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Item Motivating Self-Efficacy in Diverse Biomedical Science Post-baccalaureate and Graduate Students Through Scientific Conference Implementation(Frontiers Media, 2021) Boehmer, Kasey R.; De Souza, Suelen Lucio Boschen; Doles, Jason D.; Lachman, Nirusha; Mays, Dennis; Hedin, Karen E.; Dornink, Cheryl A.; Maher, Louis J.; Lujan, J. Luis; Anatomy, Cell Biology and Physiology, School of MedicineTactics to increase the number of underrepresented (UR) students in biomedical research PhD training programs have not yet translated to UR faculty numbers that reflect the diversity of the United States. Continued interventions are required to build skills beyond those that result in placement into a PhD program. We hypothesize that successful interventions must build skills that give UR students foundations for confident self-efficacy in leadership. We seek interventions that allow UR students to envision themselves as successful faculty. We posit that development of such skills is difficult in the classroom or laboratory alone. Therefore, novel interventions are required. As part of the NIH-funded Post-baccalaureate Research Education Program (PREP) and Initiative for Maximizing Student Development (IMSD) at the Mayo Clinic Graduate School of Biomedical Sciences, we designed and implemented a unique intervention to support development of student leadership skills: a biannual student-organized and student-led national research conference titled "Scientific Innovation Through Diverse Perspectives" (SITDP). This initiative is based on the concept that students who actively live out realistic roles as scientific leaders will be encouraged to persist to scientific leadership as faculty. Here we describe the motivation for, design of, and outcomes from, the first three pilot conferences of this series. We further discuss approaches needed to rigorously evaluate the effectiveness of such interventions in the future.Item Disrupted NOS2 metabolism drives myoblast response to wasting-associated cytokines(Elsevier, 2021) Arneson-Wissink, Paige C.; Doles, Jason D.; Anatomy, Cell Biology and Physiology, School of MedicineSkeletal muscle wasting drives negative clinical outcomes and is associated with a spectrum of pathologies including cancer. Cancer cachexia is a multi-factorial syndrome that encompasses skeletal muscle wasting and remains understudied, despite being a frequent and serious co-morbidity. Deviation from the homeostatic balance between breakdown and regeneration leads to muscle wasting disorders, such as cancer cachexia. Muscle stem cells (MuSCs) are the cellular compartment responsible for muscle regeneration, which makes MuSCs an intriguing target in the context of wasting muscle. Molecular studies investigating MuSCs and skeletal muscle wasting largely focus on transcriptional changes, but our group and others propose that metabolic changes are another layer of cellular regulation underlying MuSC dysfunction in cancer cachexia. In the present study, we combined gene expression and non-targeted metabolomic profiling of myoblasts exposed to wasting conditions (cancer cell conditioned media, CC-CM) to derive a more complete picture of the myoblast response to wasting factors. After mapping these features to annotated pathways, we found that more than half of the mapped pathways were amino acid-related, linking global amino acid metabolic disruption to conditioned media-induced myoblast defects. Notably, arginine metabolism was a highly enriched pathway in combined metabolomic and transcriptomic data. Arginine catabolism generates nitric oxide (NO), an important signaling molecule known to have negative effects on mature muscle. We hypothesize that tumor-derived disruptions in Nitric Oxide Synthase (NOS)2-regulated arginine catabolism impair differentiation of MuSCs. The work presented here further investigates the effect of NOS2 overactivity on myoblast proliferation and differentiation. We show that NOS2 inhibition is sufficient to rescue wasting phenotypes associated with inflammatory cytokines. Ultimately, this work provides new insights into MuSC biology and opens up potential therapeutic avenues