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  A common goal, a common path: Shared mechanisms between psilocybin and SSRI antidepressant actions?

  Wulff, Andreas; Thompson, Scott M.; Mathur, Brian N. (Brian Neil) (2022)

  Major Depressive Disorder is a prevalent and debilitating mental illness. While heterogenous in pathogenesis, symptomatology, and neurobiology, it is marked by reduced mood, anhedonia and severely reduced quality of life. Standard treatments such as the commonly prescribed selective serotonin reuptake inhibitors (SSRIs) are effective in approximately 70% of patients and take weeks for response and remission to occur. Psilocybin exhibits rapid antidepressant actions in clinical trials and in rodent models of depression. Due to the pan-serotonergic agonist properties of psilocin, the active metabolite of psilocybin, I hypothesized that the antidepressant actions of psilocybin may be shared with SSRIs. Serotonin 1B receptor (5-HT1BR)-induced strengthening of stress sensitive synapses mediates therapeutic effects of SSRIs. Further evidence suggests that the therapeutic signaling of 5-HT1BR may include β-arrestin. I thus, sought to test if strengthening of stress-sensitive synapses induced by 5-HT1BR-β-arrestin signaling mediated the antidepressant effects of psilocybin. I demonstrated that psilocybin may acutely potentiate stress sensitive synapses in vivo and in hippocampal slices. I further show that psilocin-induced potentiation mimics that induced by 5-HT1BR activation. Comparing 5-HT1BR agonists, I find that β-arrestin recruitment down-stream from 5-HT1BR is associated with potentiating actions at stress-sensitive synapses in hippocampal and accumbal slices. However, in behavioral experiments, I found no antidepressant actions of psilocybin in a mouse model of stress-induced anhedonia. This suggests that psilocybin’s antidepressant actions may depend on extra-drug factors or that psilocybin is limited to restore certain stress-induced neurobiological and behavioral impairments. I further describe experiments exploring behavioral mechanisms and neurophysiological heterogeneity underlying rodent models of anhedonia. Using lickometry, I record detailed drinking behavior of mice and find evidence that loss of sucrose preference in stress-susceptible mice is mediated by impaired goal-oriented behaviors. Using electrophysiological recordings of D1- and D2 expressing medium spiny neurons, I show that anhedonic behavioral phenotypes induced by fentanyl abstinence is associated with neurophysiological changes in D1- medium spiny neurons that are overlapping but distinct from those seen following stress-induced anhedonia. Combined, these data show that psilocybin may strengthen stress-sensitive synapses via 5-HT1BR-β-arrestin signaling but that this may not be sufficient to rescue all types of stress-induced anhedonia.
• Mast Cell Modulation of the Developing Hippocampus

  Blanchard, Alexa; McCarthy, Margaret M., 1958- (2023)

  Brain development requires orchestration of overlapping critical periods defined by responsiveness to endogenous or exogenous stimuli (i.e. hormones, light, touch). Simultaneously, sensitive periods represent heightened risk to perturbations by abnormal stimuli (i.e. inflammation). Though neuro-immune cooperation between resident microglia and neurons is essential during critical periods of brain development, the contribution of infiltrating immune cells to neurodevelopmental processes is largely elusive. We discovered a critical and sensitive period created by a population of peri-hippocampal mast cells (phMCs) in the lateral ventricles abundant from birth through 2-weeks-old in the neonatal rat but absent thereafter. This epoch of phMCs is maintained by self-replication inside the brain, creating a mix of mature and immature mast cells which constitutively piecemeal degranulate. phMCs harbor a unique transcriptomic identity to skin, bone marrow and other brain mast cells including transcripts encoding colony-stimulating factors (Csf1, Csf2) essential for microglial development. Pharmacological inhibition of phMC degranulation and secretion stunts microglial maturation, illuminating the role of phMC-derived factors in driving hippocampal microglial development. In contrast, activation of phMCs releases proinflammatory chemokines, compromises the blood-brain-barrier (BBB), and recruits peripheral immune cells across the CNS for at least two days. Together, these findings indicate that a transient population of peri-hippocampal mast cells creates a critical period for microglial maturation via piecemeal degranulation in the healthy brain and a sensitive period to inflammatory stimuli. The dual role of phMCs in homeostasis and inflammation highlight an essential need to understand how the immune system can adapt its function to serve neuronal populations and prevent inflammatory disturbances that lead to neurologic disease.
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  Development of an In-Cell Footprinting Method Coupled with MS for the Study of Proteins in Three-Dimensional Cancer Models

  Shortt, Raquel; Wang, Hongbing; Jones, Lisa M. (2023)

