Neuroscience research relies heavily on cell counting to assess brain injury and evaluate neuroprotective treatments. While manual methods (i.e. hand counting) have been used traditionally, automated programs offer the potential for standardized and error-free data analysis. In the context of studying hypoxic-ischemic encephalopathy (HIE), a prevalent brain injury in infants, we aimed to assess the accuracy of an automated cell counting program in an in vitro slice culture brain injury model of injury and treatment. Code templates from ImageJ/Fiji were taken and modified using ChatGPT, and other snippets were used and modified from online forums like GitHub. The program searches through folders/subfolders for images, converts them into a binary image based on fluorescence threshold data (used to stain cell nuclei), applies the ImageJ function “watershed” that breaks larger groups into smaller groups, runs the “analyze particles” function which outputs a total cell count based on the size and circularity of the cells, and then saves the final image. Fluorescence threshold, cellular size, and circularity values were determined before data collection by adjusting the values to best fit the final image of a random slice. The settings were then kept consistent within studies. Preliminary results show the program’s high accuracy and precision, with consistent results across caffeine and Azithromycin treatments in our in vitro injury model. Despite yielding higher counts than manual methods, the program remained consistent across models. Validating this automated method represents a significant advancement in research methodology. These programs offer standardized data collection, error elimination, and faster analysis compared to manual counting, potentially saving time and resources for labs. Current limitations in our research involve differentiating between healthy and dead or dying cells, which would be an important future step for automated cell counting.

Advancements in HIV prevention include current pre-exposure prophylaxis strategies (PrEP), which are effective for men, but not for women due to poor partitioning of antiretrovirals (ARVs) to the female reproductive tract. One strategy for sustained delivery of ARVs to the female reproductive tract is the integration of ARV-releasing reservoirs with established intrauterine devices (IUDs). To this end, our lab has investigated reservoirs containing polymer-drug conjugates (drugamers), where the HIV integrase inhibitor raltegravir (RAL) is covalently attached to a polymer through a hydrolysable linker. However, current drugamers release RAL too rapidly to achieve our target of 1-3 years of IUD-mediated delivery. Our current work is directed at redesigning the RAL drugamer linker to extend the duration of release from 30 days to at least one year. We hypothesize that converting the ester linker of our current drugamer design to an acetal carbonate will slow the rate of RAL release, since the rate determining step of acetal carbonate hydrolysis does not involve the particularly acidic hydroxyl of RAL (pKa = 6.6). To date, I have synthesized the required acetal carbonate monomer by forming a carbonate-linked methacrylate through acyl substitution chemistry, and conjugated RAL to this methacrylate through an SN2 reaction. We fully characterized this monomer using NMR spectroscopy and mass spectrometry. Additionally, in a release study in cell media at 37C, I measured the rate of hydrolysis to be approximately 30 times slower than in the current lab monomer. Future directions include polymerizing the monomer and measuring RAL’s rate of release from the drugamer. We plan to attach other antiretrovirals with hydroxyls to the acetal carbonate linker and measure the rate of release from these drugamers as well. These preliminary findings are promising and will inform the design of drugamers for the long-term prevention of HIV.

Mycoplasma genitalium (MG) is a sexually transmitted bacterial pathogen commonly associated with urethritis in men and cervicitis, endometritis, pelvic inflammatory disease, infertility, and preterm birth among women as it invades the upper reproductive tract. Infections can persist for months to years without effective treatment due to antimicrobial resistance. Current first-line drug choices are only successful in less than half of all patients. Preliminary in vitro data suggests that MG is susceptible to tinidazole (Tdz) and may fill the need for additional treatment options for drug resistant infections as it is already FDA-approved for other indications. As strains can vary in their susceptibility to particular drugs, we aim to identify the minimum inhibitory concentration (MIC), the concentration that inhibits growth by 99%, of Tdz against 10 MG clinical isolates. This data will determine if these strains are susceptible to Tdz, define the range of MICs, and reveal whether current strains have already developed resistance. Twofold dilutions of Tdz and doxycycline (DOX) antibiotics are added to MG clinical strains in 48-well plates and incubated at 37 C/ 5% CO2 for 21-28 days. DOX is one of the first-line drug choices with a known MIC; it is used to confirm that assays are performed correctly and to compare the effectiveness of Tdz. Four of the wells have no drugs to serve as a control to compare the number of genomes against those in the Tdz wells to determine MG inhibition. MG growth in each Tdz dilution is quantified with qPCR by isolating DNA from the wells. Calculations of the percent inhibition will dictate which antibiotic concentration is useful for treating infected patients. As physicians are already beginning to treat MG patients with Tdz, data regarding susceptibility of multiple isolates is crucial in informing these treatment regimens.

