Now showing items 1-20 of 5169

    • Association of spatial proximity to fixed-site syringe services programs with HCV serostatus and injection equipment sharing practices among people who inject drugs in rural New England, United States

      Romo, Eric; Stopka, Thomas J; Jesdale, Bill M; Wang, Bo; Mazor, Kathleen M; Friedmann, Peter D (2024-01-28)
      Background: Hepatitis C virus (HCV) disproportionately affects rural communities, where health services are geographically dispersed. It remains unknown whether proximity to a syringe services program (SSP) is associated with HCV infection among rural people who inject drugs (PWID). Methods: Data are from a cross-sectional sample of adults who reported injecting drugs in the past 30 days recruited from rural counties in New Hampshire, Vermont, and Massachusetts (2018-2019). We calculated the road network distance between each participant's address and the nearest fixed-site SSP, categorized as ≤ 1 mile, 1-3 miles, 3-10 miles, and > 10 miles. Staff performed HCV antibody tests and a survey assessed past 30-day injection equipment sharing practices: borrowing used syringes, borrowing other used injection equipment, and backloading. Mixed effects modified Poisson regression estimated prevalence ratios (aPR) and 95% confidence intervals (95% CI). Analyses were also stratified by means of transportation. Results: Among 330 PWID, 25% lived ≤ 1 mile of the nearest SSP, 17% lived 1-3 miles of an SSP, 12% lived 3-10 miles of an SSP, and 46% lived > 10 miles from an SSP. In multivariable models, compared to PWID who lived within 1 mile of an SSP, those who lived 3 to 10 miles away had a higher prevalence of HCV seropositivity (aPR: 1.25, 95% CI 1.06-1.46), borrowing other used injection equipment (aPR: 1.23, 95% CI 1.04-1.46), and backloading (aPR: 1.48, 95% CI 1.17-1.88). Similar results were observed for PWID living > 10 miles from an SSP: aPR [HCV]: 1.19, 95% CI 1.01-1.40; aPR [borrowing other used equipment]:1.45, 95% CI 1.29-1.63; and aPR [backloading]: 1.59, 95% CI 1.13-2.24. Associations between living 1 to 3 miles of an SSP and each outcome did not reach statistical significance. When stratified by means of transportation, associations between distance to SSP and each outcome (except borrowing other used injection equipment) were only observed among PWID who traveled by other means (versus traveled by automobile). Conclusions: Among PWID in rural New England, living farther from a fixed-site SSP was associated with a higher prevalence of HCV seropositivity, borrowing other used injection equipment, and backloading, reinforcing the need to increase SSP accessibility in rural areas. Means of transportation may modify this relationship.
    • Opioid Overdose Recognition: A Survey of Perceived Preparedness and Desire for Curricular Integration Among Current US Medical Students

      Walsh, Lindsay; Chapman, Brittany; Carey, Jennifer; Loycano, Kayla; Carreiro, Stephanie (2024-01-10)
      Objectives: Opioid overdose deaths remain a major health issue in the United States (US). As future physicians, medical students must receive comprehensive training to recognize and manage opioid overdoses. This study aimed to highlight training gaps at the medical student level and understand students' attitudes toward patients with opioid use disorder (OUD). Methods: We assessed baseline knowledge of and attitudes toward the management of opioid overdoses and naloxone administration among medical students in the US. Two validated survey tools (Opioid Overdose Knowledge Scale and Opioid Overdose Attitude Scale) were administered to medical students training at accredited institutions along with supplemental questions measuring knowledge and attitudes towards opioid overdose management, naloxone administration, and prior training. Results: The final sample had N = 73 participants from US medical schools with a mean age of 25.3 (range of 22-37): 72.6% of respondents were female. Although most respondents reported personal/professional experience with OUD before medical school, they expressed interest in additional training. Knowledge surrounding opioid overdoses increased insignificantly over the 4 years of medical school. However, there was a significant increase in both perceived competence in overdose recognition/management and in concerns about intervening from the first to fourth year of medical school. Female respondents had significantly lower perceived competence and readiness to intervene sub-scores than male counterparts; however, there was no significant difference in overall attitude and knowledge scores when stratified by sex. Incorporating opioid overdose prevention training (OOPT) into early medical education was favorable among respondents, who expressed an overwhelming interest in learning and supporting patients with OUD. Conclusions: Given the ongoing opioid crisis, medical students are ideally placed to identify and manage opioid overdoses. Medical students are ready to receive this training, thus strengthening the argument for OOPT integration into early medical student curricula.
    • mRNA initiation and termination are spatially coordinated [preprint]