  Fast photochemical oxidation of proteins (FPOP) is a powerful, mass spectrometry (MS)-based, biophysical method used to probe protein structure, interactions, and conformations. FPOP was recently extended into cells (IC-FPOP) and can modify thousands of proteins in a single experiment, enabling proteome-wide structural biology. Although IC-FPOP can reveal critical structural information in 2D cell culture, the conditions do not emulate an in-vivo environment. To address this, we propose to develop a mass spectrometry-based protein footprinting method that assesses the varying protein heterogeneity in 3D cell culture; Spheroid-FPOP. IC-FPOP on intact spheroids was performed using a patented PIXY platform which brought automation to IC-FPOP. Spheroid-FPOP coupled with serial trypsinization to obtain spatial resolution, revealed modifications in three distinct spheroid regions; the outer inner and core. Native oncogenic pathways were interrogated through this study showing its value in disease pathogenesis and treatment. Though progressive for FPOP, the extension into 3D model systems generated three times the samples and data compared to typical IC- or IV-FPOP experiments. This shed light to FPOP workflow limitations. The research herein responds to those challenges by developing an automated sample preparation workflow by coupling a sample handling robot with Thermo’s sample preparation kit. These modifications robustly improve the workflow by significantly reducing the manual labor, execution time, and variability of samples processed and data acquired. After workflow optimization FPOP, we apply the optimized method more complex biological sample. In our case, 3D bioprinted Huh-7 liver organoids were generated for IC-FPOP. To obtain spatial resolution within the model, we integrated cryosectioning of the top, middle and bottom layers of the organoid. Peptide level analysis revealed differences in the extent of modification for peptides identified in each region of the organoid, which confirms the acquisition of structural information. However further optimization was required to increase proteome depth. By coupling the organoid model with IC-FPOP we aim further validate its implementation for complex proteome-wide structural studies. In all, this research is focused on advancing the applications and processing workflows for IC-FPOP.
• Unraveling Cochlear Otic Mesenchyme Cells: The Role of POU3F4 During Cochlear Development

  Rose, Kevin; Hertzano, Ronna P. (2023)

  The cochlea consists of diverse cellular populations working in harmony to convert mechanical stimuli into electrical signals for the perception of sound. One such cell type are otic mesenchyme cells (OMCs), which are a specialized type of neural crest and cranial paraxial mesoderm that express multiple unique transcription factors (e.g., POU3F4), all of which are known deafness genes, highlighting the importance of OMCs in auditory function. OMCs are known to terminally differentiate into spatially and functionally distinct cell types, including fibrocytes of the lateral wall and spiral limbus, modiolar osteoblasts, and specialized tympanic border cells of the basilar membrane. Interestingly, consequences of Pou3f4 mutations are diverse and include a complete loss of endocochlear potential, shortening of the cochlear duct, and defective pathfinding and survival of spiral ganglion neurons (SGNs), indicating diverse roles of POU3F4 in each OMC-derived cell type. Here, we aim to illuminate the molecular distinctness and functionality of OMCs and show how loss of Pou3f4 impacts cochlear development. By utilizing scRNA sequencing, we elucidated that OMCs divide into four transcriptionally distinct subpopulations well before the onset of hearing, each of which corresponding to one of the OMC-derived cochlear structures. Furthermore, we show OMC subpopulations display distinct functional roles corresponding to their spatial localization. We also unravel the cochlear cellular communication pathways showcasing OMCs are the main contributors of outgoing signaling during cochlear development, including both global and subpopulation specifying signaling pathways. Finally, we indicate how Pou3f4 expression regulates gene expression in each OMC subpopulation and which signaling pathways are lost in Pou3f4 mutants which may be the cause of the defects in surrounding cell types. Our data suggest that OMC diversification occurs not long after the formation of the otocyst with further refinement until the onset of hearing, well before terminal differentiation. Our data also suggests OMCs are the main contributors of paracrine signaling during cochlear development, showcasing their importance in influencing surrounding cochlear cell types. Finally, we show how loss of Pou3f4 affects each OMC subpopulation differently, leading to diverse phenotypes in Pou3f4 mutants. Without cochlear OMCs and their later terminally differentiated cell types, normal auditory function would not be feasible highlighting the importance of tissue specific mesenchymal cells in cochlear development.
• Less is more: alcohol weakens inhibitory signaling to strengthen striatal complex output

  Mckeon, Paige; Mathur, Brian N. (Brian Neil) (2023)

  The striatal complex critically integrates a variety of inputs to regulate the motivation to perform, and the performance of, discrete action sequences executed under goal-directed and habitual strategies. Drugs of abuse such as alcohol target the striatal complex, strengthening associations between rewards and external cues and, over time, shifting intake from being goal-oriented toward compulsively automated. Our work herein examines alcohol targeting of the striatal complex under two different conditions: under the first condition, we ultimately aim to determine if and how alcohol alters the firing of a GABAergic cellular population called fast-spiking interneurons (FSIs) known to regulate striatal output to mediate habitual behaviors. To that end, we uncover a novel mechanism mediating FSI synchronous firing. Under the second condition, we examine how acute alcohol exposure influences the experience of reward. Alcohol strengthens a form of inhibitory plasticity in the NAc that is mediated by brain derived neurotrophic factor (BDNF) signaling, but whether this interaction regulates alcohol reward is unknown. We here discover the sources of BDNF that drive this form of plasticity and further find that simulating BDNF-releasing afferents in vivo is rewarding to mice. We also find that alcohol and BDNF may interact in vivo to mediate reward. As such, these findings point to a novel mechanism for alcohol reward: BDNF signaling at inhibitory synapses in the NAc.
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  Bacterial enzymatic combinatorial chemistry (BECC) enabled targeted lipid A modification of Shigella vaccine strains to reduce endotoxicity without compromising immunogenicity or invasiveness

