bluuebunny/crossref_metadata_2025_split huggingface dataset:
Formolized Cadaveric Specimens Immersion‐Recovery Protocol
Authors: Obando Fabian David Camacho , Rueda-Esteban Roberto Javier 
Published: April 2020

Cadaveric dissection, promoted by multiple anatomists and institutions as a fundamental area of medical education and training , requires embalming of the bodies to disinfect and preserve them. Therefore, despite being a known toxic and carcinogenic agent, formaldehyde is still widely used for its characteristics and results. Our objective is to introduce a Formolized Cadaveric Specimens Immersion‐Recovery Protocol developed at our laboratory, the chemical reagents used, performed processes and results. The protocol consists on specimen’s immersion in an aqueous solution of potassium hydroxide (KOH) for its maceration, this process causes chemical reactions like lipids saponification and proteins breakdown. The immersion time varies from one specimen to another; however, KOH concentration should never exceed 10% (w/v), otherwise tissue hydrolysis may occur. After a profuse wash, oxidative degradation is carried out: oxidizing formaldehyde into H2O and CO2 through the degradation of hydrogen peroxide (H2O2). It is extremely important to avoid the contact of this agent with the previously used KOH, which would cause the decomposition of H2O2 into H2O, O2 and hydroxyls, without obtaining a proper formaldehyde oxidation. For this purpose, sodium hypochlorite can also be used, with variable results. To ignore this chemical basis limits the achievement of ideal results. This processes allow to obtain 60% more joint movement on goniometry measures compared with formolized specimens, with a change from grey color generated by formaldehyde to ocher. Likewise, the degradation of formaldehyde allows obtaining minimum levels of it in treated specimens, thus reducing health impact on those who handle the specimens. After the recovery process, our specimens were preserved with a low formaldehyde solution. Anatomy students were invited to a peer‐guided extracurricular activity, they interacted with both formolized and recovered specimens, and a likert questionnaire was used to evaluate their experiences with both techniques. Students referred that recovered specimens looked more realistic and considered them better to work with. They also mentioned recovered corpses had less sensory hassles, like eye watering, airway irritation and coughing. Previous studies have compared Thiel solution and formaldehyde fixation. Thiel‐preserved bodies are considered to be a better choice on dissection and surgical training, since tissues offer a more realistic experience. Nonetheless, this technique has disadvantages such as higher costs, vacuum conservation, and the need of trained personal to handle the specimens. Moreover, some chemicals and infrastructure are not available worldwide. An example of the former scenario is present with Ammonium Nitrate, key component in Thiel’s, which is strictly regulated in several countriesSupport or Funding InformationAnatomy Laboratory, Universidad de Los Andes, School of Medicine
S369 phosphorylation of RIPK3 by PLK1 during G2/M phases enables its apoptotic function in the ripoptosome independent of necroptosis
Authors: Gupta Kartik , Liu Bo 
Published: April 2020

Receptor interacting protein kinase 3 (RIPK3) regulates a form of regulated necrosis called necroptosis. When an altered conformation is induced by via mutations in its kinase site or using chemical inhibitors, RIPK3 associates with an ensemble of proteins called the ripoptosome, containing FADD, RIPK1, Caspase 8 and cFLIP. RIPK3 now becomes decisive in the execution of apoptosis. Paradoxically, in contexts not completely understood, the ripoptosome cleaves RIPK3, highlighting a knowledge gap on how RIPK3 fulfills its role via the ripoptosome which is responsible for its own degradation. Recently, ripoptosome assembly was shown to occur physiologically during mitosis. We found RIPK3 levels to be elevated during the G2/M phases of the cell‐cycle, allowing us to study the mechanism by which RIPK3 resists degradation by the ripoptosome. We now report that polo‐like kinase 1 (PLK1) associates with RIPK3 and phosphorylates it at serine 369 before cells enter mitosis. RIPK3 in this phase has pro‐apoptotic activity but when released from ripoptosome, can elicit necroptosis. Disruption of the PLK1 site via T368A and S369A mutations causes degradation of RIPK3. RIPK3‐degradation can be prevented by the addition of pan‐caspase inhibitor zVAD.fmk, via a cis‐mutation in caspase 8 cleavage site i.e. D333A, but not by PLK1 over‐expression, suggesting that the S369 site regulates the apposing caspase cleavage site (D333) in cis‐manner. Additionally, S369D mutation on RIPK3 prevents its degradation via the ripoptosome whereas TS>>AA version of T368 and S369 is degraded. Together, phosphorylation of RIPK3 at S369 prevents its cleavage in the ripoptosome, thereby retaining its pro‐death activity during G2/M phases. The shift of cell‐death towards RIPK3 may have evolved as a mechanism by which the responsibility of balancing necroptosis vs apoptosis in mitotic cells converges on RIPK3.Support or Funding InformationThis study was supported by the National Institute of Health R01HL088447 (BL) and R01HL122562 (BL), and American Heart Association 17PRE33670082 (KG).
Systems Glycoengineering: Integrated Analyses of Glycomics, Transcriptomics and Phenotypic Data Reveal Complex Cellular Response to Glycoengineering in CHO Cells
Authors: Chiang Wan-Tien , Gazestani Vahid H. , Bao Bokan , Liang Chenguang , Sorrention James T. , Kellman Benjamin P , Ménard Patrice , Arnsdorf Jonnhy , Sukhova Zulfiya , Bjørn Sara Petersen , Brøndum Karen Kathrine , Hansen Anders Holmgaard , Yang Zhang , Joshi Hiren , Clausen Henrik , Voldborg Bjørn G. , Lewis Nathan E. 
Published: April 2020