      Calvo-Roitberg, Ezequiel; Carroll, Christine L; Venev, Sergey V; Kim, GyeungYun; Mick, Steven T; Dekker, Job; Fiszbein, Ana; Pai, Athma A (2024-01-07)
      The expression of a precise mRNA transcriptome is crucial for establishing cell identity and function, with dozens of alternative isoforms produced for a single gene sequence. The regulation of mRNA isoform usage occurs by the coordination of co-transcriptional mRNA processing mechanisms across a gene. Decisions involved in mRNA initiation and termination underlie the largest extent of mRNA isoform diversity, but little is known about any relationships between decisions at both ends of mRNA molecules. Here, we systematically profile the joint usage of mRNA transcription start sites (TSSs) and polyadenylation sites (PASs) across tissues and species. Using both short and long read RNA-seq data, we observe that mRNAs preferentially using upstream TSSs also tend to use upstream PASs, and congruently, the usage of downstream sites is similarly paired. This observation suggests that mRNA 5' end choice may directly influence mRNA 3' ends. Our results suggest a novel "Positional Initiation-Termination Axis" (PITA), in which the usage of alternative terminal sites are coupled based on the order in which they appear in the genome. PITA isoforms are more likely to encode alternative protein domains and use conserved sites. PITA is strongly associated with the length of genomic features, such that PITA is enriched in longer genes with more area devoted to regions that regulate alternative 5' or 3' ends. Strikingly, we found that PITA genes are more likely than non-PITA genes to have multiple, overlapping chromatin structural domains related to pairing of ordinally coupled start and end sites. In turn, PITA coupling is also associated with fast RNA Polymerase II (RNAPII) trafficking across these long gene regions. Our findings indicate that a combination of spatial and kinetic mechanisms couple transcription initiation and mRNA 3' end decisions based on ordinal position to define the expression mRNA isoforms.
    • Intraflagellar transport: A critical player in photoreceptor development and the pathogenesis of retinal degenerative diseases

      Gupta, Mohona; Pazour, Gregory J (2023-12-23)
      In vertebrate vision, photons are detected by highly specialized sensory cilia called outer segments. Photoreceptor outer segments form by remodeling the membrane of a primary cilium into a stack of flattened disks. Intraflagellar transport (IFT) is critical to the formation of most types of eukaryotic cilia including the outer segments. This review covers the state of knowledge of the role of IFT in the formation and maintenance of outer segments and the human diseases that result from mutations in genes encoding the IFT complex and associated motors.
    • ATXN2 is a target of N-terminal proteolysis

