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Selecting for Lysis in a Temperate Phage Population
No full textThe ongoing evolutionary arms race between bacteria and bacteriophages (phages) is a prime example of Red Queen hypothesis, where continuous adaptation drives survival. This study focuses on Escherichia coli-infecting temperate phages, which dominate natural environments and contribute to bacterial resistance mechanisms. Despite their prevalence, temperate phages are less suited for phage therapy due to their lysogenic life cycle, where they integrate into bacterial genomes instead of lysing the host. In contrast, obligately lytic phages, which directly lyse bacterial cells, are ideal for therapeutic use but occur less frequently in nature. This research aims to select for lytic behavior in temperate phages through experimental evolution. Aim 1 involves inducing temperate phages from a collection of E. coli lysogens isolated from urinary tract infections (UTIs) in females. These phages were characterized through transmission electron microscopy (TEM) and host range assays. Aim 2 applies the Appelmans\u27 Protocol to select for lytic behavior through iterative co-cultures of phage cocktails and E. coli strains over 30 days. Aim 3 uses the BRESEQ pipeline to identify mutations linked to lytic conversion, focusing on integrase genes and other loci involved in host interaction. By characterizing temperate phages and evolving them, this study addresses the urgent need for supplementation to antibiotic treatments, particularly in regards to UTIs. Beyond clinical applications, it expands our understanding of temperate phage evolution, mutation dynamics, and ecological roles. This approach offers new insights into the adaptability of phages and their potential to combat antibiotic resistance
Let-7I Mirna Antagomir as a Novel Adjuvant Intervention for the Treatment of Ischemic Stroke
No full textIschemic stroke is a leading cause of death worldwide and often results in permanent disabilities that can greatly hinder quality of life in surviving patients. Treatment options for ischemic stroke are extremely limited, emphasizing a critical need for the development of novel treatment modalities. Progesterone (P4) is a gonadal steroid hormone which has been shown to be protective in experimental models of stroke. However, the underlying protective mechanisms remain unclear. Previously, our lab has shown that inhibition of the microRNA let-7i enhances the protective effects of P4 in an animal model of stroke. In this study, we sought to evaluate the differential responses between astrocytes and neurons to P4 + let-7i antagomir treatment and assess the protective effects when administered as an intervention in vitro after ischemic insult. Furthermore, we explore a minimally invasive method of antagomir delivery using exosomes expressing the rabies virus glycoprotein (RVG), a brain-targeting peptide. Using cultures of primary cortical astrocytes, neurons, and mixed cultures (containing neurons and glia) exposed to conditions of oxygen-glucose deprivation (OGD), we report that there are cell-specific changes in expression of let-7i, as well as two key mediators of P4’s protective effects, progesterone receptor membrane component 1 (PGRMC1) and brain derived neurotrophic factor (BDNF). Our data also demonstrates that treatment with P4 + the let-7i antagomir (let-7i inhibitor), when administered 3 hours after ischemic insult, directly protects astrocytes, and indirectly protect neurons through astrocyte mediated mechanisms. Additionally, P4 + let-7i antagomir treatment does not affect BDNF protein levels in astrocytes and neurons cultured separately, but significantly increases BDNF protein levels in mixed cell cultures. Initial characterization of our exosome-mediated delivery of the let-7i antagomir revealed that RVG-expressing exosomes were non-toxic to astrocytes. However, upon evaluating the delivery of the antagomir in astrocytes exposed to OGD, we found negligible changes in expression of let-7i, PGRMC1, and BDNF. Additionally, RVG-expressing exosomes failed to produce the same protective effect seen when the let-7i antagomir was directly transfected into astrocytes, suggesting further development and/or optimization of the exosome-mediated delivery approach is warranted. In summary, we report that the protective effects of P4 and the let-7i antagomir are primarily mediated by glia, and overall, require the presence of both astrocytes and neurons to elicit a maximal protective response. These data highlight the therapeutic potential of P4 + let-7i antagomir treatment as a delayed intervention following stroke and emphasize the role of glia in mediating its protective effects. Additionally, since glia are the primary target for this intervention, our results invite further studies on effective and safe delivery methods for the let-7i antagomir, specifically to astrocytes, following ischemic stroke