  Sherman, Matthew; Ernst, Robert K. (2023)

  Shigella spp. are Gram-negative bacteria that cause severe diarrheal disease, contributing significantly to morbidity and mortality worldwide. High transmissibility and increased antibiotic resistance have propelled the development of Shigella vaccine candidates; however, no FDA-approved vaccine exists to date. We have collaborated with Walter Reed Army Institute of Research (WRAIR) to improve their live-attenuated vaccine candidates, whose only drawback during clinical trials was febrile symptoms experienced in a few individuals. In this study, we sought to resolve those unwanted side effects by detoxifying the lipid A moiety (also known as endotoxin) that is thought to contribute to the febrile symptoms observed. To do so, we employed bacterial enzymatic combinatorial chemistry (BECC), whereby lipid A modification enzymes were ectopically expressed to induce structural alterations known to dampen lipid A signaling capacity. The enzymes LpxE (phosphatase) and PagL (deacylase) were expressed in both virulent and attenuated vaccine strains of Shigella to modify the bis-phosphorylated hexa-acylated lipid A structure ordinarily present in the Shigella outer membrane. The expected modifications were confirmed using mass spectrometric and gas chromatographic analyses when the enzymes were expressed individually or in combination (both LpxE and PagL) using a construct we refer to as “Dual”. These enzyme/enzyme combinations were subsequently integrated into the chromosome using Tn7 transposition to avoid the possibility of plasmid loss during production. The impact of the induced lipid A structural alterations on innate immune signaling was assessed by stimulation of NF-?B reporter cell lines and human PBMCs with the extracted lipopolysaccharide (LPS). Additionally, in vivo reactogenicity of the LPS in a murine acute endotoxemia model was assessed. We found that dephosphorylation, but not deacylation, of lipid A, was a powerful tool to reduce LPS-mediated signaling in live-attenuated Shigella vaccines, resulting in reduced toxicity of Shigella LPS in vivo. Additionally, we found that dephosphorylation of the lipid A moiety did not impair invasion of colonic epithelia or immunogenicity in a mouse pulmonary model. Overall, this study generated Shigella vaccine candidates with reduced endotoxicity, which upon oral ingestion in humans, will ultimately have reduced reactogenicity.
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  The predictive accuracy of Shock Index in trauma outcomes in older injured patients

  Bulatao, Ilynn; Smith, Gordon S., M.B., Ch.B., M.P.H. (2023)

  The elderly is an increasing proportion of all cases treated at trauma centers. Shock index (SI) calculated as heart rate (HR) divided by systolic blood pressure (SBP), has been shown to be a good predictor of mortality and transfusion in injured patients. One limitation of SI is that its accuracy in different age groups, especially the elderly has not been fully evaluated. We studied the accuracy of admission SI in predicting early, 48-hour and in-hospital mortality, and major interventions (massive transfusion, ICU admission and surgery in 24 hours) in trauma patients admitted to a major trauma center. We examined whether age, injury severity, injury type, blood alcohol and comorbidities affected the predictive accuracy of SI. Of particular interest is the accuracy of SI in the elderly. We also compared the predictive accuracy of SI, HR and SBP. Optimal cut-points for SI were determined. SI had acceptable accuracy in predicting mortality outcomes, and ICU admission overall. Accuracy was good in the prediction of massive transfusion, and poor in the prediction of surgery in 24 hours. SI was better than HR or SBP in predicting mortality outcomes (all ages, elderly, and younger patients). However, in older patients, accuracy of SI in predicting major interventions was not different from that of SBP. Accuracy of SI in predicting 48-hour and all in-hospital mortality, and ICU admission was better in younger patients. Accuracy was also better among those with lower injury severity than in those who were more severely injured. Accuracy of SI in predicting massive transfusion was similar in older and younger trauma patients. Optimal cut-offs for predicting outcomes were lower for older patients (0.5-0.7 for mortality and major interventions) than in younger patients (0.6-0.9 for mortality and 0.6-0.8 for major interventions). Accuracy of SI in predicting all in-hospital death and massive transfusion was less among patients with elevated blood alcohol while comorbidities did not affect accuracy. In conclusion, SI is less accurate in in predicting mortality among older patients and is less accurate in predicting mortality and massive transfusion among blood alcohol-positive patients, potentially affecting its utility in triage and clinical management.
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  Nurses’ Well-being and the Work Environment During the COVID-19 Pandemic

  Baek, Hyang; Baek, Hyang; Trinkoff, Alison M. (2023)