      Chitre, Monika; Emery, Patrick (2023-12-21)
      Spinocerebellar ataxia 2 (SCA2) is a neurodegenerative disorder caused by the expansion of the poly-glutamine (polyQ) tract of Ataxin-2 (ATXN2). Other polyQ-containing proteins such as ATXN7 and huntingtin are associated with the development of neurodegenerative diseases when their N-terminal polyQ domains are expanded. Furthermore, they undergo proteolytic processing events that produce N-terminal fragments that include the polyQ stretch, which are implicated in pathogenesis. Interestingly, N-terminal ATXN2 fragments were reported in a brain extract from a SCA2 patient, but it is currently unknown whether an expanded polyQ domain contributes to ATXN2 proteolytic susceptibility. Here, we used transient expression in HEK293 cells to determine whether ATXN2 is a target for specific N-terminal proteolysis. We found that ATXN2 proteins with either normal or expanded polyQ stretches undergo proteolytic cleavage releasing an N-terminal polyQ-containing fragment. We identified a short amino acid sequence downstream of the polyQ domain that is necessary for N-terminal cleavage of full-length ATXN2 and sufficient to induce proteolysis of a heterologous protein. However, this sequence is not required for cleavage of a short ATXN2 isoform produced from an alternative start codon located just upstream of the CAG repeats encoding the polyQ domain. Our study extends our understanding of ATXN2 posttranslational regulation by revealing that this protein can be the target of specific proteolytic cleavage events releasing polyQ-containing products that are modulated by the N-terminal domain of ATXN2. N-terminal ATXN2 proteolysis of expanded polyQ domains might contribute to SCA2 pathology, as observed in other neurodegenerative disorders caused by polyQ domain expansion.
    • Genetic ablation of Sarm1 attenuates expression and mislocalization of phosphorylated TDP-43 after mouse repetitive traumatic brain injury

      Dogan, Elif O; Bouley, James; Zhong, Jianjun; Harkins, Ashley L; Keeler, Allison M; Bosco, Daryl A; Brown, Robert H; Henninger, Nils (2023-12-20)
      Traumatic brain injury (TBI), particularly when moderate-to-severe and repetitive, is a strong environmental risk factor for several progressive neurodegenerative disorders. Mislocalization and deposition of transactive response DNA binding protein 43 (TDP-43) has been reported in both TBI and TBI-associated neurodegenerative diseases. It has been hypothesized that axonal pathology, an early event after TBI, may promote TDP-43 dysregulation and serve as a trigger for neurodegenerative processes. We sought to determine whether blocking the prodegenerative Sarm1 (sterile alpha and TIR motif containing 1) axon death pathway attenuates TDP-43 pathology after TBI. We subjected 111 male Sarm1 wild type, hemizygous, and knockout mice to moderate-to-severe repetitive TBI (rTBI) using a previously established injury paradigm. We conducted serial neurological assessments followed by histological analyses (NeuN, MBP, Iba-1, GFAP, pTDP-43, and AT8) at 1 month after rTBI. Genetic ablation of the Sarm1 gene attenuated the expression and mislocalization of phosphorylated TDP-43 (pTDP-43) and accumulation of pTau. In addition, Sarm1 knockout mice had significantly improved cortical neuronal and axonal integrity, functional deficits, and improved overall survival after rTBI. In contrast, removal of one Sarm1 allele delayed, but did not prevent, neurological deficits and neuroaxonal loss. Nevertheless, Sarm1 haploinsufficient mice showed significantly less microgliosis, pTDP-43 pathology, and pTau accumulation when compared to wild type mice. These data indicate that the Sarm1-mediated prodegenerative pathway contributes to pathogenesis in rTBI including the pathological accumulation of pTDP-43. This suggests that anti-Sarm1 therapeutics are a viable approach for preserving neurological function after moderate-to-severe rTBI.
    • U.S. nursing home leadership experiences with COVID-19 and its impact on residents and staff: A qualitative analysis

