bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterizing Subsynaptic Myonuclei in Aged Muscle
Authors: Ruiz Lloyd 
Published: May 2022

IntroductionDuring aging, the neuromuscular junction (NMJ) undergoes structural fragmentation and functional deficits that alter neuromuscular communication, impair muscle contraction, and contribute to muscle atrophy. At the NMJ, myonuclei accumulate and localize in a fashion that juxtaposes three‐to‐six myonuclei in the postsynaptic region. Although the entire role of these so‐called subsynaptic myonuclei remains enigmatic, previous literature suggests they contribute to gene regulation of synapse‐specific proteins required for maintaining the structural integrity of the synaptic architecture. Interestingly, a recent report find that NMJs in muscles of aged mice contain fewer subsynaptic myonuclei compared with muscles of adult mice in parallel with degenerating or degenerated endplates. Furthermore, disturbance of the nuclear envelope by in‐vivo knock‐out of Lmna appears to accelerate NMJ degeneration and reduces the number of myonuclei confined to the endplate. Therefore, with the observed reductions in subsynaptic myonuclei in degenerated NMJs, our study aims to further examine and characterize subsynaptic myonuclei and their role in NMJ degeneration.MethodsGastrocnemius muscles were harvested from Young (6 months) and Aged (28 months) wild type mice and 6‐ and 12‐month‐old Sod1KO mice (N=3/group) and briefly fixed in 4% PFA for 10 minutes. Myofiber bundles were mechanically teased under a dissecting microscope and subjected to immunohistochemistry for visualization of motor neurons (neurofilament‐NF and synaptic vesicle glycoprotein 2‐SV2), motor end plates (alpha‐bungarotoxin), and myonuclei (nesprin 1 and 4’,6‐diamidino‐2‐phenylindole‐DAPI). Myofiber bundles were then mounted onto a slide and imaged via High‐sensitivity confocal microscopy. Captured images were then 3D projected in FIJI for assessment and quantification of subsynaptic myonuclei, innervation status, and post synaptic area assessed via aNMJ morph.ResultsIn young mice with primarily fully innervated endplates, the number of myonuclei associated with the endplate was lower than the number of synaptic myonuclei in muscles of aged and Sod1KO mice, both of which contain greater numbers of partially and non‐innervated endplates.ConclusionThere may be a greater number of subsynaptic myonuclei localized to degenerating or degenerated endplates, although whether the change in nuclear number is a consequence of NMJ degeneration or contributes to the denervation cannot be established from this study.
Measuring the precise position of transitions in transcription with PRO‐IP‐seq
Authors: Versluis Philip , DeBerardine Michael , Lis John 
Published: May 2022

Transcription is a fundamental process for all known organisms. Transcription by RNA Polymerase II (Pol II), the enzyme responsible for mRNA synthesis, is highly regulated by protein factors at every step. In multicellular eukaryotes, Pol II pauses 20‐60 bases downstream of the transcription start site in nearly all genes. This promoter‐proximal pausing is established as an important step in transcription regulation and is dependent on several pausing and elongation factors. The protein complexes DSIF (DRB Sensitivity Inducing Factor) and NELF (Negative Elongation Factor) bind to Pol II early in transcription to induce pausing. Pause release occurs upon phosphorylation of Pol II, DSIF and NELF by the Cdk9, Cyclin T1 heterodimer P‐TEFb (Positive Transcription Elongation Factor b). Although Pol II pausing is a key step in transcription regulation and has been extensively studied, the molecular mechanisms of pause and pause release are incompletely understood. Furthermore, recent studies have implicated controlled termination early in the gene as a mechanism to control transcription. A challenge to studying these key regulatory steps is that they occur in a narrow window: from initiation to Pol II pausing and release occurs in 20‐60 base range. Current approaches to study transcription factor interactions are unable to provide high enough resolution or sensitivity to address precisely where key transitions in transcription occur.To address this problem, I am developing an assay to measure the exact position of Pol II, when it is bound by a factor of interest. The technique couples our lab’s Precision Run‐on sequencing technique (PRO‐seq), which measures the position of active Pol II with single base‐pair resolution, and an immunoprecipitation enrichment step. Initial PRO‐IP‐seq (Precision Run‐on Immunoprecipitation sequencing) data shows that we can enrich for populations of Pol II bound by specific transcription factors. Utilizing this tool, we will be able to measure genomically where both stable and more transient transcription factors interact with Pol II at near single‐base resolution.
Gitelman‐like syndrome caused by pathogenic variants in mitochondrial DNA
Authors: de Baaij Jeroen H. F. , Viering Daan , Schlingmann Karl P. , Hureaux Marguerite , Nijenhuis Tom , Knoers Nine V. , Vargas Poussou Rosa , Bockenhauer Detlef 
Published: May 2022