Mycoplasma genitalium (MG) is a sexually transmitted bacteria that causes urethritis in men and cervicitis, pelvic inflammatory disease, and infertility in women. MG infections vary in length: some infections are cleared within a few weeks, while others last for years and are difficult to treat due to antimicrobial resistance observed in MG. Previous studies have determined that MG was susceptible to nitroimidazoles. Preliminary analysis of four resistant strains of the MG type strain G37 found mutations in the MG_342 gene which encodes an oxidoreductase hypothesized to react with nitroimidazoles and produce the toxic form of the drug. In order to determine the possible mechanism for nitroimidazole resistance we will amplify the MG_342 region of MG. As MG clinical strains require months to establish cultures in vitro we will develop a sensitive PCR assay so that resistance-associated mutations can be identified directly from patient specimens in future clinical trials. The PCR products will then be sequenced to determine if the MG clinical isolates have mutations that confer resistance to nitroimidazoles. Understanding how these mutations affect nitroimidazole resistance could allow for future studies on nitroimidazoles as a possible treatment for MG.

The Field Programmable Cellular Arrays (FPCA) project at the Molecular Information Systems Lab (MISL) aims to improve current biocomputing systems utilizing spatial organizations of cellular components for logical operations. This can open doors for computation to be done within biological systems where artificial computation has never before been possible. This project encompasses three aims: characterizing the properties of signal propagation within E. coli, constructing biological circuit components for spatial signal processing, and optimizing bioprinting methods for circuits. Signal propagation through molecular signaling is employed to communicate the presence or absence of a signal and truth values to specific cells. We are demonstrating logical states of "1," "0," and the absence of a signal, thereby enabling differentiation between a logical "0" and a lack of signal. Two strains of bacterial cells are capable of performing the logic of a traditional "wire" and a NOR gate. Consequently, by arranging strains in spatially organized layouts, we engineer cellular arrays capable of performing diverse complex logical functions. This research is still in progress and we are in the process of optimizing NOR gate and wire strains. My role explores bioprinting circuits into hydrogels, and I have built a bioprinter with dual extruders to bioprint biological substances into containing slurries. This required designing, printing, and assembling 3D-printed parts. I am now characterizing the behavior of 3D printed materials into various containing slurries. This requires testing the ability of different bioprinting inks to encapsulate bacteria, testing various slurry methodologies, and testing interactions between combinations of these materials over space and time. I am also computationally modeling FPCA circuits at various levels of abstraction. Computational modeling serves to further broader computational goals in this project to compile a logic circuit specification into bioprinter GCODE.

Our research aimed to investigate the microbial diversity in Pieris rapae larvae obtained from three distinct settings: wild-collected larvae, larvae fed with leaves in a laboratory setting, and larvae fed with a standard artificial diet. Additionally, this investigation sought to identify the factors contributing to developmental delays in artificial diet-fed larvae, while also investigating the underlying cause of increased cases of infection and mortality affecting the laboratory colony. Initial observations indicated that wild larvae exhibited the fastest development and appeared healthiest, followed by those fed with leaves, both settings showing the lowest instances of disease and mortality. Conversely, the artificial diet group experienced developmental delays and frequently succumbed to disease before pupation. Based on these observations, we hypothesized that variations in microbial composition within different diets and environments contributed to the disparate outcomes. Furthermore, we speculated the possible identification of bacterial strains with known associations with promoting or hindering larval success. To analyze the larval gut microbiome, a specialized contraption was designed for sterile frass (feces) collection. Frass, skin, artificial diet, and leaf surfaces were swabbed onto TSA media, enabling the isolation of distinct colonies and species. Morphological characteristics, gram staining, and Sanger sequencing data of the 16S rRNA gene were used to identify the bacterial colonies at the species level. The identification of six bacteria at the species level yielded intriguing results, with particular interest surrounding Bacillus subtilis and Bacillus thuringiensis. The presence of B. subtilis exclusively in the artificial diet group suggests its potential involvement in the developmental delay observed in the larval population fed by artificial diet. Conversely, the identification of B. thuringiensis implicates a potential bacterial infection, which could explain symptoms experienced by the colony. These findings have potential implications for enhancing larval outcomes in colonies or, in contrast, pesticidal applications in an agricultural setting. 