      Dubé, Catherine E; Nielsen, Natalia; McPhillips, Emily; Lee Hargraves, J; Cosenza, Carol; Jesdale, Bill; Lapane, Kate L (2023-12-19)
      Objectives: To explore experiences of U.S. (United States) nursing home leadership during the COVID-19 pandemic in their efforts to address resident loneliness and social isolation and to elicit stories about personal and professional impacts on themselves and staff. Design: Qualitative inquiry via three optional open-ended questions appended to a national self-administered survey of American nursing home leaders was employed. Textual data was analyzed using an iterative reflexive thematic approach. Setting and participants: A stratified sample frame defined by facility size (beds: 30-99, 100+) and quality ratings (1, 2-4, 5) was employed. Web survey links and paper surveys were sent to 1,676 nursing home directors of nursing between February and May 2022. Results: Open text responses were collected from 271 nursing homes. Broad themes included: 1) Addressing needs of residents & families; 2) Challenges; and 3) Personal experiences of nursing home leadership/staff. Respondents described trauma to residents, staff, and leadership. Resident loneliness was addressed using existing and newer technologies and innovative indoor and outdoor activities. Residents experienced fear, illness, loss, and sometimes death. Isolation from family and lack of touch were particularly difficult. Regulations were seen as punitive while ignoring emotional needs of residents. Staffing challenges and pressures to do more with less created additional stress. Leadership and staff made significant sacrifices resulting in physical, social, and emotional consequences. Beneficial outcomes included staff bonding, professional growth, and permanent implementation of new interventions. Conclusions and implications: New and creative interventions were successfully implemented to address social isolation and loneliness. Improved Wi-Fi and other nursing home infrastructure upgrades are needed to maintain them. Reimagining often conflicting overlapping federal, state, and local regulations, grounding them in good clinical judgement, and incentivizing performance improvement should be considered. Trauma experienced by staff needs to be addressed to deal with current and future workforce needs.
    • A programmable dual-targeting di-valent siRNA scaffold supports potent multi-gene modulation in the central nervous system [preprint]

      Belgrad, Jillian; Tang, Qi; Hildebrand, Samuel; Summers, Ashley; Sapp, Ellen; Echeverria, Dimas; O'Reilly, Dan; Luu, Eric; Bramato, Brianna; Allen, Sarah; et al. (2023-12-19)
      Di-valent short interfering RNA (siRNA) is a promising therapeutic modality that enables sequence-specific modulation of a single target gene in the central nervous system (CNS). To treat complex neurodegenerative disorders, where pathogenesis is driven by multiple genes or pathways, di-valent siRNA must be able to silence multiple target genes simultaneously. Here we present a framework for designing unimolecular "dual-targeting" di-valent siRNAs capable of co-silencing two genes in the CNS. We reconfigured di-valent siRNA - in which two identical, linked siRNAs are made concurrently - to create linear di-valent siRNA - where two siRNAs are made sequentially attached by a covalent linker. This linear configuration, synthesized using commercially available reagents, enables incorporation of two different siRNAs to silence two different targets. We demonstrate that this dual-targeting di-valent siRNA is fully functional in the CNS of mice, supporting at least two months of maximal target silencing. Dual-targeting di-valent siRNA is highly programmable, enabling simultaneous modulation of two different disease-relevant gene pairs (e.g., Huntington's disease: MSH3 and HTT; Alzheimer's disease: APOE and JAK1) with similar potency to a mixture of single-targeting di-valent siRNAs against each gene. This work potentiates CNS modulation of virtually any pair of disease-related targets using a simple unimolecular siRNA.
    • An exploration of influenza A virus entry factors using CRISPR-based gene editing

      Kyawe, Pyae Phyo (2023-12-18)
      Influenza A virus (IAV) is a respiratory pathogen with a segmented negative-sense RNA genome that is capable of causing epidemics and pandemics. During the 2021-2022 influenza season, approximately 9 million people in the U.S. were infected with influenza, resulting in an estimated 5,000 deaths. The error-prone nature of the IAV polymerase results in antigenic drift and antigenic shift which contribute to low vaccine efficacy and escape from antivirals. Furthermore, the host factors required for the complete IAV infectious cycle have not been fully identified. The aim of this dissertation is to examine the host factors that may contribute to IAV infectivity in human lung cells. My goal is to understand how changes in the expression levels of host factors can impact influenza infection by CRISPR-mediated knockout or overexpression of target genes. Utilizing CRISPR screens, several candidates, whose up- or down-regulation resulted in reduced IAV infection in the human A549 cell line were identified. I confirmed that the knockout of CMAS or overexpression of B4GALNT2 inhibited IAV infection. In addition, I tested whether overexpression of two candidates from the CRISPR activation screen – DEFB127 and ADAR1 – would inhibit IAV and non-IAV viruses. Surprisingly, overexpression of the two candidates had minimal impact on IAV in A549 cells, but overexpression of ADAR1 had a pro-viral effect on other viruses. Taken together, these data provide insight into host factors modulating IAV infection and how CRISPR-mediated gene modulation can be utilized to further understand the IAV life cycle and for development of therapeutic agents for flu.
    • Experimental and Computational Methods for Identifying Death-Regulatory Genes from Chemo-Genetic Profiles

