Characterization of a MicroRNA Harboring Intron for Pre-mRNA Splicing and MicroRNA Processing

Nuclear pre-mRNA splicing is an important step in eukaryotic RNA processing and can yield a variety of transcripts. Further, it is known that nuclear pre-mRNA introns are of two types, U2-dependent and U12-dependent. Recently discovered, microRNAs (miRNA) are short, endogenous, 20-22 nucleotide long, non-coding RNA molecules that base pair with target mRNAs to modulate translation. Current evidence suggests that processing of intronic miRNA does not affect the splicing. Since, intronic miRNAs are involved in essential cellular processes, we surmised if splice sites of miRNA coding introns are flexible to support productive precursor and mature miRNA processing. We constructed a minigene reporter consisting exons 26-29 including introns from MYH6 gene and an intronic miRNA, miR-208 harbored in intron 27. Using in vivo and in vitro methods, we examined splicing of MYH6 intron 27 and processing of miR-208. The predicted stem-loop structure of miR-208 and the 5\u27 splice site of the intron 27 were mutated. Stem-loop mutations had no recordable splicing defect of intron 27. The 5\u27SS mutant activated a cryptic splicing event using a sub-optimal splice site located 50 nucleotides upstream of wild-type splice site. Interestingly, conversion of U2-type 5\u27 splice site to consensus U12-type 5\u27 splice site did not affect in vivo splicing of the intron, suggesting potential conversion of intron to U12-type, albeit without a consensus U12-type branch site sequence. However, miRNA processing was affected as established by in vitro splicing assay and qRT-PCR analysis of miR-208. We mutated nucleotides from position 3 to 7 in intron 27 to study if changes in splice site will allow miRNA processing to occur and if splicing will also be affected. Our data indicates significant changes in spliced phenotype as well as in miR-208 expression level. Since, all known intronic miRNAs are harbored in U2-dependent introns of mammalian genes, we wanted to study if miRNA processing will be compatible with U12-dependent intron. With this aim,

Photodynamic Therapy As An Effective Therapeutic Approach In Mame Models Of Triple Negative And Inflammatory Breast Cancers

Introduction: Photodynamic therapy (PDT) is a minimally invasive, FDA approved therapy for treatment of several indications including endobronchial and esophageal cancers that are accessible to light. Triple negative breast cancer (TNBC) and inflammatory breast cancer (IBC) are aggressive and lethal subtypes of breast cancer that spread to chest wall and dermal lymphatics, respectively, sites that would be accessible to light. Both TNBC and IBC patients have a relatively poor survival rate due to lack of targeted therapies. Use of PDT is underexplored for breast cancers but has been proposed for treatment of subtypes for which a targeted therapy is unavailable. Methods: We optimized and used a mammary architecture and microenvironment engineering (MAME) model of IBC to examine the effects of PDT using two treatment protocols. The first protocol used the benzoporphyrin derivative monoacid A (BPD) activated at doses ranging from 45 mJ/cm2 to 540 mJ/cm2. The second PDT protocol used two photosensitizers: BPD and mono- L-aspartyl chlorin e6 (NPe6), which were sequentially activated. Effects of PDT were assessed by live-dead assays. 89 Results: Using a MAME model of TNBC and IBC, we demonstrate a significant dose-response in photokilling by BPD-PDT. We found that sequential activation of NPe6 followed by BPD is more effective in photokilling of tumor cells than is BPD alone. Sequential activation at a dose of 45 mJ/cm2 each resulted in \u3e90% cell death, a response only achieved by BPD-PDT at a dose of 360 mJ/cm2. Furthermore, our data show that volumetric measurement of 3D MAME structures reflect efficacy of PDT treatment. We also show that the mechanism of cell death after sequential activation of NPe6 followed by BPD is apoptosis. Conclusion: Our study is the first to demonstrate the potential of PDT in treating MAME structures of TNBC and IBC

Keeping PACE with the Pandemic: Experiences from and Impacts of COVID-19 on Care Provision Among North Carolina’s Programs of All-Inclusive Care for the Elderly, a Qualitative Study

Background: The novel coronavirus disease 2019 (COVID-19) pandemic has deeply affected long-term care (LTC) for older adults, particularly in Programs of All-Inclusive Care for the Elderly (PACE). Older adults are more susceptible to serious illness and/or death from COVID-19, so studying care for this population is important. The pandemic arose in the midst of a larger LTC crisis in the United States centered on an aging population, unsustainability of current financing methods, and provider and staff shortages, among other challenges. COVID-19 is an opportunity to reform LTC, and this study may help shape the future of LTC by examining the resilience of the PACE model against the health system pressures of COVID-19. Objective: This study investigates the immediate and long-term effects of COVID-19 on care provision in North Carolina’s PACE (NC PACE) programs. Since PACE is organized by state, the research team chose to investigate sites in North Carolina. Methods: NC PACE administrators were recruited and interviewed through online audio conferencing with a structured interview designed by the research team. The totality of NC PACE (N=12) was represented in the study. The interviews were transcribed, coded, and qualitatively analyzed using thematic analysis. Results: Five themes emerged from thematic analysis, each with 2-3 subthemes: insufficient access to and integration with LTC providers and medical and mental healthcare specialists, reevaluation of the core PACE model with the transition to home-based care, that the provision of high-touch care promoted participants’ psychosocial wellbeing, reorientation to pivot toward family-oriented care delivery, and that a culture of caring enabled a successful COVID-19 response. Conclusion: PACE was overall successful in mounting a COVID-19 response that upheld safety of its participants, promoted the physical and mental wellbeing of its participants, and responded to the needs of informal/family caregivers. Administrators project that PACE’s service model has permanently changed after the pandemic toward increased home-based care. Results from this study also have implications for the provision of mental health care in the PACE service population and for the federal government’s financial relationship with PACE. PACE’s success during a period in which it was difficult to uphold care quality presents a learning opportunity for LTC in the future.Bachelor of Science in Public Healt