for addressing disrupted MuSC dynamics in cancer cachexia.Item Wnt signaling mediates acquisition of blood–brain barrier properties in naïve endothelium derived from human pluripotent stem cells(eLife Sciences, 2021-11-10) Gastfriend, Benjamin D.; Nishihara, Hideaki; Canfield, Scott G.; Foreman, Koji L.; Engelhardt, Britta; Palecek, Sean P.; Shusta, Eric V.; Anatomy, Cell Biology and Physiology, School of MedicineEndothelial cells (ECs) in the central nervous system (CNS) acquire their specialized blood-brain barrier (BBB) properties in response to extrinsic signals, with Wnt/β-catenin signaling coordinating multiple aspects of this process. Our knowledge of CNS EC development has been advanced largely by animal models, and human pluripotent stem cells (hPSCs) offer the opportunity to examine BBB development in an in vitro human system. Here, we show that activation of Wnt signaling in hPSC-derived naïve endothelial progenitors, but not in matured ECs, leads to robust acquisition of canonical BBB phenotypes including expression of GLUT-1, increased claudin-5, decreased PLVAP, and decreased permeability. RNA-seq revealed a transcriptome profile resembling ECs with CNS-like characteristics, including Wnt-upregulated expression of LEF1, APCDD1, and ZIC3. Together, our work defines effects of Wnt activation in naïve ECs and establishes an improved hPSC-based model for interrogation of CNS barriergenesis.Item Prostaglandin I2 and T Regulatory Cell Function: Broader Impacts(Mary Ann Liebert, 2021) Norlander, Allison E.; Peebles, R. Stokes, Jr.; Anatomy, Cell Biology and Physiology, School of MedicineTregulatory cells (Tregs) are an important member of the adaptive immune system and function to reduce and resolve inflammation. Prostaglandin I2 (PGI2) is a lipid mediator that has potent anti-inflammatory effects on immune cells. Several studies have investigated the interplay between PGI2 and Tregs. Together, the data from these studies demonstrate that PGI2 promotes the formation and function of Tregs. This suggests that therapeutic supplementation of PGI2 may be a treatment for various autoimmune or inflammatory diseases through enhancement of Treg function.Item Electroacupuncture Alleviates Anxiety-like Behavior in Pain Aversion Rats by Attenuating the Expression of Neuropeptide Y in Anterior Cingulate Cortex(Elsevier, 2022) Shao, Fangbing; Du, Junying; Wang, Sisi; Cerne, Rok; Fang, Junfan; Shao, Xiaomei; Jin, Xiaoming; Fang, Jianqiao; Anatomy, Cell Biology and Physiology, School of MedicineBackground: Pain is considered as a multidimensional conscious experience that includes a sensory component and a negative affective-motivational component. Electroacupuncture (EA) is widely used to treat pain and pain-induced negative emotions, however, little is known about the mechanisms underlying the effect of EA. Objective: This study investigated the effect of EA on alleviating the anxiety-like behaviors in pain aversion rats and its anterior cingulate cortex (ACC) regulation mechanism. Methods: After a Freund's complete adjuvant (CFA)-conditioned place aversion (C-CPA) model was established in rats, EA treatment (2/100 Hz, 30 min, once/day, 4 days totally) was applied at bilateral Zusanli (ST36) and Kunlun (BL60) acupoints. Von Frey filaments were used to measure changes of pain withdrawal threshold (PWT) at indicated time points. Elevated zero maze (EZM) was used to investigate the changes of pain-related anxiety and CPA was used to investigate the changes of pain aversion. The protein expression levels of GAD67, PV, and NPY in ACC were detected by Western blotting. Results: Compared with the control group, the staying time in the "CFA-paired compartment" was significantly reduced, and the PWT was decreased in model group. In the EZM assessment, the distance and the time in open arm, as well as the number of open arm entries of model group were significantly lower than those in the control group. In the CPA assessment, the time spent in the "CFA-paired compartment" was significantly decreased in model group compared with control group, and EA reversed the changes in pain sensation and in pain-related emotions. Western blotting showed that the NPY