      Honeywell, Megan E (2023-12-18)
      A common approach to understanding how drugs induce their therapeutic effect is identifying the genetic determinants of drug sensitivity. This can be achieved following systematic loss- or gain-of-function genetic perturbations with CRISPR/Cas9. Because these “chemo-genetic profiles” are generally performed in a pooled format, inference of gene function is subject to several confounding influences, including variation in growth rates between clones or variation in the degree of coordination between growth and death. To overcome these issues, we developed an analysis method called MEDUSA (Method for Evaluating Death Using a Simulation-assisted Approach). MEDUSA uses time-resolved measurements and model driven constraints to reveal the combination of growth and death rates that generated the drug-treated clonal abundance. We find that MEDUSA is uniquely effective at identifying death regulatory genes, and we apply MEDUSA to determine how DNA damage-induced lethality varies in the presence and absence of p53. We find that loss of p53 switches the mechanism of DNA damage-induced death from apoptosis to a non-apoptotic form of death called MPT-driven necrosis. We find that activation of MPT by DNA damage requires high respiration, and that cell death can be exacerbated by modulating NAD+ in p53-deficient cells. These findings demonstrate the accuracy and utility of MEDUSA, both for determining the genetic dependencies of lethality and for revealing opportunities to promote the lethality of chemotherapies in a cancer specific manner.
    • Pediatric healthcare professionals' attitudes and beliefs about weight stigma: A descriptive study

      Turner, Samantha L (2023-12-15)
      Purpose: Children face weight-based stigma from their healthcare providers at a disconcerting rate, and efforts to mitigate this have been scant. This study aimed to quantify pediatric healthcare professionals' attitudes and beliefs about weight stigma and to determine stigma reduction interventions that are most supported by pediatric healthcare providers. Design and methods: Participants completed two validated instruments which measured implicit and explicit weight bias, respectively. They then completed a researcher-designed questionnaire to assess their attitudes and beliefs about weight stigma, and demographic questions. ANOVA models were used to examine associations between bias measures and participant characteristics, chi-square analyses were used to examine associations between questionnaire responses and participant characteristics, and Spearman's rank was used to determine correlations between weight bias and questionnaire responses. Results: Participants exhibited moderate-to-high levels of implicit and explicit weight bias (mean Implicit Association Test score = 0.59, mean Crandall Anti-Fat Attitudes Score = 38.95). Associations were noted between implicit bias and years in practice (p < 0.05), and implicit bias and occupation (p < 0.05). There was a significant correlation between explicit bias and multiple questionnaire items, suggesting that healthcare providers with greater weight bias are aware of those biases and are ready to take action to address them. Conclusion: Though pediatric healthcare exhibit weight-based biases, they are invested in taking steps to mitigate these biases and their impact on patients. Practice implications: The results of this study can inform the design of future interventions that aim to reduce healthcare-based weight bias, thus improving the quality of pediatric healthcare.
    • Evaluation of ChatGPT and Google Bard Using Prompt Engineering in Cancer Screening Algorithms