Glycosylation is fundamentally important to the activity, stability and pharmacokinetics of biopharmaceuticals. Move on to the future, glycoengineering is a promising way to improve protein drug efficacy. Recently, several major advances in engineering efforts have successfully led to desired protein glycoforms, but some desirable glycoengineering designs have proven toxic to the host cells (e.g., CHO cell lines). Up to date, the critical barrier in controlling the glycan structures is the diversity and complexity of glycosylation. Moreover, there has not been a systematic study unraveling how engineering efforts impact the host cell and their protein secretion system. There is a great need to bridge the knowledge gap that enables rational glycoengineering and elucidate how such strategies impact the host cell. To accomplish this, we have comprehensively studied the impact of glycoengineering on a library of more than 180 CHO cell clones, wherein dozens of glycosyltransfersases were knocked out or knocked in. We integrate data that glycoprofiled all these clones, quantified the impact of different glycosyltransferase knockouts on the bioprocessing phenotypes of the CHO cells (e.g., cell size, growth, viability, and metabolism), and expression profiling these cells using RNA‐Seq data to identify dysregulated pathways and genes following glycoengineering. Here, we deployed an innovative systems biology approach to rapidly study the changes in glycosylation across all glycoengineered mutants and to study the molecular basis of the phenotypic changes. Our key results are (1) we identified dominant glycosyltransferses responsible for the N‐linked glycosylation in CHO cells, and the glycoengineered cells differentially expressed isozymes in response to a knockout; (2) we identified groups of glycosyltransferases whose perturbations led to a more severe impact on cell glycosylation and phenotypes; (3) we also identified key metabolic and signaling pathways are modulated when different glycosyltransferase families were perturbed. This research is the first study for gaining a comprehensive view of the complex cellular response to glycoengineering on the host cells producing the recombinant protein drugs. The insights from this study provides valuable knowledge that enables the rational engineering of glycosylation to be able to control this critical quality attribute on diverse recombinant protein drugs.Support or Funding InformationThis work was supported by generous funding from the Novo Nordisk Foundation provided to the Center for Biosustainability at the Technical University of Denmark (grant no. NNF10CC1016517).
Amino acids‐induced cardiac protection by promoting mitochondrial function via mTOR and Opa‐1 signaling
Authors: Morio Atsushi , Satomi Shiho , Toyota Yukari , Watanabe Tomoyuki , Tsutsumi Yasuo M 
Published: April 2020

ObjectsAmino acids, especially branched chain amino acids (BCAAs), have important regulatory roles in protein synthesis. Recently studies revealed that BCAAs protect against ischemia/reperfusion (I/R) injury. We studied the signaling pathway and mitochondrial function affecting a cardiac preconditioning of BCAAs.MethodsCardiac myocytes were isolated by cardiac retrograde aortic perfusion and collagenase and were subjected to simulated ischemia and cell death was quantified. Immunoblot and real time PCR were performed as biochemical assay and mitochondrial function was assessed using Seahorse XF cell Mito test.ResultsIn myocytes undergoing simulated I/R, BCAA treatment significantly preserved cell viability (71.7 ± 2.7% vs. 34.5 ± 1.6%, respectively, p < 0.0001), whereas rapamycin prevented this BCAA‐induced cardioprotective effect (43.5 ± 3.4% vs. BCAA, p < 0.0001). To elucidate the mechanism, we assessed changes in the mitochondrial fusion regulators Opa‐1, Mfn‐1, and Mfn‐2. BCAA treatment increased expression of Opa‐1, but not Mfn‐1 or Mfn‐2. Enhanced mitochondrial fusion also improved mitochondrial function including oxygen consumption rate.ConclusionsBCAA acutely regulates mitochondrial metabolism in cardiomyocytes and increased mitochondrial fusion, Opa‐1, and the Akt‐mTOR pathway, offering a novel target for prevention and treatment of coronary disease.Support or Funding InformationThis study was supported by Japan Society of Promotion Science #19K09353 to YMT.
Manipulating Levels of Stress‐Response Proteins in a Drosophila Model of Myosin‐Based Inclusion Body Myopathy 3 Worsens Muscle Dysfunction
Authors: Suggs Jennifer A. , Manalo Kimberley , Melkani Girish C. , Melkani Anju , Foster D. Brian , Bernstein Sanford I. 
Published: April 2020

