CoHP - SoP - Student Works

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INVESTIGATING BLUEBERRY-DERIVED EXOSOMES AS A NOVEL APPROACH IN NEURODEGENERATIVE DISEASE THERAPY
(2024-04-18) Warner, April; Canham, Spencer; Yang, Tianzhi; Bai, Shuhua
Blueberries are renowned for their neuroprotective properties, including their ability to mitigate oxidative stress and enhance cognitive function, making them promising candidates for combating neurodegenerative diseases. This study investigates the potential of blueberry-derived exosomes (BBDExo) as a novel nanosized antioxidant system for neurodegenerative disease therapy. BBDExo were isolated from blueberries through sequential centrifugation and analyzed for protein and polyphenol content. Their stability under gastrointestinal conditions and their uptake by brain endothelial bEND.3 cells were assessed, along with their effects on neuronal SH-SY5Y cells, to ascertain their therapeutic potential. The results showed that BBDExo displayed a nanosized, oval-shaped morphology, averaging 82.7±6.4 nm in size. They demonstrated stability in simulated gastrointestinal environments, maintaining their polyphenol content for up to 4 hours. The uptake of BBDExo by bEND.3 cells was dose-dependent, indicating potential for blood-brain barrier transport. Furthermore, BBDExo significantly enhanced the proliferation of SH-SY5Y cells, with a peak increase of 158.8±1.8% at a concentration of 25 µg/mL. Additionally, BBDExo notably reduced the release of the pro-inflammatory cytokine interleukin-8 in stressed SH-SY5Y cells. These findings suggest that BBDExo offer improved stability and enhanced cellular uptake of bioactive compounds, potentially increasing their effectiveness in neurological applications. The ability of BBDExo to interact with targeted brain inflammatory cells and regulate anti-inflammatory responses demonstrates their promise as an innovative nanomedicine for neurodegenerative diseases, potentially leading to improved therapeutic efficacy and disease management.
METHYLMERCURY ALTERATION IN THERMOGENESIS IS DIET DEPENDENT IN CAENORHABDITIS ELEGANS
(2024-04-18) Varney, Abigail; Radzimirski, Anthony; Caito, Samuel
Thermogenesis is the process by which adipocytes metabolize triglycerides (TAG) to release energy as heat. This process is disrupted in metabolic syndrome, leading to increased TAG levels and weight gain. Methylmercury (MeHg) is an environmental toxin that has both neurotoxic and metabolic effects. We have previously shown that MeHg disrupts lipid homeostasis, leading to increased TAG content and storage sites in Caenorhabditis elegans. Furthermore, we have shown that TAG content in response to MeHg is dependent on their Escherichia coli diet. Worms fed a low lipid containing strain (HT115) showed less lipid dysregulation than worms fed a high lipid containing strain (OP50). As we have seen accumulation of TAG in response to MeHg, we hypothesized that MeHg could reduce thermogenesis in C. elegans. Worms were treated with environmentally relevant doses of MeHg, fed either OP50 or HT115, and were grown to adulthood at 15 or 25˚C. Worms were then placed at 4˚C for 48 hours and scored for survival. Untreated worms maintained at 15˚C and fed either diet were able to survive the temperature shift. MeHg significantly decreased survival of worms fed OP50 diet following the 15 to 4˚C shift, suggesting that thermoregulation was inhibited by MeHg. In contrast, MeHg had minimal effects on the survival of worms fed HT115 diet following the 15 to 4˚C shift. Shifting worms from 25 to 4˚C is lethal to all worms fed OP50. However, the HT115 diet prevented lethality in untreated or MeHg treated worms shifted from 25 to 4˚C. These data suggest that the HT115 diet is protective and can induce thermogenesis. Heat generation derives from mitochondria. HT115 fed worms had improved mitochondrial health in response to MeHg than OP50 fed worms. Taken together, our data suggests that MeHg-dependent mitochondrial damage is diet dependent leading to alterations in thermogenesis.
