MODULATION OF PULMONARY EPITHELIAL PARACELLULAR PERMEABILITY BY PNEUMOLYSIN FROM STREPTOCOCCUS PNEUMONIAE

Pneumolysin (PLY), a key virulence factor of Streptococcus pneumoniae (S. pneumoniae), disrupts airway epithelial barriers, yet its cellular and molecular mechanisms remain poorly understood. We aim to elucidate recombinant pneumolysin (rPLY)’s effects on Calu-3 bronchial epithelial barrier integrity and underlying pathways. This study investigates the effects of non-lytic and lytic PLY concentrations (5, 15, 30 µg/mL) to test the hypothesis that PLY increases epithelial paracellular permeability by affecting the anatomy and physiology of epithelial tight junctions. To address the hypothesis that PLY increases paracellular permeability through disruption of tight junction integrity in Calu-3 cells in a time- and dose-dependent manner, genomic DNA was isolated from virulent S. pneumoniae strain D39, and specific regions were amplified using PCR. The amplified DNA was ligated into the pET28b plasmid, and Escherichia coli (E. coli) BL21 cells were transformed to support the recombinant vector and produce a viable pneumolysin product. The PLY protein was purified via FPLC-NGC and further purified via the De-Salting column. Analysis by SDS-PAGE revealed a final product of ~53 kDa, corresponding to the molecular weight of the natural pneumolysin. The non- lethal concentration of the purified recombinant protein was determined via LDH assay. Confluent monolayers of Calu-3 cell line were cultured on porous membrane inserts and used to investigate PLY\u27s impact on epithelial paracellular permeability. Transepithelial electrical resistance (TEER) was used to evaluate the integrity of epithelial junctional complexes. The TEER data revealed a significant reduction in resistance post-PLY exposure with various concentrations of PLY (5, 15, and 30 µg/mL) for up to 50 minutes. TEER data indicated a significant decrease in epithelial resistance at 50 minutes for the highest concentration (Triplicate inserts, 4 independent experiments, t-test, p\u3c 0.01), indicating a compromised barrier integrity. Fluorescence microscopy images indicated subtle alterations in the expression and localization of the tight junction protein occludin. In conclusion, our data revealed the potential mechanism of S. pneumoniae-induced pulmonary injury at the cellular level

PHOTONIC CRYSTAL-BASED HYPERSPECTRAL IMAGING FOR IN VIVO SENSING AND ENDOSCOPY APPLICATIONS

Photonic crystal slab sensors are extensively researched for label-free detection due to their sensitivity to changes in the refractive index of surrounding media. However, their application in imaging and endoscopy remains relatively underexplored. This thesis introduces a novel approach to endoscopic technology by designing a photonic crystal slab-based spectral sensor that can be incorporated at the tip of an endoscopic probe. The system incorporates hyperspectral imaging to analyze the optical properties of targeted biological or agricultural tissues in real time. In biomedical diagnostics, it can aid in the early detection of diseases, including the identification of cancer cells. In agriculture, it allows for non-invasive imaging of plant roots to identify plant disease conditions, root-microbial interactions, nutrient uptake, etc. What makes this sensor unique is its ability to capture the reflection spectrum from biological tissues and reconstruct it into detailed spectral mapping for further analysis. The photonic crystal slab itself is highly miniaturized—measuring 34 µm × 34 µm—making it compatible with navigating narrow human arteries such as terminal arterioles, which are as small as 50 µm in diameter. To enhance its sensing capability, the design employs a 3×3 matrix of photonic crystal slab arrays, each with precisely engineered geometrical parameters such as periodicity and structural dimensions. These parameters determine the distinct spectral response of each array. The reflection spectra collected from these arrays are processed through an image reconstruction algorithm to create detailed spectral maps. Simulations of the sensor in a gastrointestinal environment were conducted using COMSOL Multiphysics 5.6. Results revealed that cancer cells exhibit higher light absorption in the visible spectrum range (400 nm to 700 nm) than normal cells due to their higher refractive index, providing a basis for distinguishing between healthy and cancerous tissues. For agricultural applications, the photonic crystal-equipped endoscopic probe is capable of reaching root zones as narrow as 0.04 mm, enabling simultaneous collection of spectral and visual data from delicate plant root areas, which are difficult to reach otherwise. This capability supports early disease detection, stress assessment, and evaluation of nutrient uptake in plants. In conclusion, the integration of a photonic crystal slab-based spectral sensor into an endoscopic system marks a significant advancement for both biomedical diagnostics and precision agriculture. Future developments will focus on physical implementation of this technology for widespread use in clinical and agricultural applications