Gitelman syndrome is the most frequent hereditary salt‐losing tubulopathy characterized by hypokalemic alkalosis and hypomagnesemia. Gitelman syndrome is caused by biallelic pathogenic variants in SLC12A3, encoding the Na+‐Cl‐ cotransporter (NCC) expressed in the distal convoluted tubule. Pathogenic variants in CLCNKB, HNF1B, FXYD2 or KCNJ10 may result in renal phenocopies of Gitelman syndrome, as they can lead to reduced NCC activity. Nevertheless, ±10% of patients with a Gitelman syndrome phenotype remain genetically unsolved.After identification of mitochondrial DNA (mtDNA) variants in three families with Gitelman syndrome‐like electrolyte abnormalities, 156 families were investigated for variants in MT‐TI and MT‐TF, encoding the transfer RNAs for phenylalanine and isoleucine. Mitochondrial respiratory chain function was assessed in patient fibroblasts. In NCC‐expressing HEK293 cells, mitochondrial dysfunction was induced to assess the effect on thiazide‐sensitive 22Na+ transport.Genetic investigations revealed four mtDNA variants in 13 families: m.591C>T (n=7), m.616T>C (n=1), m.643A>G (n=1) (all in MT‐TF) and m.4291T>C (n=4, in MT‐TI). Variants were near homoplasmic in affected individuals. Importantly, affected members of six families with an MT‐TF variant additionally suffered from progressive chronic kidney disease. Maximal mitochondrial respiratory capacity was reduced in patient fibroblasts, caused by dysfunction of oxidative phosphorylation complex IV. In vitro pharmacological inhibition of complex IV, mimicking the effect of the mtDNA variants, demonstrated an inhibitory effect on NCC phosphorylation and NCC‐mediated sodium uptake.Pathogenic mtDNA variants in MT‐TF and MT‐TI can cause a Gitelman syndrome‐like syndrome. Genetic investigation of mtDNA should be considered in patients with unexplained Gitelman syndrome‐like tubulopathies.
NMDA Receptor Neuropharmacology: NMDA Receptor‐mediated Calcium Transients are Attenuated by Agmatine in Mouse Spinal Cord Dorsal Horn
Authors: Xie Tongzhen 
Published: May 2022

NMDA receptor (NMDAr) plays an important role in synaptic plasticity and one of the targets for chronic pain management is antagonism of the NMDA receptors (NMDAr). Agmatine is an endogenous aminoguanidine that preferentially antagonizes GluN2B‐containing NMDArs. Our previous studies showed that the intrathecal administration of agmatine, an endogenous aminoguanidine that selectively antagonizes GluN2B‐containing NMDArs, reverses the mechanical hypersensitivity without motor side effects. In this study, we demonstrated the spinal modulatory effect of agmatine and other NMDAr antagonists utilizing calcium imaging.The aim of this study is to use a newly developed NMDAr‐mediated calcium transient assay (Figure 1.) in ex vivo mouse spinal cord slices and examine the effect of agmatine and other NMDAr antagonists in this assay. We hypothesize that agmatine and other NMDAr antagonists can dose‐dependently inhibit NMDAr‐mediated calcium transient in mouse spinal cord dorsal horn. Female and male ICR mice (4‐6 weeks) were perfused before spinal cord extraction and ex vivo spinal slices were incubated with the calcium indicator dye Fluo‐4. Intracellular Ca2+ was visualized by single plane two‐photon microscopy. Time‐lapse of images were acquired and the peak amplitude of fluorescence intensity was analyzed by Student’s t‐test.We found that NMDAr‐mediated calcium transients were elicited by an NMDA drug mixture containing NMDAr agonists (NMDA, glycine) and AMPA receptor antagonists (NBQX). 5mM agmatine incubation significantly attenuated the NMDAr‐mediated calcium transients compared to the control. Increasing agmatine concentration dose‐dependently attenuates NMDAr‐mediated calcium transients. Other NMDAr antagonists including AP5 and ifenprodil, showed dose‐dependent inhibition of NMDAr‐mediated calcium transients, supporting that the newly developed calcium imaging assay is NMDAr specific. These data suggest that agmatine attenuates NMDAr‐mediated calcium transients in the spinal cord dorsal horn, which is consistent with our previous research demonstrating that agmatine is an effective inhibitor of NMDAr in the spinal cord.
Objectification, Desensitization, and Personification: Medical Student Strategies for Dealing with Human Body in the Anatomy Lab
Authors: Azim Homaira M. 
Published: May 2022