SYNERGISTIC NEUROTOXIC EFFECTS OF METHYLMERCURY AND PER-AND-POLYFLUOROALKYL SUBSTANCES (PFAS) IN CAENORHABDITIS ELEGANS
(2024-04-18) Radzimirski, Anthony; Varney, Abigail; Ireland, Nicholas; Caito, Samuel
Methylmercury (MeHg) is a well-known neurotoxic metal that is a major contaminant of our fish supply. Developmental exposure to MeHg causes cognitive and behavioral dysfunction in children, and cumulative exposure to MeHg has been linked to the development of neurodegenerative diseases, such as Parkinson’s disease. Recently, it has been determined that fish are becoming increasingly contaminated with per- and polyfluoroalkyl substances (PFAS). PFAS are a group of amphipathic compounds which have been used in industry for their unique property to repel both water and oils. While specific PFAS have been phased out of use and production in the United States, the environmental degradation of PFAS is slow. Both MeHg and PFAS have similar characteristics, particularly bioaccumulation and biomagnification up the food chain, ability to accumulate in the brain, and alteration in synaptic transmission of glutamate and dopamine. We therefore hypothesized that MeHg and PFAS co-exposures may synergize and produce more damage to the dopaminergic and glutamatergic nervous systems in Caenorhabditis elgans than single exposures alone. Worms were treated for 72 hours with increasing concentrations of PFOS, PFOA, or PFBS in the presence or absence of a low nontoxic dose of MeHg. Dose-response curves were generated and the lethal dose 50 (LD50) were calculated for each curve. Co-exposure of MeHg with either of the PFAS compounds shifted the dose-response curve to the left of the dose-response curve for PFAS. This suggests that the co-exposure was more toxic than PFAS exposure in worms. Glutamatergic and dopaminergic behaviors were assayed in worms treated with MeHg, PFAS, or MeHg + PFAS combination. For both behaviors, the co-exposure caused more behavioral deficits than MeHg or PFAS alone. Furthermore co-exposure to PFAS and MeHg altered both dopamine and glutamate neurotransmitter content. Taken together, our results suggest that there is a synergistic relationship between exposure to MeHg and PFAS compounds in C. elegans.
DISCIPLINARY OUTCOMES FOR MAINE BOARD OF PHARMACY COMPLAINTS IN 2022
(2024-04-18) White, Cassandra; Dumont, Erica; Mroz, Katherine
The purpose of the licensing system regulated by the Maine Board of Pharmacy (MeBOP) is to protect the public against 1. dishonest or unethical licensees, and 2. licensees who have fallen below minimum standards of competence in the practice of pharmacy. Violators are subject to disciplinary action that can range from a formal warning to license revocation, with or without civil penalties. The purpose of this study was to quantify and compare complaints submitted to the MeBOP for the 2022 calendar year. The majority of complaints in 2022 were against pharmacists (57.9%, n = 66 of 114 complaints), consistent with disciplinary records for all United States (U.S) Boards of Pharmacy (BOP)1. 78.8% were licensed as a pharmacist-in-charge (PIC). The MeBOP had far less complaints against pharmacy technicians (1.8%, n = 2 of 114) compared to U.S. BOPs (25.2%, n = 1,309 of 5,196)1. There was a high number of dismissed complaints (53.5%), while 14% resulted in a letter of guidance and 32.5% were offered consent agreements. 21.9% of complaints were against licensees with prior discipline. The average civil penalty (n = 34 of 114) was $12,000 (median = $1,625, range = $250 - $175,000). Investigation into complaint data for U.S. BOPs is necessary in order to establish best practices for the management of complaints, complaint outcomes, and the impact of complaints on licensees. The MeBOP and other U.S. BOPs can collect and use complaint data to contemplate ways to promote a more just culture. Future studies are needed to evaluate the number of complaints each U.S. BOP receives, PIC designation and license status, types of violations (e.g. prescription misfill, drug diversion), ways to promote the reduction of recidivism, as well as the civil penalties and how these compare across states. 1. NABP Clearinghouse Update - 2022. Innovations. March 2023;52(3):7.