      Nguyen, Daniel; Swanson, Daniel; Newbury, Alex; Kim, Young H (2023-12-15)
      Large language models (LLMs) such as ChatGPT and Bard have emerged as powerful tools in medicine, showcasing strong results in tasks such as radiology report translations and research paper drafting. While their implementation in clinical practice holds promise, their response accuracy remains variable. This study aimed to evaluate the accuracy of ChatGPT and Bard in clinical decision-making based on the American College of Radiology Appropriateness Criteria for various cancers. Both LLMs were evaluated in terms of their responses to open-ended (OE) and select-all-that-apply (SATA) prompts. Furthermore, the study incorporated prompt engineering (PE) techniques to enhance the accuracy of LLM outputs. The results revealed similar performances between ChatGPT and Bard on OE prompts, with ChatGPT exhibiting marginally higher accuracy in SATA scenarios. The introduction of PE also marginally improved LLM outputs in OE prompts but did not enhance SATA responses. The results highlight the potential of LLMs in aiding clinical decision-making processes, especially when guided by optimally engineered prompts. Future studies in diverse clinical situations are imperative to better understand the impact of LLMs in radiology.
    • Advancing RNA-Targeting Therapeutics by Oligonucleotide Engineering: RNA Activation, Off-Target Effect, and Co-Targeting Nuclear and Cytoplasmic RNA

      Wang, Feng (2023-12-15)
      Traditional protein-targeting therapeutics by small molecules can target ~3000 proteins, representing <2% of the human genome. In comparison, ~80% of human genome is transcribed into RNAs, which are mostly targetable by oligonucleotides. Thus, RNA-targeting therapeutics enable a much bigger spectrum of drug targets. With nearly 20 FDA-approved oligonucleotide drugs and numerous ongoing clinical trials, RNA-targeting therapeutics by oligonucleotides have significantly addressed unmet medical needs. However, challenges persist in gene expression enhancement and comprehensive dual-RNA modulation. Here, I set out to address these two challenges. Firstly, I identified two ASOs designed to target intron 1 of FXN pre-mRNA, leading to a ~2-fold increase in FXN mRNA levels in multiple cell models. Despite rigorous controls such as two RNA quantification assays and normalization by multiple housekeeping genes, the FXN activation by these two ASOs wasn’t driven by direct binding to the FXN pre-mRNA or a mutual off-target transcript. Surprisingly, it’s found dependent on guanosine-rich motifs in the full PS backbone, suggesting non-base-paired off-target effects. Secondly, we developed siRNASO, an oligonucleotide scaffold integrating the functionalities of siRNA and ASO into a single molecule. siRNASOs demonstrated potent single RNA silencing and comprehensive dual-RNA modulation, including RNA silencing, splicing modulation, and RNA editing in vitro and in vivo. Notably, siRNASO exhibits excellent tolerability in the mouse CNS, suggesting its potential as a therapeutic platform for CNS disorders. Overall, the research in this thesis will serve as a valuable foundation for future research and therapeutic applications, significantly contributing to the advancement of RNA-targeting therapeutics.
    • A role for mutations in AK9 and other genes affecting ependymal cells in idiopathic normal pressure hydrocephalus

      Yang, Hong Wei; Lee, Semin; Berry, Bethany C; Yang, Dejun; Zheng, Shaokuan; Carroll, Rona S; Park, Peter J; Johnson, Mark D (2023-12-15)
      Idiopathic normal pressure hydrocephalus (iNPH) is an enigmatic neurological disorder that develops after age 60 and is characterized by gait difficulty, dementia, and incontinence. Recently, we reported that heterozygous CWH43 deletions may cause iNPH. Here, we identify mutations affecting nine additional genes (AK9, RXFP2, PRKD1, HAVCR1, OTOG, MYO7A, NOTCH1, SPG11, and MYH13) that are statistically enriched among iNPH patients. The encoded proteins are all highly expressed in choroid plexus and ependymal cells, and most have been associated with cilia. Damaging mutations in AK9, which encodes an adenylate kinase, were detected in 9.6% of iNPH patients. Mice homozygous for an iNPH-associated AK9 mutation displayed normal cilia structure and number, but decreased cilia motility and beat frequency, communicating hydrocephalus, and balance impairment. AK9+/- mice displayed normal brain development and behavior until early adulthood, but subsequently developed communicating hydrocephalus. Together, our findings suggest that heterozygous mutations that impair ventricular epithelial function may contribute to iNPH.
    • Acute Care Advanced Practice Providers' Use of Telehealth During the COVID-19 Pandemic