level, but not the levels of GAD67 and PV, was significantly increased in the ACC of the model group compared to that of the control group. The increased expression of NPY in the ACC was significantly downregulated by EA, while sham EA produced no such effect. Conclusion: EA can effectively relieve the pain and pain-related emotions, and its mechanism may be achieved by down-regulating the expression of NPY in the ACC.Item First patient project: Engaging pathology through the donor dissection experience and its role in professionalism(Wiley, 2024-01) Robertson, Kyle A.; Organ, Jason M.; Yard, Michael; Byram, Jessica N.; Anatomy, Cell Biology and Physiology, School of MedicineThe peer-reviewed anatomical education literature thoroughly describes the benefits and drawbacks of donor dissection. Gross anatomy laboratory environments utilizing donor dissection are generally considered to be a premier environment where students foster non-traditional discipline-independent skills (NTDIS), including the acquisition of professionalism, empathy, resilience, emotional intelligence, and situational awareness. Therefore, this IRB-approved study explored the impact of a formal humanism and pathology thread, the first patient project (FPP), on the personal and professional development of pre-professional undergraduate students in a gross anatomy dissection-based course. Five reflections from each student were collected across four cohorts (n = 74 students, 370 reflections). A post-course questionnaire collected data on student perceptions of the project. The framework method was used to analyze reflection and free response data and descriptive statistics were performed on Likert-style items using Excel. Three themes were identified to encompass the impacts of the FPP on professional development and include: Socialization (through collective dissection experience and pathology), Humanistic Qualities (respect for the donor and their history, and introspection), and Content and Skills (technical and NTDIS, anatomical knowledge). The end of course FPP survey was completed by 29 students across three cohorts (65%) and their perspectives were generally favorable regarding the promotion of respect, empathy, and humanization of their donors. This study underscores the value of incorporating humanism, pathology, and reflection, facilitated through formal curriculum for pre-professional undergraduate students. It provides evidence of the positive impact on their personal and professional development, supporting the integration of NTDIS in curricula across various disciplines.Item Succinate dehydrogenase-complex II regulates skeletal muscle cellular respiration and contractility but not muscle mass in genetically induced pulmonary emphysema(American Association for the Advancement of Science, 2024) Balnis, Joseph; Tufts, Ankita; Jackson, Emily L.; Drake, Lisa A.; Singer, Diane V.; Lacomis, David; Lee, Chun Geun; Elias, Jack A.; Doles, Jason D.; Maher, L. James, III; Jen, Annie; Coon, Joshua J.; Jourd’heuil, David; Singer, Harold A.; Vincent, Catherine E.; Jaitovich, Ariel; Anatomy, Cell Biology and Physiology, School of MedicineReduced skeletal muscle mass and oxidative capacity coexist in patients with pulmonary emphysema and are independently associated with higher mortality. If reduced cellular respiration contributes to muscle atrophy in that setting remains unknown. Using a mouse with genetically induced pulmonary emphysema that recapitulates muscle dysfunction, we found that reduced activity of succinate dehydrogenase (SDH) is a hallmark of its myopathic changes. We generated an inducible, muscle-specific SDH knockout mouse that demonstrates lower mitochondrial oxygen consumption, myofiber contractility, and exercise endurance. Respirometry analyses show that in vitro complex I respiration is unaffected by loss of SDH subunit C in muscle mitochondria, which is consistent with the pulmonary emphysema animal data. SDH knockout initially causes succinate accumulation associated with a down-regulated transcriptome but modest proteome effects. Muscle mass, myofiber type composition, and overall body mass constituents remain unaltered in the transgenic mice. Thus, while