SEROTONERGIC AND DOPAMINERGIC-DEPENDENT BEHAVIORS ARE ALTERED BY LANTHANIDE SERIES METALS IN Caenorhabditis elegans
(2023-04-20) Radzimirski, Anthony; Ireland, Nicholas; Miller, Lydia; Croft, Michael; Newell-Caito, Jennifer; Caito, Samuel
The lanthanide series elements are transition metals used as critical components of electronics, as well as rechargeable batteries, fertilizers, antimicrobials, contrast agents for medical imaging, and diesel fuel additives. Furthermore, as electronics are limited in lifespan, lanthanides are found in electronic waste. With the surge in their utilization, lanthanide metals are being found more in our environment. However, little is known about the health effects associated with lanthanide exposure. Epidemiological studies as well as studies performed in rodents exposed to lanthanum (La) suggest neurological damage, learning and memory impairment, and disruption of neurotransmitter signaling, particularly in serotonin and dopamine pathways. Unfortunately, little is known about the neurological effects of heavier lanthanides. As dysfunction of serotonergic and dopaminergic signaling are implicated in multiple neurological conditions, including Parkinson’s disease, depression, generalized anxiety disorder, and post-traumatic stress disorder, it is of utmost importance to determine the effects of La and other lanthanides on these neurotransmitter systems. We therefore hypothesized that early life exposure of light (La or cerium (Ce)) or heavy (erbium(Er) or ytterbium (Yb)) lanthanides in Caenorhabditis elegans could cause dysregulation of serotonergic and dopaminergic signaling upon adulthood. Serotonergic signaling was assessed by measuring pharyngeal pump rate, crawl-to-swim transition, as well as egg laying behaviors. Dopaminergic signaling was assessed by measuring locomotor rate, egg laying, and swim-to-crawl transition behaviors. Treatment with La, Ce, Er, or Yb caused deficits in serotonergic or dopaminergic signaling in all assays, suggesting both the heavy and light lanthanides disrupt these neurotransmitter systems. Concomitant with dysregulation of neurotransmission, all four lanthanides increased reactive oxygen species (ROS) generation, while decreased glutathione and ATP levels. This suggests increased oxidative stress, which is a known modifier of neurotransmission. Altogether, our data suggest that both heavy and light lanthanide series elements disrupt serotonergic and dopaminergic signaling and may affect the development or pharmacological management of related neurological conditions.
NANOVESICLES IN BROCCOLI SPROUTS AS A NATURAL POTENTIAL MEDICINE FOR THE TARGETED TREATMENT OF INFLAMMATORY BOWEL DISEASE
(2023-04-20) Palmer, Emilie; Kaserman, Elise; Dunham, Kaitlyn; Kuhn, Brian; Yang, Tianzhi; Bai, Shuhua
Inflammatory bowel disease (IBD) is an incurable small intestine and colon disorder. While exerting anti-inflammatory effects, bioactives in broccoli sprouts cannot treat IBD well probably due to their instability in the gastrointestinal (GI) tract and/or inadequate transit into colon cells. The study aimed to investigate if broccoli sprout-derived exosome (BSDExo) nanovesicles can protect bioactives from the upper GI extreme environments and confer selectively targeted delivery of bioactives to inflamed colon cells in IBD. Microparticles with 1198.3±86.2 nm and nanovesicles with 40.1±17.2 nm were isolated from broccoli sprout juice using differential centrifugations and measured by a DelsaTM Nano C nanosizing system. They showed different morphologies under a scanning electron microscope (SEM). Total protein and RNA were characterized in both broccoli sprout-derived microparticles and nanoparticles. Exosomes expressed the highest level of transmembrane protein CD63 evaluated by an Exo-Check array. These nanosized and identified proteins are known to be associated with exosomes. BSDExo protected bioactive sulforaphane for 2 hours in stomach-mimicking acid and 24 hours in intestine-mimicking conditions. BSDExo with a total protein level of 25 ng/mL could promote the proliferation of human normal colon epithelial CCD841 CoN cells with a cell viability of 154±5% (p<0.05). Both colon CCD841 CoN and Caco-2 cellular uptake of fluorescence-labeled BSDExo significantly increased with more severe inflammation stimulations. Interleukin 8 (IL-8) secretion from inflammation-stimulated normal colon cells was significantly reduced by the BSDExo treatment (p<0.05). BSDExo also significantly recovered the decreased transepithelial electrical resistance (TEER) values caused by inflammations in Caco-2 cells (p<0.05). Overall, the isolated BSDExo improved the stability, inflammation-targeting, and therapeutic efficacy of bioactives in colon epithelial cells via a naturally formed nanostructure. Demonstrating that BSDExo interacts with the targeting gut inflammatory cells and regulates anti-inflammatory responses would be a significant step forward in treating IBD.