Forage preference in two geographically co-occurring fungus gardening ants: A dietary DNA approach

Traditional methods of forage identification are impractical with non-leafcutting fungus gardening ants, making diet-related ecological and life history questions difficult to study. To address this limitation, we utilized dietary DNA metabarcoding on excavated ant fungus gardens to generate forage diversity metrics for the two co-occurring species Trachymyrmex septentrionalis and Mycetomoellerius turrifex. Ten fungus garden samples from each species were collected from a 60x70 m plot in East Texas. Each of the colonies we sampled was paired with a colony from the other species within 3 m of it. Plant forage diversity was assessed with chloroplast trnL primers, and insect frass forage diversity was assessed with mitochondria COI primers. DNA metabarcoding identified a total of 44 plant taxa across all samples, but performed poorly when characterizing foraged insect frass. Plant beta diversity was significantly different between the gardens of T. septentrionalis and M. turrifex colonies, as well as paired colonies. Colony pairs also had significantly different plant alpha diversity. This indicates that diet preference is likely driven both by ant species-specific plant preference, and colony location-specific plant resource availability. Overall, our results show that dietary DNA techniques are a promising tool for the identification of plant forage in ant fungus gardens, enabling the study of future diet-based ecological and natural history questions

Risk Communication and Public Health Emergency Responses During COVID-19 Pandemic in Rural Communities in Kenya: A Cross-Sectional Study

Background: The COVID-19 pandemic highlighted the crucial role of community preventive behaviors in controlling the virus’ spread. Studies show that people’s risk perceptions and awareness significantly contribute to the containment and prevention of infections by motivating adoption of desired actions and behaviors. This study aimed at assessing the role of risk communication and factors that influenced responses during the COVID-19 pandemic in rural communities in Western Kenya. Methods: A descriptive cross-sectional study was conducted using a quantitative research approach, collecting data from 806 individuals across Kisumu, Vihiga, and Kakamega counties. Descriptive statistics were used to detail the demographic characteristics of the study population, while logistic regression analysis estimated the associations between risk communication and demographic characteristics on COVID-19 vaccine acceptance, compliance with mitigation behaviors, perceived severity, and perceived susceptibility. Results: The results showed that 55% of participants were male and 45% were female, with an average moderate compliance with safety measures (mean = 5.15). A significant portion of participants wore face masks (85.3%), practiced hand hygiene (78.9%), and avoided close contact behaviors (66.6%). Most respondents received information through mass media (86.1%) and health workers (72.9%). Compliance with COVID-19 mitigation measures was highest among those who trusted information from official institutions, health professionals, and mass media, compared to social media, with increased odds of 2.7 times and 2.5 times, respectively. Higher risk perception was significantly associated with older age groups (above 50 years), being male, and working in the private sector. Effective risk communication significantly influenced risk perception, compliance with COVID-19 measures, and vaccination acceptance. Conclusions: The findings suggest that effective risk communication strategies are critical during public health emergencies and hence implications for future public health crises. The results underscore the importance of targeted communication and tailored interventions to improve compliance and vaccine acceptance among different demographic groups, ensuring a more robust public health response during outbreaks and pandemics

Disruption of Pulmonary Epithelial Barrier Function by Pneumolysin from Streptococcus pneumoniae