SDH regulates myofiber respiration in experimental pulmonary emphysema, it does not control muscle mass or other body constituents.Item Protocol for the isolation and proteomic analysis of pathological tau-seeds(Elsevier, 2024) Martinez, Pablo; Patel, Henika; You, Yanwen; Doud, Emma H.; Mosley, Amber L.; Lasagna-Reeves, Cristian A.; Anatomy, Cell Biology and Physiology, School of MedicineThe aggregation and spreading of "tau-seeds" are key for the development and progression of tauopathies, including Alzheimer's disease. Here we describe the steps to isolate and analyze biochemically active tau-seeds from human, mouse, and cell origin. We detail the procedure to isolate soluble tau-seeds by size exclusion chromatography and seeding assay. The isolated tau-seed can be further analyzed to determine the interactome by mass spectrometry. This workflow identifies protein-protein interactors of tau-seeds, providing a useful tool for finding new therapeutic targets. For complete details on the use and execution of this protocol, please refer to Martinez et al.1.Item Both enantiomers of β-aminoisobutyric acid BAIBA regulate Fgf23 via MRGPRD receptor by activating distinct signaling pathways in osteocytes(Elsevier, 2024) Sakamoto, Eijiro; Kitase, Yukiko; Fitt, Alexander J.; Zhu, Zewu; Awad, Kamal; Brotto, Marco; White, Kenneth E.; Welc, Steven S.; Bergwitz, Clemens; Bonewald, Lynda F.a; Anatomy, Cell Biology and Physiology, School of MedicineWith exercise, muscle and bone produce factors with beneficial effects on brain, fat, and other organs. Exercise in mice increased fibroblast growth factor 23 (FGF23), urine phosphate, and the muscle metabolite L-β-aminoisobutyric acid (L-BAIBA), suggesting that L-BAIBA may play a role in phosphate metabolism. Here, we show that L-BAIBA increases in serum with exercise and elevates Fgf23 in osteocytes. The D enantiomer, described to be elevated with exercise in humans, can also induce Fgf23 but through a delayed, indirect process via sclerostin. The two enantiomers both signal through the same receptor, Mas-related G-protein-coupled receptor type D, but activate distinct signaling pathways; L-BAIBA increases Fgf23 through Gαs/cAMP/PKA/CBP/β-catenin and Gαq/PKC/CREB, whereas D-BAIBA increases Fgf23 indirectly through sclerostin via Gαi/NF-κB. In vivo, both enantiomers increased Fgf23 in bone in parallel with elevated urinary phosphate excretion. Thus, exercise-induced increases in BAIBA and FGF23 work together to maintain phosphate homeostasis.Item The SOD1 Inhibitor, LCS-1, Oxidizes H2S to Reactive Sulfur Species, Directly and Indirectly, through Conversion of SOD1 to an Oxidase(MDPI, 2024-08-15) Olson, Kenneth R.; Takata, Tsuyoshi; Clear, Kasey J.; Gao, Yan; Ma, Zhilin; Pfaff, Ella; Mouli, Karthik; Kent, Thomas A.; Jones, Prentiss, Jr.; Fukuto, Jon; Wu, Gang; Straub, Karl D.; Anatomy, Cell Biology and Physiology, School of MedicineLCS-1, a putative selective inhibitor of SOD1, is a substituted pyridazinone with rudimentary similarity to quinones and naphthoquinones. As quinones catalytically oxidize H2S to biologically active reactive sulfur species (RSS), we hypothesized LCS-1 might have similar attributes. Here, we examine LCS-1 reactions with H2S and SOD1 using thiol-specific fluorophores, liquid chromatography-mass spectrometry, electron paramagnetic resonance (EPR), UV-vis spectrometry, and oxygen consumption. We show that LCS-1 catalytically oxidizes H2S in buffer solutions to form RSS, namely per- and polyhydrosulfides (H2Sn, n = 2-6). These reactions consume oxygen and produce hydrogen peroxide, but they do not have an EPR signature, nor do they affect the UV-vis spectrum. Surprisingly, LCS-1 synergizes with SOD1, but not SOD2, to oxidize H2S to H2S3-6. LCS-1 forms monothiol adducts with H2S, glutathione (GSH), and cysteine (Cys), but not with oxidized glutathione or cystine; both thiol adducts inhibit LCS-1-SOD1 synergism. We propose that LCS-1 forms an adduct with SOD1 that disrupts the intramolecular Cys57-Cys146 disulfide bond and transforms SOD1 from a dismutase to an oxidase. This would increase cellular ROS and polysulfides, the latter potentially affecting cellular signaling and/or cytoprotection.