EXAMINATION OF THE POTENTIAL ROLE OF UDP IN INTERLEUKIN-8 EXPRESSION BY FIBROBLASTS DURING CELL INJURY
(2023-04-20) Kennard, Emma; Lindblad, William J.
Prior studies from our lab show scraping a monolayer of fibroblasts, an in vitro wound model, results in cell migration and enhanced expression of inflammatory mediators including interleukin-8 (IL-8) by residual cells. This effect is more pronounced in fibroblasts from the skin of diabetic people than non-affected people. This expression is seen in the cells adjacent to the scraped area but also in cells 2 and 3 cells removed from the scraped edge. Studies in epithelial cells suggest that nucleotides, specifically uridine 5’-diphosphate (UDP) released from cells upon wounding in the same model bind to P2Y6 receptors on cells inducing cell migration. We hypothesized that UDP may serve as a wound mediator for fibroblasts and that it may induce the expression of IL-8. Human dermal fibroblasts (GM 1872 cells, Coriell Institute) were cultured in 100 mm dishes and grown to confluence in DMEM high glucose media containing 10% fetal calf serum and penicillin-streptomycin. Cell monolayers were scraped in a grid pattern and cultured for 24 hrs. with/out additives. UDP at 100 and 10 µM was added to non-scraped and scraped cultures of fibroblasts to determine the potential involvement of UDP that we have seen in IL-8 expression. Following 24 hr. incubation, total RNA was extracted and quantitation of IL-8 mRNA content was assessed by qPCR. Modified culture media used for the 24 hr. culturing was analyzed for UDP content by ion-pairing HPLC. UDP, immediately released from scraped cell monolayers, was determined by HPLC following a 10X concentration of PBS used to maintain cell viability during scraping. Despite a lower limit of detection of 20 ng, no UDP could be detected and further studies with LC/MS are continuing to ascertain if any quantifiable UDP is released from the scraped cells. Cell levels of mRNA for IL-8 normalized to GAPDH are being analyzed to determine if exogenous UDP enhances the expression of this cytokine. These studies will increase our understanding of what factors during fibroblast injury promote migration and the expression of pro-inflammatory mediators.
INVOLVEMENT OF DIVALENT METAL TRANSPORTER HOMOLOGS IN THE TOXICITY OF LANTHANIDE SERIES ELEMENTS IN Caenorhabditis elegans
(2023-04-20) Ireland, Nicholas; Miller, Lydia; Caito, Samuel
Lanthanide series elements are transition metals that are used in a variety of electronics, including superconductors, electronic polishers, hybrid car components, and rechargeable batteries, as well as in fertilizers, antimicrobials, contrast agents for medical imaging and diesel fuel additives. Lanthanides are also present in electronic waste and are released during electronic cigarette smoking. Levels of lanthanides have risen significantly in both industrial areas and environmentally. While we are starting to recognize health effects of lanthanide exposure, it is currently unknown how lanthanide metals enter cells. If we are to understand their toxicokinetics in the human body, it is imperative to determine mechanisms by which lanthanides are distributed. We hypothesized that endogenous divalent metal transporters (DMTs) are responsible for lanthanide entry into cells, and that genetic ablation of DMT transporters in Caenorhabditis elegans would protect the worms from lanthanide-induced toxicity. In this study, we performed a competitive inhibition survival assay by pretreating wildtype worms with nonlethal doses of metals that enter cells through a variety of mechanisms, then treating the worms with increasing concentrations of lanthanum (La). Lethality was assessed 48 hours post exposure. Worms treated with either manganese or iron, metals that enter cells via DMTs, shifted the La dose-response survival curve to the right, suggesting La was competing for the same transporter as iron or manganese. However worms pretreated with zinc, magnesium, or chromium could not block lanthanum-mediated lethality. These data suggest that DMTs are responsible for lanthanides to enter cells. We then treated transgenic worms that lacked DMT homologs smf-1, smf-2, or smf-3, with increasing concentrations of La or ytterbium (Yb). Knock out of either of the smf genes shifted the dose-response curve for La or Yb to the right, showing protection. These results suggest that DMTs are important mediators for lanthanide series elements to enter eukaryotic cells.