  Problem: The Coronavirus Disease 2019 pandemic, despite the improved current situation, has lasted longer than expected and has had a severe impact on the nursing profession. Nurses continue to face heightened levels of stress, frustration, exhaustion, and overwork. As a result, many nurses have either retired or left their positions, further worsening the existing nursing shortage. This shortage poses a significant threat to the efficiency and resources of the healthcare system. Therefore, it is crucial to gain a better understanding of how work-related factors affect the well-being of nurses. Purpose: The aims of this study were to 1) Examine the relationship between work-related factors and personal well-being during the pandemic; 2) Examine the degree and severity of workplace bullying in nurses and work-related factors; and 3) Examine the relationship between well-being and intention to stay while examining the potential moderating effect of workplace support on this relationship. Methods: This cross-sectional study analyzed survey responses from 1170 registered nurses across the US. The study data are from the Nurse Worklife and Wellness Study, 2020-21 (Trinkoff et al., 2021a). Results: Nurses who were younger, less experienced, working in hospitals, a staff or charge nurse, or on the frontlines reported lower well-being than their counterparts. Factors such as sufficient staffing and enough time to complete tasks significantly influenced nurses’ well-being. In aim 2, over 40% of nurses experienced workplace bullying, with 13.4% experiencing severe bullying in the past year. Inadequate staffing, insufficient time to complete tasks, and lack of breaks away from the work area were significantly related to severe bullying. For aim 3, only 75% of the nurses expressed their intention to stay at their current job for the next year. The intention to stay significantly differed by age and work-related factors, but workplace support had a significant moderating effect. As workplace support increased, the positive relationship between well-being and intention to stay was strengthened. Conclusion: This study provides guidance for organizations and management to address adverse work-related factors and to establish a healthy work environment. By ensuring sufficient staffing, providing adequate time for tasks, and actively addressing workplace bullying, nursing management can create an improved environment, ultimately promoting nurse retention.
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  The Relationship of Resilient Factors to Chronic Orofacial and Patient Expectations of Analgesia

  Thomas, Sharon L; Colloca, Luana (2023)

  Background: Adults with chronic orofacial pain experience significantly impaired daily functioning and reduced quality of life. Patient expectations are recognized as useful in medical treatment. However, we know little about coping behaviors, beliefs, and attitudes influencing disability or expectations in adults with chronic orofacial pain. Purpose: Positive psychological factors are protective against functional impairment and are implicated in placebo and expectations of analgesia. Examining the relationships between psychological factors, pain-related disability, and patient expectations will help identify correlates of disability, and individual susceptibility to placebo effects, a useful pain management modality. Methods: Three cross-sectional studies explored the correlates of pain-related disability and determined the influence of resilience on pain-related disability and patient expectations in adults with temporomandibular disorder. This study used secondary data from a clinical trial of placebo manipulation to examine the relationships between patient expectations of analgesia and psychological factors (resilience, dispositional optimism ) and a correlational observation design to explore the relationship between personal characteristics and psychological factors and pain-related disability in adults with TMD. The Aims were to 1) determine how positive and negative psychological constructs, jaw function, and chronic overlapping pain conditions contribute to pain-related disability; 2) describe the coping strategy patterns which patients engage in and compare the differences in these patterns based on pain characteristics; and 3) determine the relationship between pain resilience, pain interference, and patients expectations. Results Study 1 found that Resilience factors, optimism, and positive affect had a positive relationship with pain-related disability. Study 2 demonstrated chronic orofacial pain patients used both wellness and illness coping strategies. High Pain interference, Pain intensity, and Pain catastrophizing were associated with higher frequency use of all coping strategies. Study 3 revealed that pain resilience had an inverse relationship with pain disability and a direct and positive relationship with patient expectations. Conclusion: Psychosocial factors are important targets for clinical management of chronic pain. The effect of pain resilience on pain interference and reinforced expectations of analgesia means that clinicians focus efforts on building pain-resilient mechanisms, which can result in improved pain outcomes, augment its effect on expectations, and directly influence placebo treatment outcomes.
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  TLR4-SNP Mice Reveal the Role of M2a Macrophages in Resolution of Chemically-Induced Colitis

  Vlk, Alexandra; Vogel, Stefanie N. (2023)