Pneumolysin (Ply), a cholesterol-dependent pore-forming toxin produced by Streptococcus pneumoniae, compromises epithelial barrier integrity and facilitates dissemination of infection. we hypothesize that Ply disrupts tight junction integrity in Calu-3 monolayers by modulating occludin expression and its interactions with other junctional components, including ZO-1 and actin filaments. To test this hypothesis, we utilized confluent bronchial epithelial monolayers of Cancer Lung cell line, clone 3 (Calu-3) cultured on a porous membrane insert that separates the apical and basal domains of a 3-dimensional (3D) cell culture model. The monolayers were exposed apically to 5 µg/mL of in-house purified recombinant pneumolysin (rPly). Transepithelial electrical resistance (TEER) was used to monitor Calu-3 monolayers’ barrier integrity over time, and tight junction proteins localization and actin remodeling were examined with a fluorescence microscope. Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western blot analysis were used to quantify protein expression levels. We found that rPly (5 µg/mL) induced a significant drop in TEER within 0.5 hour, sustained up to 1 hour, with transient recovery at 5 hours and subsequent decline at 10 hours. Removal of rPly from the monolayers at 5 hours and subsequent incubation in fresh serum-free medium led to progressive TEER restoration over 24 hours. Immunofluorescence analysis at recovery time points revealed redistribution of occludin and Zonula Occluden (ZO-1) from the cell junction into the cytoplasm, stress fiber formation at 5-10 hours, and partial restoration of occludin and ZO-1 to the cell junction, alongside cortical actin reorganization at 24 hours. Western blot analysis confirmed decreased occludin expression after 1 and 5 hours of rPly exposure, with partial restoration after 24 hours post-toxin removal. These findings suggest that rPly-induced epithelial barrier disruption is reversible through junctional reorganization and cytoskeletal remodeling, highlighting potential therapeutic avenues to prevent exacerbated pneumococcal lung injury

POPULATION STRUCTURE ANALYSIS OF FOUR BASAL HIGHER-ATTINES USING BIOINFORMATIC APPROACHES

Metapopulations within the family Formicidae are unique among other animals due to the large colonies they build, and the mating strategies required to fertilize a specialized reproductive caste that will often produce young for the life of the colony. One group of ants, the fungus-gardeners (tribe Attini). The population structure of North American non-leafcutting, fungus-gardening ants has been understudied, especially in the southwest of the United States. Additionally, not much is known about their dispersal biology, so the dynamics of dispersal of these species and how they affect population structure is likewise not well known. To shed light on the structure in these species, four species of non-leaf-cutting ants in the genus Trachymyrmex and Mycetomoellerius were obtained from two other studies for population analysis. The GBS reads of two species sampled in Texas and Oklahoma, Trachymyrmex septentrionalis and Mycetomoellerius turrifex, and two from Arizona, Trachymyrmex arizonensis and Trachymyrmex pomonae, were analyzed using a custom pipeline of bioinformatic software. This pipeline separated the GBS read into two paths: one calling SNPs from the nuclear genome, and the second extracting the mitochondrial genome with MitoFinder. The SNP markers were used to answer the question of population structure by using common tests of structure such as STRUCTURE, FST, AMOVA, and PCA. The extracted mitochondrial markers were used to compare with the nuclear SNPs to determine if there is a discordance between the two-genome suggesting one sex disperses more than the other. The results showed that there is significant structure in T. septentrionalis, M. turrifex, and T. arizonensis; however, no structure was found in T. pomonae. Further, comparison of the nuclear and mitochondrial genomes found evidence of male-biased dispersal within the same T. septentrionalis, M. turrifex, and T. arizonensis

Electrostatic Effects on the Transport and Dispersion of Micro-Nano Pharmaceutical Powders

A predetermined amount of micro-nano powder particles contributes to the treatment during a patient’s inhalation of the bolus of respiratory micro-nano pharmaceutical powders in an aerosolized state from commercially available inhalers. The most popular inhalers are the metered dose inhalers (MDIs) and dry powder inhalers (DPIs). While inhaling, the complicated inlet velocity profile and turbulent flow in the mouth-throat significantly impact the gaining of electrostatic charge through triboelectric charging. The inhaled particles are not only varying sizes but also polarities of charges. However, few studies have been conducted to investigate electrostatic charge force effects on particle losses in the oral, pharyngeal, and laryngeal (OPL) regions before inhaling into the trachea and subsequent deeper lung. This research, however, has shed light on the integrated impact of powder particles’ size and electrostatic charge on the deposition of inhaled pharmaceutical particles in a polyester resin cast of a cadaver-based mouth-throat, a replicate of human OPL regions. Each powder aerosol particle was characterized for its aerodynamic diameter (size) and charge by an electronic single-particle aerodynamic relaxation time analyzer before and after passing through the mouth-throat cast. The study findings show that particles’ charge-to-mass ratio and electrical mobility varied with size. Electrostatic properties played a significant and enlightening role in particle losses in the mouth-throat cast. Deposition fraction or losses of powder particles were comparatively higher for DPIs than for MDIs