Utilizing a Drosophila model of inclusion body myopathy 3 (IBM‐3), we identified a subset of stress‐response proteins essential for skeletal muscle proteostasis in both health and disease. Inclusion body myopathy 3 (IBM‐3) is a rare, dominant skeletal muscle disease caused by an E706K substitution in the SH1 helix of myosin heavy chain IIa. We produced a model of the human disease in the indirect flight muscle (IFM) of Drosophila. As in the human disease, progressive degradation occurs in muscle expressing the IBM‐3 mutant myosin. Homozygotes assemble defective sarcomeres that accumulate thermally unstable myosin and autophagic vesicles. To further investigate the mechanism of pathogenesis in IBM‐3 muscle fibers, we extracted insoluble proteins from IFM of young and old homozygotes and identified aggregated proteins within inclusions. Quantitative iTRAQ proteomics defined 18 proteins with >1.5‐fold difference in relative abundance at both ages in mutants vs. controls. Two of 6 proteins with increased levels in IBM‐3 have small heat shock protein domains (Hsp20, CG7409), while the 12 proteins with decreased levels include Hsp23, Hsp60, and Trim32 (abba/thin), which has ubiquitin‐protein transferase activity. Using the GAL4‐UAS system for over‐expression or knock down (RNAi), we tested the role of these proteins in wild‐type muscle and determined how altering expression affects the IBM‐3 heterozygote phenotype. While knockdown of Hsp20, Hsp23 or CG7409 yielded normal flight in wild type, Hsp23 or CG7409 over‐expression resulted in age‐dependent reduction in flight ability. Knockdown or over‐expression of these chaperones typically exacerbated the IBM‐3/+ phenotype. As expected, Trim32 knockdown eliminated flight in wild‐type or IBM‐3/+ flies and correlated with less organized, thinner myofibrils. Overexpression of Trim32 also eliminated flight and yielded deteriorating myofibrils that hypercontracted by 3 weeks of age. Remarkably, electron microscopy shows the ultrastructure of this phenotype in IBM‐3/+ resembles the dramatic destruction of myofibrils observed in IBM‐3 homozygotes. Overall, each of the stress response proteins assessed is critical for proper muscle development, performance, or maintenance. Altering levels of these key proteins in muscle fibers challenged with the IBM‐3/+ phenotype has detrimental effects. Our findings add to evidence that expression levels of stress response proteins modulate alternative pathways to respond to challenges to proteostasis.Support or Funding InformationThis work was supported by grants from the Muscular Dystrophy Association (MDA217900)and the National Institutes of Health (NIGMS R37 32443) to S.I.B. The authors acknowledgethe Bloomington Drosophila Stock Center (NIH P40OD018537), the Vienna DrosophilaResource Center and Dr. Richard Cripps (SDSU) for Drosophila stocks.
Manganese‐Induced Central Precocious Puberty (MnPP) Increases Hormone Receptor Expression (HR+) in Proliferating Mammary Epithelial Cells in Adult Female Rats
Authors: Dearth Robert K. , Hamilton Alina M. , Srivastava Vinod , Hiney Jill K. , Dees W. Les 
Published: April 2020