      Winterbottom, Fiona; Katz, Adam W; Skinner, Sarah; Carpenter, Dawn; Williams, Lisa-Mae; Kleinpell, Ruth (2023-12-15)
      Advanced practice registered nurses and physician assistants, collectively termed advanced practice providers (APPs), have been part of telehealth for many years. During the COVID-19 pandemic, APPs experienced the growth in roles, responsibilities, and tools used for telehealth care delivery. This article uses examples from 3 health systems to highlight the ways in which telehealth use was expanded due to the pandemic, how APP roles were altered across the United States during and after the pandemic, and implications for future practice.
    • In Our World: Uncovering the Meaning of Parent-Nurse Relationships in Childhood Cancer Care

      Costa, Jennifer (2023-12-14)
      Background: The diagnosis of cancer in a child thrusts parents into a complex healthcare system where they find themselves developing new relationships with pediatric oncology nurses. While parents and nurses acknowledge the meaningfulness and complexity of these relationships, many find it difficult to articulate. The purpose of this study was to understand the meaning and experience of the parent-nurse relationship in childhood cancer care. Methods: This study employed the tenets of Gadamerian hermeneutic philosophy. Data analysis looked to articulate meaningful interpretations of the interviews with parents and nurses of children with cancer through interpretive data analysis and utilized the hermeneutic circle as a way of conceptualizing and understanding the interpretive process. Results: Sixteen interviews (n = 8 parents, n = 8 nurses) were completed. The findings suggested that both parents and nurses of children with cancer navigate through an evolving inner world unique to the cancer experience. The parent-nurse relationships were mutually identified as dynamic, complex, and emotionally laden, and drew on the shared strength, trust, humanity, and interconnectedness of one another. The parent-nurse relationships played a significant role in traversing the challenges encountered with entry into and navigation through the pediatric cancer world. Discussion: The findings provide practical knowledge to inform and enhance relationship development at the clinical level, impact how relationship development is taught at the academic level, as well as inform professional development. This study contributes to the understanding of parent-nurse relationships and opens the door for integration of innovative practices in nursing education and clinical care.
    • A Fluorescent Reporter Mouse for Assessment of Genome Editing with Diverse Cas Nucleases and Prime Editors

      Chen, Zexiang; Kwan, Suet-Yan; Mir, Aamir; Hazeltine, Max; Shin, Minwook; Liang, Shun-Qing; Chan, Io Long; Kelly, Karen; Ghanta, Krishna S; Gaston, Nicholas; et al. (2023-12-13)
      CRISPR-based genome-editing technologies, including nuclease editing, base editing, and prime editing, have recently revolutionized the development of therapeutics targeting disease-causing mutations. To advance the assessment and development of genome editing tools, a robust mouse model is valuable, particularly for evaluating in vivo activity and delivery strategies. In this study, we successfully generated a knock-in mouse line carrying the Traffic Light Reporter design known as TLR-multi-Cas variant 1 (TLR-MCV1). We comprehensively validated the functionality of this mouse model for both in vitro and in vivo nuclease and prime editing. The TLR-MCV1 reporter mouse represents a versatile and powerful tool for expediting the development of editing technologies and their therapeutic applications.
    • The Role of Binding Sequence, Position, and Promoter Strength on the Regulatory Modes of E. coli Transcription