Introduction & PurposeCadaver labs play a significant role in supporting learners form a professional attitude towards human body. Most medical students have their first authoritative encounter with human body in a cadaver lab where they get a right to manipulate and cut through the body. Dealing with death and dissection of the body may simply be a unique learning experience for some but it can be an emotional roller coaster for others. A few student strategies for dealing with cadaver bodies (such as objectification of the body) are documented in the literature but a full range of such strategies is rarely investigated. This study sought to understand the range of strategies medical students use for dealing with cadaver bodies in the anatomy lab at one medical school.MethodsTwenty semi‐structured interviews and four focus groups were conducted with a total of 44 students from all four years of undergraduate medical education at Indiana University School of Medicine, in 2019. All sessions were audio‐recorded and later transcribed verbatim. A qualitative thematic analysis was performed by developing an inductive coding scheme which resulted in emergence of salient themes from the data.ResultsThe full spectrum of student strategies for dealing with cadaver bodies included: 1) complete, effortless objectification of cadaver’s body as a non‐living thing, 2) actively desensitizing oneself toward cadaver’s body as so to be able to work with it, and 3) paralyzing personification of the cadaver’s body that impedes learning from it let alone dissecting it. Falling within this spectrum were also the so‐called middle ground strategies that were chosen by students attempting to adopt a more balanced behavior between desensitization and personification but still found themselves alternating between those two positions at certain times.ConclusionStudents employ certain strategies for handling human body in the cadaver lab. These strategies are based on their personal values and/or actively chosen to help them make the most out of their experience working with cadavers in the anatomy lab.Significance/ImplicationsLearning to deal with cadaver bodies in a professional manner is an impactful part of medical students’ professional identity formation process. The way students handle those bodies is likely to impact their attitude toward their patients’ bodies in the future as a doctor. Understanding the full range of student strategies for handling bodies in cadaver lab is a critical step for anatomy lab educators to facilitate a healthy, professional, ethical, and at the same time humane relationship between students and their cadaver’s body. Future studies should dig deeper into each of the strategies identified here and explore the best ways to oversee them for an effective medical professional identity formation in medical students.
Sustained Exposure to the SGLT2 Inhibitor Ertugliflozin, But Not NHE‐1 Inhibition By Cariporide, Attenuates Adrenergic Stimulation of Cytosolic CA2+ Levels in Spontaneously Contracting Cardiac Myocytes
Authors: Alam Sifat M. , Gierden Kyle J. , Zhao Tengteng , Lu Sun S. , Dixit Mihir , Babicheva Aleksandra , Yuan Jason , Vallon Volker , Zemljic‐Harpf Alice E. 
Published: May 2022

IntroductionInhibitors of sodium‐glucose cotransporter 2 (SGLT2i) target renal SGLT2 to reduce reabsorption of glucose, facilitate urinary glucose excretion, and lower blood glucose levels in patients with Type II diabetes (T2DM). The drugs, however, also decrease mortality and heart failure (HF) hospitalizations in T2DM patients. Patients with HF exhibit sympathetic overdrive and cytosolic Ca2+ overload in cardiac myocytes. Because SGLT2i harbor cardioprotective effects, we hypothesize that the SGLT2i ertugliflozin (ERTU) will lower cytosolic Ca2+ concentration ([Ca2+]cyt) in metabolically active and contracting cardiac myocytes after adrenergic stimulation.MethodsERTU was reconstituted in DMSO based on manufacture’s recommendation. Cardiac myocytes were isolated from 0–2‐day old mice and plated on coated (0.2% v/v gelatin, 1 μg/ml fibronectin, and 1 μg/ml laminin) glass cover slips. After two days, spontaneous contractions were observed, and fetal bovine serum content reduced to limit fibroblast overgrowth. By changing the media daily, spontaneously contracting cardiac myocytes were treated for 72 hours with either 1 µM ERTU, 10 µM cariporide (CARI), ERTU and CARI, or vehicle (0.01% v/v DMSO). After 72 hours, cells were loaded with FURA‐2/AM for 1 hour, a steady baseline [Ca2+]cyt was first recorded, then cells were stimulated with phenylephrine (100 µM), and [Ca2+]cyt was recorded for additional 30 minutes.ResultsERTU exposure for 72 hours did not alter baseline [Ca2+]cyt in beating cardiac myocytes when compared to vehicle treated controls but reduced the increase in [Ca2+]cyt after adrenergic stimulation with phenylephrine. In contrast, 72‐hour‐long NHE1 inhibition with CARI increased [Ca2+]cyt at baseline when compared to vehicle and ERTU, and further enhanced the increase in [Ca2+]cyt after phenylephrine stimulation. Interestingly, concomitant administration of ERTU and CARI for 72 hours reduced baseline [Ca2+]cyt and inhibited phenylephrine‐mediated increase in [Ca2+]cyt.ConclusionBecause sustained exposure to the SGLT2i ERTU reduced, but the NHE‐1 inhibitor CARI increased [Ca2+]cyt after acute adrenergic stimulation of metabolically active cardiac myocytes, these data indicate for the first time that ERTU’s beneficial effect on [Ca2+]cyt homeostasis is distinct from NHE1 inhibition. Reduced [Ca2+]cyt levels after sympathetic stimulation may contribute to the cardioprotective effect of SGLT2i. Further research is needed on the molecular mechanisms behind ERTU’s influence on cardiac [Ca2+]cyt.
Chronic Stress Accelerates the Progression of Cerebrovascular Dysfunction with Alzheimer's Disease
Authors: Burrage Emily N. , Prabhu Saina , Frazier James , Simpkins James , Kelley Eric E. , Chantler Paul D. 
Published: May 2022