In the developing mammary gland (MG), hormone receptor expressing (HR+) cells very rarely proliferate. The actions of estrogen (E2) and progesterone (P4) on ductal morphogenesis are paracrine and require mediators to stimulate growth of HR− adjacent cells. However, in premalignant and invasive human breast lesions, proliferating cells are HR+. Thus, a dramatic change occurs in MG cell fate with a suspected increased sensitivity to steroid mediated growth. Studies have theorized that early pubertal onset alters MG cell fate increasing the risk of breast cancer later in life. Yet, it is not known if early puberty can cause this conversion to HR+ proliferating MG cells and if this phenotype is sustained in the adult gland. We previously demonstrated that manganese‐induced precocious puberty (MnPP) accelerates E2‐regulated MG development, resulting in sustained proliferation of MG epithelial cells and adult hyperplasia. In the current study, we characterized the normal distribution of HR+ proliferative cells in the MG of prepubertal and adult female rats, determined if MnPP could increase this population and identify cellular proteins that may be involved. Sprague Dawley female rats were exposed daily to 10mg/kg manganese chloride (MnCl2) or saline (control) via gastric gavage from post‐natal day (PND) 12 to PND 30. MGs were collected on PNDs 30 and 120. Using dual labeling immunofluorescence (IF), only 2% of total MG epithelial cells co‐express HRs (E2 or P4 receptor) and the proliferative marker PCNA in the prepubertal MG. As expected, MnPP significantly (p<0.01) increased the percentage of proliferating (PCNA+) MG cells. The percentage of proliferating cells co‐expressing progesterone receptor (PR+/PCNA+) increased by more than 2‐fold in MnPP females relative to controls at PND30 (p<0.01). The proportion of proliferating HR+ cells (ER+/PCNA+ or PR+/PCNA+) remained elevated in PND120 glands of MnPP females compared to controls (p<0.001). Western blot analysis revealed a significant switch in expression levels of the cell cycle regulator, p27, and the mitotic stimulator EREG in MGs that corroborated changes in PR+ proliferation between PND 30 and 120. Furthermore, histologically normal human breast tissue adjacent to ductal carcinoma in situ (DCIS) from The Human Protein Atlas show ER and PR expression similar to expression levels observed in our MnPP rat MGs at PND120. Collectively, these data show that MnPP results in persistent HR expression in proliferating MG cells. Thereby possibly sensitizing it to the hormonal milieu and making the MG more responsive to steroid mediated growth.Support or Funding InformationThis work was supported by NIEHS grant ESO13143 awarded to WLD and NIGMS grant 1R25GM100866 awarded to RKD.
Pharyngeal Pumping Variability Assessment Reveals Subtle Mutant and Treatment Effects in the Nematode C. elegans
Authors: Harris Michael B. , Barrientos Christopher , Bui Andy , Co Mary , Gill Sunny , Lim Shelby , Ly Lily , Nguyen Micayla , Ortiz Brianna , Parabo Ashley , Phillips Derek , Rieger Iris , Shilleh Ahmad , Suree Nathan , Tran Adrianna , Valdez Vanessa , Vu Tien , Taylor Barbara E. , Berlemont Renaud 
Published: April 2020

In C. elegans, feeding is achieved through a well characterized behavior, pharyngeal pumping. This pumping is controlled by two pairs of pharyngeal motor neurons (MC and M3). Each pump cycle corresponds to the propagation of a single pharyngeal muscle action potential, initiated by MC and transmitted across a single cholinergic neuromuscular junction. The eat‐2 gene is required for nicotinic neurotransmission at the pharynx. We assessed pumping patterns exhibited by wild type (N2) and a series of eat‐2 mutant strains, each identified as possessing deficits in pumping, including: (Strain: genotype) DA465: eat‐2(ad465) II; DA1113: eat‐2(ad1113) II; DA1116: eat‐2(ad1116) II. We also assessed pumping patterns in N2 strain animals incubated in the anthelmintic and acetylcholine receptor antagonist Levamisole ((−)‐Tetramisole hydrochloride; 0 – 50 uM). All studies we done in animals incubated in 10mM serotonin in M9 buffer. We recorded electropharyngeograms (EPG) using the recently developed ScreenChip system (NemaMetrix) to detect individual pump events, and analyzed recordings using an algorithm (WormBeat) to distinguish and quantify neuromuscular fidelity of the pharynx and pharyngeal pumping pace variation. As expected, mutant strains and Levamisole‐exposed N2 worms each illustrated pump pattern defects. Results show that the WormBeat algorithm can identify subtle treatment effects and distinguish phenotypic variation between mutant strains. Pump pattern assessment distinguished individual strains and allowed comparison of defect to the Levamisole dose‐responses of N2s.Support or Funding InformationWork reported in this publication was supported by the National Heart Lung and Blood Institute and National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers 1R15HL126105, 1SC2GM112570, UL1GM118979, TL4GM118980, and RL5GM118978. The work is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health or any other funding body.
The Evolution of High‐fidelity Patient Simulation in Medical Education: A Literature Review
Authors: Klein Barbie Ann 
Published: April 2020