Intracellular Persistence of Cytotoxic Strains of P. Aeruginosa Isolates Is Enabled by Bistable Expression of the Type Three Secretion System
No full textPseudomonas aeruginosa is a leading contributor to nosocomial infections. Generally thought of as an extracellular pathogen, a growing number of studies have observed P. aeruginosa surviving and replicating within host cells. Investigations characterizing the intracellular lifestyle of P. aeruginosa found two populations: cytoplasmic bacteria replicating in a manner requiring the type-three secretion system (T3SS) and the effector ExoS, while others remain in a T3SS negative state and reside within host cell vacuoles without inducing host cell death. Here, we used experimental models to test our hypothesis that P. aeruginosa strains encoding the phospholipase ExoU (instead of ExoS) may also be internalized and are able to persist within compartments if they remain negative for T3SS effector secretion. We primarily investigated two lab strains (PA103 and PA14) and two clinical isolates from keratitis: 6206 and 6452. Using time-lapse microscopy, we demonstrate that some cytotoxic strains of P. aeruginosa are capable of persisting within intracellular compartments of host cells for at least 20 hours post-infection (PA14, 6452), while others do not (PA103, 6206). Using a fluorescent reporter plasmid to quantify T3SS transcriptional activation by flow cytometry, we found correlations between the T3SS status of clinical isolate strains and the tendency of each isolate to become intracellular. Populations of strains capable of invading (PA14 and 6452) remained with the majority T3SS-off even when exposed to an in vitro stimulus, whereas classically cytotoxic strains (PA103 and 6206) were comprised of more T3SS-on bacteria. Strains able to invade also exhibited lower basal transcriptional activity of the T3SS effectors, with non-invasive strains displaying up to a 10-fold increase in transcriptional activity of T3SS effectors even when uninduced. Interestingly, the mutation of a known regulator of the T3SS in PA103, vfr, lead to an increase in bacterial invasion and persistence; the same mutation in 6206, however, did not yield similar results, indicating that other factors may be in play and differ between strains. Taken together, our results reveal a potential intracellular lifestyle for some cytotoxic strains of P. aeruginosa, which may be related to intrinsic strain differences in bistability of T3SS activity
The De Mau Mau Conspiracy: The Specter of Black Vietnam Veterans in the Exercise of Police Power, 1968-1976
No full textA forgotten and bizarre event encapsulates the violent decay that characterized the pivotal years the United States endured in the wake of the Vietnam War. Black veterans—guerrilla combat-trained, politically awakened by war, and disillusioned with the entrenched racial order at home—emerged as totems in a fearful, cynical political culture wracked by violence. Men overrepresented in the projection of American power abroad now stood as symbols of its fragility within, potential vessels of retribution for centuries of domestic and global oppression. This anxiety, merging with widespread protests and political upheaval, intensified the belief that American cities stood on the brink of insurrection. This dissertation deploys microhistory to sit-uate the De Mau Mau Conspiracy (1972-1976)—when black Vietnam veterans and their associates were accused of wantonly murdering whites—and its contexts, as representative of the post-1968 unraveling in American society before the consolidation of the War on Drugs regime. In this moment of fracture, a multifaceted specter of the re-turning black veteran was central to several discourses on the racial and civil order of the United States. Structured as a layered political, intellectual, and policing history, the dissertation moves from an examination of media hysteria and electoral manipulation to microhistorical treatment of the forensics of the murder cases at the center of the conspiracy, concluding with a close reading of the accused men’s writings. Through a detailed analysis of police records, court documents, print and television media coverage, and an array of periodicals, including GI journals, the study explores two parallel developments: how law enforcement and public officials weaponized fear of black veterans to consolidate carceral power; and how the profound demonization, alienation, and neglect of these young men effectively radicalized a small segment of the population. It concludes that the De Mau Mau Conspiracy functions as both a cautionary tale and a political instrument—illuminating broader strategies of racial containment and the enduring consequences of war, both for those conscripted into imperial folly and for the nation that disavowed them upon their return
Development of Novel Force Fields for Metal Ions