Vascular contributions to cognitive impairment and dementia are a key pathology associated with Alzheimer’s disease (AD). Past work in our lab has shown that chronic stress induces cerebrovascular dysfunction due to a pro‐oxidative environment while epidemiological studies suggest a link between chronic stress and dementia. With this in mind, we tested the hypothesis that chronic stress accelerates the vascular pathology associated with AD potentially via the xanthine oxidase pathway.Here, we utilized a triple transgenic mouse model of AD (3xTg AD) combined with our unpredicted chronic mild stress paradigm (UCMS) and a xanthine oxidase inhibitor, Febuxostat, to examine middle cerebral artery (MCA) function. Beginning at 4 months of age all mice underwent the UCMS paradigm for 8 weeks total while a subset of mice were given Febuxostat treated water (50mg/L). Mice were either euthanized immediately following the 8 weeks of UCMS (at 6 months‐of‐age) or euthanized 3 months post‐UCMS (at 9 months‐of‐age). Following euthanasia, the MCA’s were removed and positioned in a pressurized myobath. The MCA was exposed to increasing concentrations of acetylcholine (ACh; 10‐9M to 10‐4M), phenylephrine (PE; 10‐9M to 10‐4M), and sodium nitroprusside (SNP; 10‐9M to 10‐4M).At 6 months‐of‐age, AD mice displayed impaired MCA dilation to ACh compared to WT control mice (8.2±1.4mm to 17.5±2.1mm, respectively; p<0.05). WT and AD mice that underwent UCMS displayed similar response to ACh compared to AD control mice (8.25±0.3mm and 7.125±1mm to 8.2±1.4mm, respectively). Treatment with Febuxostat prevented MCA dysfunction in AD control mice, WT UCMS mice, and AD UCMS mice (8.2±1.4mm vs 17.1±1mm, 8.25±0.3mm vs 18.25±1.1mm, 7.1±1mm vs 17.2±0.9mm, respectively; p<0.05). No differences were found between groups in the PE or SNP response. Interestingly, at 9 months‐of‐age, the majority of these trends persisted with the one exception being a compounded impairment in the AD UCMS group compared to both AD control or WT UCMS mice (3.5±1.2mm vs 8.5±1mm, and 9.3±2.2mm, respectively; p<0.05).Overall, these data suggest that chronic stress does accelerate the cerebrovascular pathology associated with AD and potentially via a pro‐oxidative state driven by activation of the xanthine oxidase pathway.
Pharmacologic or Genetic PDE4 Inactivation Reduces Obesity and Improves Glucose Handling in Mice
Authors: Irelan Daniel , Boyd Abigail , Aragon Ileana , Fiedler Edward , Richter Wito 
Published: May 2022