Cannibalism is a widespread phenomenon among arthropods with consequences for population dynamics, stability, pathogen transmission, and epidemiology; while it is common in carnivorous arthropods, incidents of cannibalism among non-carnivorous species are less frequent, and the mechanisms behind cannibalism in these species are still poorly understood. Laboratory observations of Pieris rapae (cabbage white) larvae have found that larvae will engage in cannibalistic behavior under certain conditions. The objective of our research was to better understand the conditions that may cause this herbivorous larva to become cannibalistic. Experiments were conducted on variables of density, food source, food scarcity, instar, instar gaps, sex, and prior experience as possible influencing factors in the likelihood that a larvae will engage in cannibalism. Experiments in density, food source, and food scarcity had groups of larvae monitored for 48 hours at differing densities with a collard leaf, artificial diet, or withheld food. For experiments in sex, instar, and instar gaps inexperienced larvae were monitored in sets of two for 72 hours for cannibalism. For prior experience experiments, larvae that had previously engaged in cannibalism were paired with inexperienced larvae and monitored for another 72 hours. We found that higher larvae densities had a greater percentage of cannibalization. Furthermore, a lack of food source produced significantly higher cannibalization than either artificial or leaf diets. A higher rate of cannibalism was also seen in early instars. Prior cannibalistic behavior increased the rate of new cannibalism events within the first 24 hours of observations.These findings provide insights into cannibalistic behavior in P. rapae, with potential applications to prevent cannibalism in laboratory settings and related disease transmission, understand fluctuations in wild populations, and in agricultural settings to leverage cannibalism as a form of natural population control. Future research aims to isolate other possible factors contributing to cannibalism in P. rapae.

Temperature has been shown to impact the physiology and performance of ectothermic organisms including metabolic rate with enzymatic activity. The majority of studies have focused on thermal performance at constant temperatures although organisms experience a range of fluctuating temperatures. Understanding which aspects of performance are affected by fluctuations in temperature can be crucial for predicting how ectothermic species will respond to changes in their environment. Research has shown that ectotherms are more vulnerable to climate change and their growth rates are often temperature-dependent. Previous studies on the cabbage white butterfly (cabbageworm), Pieris rapae, have shown that caterpillars maximize growth at higher temperatures even when higher temperatures are infrequent. Physiological response of P. rapae on fluctuating temperature change will provide an important insight into its ability to adapt and survive to changing temperature ranges. For our study eggs from a lab colony were reared at two different fluctuating temperature regimes (11-35℃ and 18-24℃) until pupation. Our studies measured the overall performance of P. rapae, including egg hatch percentage, growth rate of 4th instar caterpillars, development time from 4th instar to pupation, percent survival, and mass gain at two fluctuating temperature regimes of 11-35°C and 18-24°C. We found that the percent survival was significantly higher at 18-24°C. There was significant mass gain and shorter development time of 4th instar to pupation at 11-35°C. Our results differed from other studies that found growth rate to be greater at 11-35°C. Many studies have found a significant genetic variation in growth rate, development rate, and pupal mass. Future studies using a split sib-family design, may provide insight into the mechanisms of thermal performance in a fluctuating thermal environment. Understanding how fluctuating temperatures impact ectothermic organisms, such as the cabbageworm, can contribute to a deeper understanding of organismal responses to climate change.

Ferrate is an effective technology for water treatment applications because of its capabilities as an oxidant, coagulant, and disinfectant. Furthermore, ferrate is an environmentally benign chemical derived from a ubiquitous mineral on the Earth’s surface. However, ferrate rapid reduction to ferric species reduces its oxidation capacity. Ferrate-coated sand has been proposed as a better deployable method for ferrate in water treatment applications. Sand has a high composition (>80%) of silica (SiO₂) which has been demonstrated to stabilize ferrate reactivity and increase its oxidation capacity. A previous study on the treatment of phenol, a common surface water contaminant, showed that ferrate-coated sand was better at degrading phenol than ferrate only (in the absence of sand). However, the study was conducted in pure water matrices. Here, we are evaluating the oxidation of phenol by ferrate-coated sand in the presence of effluent organic matter and trace metals (i.e. copper). Organic matter is ubiquitous in the environment and can impact contaminant remediation efficiency. Studies have detected trace metals in surface waters which can pose environmental and health risks. Through batch tests, we observed that effluent organic matter hinders the stability of the ferrate-coated media and reduces its oxidation capacity. The results of this study will provide information about the ferrate-coated sand reactivity and capacity for the treatment of complex water matrices.

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.