  Toll-like receptor 4 (TLR4) is an innate immune receptor responsive to Gram negative lipopolysaccharide (LPS). Single nucleotide polymorphisms (SNPs) in human TLR4 that encode an A896G transition at SNP rs4986790 (D299G) and a C1196T transition at SNP rs4986791 (T399I) render individuals hyporesponsive to LPS. In humans, these SNPs have also been associated with increased susceptibility to inflammatory bowel disease (IBD). IBD, including Crohn’s Disease (CD) and Ulcerative Colitis (UC), impacts millions of individuals worldwide and severely impairs quality of life for these patients. While multiple treatments are available for IBD, there are several problems: (1) not all patients respond, (2) responses may diminish over time, and (3) treatments often have undesirable side effects. Some patients with IBD express these SNPs and, using knock-in mice engineered to express the murine homologues of these human TLR4 mutations (“TLR4-SNP” mice), we have shown that TLR4-SNP mice develop significantly more severe colitis induced by dextran sodium sulfate (DSS) than wild-type (WT) mice. Previous studies have provided indirect evidence for a role of “tissue repair” M2 macrophages (Mφ) in the resolution of colitis. Signaling through the shared IL-4/IL-13 receptor, IL-4Rα, leading to activation of the transcription factor peroxisome proliferator-activated receptor (PPARγ) has been shown to be required for induction of M2a Mφ and our data provide direct evidence for the involvement of both in repair of DSS-induced colonic damage. In response to DSS, colons of TLR4-SNP mice produced reduced levels of M2a Mφ marker mRNA and protein. Additionally, PPARγ protein levels were reduced in colons from DSS-treated TLR4-SNP mice and therapeutic administration of the PPARγ agonist ligand, rosiglitazone, ameliorated colitis in TLR4-SNP mice. Together, these data indicate that the failure of TLR4-SNP mice to resolve DSS-induced colitis may be secondary to their failure to induce “tissue repair” M2a Mφ. Our findings provide insight into the potential development of novel therapies targeting Mφ signaling pathways that aim to alleviate the debilitating symptoms experienced by individuals with IBD.
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  From What to How: An Exploration of How Genetic Counseling Program Directors Learn to Teach

  Dixon, Shannan; Kulo, Violet; Cestone, Christina (2023)

  The pathway from medical practitioner to academic teacher is not-well defined, specifically, there is a lack of clarity in how clinicians learn to teach, particularly within the field of genetic counseling. While there is a defined curricular pathway for entry into clinical practice there is not a defined pathway for instruction of health professionals who want to teach. While healthcare professionals enter an academic role with a defined set of clinical skills, they often lack formal training in how to teach. This qualitative phenomenological study, using a community of practice (CoP) theoretical model, explored the pathway to teaching for genetic counseling education program directors. Thirteen study participants shared their professional journey of learning to teach. Data from this study support the three phases of CoP in genetic counseling education program director development: engagement, imagination, and alignment. Participants reported content, instructional, and pedagogical reflection as they progressed in their understanding and development of their own teaching practice. Engagement with genetic counseling education programs was reported in three different ways: planned, sequential, and unplanned. All respondents acknowledged content reflection as a part of their early practice of learning to teach. The imagination phase is demonstrated as individuals expanded their understanding of teaching, program leadership, and responsibilities required to serve as a program director – in other words they began a practice of instructional reflection. Those who reported alignment did so based on mentorship and contribution to the community of genetic counseling education program directors and shared instances of pedagogical reflection. The findings of this study support the premise that genetic counseling education program directors, although not formal scholars in education prior to entry into their educator role, practice the scholarship of teaching and learning through a CoP. They are committed to the practice of evaluating how students learn to improve their own teaching. Further, they are committed to modeling professional development and learning as a member of a CoP. What they lack is the formal understanding of educational theory as it relates to genetic counseling instruction – without this knowledge it is difficult to conduct theoretically grounded educational research and advance the profession.
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  The Effect of Medication Information Delivery Format on Cognitive Load and Knowledge Retention of Informal Caregivers

  McPherson, Mary Lynn M.; Kulo, Violet A; Cestone, Christina (2023)

  Informal caregivers (IFCs) are tasked with many responsibilities in patient care, including medication management. Many IFCs feel ill-prepared for this responsibility, and it is incumbent on health care professionals to provide education and ensure IFCs competence in medication management. One common strategy is to provide a medication information leaflet to the IFC to prepare them for this role. Designing medication information leaflets using sound educational principles, such as an infographic designed according to the cognitive theory of multimedia learning (CTML), may optimize knowledge retention and decrease cognitive load for IFCs. The purpose of this randomized, experimental study was to investigate the impact of medication information delivery format on immediate retention of medication information and cognitive load of IFCs of patients with a serious illness. Using purposive sampling, 120 IFCs who have provided some element of medication management for patients diagnosed with a serious illness, including patients who may have been receiving hospice or palliative care services were recruited. Study participants were randomly assigned in either the experimental group or the control group. The experimental group viewed an infographic on the medication hydromorphone, followed by a knowledge quiz, and a self-assessment of cognitive load. This was followed by a second infographic on hydroxyzine, the quiz, and cognitive load assessment. The control group went through the same steps but viewed a text-only medication leaflet. Statistical analyses included descriptive statistics, independent samples t-test, one-way analysis of variance, and one-way multivariate analysis of variance. Statistically significantly higher quiz scores were observed among those who viewed the infographics than those who viewed text-only medication leaflets, indicating better immediate knowledge retention of medication information. Those who viewed the infographic also had statistically significantly lower intrinsic and extraneous cognitive load, and higher germane cognitive load. These findings are consistent with the hypothesis that infographics prepared using the CTML result in better and more efficient learning. Limitations of this research include use of nonprobability sampling, examining only two medications that are commonly used in serious illness, and lack of systematic randomization. Additional research is needed to continue determining best practices for instructing and supporting IFCs in medication management. 
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  Evaluation of Salmonella enterica serovar Typhimurium Vaccines in the Context of Immunosenescence

  Allen, Jessica; Tennant, Sharon M. (2023)