PurposeType 4 cyclic nucleotide phosphodiesterases (PDE4s) comprise a group of four isoenzymes (PDE4A to D) that hydrolyze the second messenger cAMP. Non/PAN‐selective PDE4 inhibitors exert potent anti‐inflammatory properties and are approved for the treatment of COPD and psoriasis, but are also associated with significant side effects, including nausea, emesis and weight loss. Here, we explored the idea that the weight loss associated with PAN‐PDE4 inhibitor use may be pursued as a desirable therapeutic outcome in the treatment of obesity‐related metabolic syndromes. As a first step, we tested whether treatment of mice with a PAN‐PDE4 inhibitor would replicate clinical data and reduce ageing‐ and/or high‐fat diet‐induced obesity in the animals. As each of the four PDE4 subtypes plays unique physiological roles, targeting individual PDE4s is a promising approach to improve the tolerability of PDE4 inhibitor therapy. To this end, we determined the metabolic phenotypes of individual PDE4 subtypes in mice.MethodsBody and tissue weights, food and water consumption, glucose and insulin tolerance, serum insulin and glucagon levels, and locomotor activity and exercise capacity were assessed in aged mice, as well as in young mice fed a high‐fat diet. To delineate the role of PDE4s, the phenotypes of mice genetically deficient (KO) in each of the four individual PDE4 subtypes, PDE4A to PDE4D, were compared to their wildtype littermates, and mice treated with the PAN‐PDE4 inhibitor Roflumilast were compared to solvent controls.ResultsTreatment with the PAN‐PDE4 inhibitor Roflumilast reduced high‐fat diet‐induced obesity in mice, as reflected by reduced body weight and white fat pads, without lowering food consumption or increasing physical activity (unchanged locomotor activity and exercise capacity) suggesting that the weight‐loss effect of PDE4 inhibition observed in clinical trials is replicated in the mouse. Genetic ablation of PDE4B or PDE4D in mice replicated the effects of PAN‐PDE4 inhibition by reducing body‐ and adipose tissue weights in mice, whereas ablation of PDE4A or PDE4C had no effect. Reduced adiposity as a result of PDE4 inactivation was associated with improved glucose handling and insulin sensitivity, while serum levels of insulin and glucagon were unchanged. These data suggest that PDE4 inactivation does not act via pancreatic hormone release but acts in downstream target tissues to enhance glucose utilization.ConclusionsInactivation of PDE4s represents a promising approach to tackle obesity and associated metabolic abnormalities such as elevated blood glucose levels. Targeting PDE4B and/or PDE4D with subtype‐selective PDE4 inhibitors appears sufficient to mediate these therapeutic benefits and may be free of the adverse effects associated with the PAN‐PDE4 inhibitors available to date.
Sequential Model of Teaching in Anatomy Lab Aims to Improve Student Learning
Authors: Dugan Lauren , Thomas Kyle , Scali Frank , Hassan Sherif S. 
Published: May 2022

IntroductionAt the California University of Science and Medicine, School of Medicine, the anatomy department has developed a sequential model of teaching in which anatomical concepts are gradually introduced and integrated with increasingly complex modalities. This teaching pedagogy aims to establish strong anatomical foundations to equip students with the critical thinking skills to identify landmarks on prosected cadaveric specimens and radiographic imaging.MethodsAnatomy labs are divided into virtual and in‐person components. These lab sessions have been developed to teach anatomical structures to students in an explicitly stepwise progression. During virtual anatomy sessions, students are initially taught to draw simple line drawings of anatomical structures. Students progress from drawing anatomical structures to studying more complex, professional illustrations of those same structures, such as those in Netter's Atlas of Human Anatomy. Following exposure to illustrations, students are introduced to three‐dimensional images of the structures using virtual anatomy software. During independent in‐person lab sessions, students apply knowledge acquired from each of these prior learning modalities to identify structures on prosected specimens. Lastly, students implement critical thinking skills to distinguish anatomical landmarks and pathological changes in radiographic images.ResultsThis systematic progression of teaching aims to allow students to engage in higher‐level thinking in which they apply newly acquired knowledge to increasingly more complex material. By doing so, the following learning outcomes of each anatomy session can be successfully achieved:

Identifying anatomical structures on plastinated specimens
Identifying anatomical structures in various imaging modalities, such as X‐rays, CT, and MRI
Discerning pathological structures from healthy structures in various imaging modalities
Prospective data collection through course evaluation forms will be analyzed to assess the sequential teaching model's efficacy.Conclusion & SignificanceUsing the sequential model of teaching in anatomy lab can effectively help medical students understand, rather than memorize, anatomy. The systematic scaffolding of anatomical concepts with increasingly complex material, progressing from line drawings to imaging interpretation, encourages critical thinking and reinforces learning. We expect these skills will prepare students for the challenges presented in their clinical years and beyond.