      Guharajan, Sunil (2023-12-11)
      The activity of Transcription Factors (TFs) is essential to gene regulation, facilitating the transfer of information encoded in cis-regulatory elements into temporal and spatial control of gene expression. Despite progress in mapping the genomic binding sites of TFs, the regulatory role of TFs once bound is often convoluted. Here, we utilize a synthetic biology approach that allows for precise manipulation of TF concentration in vivo and interrogation of the role of promoter features on the regulatory function of E.coli TFs. Using thermodynamic models of gene regulation, we decouple TF occupancy from its maximal regulatory output and probe its regulation on two steps of the transcription process: stabilization of RNA polymerase (RNAP) binding and modulation of transcription initiation. Profiling the CpxR activator, we discover universal stabilization across regulated positions, with differences in strong and weak activation set primarily by regulation of the initiation rate. Formulating a high-throughput approach, we probe the regulation of 93 E.coli TFs to find that the relative contributions of binding sequence and position on the mode of action are decoupled, with position playing a dominant role for select factors. Building on this information, we assessed the interplay between binding position, TF identity, and basal promoter strength - uncovering a conserved mode of stabilizing regulation across TFs with diverse regulatory outcomes. Taken together, our work delineates the effect of promoter architecture on the quantitative regulatory activity of TFs, with implications for design of synthetic gene circuits and understanding natural promoters
    • A neuronal coping mechanism linking stress-induced anxiety to motivation for reward

      Klenowski, Paul M; Zhao-Shea, Rubing; Freels, Timothy G; Molas, Susanna; Zinter, Max; M'Angale, Peter; Xiao, Cong; Martinez-Núñez, Leonora; Thomson, Travis; Tapper, Andrew R (2023-12-06)
      Stress coping involves innate and active motivational behaviors that reduce anxiety under stressful situations. However, the neuronal bases directly linking stress, anxiety, and motivation are largely unknown. Here, we show that acute stressors activate mouse GABAergic neurons in the interpeduncular nucleus (IPN). Stress-coping behavior including self-grooming and reward behavior including sucrose consumption inherently reduced IPN GABAergic neuron activity. Optogenetic silencing of IPN GABAergic neuron activation during acute stress episodes mimicked coping strategies and alleviated anxiety-like behavior. In a mouse model of stress-enhanced motivation for sucrose seeking, photoinhibition of IPN GABAergic neurons reduced stress-induced motivation for sucrose, whereas photoactivation of IPN GABAergic neurons or excitatory inputs from medial habenula potentiated sucrose seeking. Single-cell sequencing, fiber photometry, and optogenetic experiments revealed that stress-activated IPN GABAergic neurons that drive motivated sucrose seeking express somatostatin. Together, these data suggest that stress induces innate behaviors and motivates reward seeking to oppose IPN neuronal activation as an anxiolytic stress-coping mechanism.
    • Microfluidic High-Throughput Methods for the Induction and Characterization of Repeatable, Titratable Traumatic Neural Injury in the Nematode Caenorhabditis elegans

      White, Hamilton (2023-12-06)
      Traumatic brain injury (TBI) causes polymodal trauma leading to persistent changes in brain function, behavior, cellular structure, and is a known risk factor for neurodegenerative disease. Current injury models correlate the presence and duration of injury conditions with animal behavior, but they do not reveal underlying effects on brain function at the cellular and subcellular scale in a continuous, longitudinal manner. To identify underlying mechanisms relating acute brain injury with functional outcomes, we sought to develop a reliable, scalable TBI model in C. elegans to directly observe injury progression at behavioral, neurofunctional and structural levels, both immediately and over hours to days. Previously, ultrasonic shock waves and vortex-induced blunt force trauma caused paralysis in thrashing animals, with broad population variability. We investigated ultrasonic cavitation as a repeatable and titratable TBI induction method using a bath sonicator modified for precise, sub-second timing control. Video recordings during sonication revealed animals near a rigid surface were injured in a dose-dependent manner, whereas those in bulk liquid were relatively unharmed. Using an expanded and flexible optogenetic and chemical stimulation platform, repeated assessment of neural function of up to 24 hours allowed examination of structural degeneration and neurofunctional recovery. To show the platform’s usefulness, we identified sexually dimorphic outcomes in injury response, a channel inhibitor that modulates neural activity recovery post-injury via genetic and pharmacological means, and assess an ortholog of FHM1 for sensitizing animals to injury. Overall, sonication-induced TBI provides repeatable assays for real-time, in vivo recording of neuronal structure, function, and behavior, before and after single or repeated injury, enabling further study on injury mechanisms, progression, and potential therapies to minimize damage and enhance recovery.