  Non-typhoidal Salmonella (NTS) is responsible for a high burden of foodborne infections and deaths worldwide. In the United States, NTS infections are the leading cause of hospitalizations and deaths due to foodborne illnesses, and older adults (≥65 years) are disproportionately affected by Salmonella infections. Due to this public health concern, we have developed a live attenuated vaccine, CVD 1926 (I77 ΔguaBA ΔclpP ΔpipA ΔhtrA), against Salmonella enterica serovar Typhimurium, a common serovar of NTS. The effects of age on parenteral vaccination are well documented, however, how advanced age impacts oral vaccine responses is less understood. In this dissertation, systemic and mucosal immune responses to CVD 1926 were evaluated in adult (six-to-eight-week-old) and aged (18-month-old) mice. We demonstrated that aged mice immunized with CVD 1926 failed to reduce bacterial burden upon challenge with wild-type S. Typhimurium, which was associated with lower vaccine-induced antibody titers and weaker T cell responses. Notably, characteristics of a successful mucosal vaccine response were weak in aged mice, suggesting that mucosal responses to oral vaccines decrease with advanced age. In efforts to develop a NTS vaccine that is effective for older adults, two vaccine approaches were evaluated: (i) a novel live-attenuated vaccine strain, CVD 1926 ΔsteD and (ii) heterologous prime boost strategy involving a mucosal prime with CVD 1926 that was followed by a parenteral boost with a conjugate S. Typhimurium vaccine. SteD is a Salmonella effector that suppresses CD4+ T cell responses during infection with wild-type S. Typhimurium. Using in vitro and in vivo assays, we demonstrated that CVD 1926 ΔsteD immunization elicits enhanced MHC-II expression, increased flagellin-specific CD4+ T cells, robust serum IgG and fecal IgA responses, and protection against S. Typhimurium colonization of the spleen, cecum, and small intestine upon challenge in aged mice. While the heterologous prime boost strategy induced robust Salmonella-specific antibody responses in aged mice, only modest protection against S. Typhimurium colonization was observed, suggesting that this vaccination approach cannot overcome immunosenescence. Taken together, these studies identify the age-associated deficits in mucosal vaccine responses and presents a promising prototype vaccine strain that may be effective for older adults.
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  The Effects of Graded Versus Ungraded Individual Readiness Assurance Tests on Pharmacy Students’ Assessment Performance and Achievement Goals in a Team-Based Learning Classroom

  Noel, Zachary; Cestone, Christina; Gordes, Karen L. (2023)

  Individual readiness assurance tests (iRATs) are frequently graded in team-based learning (TBL) classrooms, with the goal of incentivizing individual pre-class preparation. The purpose of this study was to determine whether shifting to an ungraded iRAT process affects student preparation and learning, as measured using assessment scores, and whether this is accompanied by a change in achievement goals. Using a crossover design in a required second-year Doctor of Pharmacy pharmacotherapy course, students were assigned to one of two iRAT grading sequences: graded/ungraded (G/UG) or ungraded/graded (UG/G). In the G condition iRATs were graded based on correctness and in the UG condition based on completion. Each period consisted of four iRATs and one examination. Students completed the Achievement Goal Questionnaire at the conclusion of each period. A one-way repeated measures multivariate analysis of variance (MANOVA) was used to test within-subject differences of mean iRAT and examination scores across grading conditions. A separate one-way repeated measures MANOVA was used to analyze differences in achievement goal scores. A total of 91 doctor of pharmacy students were included in the study. There was a statistically significant main effect for iRAT grading condition on assessment scores, F(2,88) = 3.851, Wilks’ Λ = .992, p = .025. Univariate testing using one-way analysis of variance with Bonferroni correction demonstrated a significant difference only in iRAT scores, with the mean score higher in the G condition (72.51% versus 67.99%; p = .011). Examination scores were similar in the G and UG conditions (81.07% versus 80.32%, p = .397). There was not a statistically significant difference in achievement goals based on iRAT grading condition, F(4,85) = 1.109, η2 =.050, p = .358. In conclusion, a modest reduction in iRAT performance was observed when shifting from a graded to ungraded iRAT; however, this had no effect on examination performance. Achievement goals were unaffected by the change in iRAT grading condition.
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  The quality of information on oral hygiene instructions for orthodontic patients in TikTok videos

  Dorfmann, Sabrina; Schneider, Monica, D.D.S, M.S. (2023)

  Objective: The study aimed to assess the reliability, content, quality, and overall usefulness of orthodontic oral hygiene instructions in TikTok videos. Methods: The final videos were evaluated for content, reliability, quality, and usefulness by using a content domain checklist, DISCERN questionnaire, and Global Quality Scale (GQS). Descriptive video characteristics were also recorded. Results: Dental professionals uploaded 70.00% of the TikTok videos, with orthodontists responsible for 39%. The videos had average scores of 2.19 for DISCERN, 3.96 for content, 2.14 for GQS, and 6.10 for usefulness. Video length was significantly associated with content scores (r = 0.3553, P = 0.0003), usefulness (r=0.3553, P = 0.0003), and quality (GQS) (r=0.2620, P=0.0085). Conclusion: Most TikTok videos on orthodontic oral hygiene were uploaded by dental professionals. Reliability, content, and quality of the videos were all poor. Videos were considered slightly useful. Longer videos were significantly correlated with higher content, higher quality, and increased usefulness.
• Predictors of Traumatic Stress in Hospital Nurses in the Third Year of COVID-19 and Their Lived Experiences: A Mixed Methods Study