Hypoxia-ischemia (HI), or brain injury caused by a lack of blood flow and oxygen to the brain, is a leading cause of infant mortality and morbidity. Contrasting between ages, the effects of HI tend to be more severe in younger neonates. Curcumin, a dietary compound derived from turmeric, exhibits anti-inflammatory, antioxidant, and antiapoptotic properties, but is not bioavailable in molecular form, thus may serve as a neuroprotective treatment when loaded into synthetic nanoparticles to allow for effective absorption and crossing of the blood-brain barrier, forming the treatment NanoCurc. Gestational ages of 37 weeks through 42 weeks are all considered term neonates, yet their brain continues to develop and differ significantly in response to treatments against HI. Using the rat Vannucci model of unilateral hypoxic-ischemic brain injury, we studied the in vivo effects of NanoCurc in neuroprotection, in P7, P10, and P13 rats, equivalent to 34, 38, and 42 weeks’ gestation, respectively. Tissue is collected 72 hours after unilateral carotid artery ligation surgery, followed by tissue staining and analyzed by tracing the healthy tissue versus damaged tissue, to calculate the average percent area loss in treated and untreated rats. I hypothesize that in all ages, neonatal rats treated with NanoCurc will have lower injury in comparison to those treated with saline (vehicle), while the treatment will be more effective in younger rats in comparison to older ages. In the future, NanoCurc treatment may be used as a neuroprotective agent in reducing the effect of HI in preterm and term infants. If NanoCurc provides a stronger neuroprotective effect in the younger population, it may serve to target infants most severely affected by HI, potentially creating personalized treatment for gestational ages.

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 infant morbidity and mortality worldwide, with a strong association between the degree of prematurity and the likelihood of death or neurodevelopmental impairment. Intracranial hemorrhage (ICH) is one of the most common neurological injuries for extremely preterm infants (born less than 28 weeks’ gestation). During the last trimester of pregnancy, neurons and glial cells develop in the germinal matrix requiring vast amounts of vascular support. In preterm infants, disturbances to blood and hydrostatic pressure are thought to rupture the immature vessels of the germinal matrix, leading to the bleeding in and around the ventricles. ICH is rated on a scale of I to IV, with severe ICH being grade III-IV. Mortality associated with ICH ranges from 30-60 percent, increasing with ICH severity, and survivors have an increased risk of cerebral palsy, seizures, and neurodevelopmental delay. From 2018-2020 the University of Washington (UW) neonatal intensive care unit (NICU) implemented an ICH Prevention Bundle, which focused on minimizing blood pressure disturbances during the first 72 hours after birth in infants born extremely premature, and appeared to result in a decrease in severe ICH. This study will evaluate the incidence rate of ICH at the UW NICU over a ten-year period. In a retrospective analysis of the UW NICU’s admissions, we will investigate extremely preterm infants born during the time periods of December 2013-September 2016 versus January 2017-December 2023 and record the incidence of ICH. Our primary outcomes will be ICH, by grades I-IV, as well as ICH complications such as posthemorrhagic ventricular dilatation with and without need for intervention, and death before discharge. We hypothesize that with improved prevention methods, such as the implementation of the ICH Prevention Bundle, we will see an associated long-term decrease in the incidence rate of ICH.

My research explores the metabolic flexibility and longevity of Rhodopseudomonas palustris (R. palustris). This alpha-proteobacterium has become a model organism for studying bacterial survival in non-growing states. R. palustris can endure long-term starvation in a growth-arrested state without forming dormant structures, prompting a comprehensive investigation of the molecular basis of growth-arrest and metabolic modes under these conditions. Recent studies have demonstrated that R. palustris can enter the growth-arrested state due to nutrient limitation but not energy limitation. R. palustris utilizes cyclic phosphorylation to generate ATP, allowing it to sustain viability for an extended period, even in the absence of nutrients including carbon and nitrogen. Earlier research examined the molecular response of R. palustris to growth arrest induced by carbon starvation under light and dark anaerobic conditions. Results indicated that light-incubated cells remained viable for months while dark-incubated cells exhibited a significant decrease in viability following growth arrest. The decline in viability was associated with ATP depletion, which underscores the critical role of ATP in R. palustris’s survival during growth arrest. To further investigate the versatile metabolism of R. palustris, we conducted anaerobic growth experiments using wild-type strain CGA009. We manipulated casamino acids concentrations in nitrogen-rich medium (PM) and nitrogen-free medium (NFM). Results revealed that R. palustris CGA009 utilizes casamino acids as both carbon and nitrogen sources. Our experiments also confirmed that R. palustris CGA009 can grow in the amino acid L-Leucine. Currently, we are researching the capacity of R. palustris CGA009 to utilize diverse carbon substrates through aerobic and anaerobic cultivation on Gelrite medium. Distinct growth patterns provided insights into specific concentrations of carbon substrates tolerated by R. palustris. This ongoing research aims to identify additional carbon substrates supporting R. palustris’s growth, with implications for harnessing its unique metabolic capabilities and expanding our understanding of R. palustris’s metabolic versatility.