INTRODUCTIONThe use of patient simulation in healthcare education is extensive. This literature review identified and discussed the origins and current state of high‐fidelity patient simulation (HFPS) in medical education.AIMThe aim of this research was to piece together a comprehensive history of clinical simulation and to compile results from past and present HFPS investigations in order to develop evidence‐based recommendations for best practices when incorporating HFPS into medical curricula.METHODSData was obtained from an iterative review of historical literature, primary resources and references, as well as personal communication with authors of articles and texts related to the clinical application of simulation. Databases consulted included PubMed, Web of Science, ERIC, ProQuest Dissertations & Theses, EBSCOhost, Google Scholar, and the Indiana University library archive collections. As human subjects were not directly involved in this study, ethical approval was not applicable.RESULTSOver 110 different sources were reviewed and synthesized for data regarding the history, fidelity, advantages, challenges, and learning theories associated with HFPS in medical education. A review of current clinical HFPS research is organized by discipline and includes undergraduate medical education, continuing medical education, nursing education, and undergraduate pre‐professional education.DISCUSSION AND CONCLUSIONThis review spanned historical applications of clinical simulation dating back to Mesopotamia between the fourth and first millennia BCE and concludes with a review of computer‐controlled high‐fidelity simulators available today to train healthcare providers. Recommendations based on the literature include: formally integrating HFPS throughout the medical curriculum; providing authentic interprofessional education (IPE) opportunities for medical and other allied health professionals to develop essential team skills; and investing in the initial and continued training for those delivering the simulated experience and post‐simulation debrief.
G‐protein coupled receptor kinases’ (GRKs) role in APC/PAR1‐induced endothelial cells
Authors: Ramirez Monica Lizeth Gonzalez , Lin Ying , Molinar-Inglis Olivia , Birch Cierra , Trejo JoAnn 
Published: April 2020

Endothelial dysfunction is a hallmark of inflammation and prevalent in vascular diseases. Recent efforts are focused on developing new strategies to enhance endothelial resistance to inflammation. One promising candidate is activated Protein C (APC), which has been shown in preclinical studies to increase endothelial barrier protection and reduce inflammatory responses. APC enhances endothelial cytoprotection by activation of PAR1 through cleavage at an N‐terminal arginine (R)‐46 site, that is distinct from the canonical thrombin cleavage site at R‐41. This results in the generation of a distinct tethered ligand that promotes PAR1 bias signaling. G‐protein coupled receptor kinase (GRK) family plays a role in phosphorylating PAR1 and recruiting b‐arrestins, however, the contribution of GRKs to b‐arrestin driven cytoprotection is unknown. Our study focuses on determining the role and mechanisms by which GRKs regulate APC/PAR1‐induced endothelial cytoprotection to delineate their function in this pathway. Here we will present studies examining the function and mechanism by which GRKs regulate endothelial cytoprotective responses using GRK‐specific siRNA to assess function as well as confocal immunofluorescence imaging and biochemical sucrose fraction to examine GRK localization and compartmentalization in caveolae. The work is expected to provide important new insight into the role of GRKs in regulating APC/PAR1 cytoprotection in human cultured endothelial cells.Support or Funding InformationThis work is funded by NIH R01 GM090689, NIH R35GM127121, and IRACDA K12 GM068524.
Expression and Characterization of the Drosophila melanogaster (Dm)IKKβ:γ complex
Authors: Cohen Samantha , Wu Sheri , Huxford Tom 
Published: April 2020

In Drosophila, the IMD pathway is indispensable for proper innate immune responses. Infection by gram‐negative bacteria elicits a signaling cascade culminating in the rapid induction of antimicrobial peptide gene expression by the NF‐κB transcription factor Relish. Signal‐dependent activation of Relish is an essential component in the IMD pathway and is regulated by the Drosophila melanogaster IκB Kinase (DmIKK) complex. DmIKK is composed of two subunits: the catalytic subunit IKKβ and non‐catalytic subunit IKKγ. Like many protein kinases, DmIKK must be activated for proper catalysis. Although there has been extensive research into the signaling pathways of the Drosophila innate immune response, little is known about the molecular mechanisms that lead to DmIKK activation. Here we report expression, purification, and in vitro characterization of a catalytically active multi‐subunit DmIKK complex.Support or Funding InformationThis work is supported in part by the California Metabolic Research Foundation. S. Cohen is a recipient of the Arne N. Wick predoctoral fellowship.