  Storm, Asha; Friedmann, Erika (2023)

  Background: Research on nurses early in the COVID-19 pandemic shows elevated severity of trauma-related stress, depression, anxiety, and poorer well-being than before the pandemic. Fewer studies examined nurses’ experiences three years into the pandemic and the relationships of the experiences that predict increased post-traumatic stress severity. Objectives: This study evaluates the relationships among peritraumatic distress, moral distress, resilience, and post-traumatic stress severity as its primary objective. The study also examined the relationships of depression, anxiety, sleep quality, and nurses’ perceived work environment to post-traumatic stress as its secondary objective. The overall goal is to understand targets for intervention on the path to the development of post-traumatic stress that could potentially reduce the impact of crises on nurses. Methods: This study evaluates the interrelationships of these variables using a concurrent triangulation mixed methods framework. Nurses participated in multiple surveys, and a subset of these nurses participated in semi-structured interviews. A structural equation model (SEM) examined the relationships of the primary outcomes, and multiple regression analyses investigate the independent predictive ability of the variables on post-traumatic stress. The interviews utilized a descriptive phenomenological methodology to describe the lived experiences of traumatic stress during the pandemic for these nurses. Results: In the SEM, moral distress partially complementarily mediated the direct positive effect of peritraumatic distress on post-traumatic stress and negatively moderated the direct negative effect of resilience on post-traumatic stress. A multiple regression with all variables excluding resilience and sleep quality (not significant at p < .20) accounted for 62.6% of the variability in post-traumatic stress symptom severity. The interviews revealed that nurses exist in three interrelated worlds: their “Internal World” (emotions and personal well-being), their ‘Hospital World” (coworkers, leadership, environment, etc.) and the “Outside World” (the public, social media, current events, etc.). Conclusion: Nurses require support for their mental health at work and outside the hospital during a crisis, and hospital policies must consider all three. Reduction in peritraumatic stress and moral distress and support for nurse resilience are some of the most critical areas to focus on to reduce the post-traumatic stress severity in nurses during a long-term crisis.
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  Calcium-Dependent Mechanisms of Microtentacle Regulation in Mammary Epithelial and Breast Tumor Cells

  Chang, Katarina; Martin, Stuart S. (2023)

  Remodeling of cellular cytoskeletal structures in circulating tumor cells facilitates metastatic spread. Rearrangements of actin and tubulin in a nonadherent environment such as the vasculature produces microtubule-based protrusions termed microtentacles. Microtentacles are highly dynamic tubulin structures formed on free-floating cells when the physical outward force generated by the microtubule network overcomes the inward contractile force of the actin cortex. When cells enter a free-floating environment, microtentacles form within minutes, along a similar timeline to the rapid initiation of calcium signal transduction after mechanical stimulation. Historically, many studies focus on aberrant calcium-mediated oncogenic signaling pathways through receptor overactivation or overexpression to examine long-term (>24 hours) functions and phenotypes. Thus, the acute cellular response that occurs within seconds to minutes of stimulation is often overlooked in cancer biology. This study directly examines the cytoskeletal response of breast cancer cells to fluctuations in intracellular and extracellular calcium content. With the use of TetherChip technology in tandem with confocal microscopy, live cell time course imaging, and quantitative cell-based assays we have defined the rapid signaling mechanisms that mediated the cytoskeletal changes in detached and suspended breast epithelial and tumor cells. These findings reveal an induction of acute elevations of cytoplasmic calcium in tumorigenic and metastatic breast tumor cells. We observed that cells undergo rapid actomyosin contraction and rearrangement through phosphorylation on myosin light chain 2 and dephosphorylation of cofilin to suppress the microtentacle phenotype and functions. Moreover, rapid fluctuations from physiological to low intracellular and extracellular calcium conditions profoundly affect microtentacles in mammary epithelial and breast tumor cells. Although the probability of cancer cells encountering extreme environmental calcium gradient changes, these cells modulate their cytoplasmic calcium homeostasis through various calcium sensitive upstream regulators. Therefore, examining these specific channels, pumps, and receptors could have interesting implications for cancer cell behaviors in the tumor microenvironment. By understanding the acute molecular responses to calcium-mediated signal transduction, we can better develop and profile calcium modulators to target circulating tumor cells for the eventual application of rapidly profiling patient circulating tumor cells.
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  The ACE Project: A Pilot Study of an Afterschool Program for School-Aged Racial Minority Children Living in an Under-Resourced Community

  Klumpner, Susan T; Rose, Theda; Woolley, Michael E. (2023)