CHARACTERIZATION AND ANALYSIS OF BIOACTIVES IN BLUEBERRY-DERIVED EXOSOMES: NEW INSIGHTS INTO A POTENTIAL ANTIOXIDATION NANOMEDICINE
(2023-04-20) Dunham, Kaitlyn; Canham, Spencer; Palmer, Emilie; Bai, Shuhua; Yang, Tianzhi
Blueberries provide evident benefits of reducing oxidative stress and inflammation, improving cognitive function, and protecting against neurodegenerative diseases as a “super” fruit. However, high levels of bioactive molecules in blueberries, such as anthocyanin, have poor stability and absorption, leading to low bioavailability in the brain. Exosomes attract strong interest as an important vehicle of intercellular communication and as a delivery carrier of bioactive molecules. Herein, the study focuses on characterizing blueberry-derived exosomal nano-vesicles that contain bioactive molecules, and determining if they have better stability and are more readily taken up by cells. Blueberry juice was directly extracted with an electric blender and passed through filter papers. The collected juice was sequentially centrifuged at 1,000 × g for 10 min, 3,000 × g for 20 min, and 10,000 × g for 40 min at 4 °C to remove large particles and debris. The final supernatant was centrifuged at 100,000 × g for 30 min to obtain exosomes. The exosome morphology was observed with scanning electron microscopy (SEM) and the particle size was determined with a Nano Sizing Analyzer. The exosomes had a size of 82.7±6.4 nm and appeared as individually sphere-shaped morphology as shown in SEM images. Exosomes were analyzed and quantified for total proteins and RNAs. Well-known exosome-unique markers, including Cis-Golgi matrix protein GM130, adaptor protein and sort cargo ALIX, tumor susceptibility gene TSG10, intercellular adhesion molecule 1 (ICAM-1), apoptosis ANXA5, integral membrane protein FLOT, epithelial cell adhesion molecule (EpCam), and transmembrane tetraspanin family CD63 and CD81, were detected by an Exo-Check array. MicroRNA sequencing analysis revealed that the intersection between differentially expressed genes and miRNAs contained in exosomes could unveil a set of candidate target genes. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) study demonstrated that the exosomes showed cytotoxicity in neural cancerous cells. The characterized exosomes with biomolecules may deliver therapeutic molecules in the brain and target neural cells, leading to improved efficacy in the treatment of neurodegenerative diseases.
HOMELESS AND HUNGRY IN BANGOR: RX 504 SERVICE LEARNING CAPSTONE PROJECT
(2023-04-20) Johnson, Jessica; Doctor of Pharmacy Candidates, Class of 2024
Husson University School of Pharmacy Class of 2024 enrolled in RX 504 - Clinical Pharmacy Capstone, a required service-learning course that allows students to apply knowledge developed over the course of their studies to a real-world problem in their local community. Doctor of Pharmacy candidates established a goal of assessing the needs of individuals experiencing homelessness in Penobscot County. First, students held a discussion surrounding the documentary “Hungry Now,” which explored the current homelessness crisis in the state of Maine. Second, students read published scientific literature evaluating the barriers to healthcare and medication adherence unique to persons experiencing homelessness. Students proposed possible solutions and explored how their various ideas might help individuals to overcome barriers to accessing needed medical care. Third, we visited the Bangor Area Homeless Shelter on two separate occasions with the goal of sharing a nutritious meal and speaking with residents to explore their needs and concerns regarding their medication regimens. Additionally, students advocated for expanded access to vaccinations in Maine by writing and delivering verbal testimonies at the Maine State House and provided community education at the Maine Science Festival. Through this community-engaged service-learning experience, Pharmacy students explored themes of access, wellness, identity, responsibility, and sustainability; developed a better understanding of the daily struggles faced by unhoused individuals in their local community; and were empowered to take evidence-based action in response to identified health disparity.