Get PDFMany biological proteins require the presence of a metal ion in order to properly function. However, metal ions’ complex nature charge poses several challenges to accurately and efficiently simulate computationally. For example, metal ions can exhibit multiple oxidation states, electronic state degeneracy, flexible coordination numbers, and significant polarization effects. To address these problems, this dissertation aims to enhance force fields for modeling metal ions in molecular dynamics simulations. First, a comprehensive set of van der Waals radii for metal ions is derived, demonstrating the importance of using physically meaningful parameters in force fields. Second, a comprehensive set of atomic and ionic polarizabilities across the periodic table is established, exploring their relationship with van der Waals radii. These studies lay the foundation for developing a general polarizable force field for metal ions. Third, we developed a strategy to parametrize the C4 parameters in the 12-6-4 model using an energy decomposition approach based on quantum mechanical calculations, exemplified in metal-imidazole systems. Finally, a practical study of a metalloprotein simulations using conventional molecular dynamics and free energy calculations is presented. Overall, this dissertation contributes to the metal-ion modeling community by providing comprehensive sets of polarizabilities and van der Waals radii for ions, contributing to ion-imidazole parameters, and providing key insights into protein-ion binding affinity regulation
Development of a Humanized SCFV Antibody: Step Towards Treatment of Neuropathic Pain
No full textNeuropathic pain, caused by damage to the somatosensory nervous system, remains a significant medical challenge due to limited efficacy and side effects of current treatments. The goal of this research was to develop a novel, non-opioid therapeutic strategy targeting the cholecystokinin B receptor (CCKBR), which is implicated in chronic pain propagation and opioid resistance. By designing a humanized single-chain variable fragment (scFv) antibody against CCKBR, this study aims to address the limitations of existing pain management approaches as CCKBR plays a critical role in modulating pain pathways by opposing μ-opioid receptor (MOR) signaling, contributing to persistent pain and reduced opioid efficacy. Immunohistochemical analyses confirmed the co-localization of CCKBR and MOR in pain-related regions of the brain, including dorsal root ganglia (DRG) and spinal cord tissues from rats, mice, and humans. Functional studies using live-cell calcium imaging demonstrated that CCKBR activation antagonizes MOR signaling by increasing intracellular calcium levels. To target this receptor, a murine anti-CCKBR scFv was generated using ribosomal display technology and subsequently humanized to reduce immunogenicity while preserving high specificity and affinity. The lead humanized scFv exhibited robust binding to CCKBR and functional assays confirmed its ability to inhibit CCKBR-mediated pronociceptive signaling in vitro. The findings highlight the potential of humanized anti-CCKBR scFv antibodies as a promising therapeutic approach for neuropathic pain. By specifically targeting CCKBR, these antibodies may restore MOR function and provide effective analgesia without the risks associated with opioids. Future directions include further preclinical development and clinical translation of this innovative treatment strategy
Loss of Neuropeptide F (NPF) Signaling Reduces the Strength of Circadian Rest-Activity Rhythms but not Feeding-Fasting Rhythms
Get PDFThe circadian system governs crucial physiological and behavioral processes, dictating activities like movement, hormone release, and sleep patterns over a 24-hour cycle. Disruptions to these rhythms can lead to various health problems, including cardiometabolic dysfunction. Research has demonstrated the presence of circadian rhythms of feeding behavior in diverse organisms, but the specific molecules involved are not yet identified. Neuropeptide Y (NPY) influences metabolic activity in mammals, and its counterpart, NPF, affects locomotor activity and feeding in Drosophila melanogaster. Our study aims to investigate whether NPF, its receptor (NPFR), and NPF-expressing cells play a role in regulating circadian rest-activity and feeding-fasting rhythms. We use behavioral analysis tools, such as the Drosophila Activity Monitor (DAM) and Fly Liquid-Food Interaction Counter (FLIC) systems, to study locomotor activity and feeding behavior in mutant, cellular ablation, and control flies. Our results indicate that flies lacking functional NPF or NPFR exhibit abnormal rhythms in locomotor activity behavior but not feeding behavior, suggesting a differential disruption in circadian regulation. However, when NPF-expressing cells are ablated in the brain, we observe normal locomotor activity and feeding rhythms, implying that the ablated cells are not involved in regulating the rhythmicity of these behaviors. Understanding these mechanisms will not only shed light on circadian biology in fruit flies but also have implications for addressing circadian rhythm-related health issues in humans