  Of the 91 million children living in the United States, approximately 25 million children are not able to access after school programs (ASPs). ASPs are becoming an increasingly ubiquitous feature in the U.S. public school system; however, demand outweighs the system’s ability to serve all children who want and could benefit from participation. ASPs and the promotion of youth development are considered a promising solution to closing the documented inequities and educational gaps among at-risk children. Guided by a strengths-based, developmental-ecological framework, this mixed methods study examined the influence of one after school program on positive youth development (PYD) outcomes during the coronavirus (herein referred to as COVID) pandemic. This pilot evaluation of The ACE Project, an ASP which was designed and developed by the author of this study, was implemented in Fall 2020 during the height of COVID restrictions. This forced several changes to implementation (e.g., shifting from a school- to a community-based setting, social distancing, mask wearing, increased sanitization) and evaluation (e.g., fewer measures administered, delayed timeline, and smaller sample size). These modifications were substantial and affected the study’s methods, findings, and implications. In the quantitative phase of this study, 8 youth from Chicago completed surveys that assessed individual and youth development characteristics (i.e., Competence, Character, Connection, and Caring). In the qualitative phase, 15 program staff completed semi-structured interviews addressing issues related to implementation and observable behavioral changes among youth. Results from paired samples t-tests suggest that there were no statistically significant changes between pre- and post- test; meaning, there were no PYD changes from the beginning to the end of the program. Interviews with program staff revealed anecdotal evidence of PYD changes within program participants; though, these positive effects were mitigated by aspects of the running a youth program during COVID. Furthermore, more sports-based (i.e., tennis) or physical activity components, formalized human resource policies and procedures, and supplementary staff training to address preparedness and professional development was implicated as necessary improvements to the program. Additional research must be conducted to fully understand the influence of individual-level factors and program context and its impact on PYD outcomes.
• RTS,S/AS02A/1B induces antibodies to a novel Plasmodium falciparum circumsporozoite protein epitope with sequence similarities to the junctional region

  Friedman-Klabanoff, DeAnna; Berry, Andrea A. (2023)

  Background: Plasmodium falciparum circumsporozoite protein (CSP) is the target of multiple vaccines in development. New promising monoclonal antibodies target the CSP junctional region, which is not contained in the RTS,S vaccine. Methods: We compared CSP antibody responses on a diversity-reflecting peptide array between adults receiving a full-length CSP (rCSP) vaccine and unprotected after controlled human malaria infection (CHMI), adults receiving RTS,S and protected after CHMI, and adults receiving RTS,S and unprotected after CHMI. Results: Overall, the rCSP group had lower anti-CSP antibody responses compared to the RTS,S-protected group. An epitope of interest in the C-terminal region was identified for both RTS,S groups that had sequence similarities to junctional region epitopes, and the RTS,S-protected group responded to more diverse peptides at this epitope than the RTS,S-unprotected group. Conclusions: Sequence similarities between the identified C-terminal epitope and junctional region epitopes warrant further investigation into whether these are cross-reactive antibodies driving protective responses.
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  Clinician Resilience after Traumatic Child Birth Exposure

  Robinson, Keisha; Ogbolu, Yolanda (2023)

  Background: Traumatic Childbirths (TCE) involving actual or threatened serious injury or death to a mother or child are documented sources of occupational stressors. Maternity care is a profession often perceived as filled with joy. However, TCEs may affect both professional practice and personal life and can contribute to burnout. Clinician resilience may play an important role in coping with TCEs. Purpose: The aims of this study were to describe and explore the frequency and severity of TCE experiences in OB clinicians, assess associations between TCEs and levels of resilience, and determine if TCE exposure and resilience are associated with OB clinician burnout. Methods: An anonymous survey was administered via a web link to obstetrical clinicians in five Maryland hospitals. The survey had four sections: demographics, TCE (frequency, severity, and influence on personal and professional practice), resilience (25-item Connor-Davidson Resilience Scale), and burnout (Maslach Burnout–Human Services Survey for Medical Personnel). Multi-level modeling explored factors influencing resilience and burnout nested within hospitals. Results: The 160 usable surveys included registered nurses (N=104), certified nurse-midwives (N=17), attending physicians (N=28), and resident physicians (N=11). Nearly all had experienced at least one TCE during their careers, with shoulder dystocia and stillbirth being the most frequent. The frequency of TCE exposure was associated with influencing the OB clinician’s professional practice (r=.415, p<.001) and personal life (r=.386, p<.001). Perception of severity strongly influenced professional practice (=.52, p<.001) and personal life (=.46, p<.001). Resilience scores were significantly lower in clinicians aged 35-54 years compared to the 55 or older group (B=-7.60, p=.011). TCE exposure was not associated with burnout. However, nearly a third (31%) of the convenience sample reported high emotional exhaustion, and 13% reported high depersonalization. Conclusion: TCE exposure can affect the professional practice and personal life of maternity care clinicians. While TCE did not have a strong relationship with resilience and burnout, a third of the respondents reported high emotional exhaustion, a dimension of burnout. Longitudinal research is needed to understand the short and long-term effects of TCEs and the role of resilience in helping clinicians cope with occupational stress.

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