EXCLUSIVE BREASTMILK PUMPING: REFINING KNOWLEDGE AND EXPLORING FAMILIES’ EXPERIENCES

Breastmilk is the biological normative substance for infants, yet some families cannot breastfeed, choose not to breastfeed, or attempt to breastfeed and then transition to formula when they encounter problems. Some parents are feeding their infants breastmilk produced from the process of exclusive expression (EE). This dissertation portfolio includes a definition of EE derived from a concept analysis and an integrative review of the current literature, together providing an essential foundation for all future EE-related research. Additionally, this portfolio includes the report of original qualitative research, which was guided by the methodology of Interpretive Description. The study entailed conducting interviews with and reviewing social media posts from EE parents. The aim was to determine what we can learn from parents who utilize EE in the home environment to provide nutrition for their full-term, healthy babies. Some findings were consistent with the published literature, while others were novel. Unique findings in this study included the parents’ experiential wisdom transforming into guidance for both healthcare providers and for future parents who may choose to provide breastmilk using EE. In addition, within the theme of experiential wisdom, there are clear examples of parents having learned to “trust their gut” and the reassuring results that followed. Clinical insights were gleaned from hearing parents recount their antepartum, intrapartum, and EE experiences. The portfolio concludes with a summary of the dissertation documents and an overview of next steps in the author’s anticipated program of EE research

MYOCARDIN EFFECTOR PROTEINS AFFECT TGF-BETA-INDUCED MYOFIBROBLAST DIFFERENTIATION IN IPF CELLS

Idiopathic pulmonary fibrosis (IPF) is an incurable interstitial lung disease defined by the progressive, irreversible scarring of the tissues between the lung’s alveoli. To date, only two drugs have received approval for treatment. However, these medications are not considered cures since they do not stop the progression of the disease. A deeper understanding of the mechanisms underlying IPF could aid in discovering a cure. We recently found that myocardin (MyoCD) regulates SMAD2/3 transcription factors in the TGF-β signaling pathway and governs the genes involved in pleural fibrosis. However, it is unclear whether MyoCD plays a critical role in IPF by regulating various genes, leading to the differentiation of myofibroblasts. In this study, six novel MyoCD effector genes were examined to determine their potential roles in myofibroblast differentiation and cytoskeletal reorganization of IPF cells stimulated with TGF-β. At the mRNA and protein levels, MyoD Family Inhibitor (MDFI) knockdown (KD) decreased the expression of MyoCD, α-SMA, PAI-1, CNN-1, and FN-1, while COL-1 remained unaffected. This was the main MyoCD effector protein that showed significance in the data. Results from immunocytochemistry confirmed that MDFI influences cytoskeletal changes by reducing the formation of stress fibers. Furthermore, secretion was also affected by MDFI KD. This data suggests that MDFI impacts myofibroblast differentiation and cytoskeletal remodeling in IPF fibroblast cells through the TGF-β/MyoCD signaling pathway; therefore, it may contribute to the progression of IPF

Tyler Metropolitan Statistical Area (MSA): A Texas Veteran-Friendly Community

The Hibbs Institute\u27s November 2024 brief, Tyler Metropolitan Statistical Area (MSA): A Texas Veteran-Friendly Community, outlines Tyler\u27s application for designation as a Texas Veteran-Friendly Community through the East Texas Council of Governments (ETCOG). The brief highlights Tyler\u27s strong economic environment, community support, employment opportunities, healthcare services, educational and career prospects, quality of life, and future plans that collectively honor and support veterans. Notable recognitions include Tyler\u27s designation as a Purple Heart City (2014), multiple Military Friendly Schools® awards, and being ranked No. 8 in the 10 Best Cities for Veterans by Livability.com (2016). The economic impact of veterans in the region is significant, with contributions estimated between $427.4 million and$628.6 million. The brief also emphasizes the availability of world-class healthcare facilities, such as the Tyler Centennial VA Clinic, and various community organizations like CampV that provide resources to veterans. The results of the designation application are expected on January 2, 2025, with a comprehensive report to follow in spring 2025