Two Leading Female Figures of the Anti-Colonial Struggle in Africa
Get PDFThe Eurocentric and male narrative of the anti-colonial struggle in Africa during the twentieth century marginalized women’s contributions and presented them as passive victims of colonialism. Therefore, this study offers a more comprehensive historical account of women\u27s leadership in Africa and a more inclusive and liberated narrative of the anti-colonial movement. Achieving this goal involves delving into the roles and contributions of two prominent female figures in the anti-colonial resistance in Northern and Sub-Saharan Africa during the twentieth century: Huda Shaarawi from Egypt and Funmilayo Ransome-Kuti from Nigeria. Furthermore, this study aims to demonstrate that the actions and strategies of these female leaders were rooted in Black feminist principles. This research is crucial not only for its contribution to academia through the exploration of a new topic and the generation of fresh insights but also for empowering African men and women to share their own narratives and challenge the Eurocentric knowledge that has shaped the understanding of Africa\u27s history. Additionally, this work is vital for presenting the Black feminist activism of African women as they fight for their communities\u27 political and patriarchal liberation. The theoretical framework will be informed by Black feminism and Afro-decolonialism, while the methodological approach will incorporate textual analysis
The Effectiveness of a Race-Based Stress Reduction Intervention on Improving Stress-Related Symptoms and Inflammation in African American Women at Risk for Cardiometabolic Disease: Protocol for Recruitment and Intervention for a Randomized Controlled Trial
Get PDFBackground: In recent years, the prevalence of cardiometabolic disease (CMD) in African American women has risen; the risk also increases with age, in comparison to men. Evidence demonstrates that stressful life events, including experiences of racism and perceived discrimination, contribute substantially to inflammatory diseases, such as CMD. Despite this evidence, few evidence-based interventions are available to assist individuals from minoritized communities in coping with the chronic stress related to their racial or ethnic identity.
Objective: Our proposed randomized controlled trial will test a novel, race-based intervention tailored to African American women, called Resilience, Stress, and Ethnicity (RiSE).
Methods: In this randomized controlled trial, we will randomize participants 1:1 to the 8-week, group-based RiSE program (intervention) or a health education program (active control group). Both programs will consist of synchronous classes on Zoom and will be led by experts. The primary end point will be stress at 6 months after the intervention, and the efficacy of RiSE will be evaluated for improving stress-related symptoms (current perceived stress, depressive symptoms, fatigue, and sleep disturbance), improving coping strategies, and reducing inflammatory burden in African American women at risk for CMD. Validated survey measures and inflammatory biomarkers will be assessed at baseline, midintervention, intervention completion, and 6 months after the intervention, and differences over time by intervention will be evaluated using mixed effects models.
Results: This study was funded by the National Institute on Aging on March 30, 2023, with recruitment and enrollment beginning in October 2023. The study is underway, with 120 participants enrolled as of March 2025.
Conclusions: This study will be one of the first to examine a race-based stress reduction intervention in African American women and has the potential to improve the health of minoritized groups faced with chronic stress associated with experiencing racism and discrimination. We anticipate that RiSE will reduce stress-related symptoms, enhance adaptive coping, and reduce inflammation
Endings of Half-Lines in the Middle English Alliterative Joseph of Arimathea
Get PDFThe Middle English alliterative poem Joseph of Arimathea survives in a single copy, lacking verse lineation and the regular alliteration expected of this form. Several explanations have been advanced, but the poem has been treated with reserve: scholars and critics have been reluctant, perhaps, to attend closely to a text that has seemed unfinished, careless, or corrupt. The present study counters these impressions with a narrow metrical test. Joseph agrees very well with one of the ‘finer rules’ described in recent scholarship on alliterative metrics, namely the requirement that lines end with a trochaic constituent. Regular line endings support the view that the text of Joseph, as transmitted, is deliberate and mostly accurate. Supporting data are available at doi.org/10.5281/zenodo.15724220