age of assessment altering GI relative to control animals. Contextualizing age differences in GI could help inform future therapy regimens to treat infants with premature brain injury.

Preterm neonates frequently undergo episodes of hypoxia, hypotension, and hyperoxia due to their underdeveloped cardiorespiratory system. As a result, preterm infants are particularly susceptible to brain injury from lack of sufficient oxygen or blood flow. In this study, we use a neonatal ferret model of hypoxic-ischemic-hyperoxic (HIH) brain injury to examine the neuroprotective efficacy of azithromycin (Az), a safe and accessible antibiotic with anti-inflammatory properties that crosses the blood-brain barrier in premature infants. Our HIH ferret surgery model produces global brain injury in Postnatal day (P)14 ferrets, which are developmentally equivalent to extremely preterm neonates. Ferret kits in the surgical group will receive a pro-inflammatory stimulus (E. coli lipopolysaccharide), followed by a bilateral carotid artery ligation and exposure to HIH. Then, surgical animals will be randomized to receive intraperitoneal injections of saline vehicle (Veh) or Az. Control animals will not receive LPS or undergo surgery. We will use a Kruskall-Wallis test to compare P21 ferret weights and brain measurements seven days after surgery to test whether Az is neuroprotective. We will assess pathology using quantitative immunohistochemistry with Iba-1 (microglia) and GFAP (astrocytes) stains. We have two main hypotheses. We hypothesize that 1) ferrets treated with Az will lose less body weight after exposure to inflammation-sensitized HIH than the Veh group, with Az-treated animals being similar in weight to non-injured controls, and 2) normalized brain measurements, corrected for body weight and sex, will show that Veh animals have smaller gyri and sulci measurements than controls, and Az-treated animals will have brain measurements that are more similar to controls. We are optimistic that our experiments will provide evidence that azithromycin is potentially neuroprotective and will offer a promising treatment option for preterm babies with brain injury.

Traumatic brain injury (TBI) is caused by an external force to the head, resulting in brain injury and is a major cause of death, particularly in adults 75 years and older who are at increased risk of falls which can lead to disability. Humans have a natural response to impact and strain called the Valsalva maneuver, which leads to an increased pressure in the chest and abdomen, which can result in a neuroprotective increase in intracranial pressure (ICP). However, most people are unable to anticipate TBIs and cannot perform their own Valsalva maneuver. Using a ferret model of TBI, the neuroprotective effects of externally-stimulated Valsalva-like response will be assessed. Ferrets are used to model human TBIs because the cortical thickness and layer distribution of their brains are more akin to humans in the ferret compared to rodents. The ferrets will be randomized to one of the three groups: control, TBI+sham valsalva, and TBI+valsalva. To show that the intracranial pressure of ferrets can be transiently increased, an inflatable cuff will be utilized to exert pressure on the abdomen, resulting in a partial Valsalva maneuver. TBI will be induced in the ferrets using a closed-head impact, and the neuroprotective effects of increased ICP from the inflatable abdominal cuff will be assessed using a battery of motor and cognitive tests before and after the TBI event, additionally, brain injury and neuroprotection will be evaluated using histopathology. I hypothesize that the Valsalva maneuver induced by the inflatable abdominal cuff will reduce behavioral deficits resulting from impact. If the behavioral deficits are reduced, this study can work to inform future interventions for TBI, such as environment-sensing wearable devices for high risk populations.

Preterm birth is one of the leading causes of neurological deficits and mortality in neonates. Hypoxia-ischemia (HI), when the brain receives insufficient oxygen and blood supply, is a commonly observed condition among preterm infants. Preterm birth and HI affect the structure of the developing brain, particularly the formation of cortical folds (gyri) of the brain. This process is difficult to study in rodent models as they are lissencephalic and do not have gyrified brains. Ferrets, unlike rodents and more similar to humans, have gyrification – the folding of the cerebral cortex, creating gyri and sulci. Using the ferret as a model, examining changes in gyrification after injury can help us identify and further understand brain injuries like HI and its effects. Preterm-equivalent postnatal day (P) 17 ferrets received lipopolysaccharide intraperitoneally and underwent bilateral carotid artery ligation followed by hypoxia-ischemia plus hyperoxia (HIH). Animals were either non-HIH exposed (controls) or HIH-exposed and randomized to receive saline vehicle, erythropoietin, or therapeutic hypothermia. At P42, ex vivo (n=74) magnetic resonance images were collected. Using ImageJ software, the inner to outer hemispheric ratio was used to set the gyrification index (GI). We assessed the average GI across the whole brain and the peak GI of left and right hemispheres. Kruskal-Wallis test will be used to measure the differences in average and peak GI across treatment groups. We hypothesize that the average and peak GI of injured animals treated with Epo, TH, and saline will be lower compared to control animals. Gyrification can be an essential metric for researchers to analyze brain injuries like HI in preterm neonates. As preclinical and clinical studies for HI continue to improve, gyrification can inform the relationship of folding patterns in the cerebral cortex area, assisting us to examine the potential shared developmental processes between animals and neonates.

Mobile genes are commonly found in bacteria, and they are capable of being transferred between unrelated bacterial cells via a process termed horizontal gene transfer (HGT). Mobile genes that undergo HGT can evolve in various “host” bacteria and thereby evolve in several different genomic backgrounds. In a recent publication, my lab constructed a mathematical model to assess the effects of HGT (i.e “host-switching”) on mobile gene evolution. I have built upon this work by probing additional factors that may influence mobile gene evolution. In phase one of my project, I compared the original evolutionary model used in the publication to a newly expanded, more ecologically realistic model in which growth rates of the bacteria depend on resource availability. To compare the models, I ran simulations with both models on a set of bacterial-host pairs and compared the resulting evolutionary outcomes from both models. I found that the categorical effect of HGT occurring (i.e. HGT confers a higher, lower, or neutral change in host fitness) was the same across both models, indicating that ecological factors are less predictive of mobile gene evolutionary outcomes. For the second phase of my experiment, I assessed the effect of variable HGT rate on mobile gene evolutionary outcomes. I ran simulations using a set of bacterial host-pairs while varying the HGT rate along a biologically relevant range, and found a positive correlation between HGT rate and the magnitude of positive fitness effects conferred by a mobile gene that has undergone HGT. This indicates that HGT rates play an important role in governing the evolutionary outcomes of mobile genes. Using evolutionary simulations has allowed us to gain insight into the predictive factors governing mobile gene evolution and thereby mobile gene-containing bacterial evolution. This is especially important, as many genes conferring antibiotic resistance are mobile.