COMPENSATION FOR BOARD OF PHARMACY MEMBERS IN THE UNITED STATES
(2023-04-20) Pham, Hannah; Ifeji, Chidubem; White, Cassandra
Background: No published data exists regarding compensation and demographic differences between the memberships of the 50 United States (U.S.) Boards of Pharmacy. Objectives: The purpose of this study was to quantify and compare the per diem pay rate for U.S. Board of Pharmacy members. Board member demographics and compensation for mileage and meals were also evaluated. Methods: In June 2022, each state Board of Pharmacy was contacted to gather data including per diem pay, mileage and meal compensation, number of meetings per year, number and gender of Board members, length of appointment, and regulatory statutes. Results: The average per diem pay for Board members was $75.86 (median = $50.00, range = $0.00 to $250.00, n = 48 states). Most states report paying Board members for mileage (95.1%, n = 39 out of 41) and meals (80.0%, n = 28 out of 35). On average, Boards are composed of 8.3 members (median = 7.5, range = 5-17, n = 50), meet 8.3 times annually (median = 8, range 3-16, n = 47), and have a 4.5 year length of appointment (median = 4, range = 3-6, n = 47). Males represented 61.2% of occupied Board positions and pharmacists accounted for 74.1% of all positions. The average year for pharmacy statute update was 2002. Conclusion: Compensation for U.S. Board of Pharmacy members varies greatly, from unpaid (n = 8 states) to a high of $250.00 per diem. Only 38.8% of occupied Board positions are held by women and pharmacy technicians represent just 3.6% of all positions. Fair compensation, increased female and pharmacy technician representation, and more timely pharmacy statute updates are necessary in order to achieve inclusion, diversity, and equity between state Boards of Pharmacy.
METFORMIN REDUCES ATYPICAL ANTIPSYCHOTIC-ASSOCIATED WEIGHT GAIN IN ADULTS
(2023-04-20) Bosquet, Randy; Caron, Lindsey; James, Shannon
Purpose: Metformin is an underutilized management strategy in preventing and treating antipsychotic-associated weight gain (AAWG). Pharmacists may be in a unique position to educate providers and drive implementation into current clinical practice. This review analyzes recent literature to identify the impact of metformin treatment on AAWG in adult patients. Methods: We performed a review using PubMed with search criteria (antipsychotic induced weight gain) AND (metformin) which yielded 125 results. Articles were included if they discussed AAWG for adult patients along with metformin use for prophylaxis or treatment. Articles were excluded if they focused on the pediatric population or did not include management strategies for AAWG. There were 21 studies that were analyzed and included in this review. The primary endpoint was weight loss or change in weight in patients taking metformin with an atypical antipsychotic. Results: Although AAWG is common amongst all medications within the class, studies showed that some atypical antipsychotics have a much larger incidence rate without metformin use, such as olanzapine, where 30% of individuals were shown to have greater than 7% increase in body weight from baseline. Both short and long-term studies show metformin prevents an increase in weight gain by about 5 kg when it is started at the initiation of an atypical antipsychotic. Treatment based metformin use showed an approximate 3 kg reduction in body weight when started after AAWG occurred. Studies showed that higher doses of metformin (>500 mg) had a greater effect on weight decrease when compared to other dosages. Doses of metformin ranged from 500 to 2250 mg daily for AAWG prophylaxis and treatment ranged from 6 weeks to 18 months long. Conclusion: Use of metformin prophylactically could decrease premature discontinuation of atypical antipsychotics and prevent associated morbidity. Future studies could assess the impact of pharmacist led metformin protocols in patients initiating atypical antipsychotics.

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