bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic correction of structural variations in patient‐derived iPSCs using CRISPR/Cas9
Authors: Park Chul‐Yong , Kim Dong‐Wook 
Published: June 2021

Genome engineering technology using engineered nucleases has been rapidly developed, enabling the efficient correction of simple mutations. However, the precise correction of structural variations such as large inversions remains limited. Here, we describe a detailed procedure for the modeling or correction of large chromosomal rearrangements and short nucleotide repeat expansions using engineered nucleases in human induced pluripotent stem cells (hiPSCs) from a healthy donor and patients with structural variations. This protocol enables the correction of large inverted segments and short nucleotide repeat expansions in diseases such as hemophilia A, fragile X syndrome, Hunter syndrome, and Friedreich's ataxia.Support or Funding InformationC.Y. P. was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2016R1C1B1008742). D.W.K. was supported by grants from the National Research Foundation of Korea funded by the Ministry of Science & ICT (the Bio & Medical Technology Development Program, 2017M3A9B4042580) and from Korean Ministry of Health & Welfare (HI15C0916).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mass Spectrometry based Proteomics Investigation of Induced Obstructive Sleep Apnea (OSA) in Rat Atria
Authors: Darie Costel C , Channaveerappa Devika , Wormwood Kelly L. , Lux Jacob , Panama Brian 
Published: June 2021

IntroductionObstructive sleep apnea (OSA) affects up to 24% of the adult population and is associated with several atrial diseases. OSA is characterized by transient cessations in respiration lasting >10 seconds as a result of narrowing of the upper airway during sleep. An estimated 20% of adults have mild OSA and 7% of adults have moderate to severe OSA, with 85% of patients remaining undiagnosed. Although clinical evidence linking OSA to proarrhythmaic atrial changes is well known, the specific molecular mechanisms by which OSA causes atrial disease remain elusive. To study the OSA‐induced cardiac changes, we have initiated a recently developed rat model which closely recapitulates the characteristics of OSA.MethodsMale Sprague Dawley rats, aged 50–70 days, received surgically implanted tracheal balloons which were inflated to cause transient airway obstructions. Apnea groups experienced 60 apneas per hour of either 13 seconds (moderate apnea) or 23 seconds (severe apnea) for 2 weeks and 8 hours per day. Control rats received surgeries but no inflations. Proteomics analysis was done on the rat atria homogenates to identify dysregulated proteins in moderate and severe apnea when compared to control. SDS‐PAGE was performed to separate the proteins and the peptide mixtures after trypsin digestion were analyzed by a Nano Acquity UPLC coupled with Xevo G2 Mass Spectrometer. Data analysis was done using ProteinLynx Global Server (PLGS 2.4), Mascot server and Scaffold 4.1 software.ResultsRats were given transient apneas for two durations: 13 and 23 s, we termed ‘moderate’ and ‘severe,’ respectively. These two apnea durations are within the range of those experienced by people with OSA. ECG recordings showed that the P wave durations (associated with left and right atrial depolarization) and T wave amplitudes (associated with ventricular repolarization) were increased by 2 weeks of chronic OSA. This was important since OSA patients show atrial electrical remodeling. The apnea model recapitulated the pathophysiological characteristics of OSA. The proteomics analysis revealed that 3 of the 9 enzymes in glycolysis and 2 proteins related to oxidative phosphorylation were down‐regulated in the severe apnea group. In contrast, several structural and pro‐hypertrophic proteins were up‐regulated with chronic OSA. The data suggests the chronic OSA causes proteins changes which lead to cessation of glycolysis, a diminished capacity to generate reducing equivalents (i.e. NADH) as well as promotion of cardiac hypertrophy.Support or Funding InformationCAMP matching fundsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring the intersection of cellular zinc and kinase activity with single cell imaging
Authors: Anson Kelsie J , Palmer Amy 
Published: June 2021

Zinc is an essential metal in biology, playing a structural or functional role in up to 10% of the human proteome. Certain cell types experience fluctuations in labile zinc, and these zinc dynamics have been shown to play a role in cellular signaling. Neurons in the hippocampus concentrate synaptic zinc, but its function in signal transduction and neuronal development is not fully understood. We use and develop fluorescent tools to explore how physiologically‐relevant changes in labile zinc impact intracellular kinase signaling in single cells and in the context of neuronal signaling. In this work, we demonstrate that increases in labile cellular zinc lead to greater activation of ERK kinase and explore the mechanism of this activation. The impact of zinc on other signaling pathways are also explored using fluorescent biosensors and kinase activity profiling. Understanding how zinc impacts cell signaling pathways is fundamental to characterizing the effects of zinc fluctuations in a variety of cell types.Support or Funding InformationNSF Graduate Research Fellowship and NIH T32 Biophysics Training GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Double‐stranded RNAs Attenuate Interferon Response via Parkin‐mediated MAVS Ubiquitination
Authors: Kim Seong‐Jun , Ahn Dae‐Gyun , Syed Gulam Hussain , McMillan Aimee , Thomas Sunil , Gale Michael , Kim Bum‐Tae , Siddiqui Aleem 
Published: June 2021

Mitochondria play a central role in innate immune signaling and are primarily targeted by many viruses which have evolved elaborate mechanisms to evade host antiviral response. Recent studies demonstrate that viruses explicitly alter mitochondrial dynamics and mitophagy to evade innate immunity and maintain viral persistence. Here we report that Parkin and an upstream regulator, PTEN‐induced putative kinase 1 (PINK1), beyond their recognized role in mitochondrial quality control and Parkinson's disease, also serve as modulators of mitochondrial antiviral signaling protein (MAVS)‐mediated host innate immune response. MAVS serves as a mitochondrial platform for transduction of signal from various RIG‐I‐like receptors (RLRs) that sense pathogen‐associated molecular patterns (PAMPs) to orchestrate type I interferon (IFN)‐α/β production in response to viral infection. We show that the synthetic double‐stranded (ds) RNA mimics, poly(I:C) and 5′ppp‐dsRNA, potently induce Parkin translocation to the mitochondria, leading to Parkin‐dependent K63‐linked polyubiquitination of MAVS, eventually resulting in the attenuation of type I IFN production. Interference of Parkin and PINK1 expression led to a distinct elevation of type I IFN production on stimulation with viral RNA mimics or infection with RNA viruses. In agreement, primary mouse embryonic fibroblasts (MEFs) derived from PINK1 knockout mice (PINK1−/−) staged more robust type 1 IFN response compared to wild type PINK1 MEFs on stimulation with viral RNA mimics. This study unravels the yet unidentified function of Parkin and PINK1 in the modulation of MAVS‐dependent innate immune signaling and depicts how the RNA viruses exploit them to evade innate immunity.Support or Funding InformationThis work was supported in whole or in part by the National Institute of Health Grants DK077704 (to A.S.), AI085087 (to A.S.), AI104002 (to M.G.), AI100625 (to M.G.), Michael J Fox Foundation grants (to A.S. and S.J.K.), and National Research Council of Science & Technology (NST) grant by the Korea government (MSIP) CRC‐16‐01‐KRICT (to S.J.K. and B.T.K.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phenotype analysis of Myo10 knockout mice lacking the motorized full‐length, but not the brain‐specific headless isoform
Authors: Bachg Anne Christina , Horsthemke Markus , Skryabin Boris V. , Klasen Tim , Nagelmann Nina , Faber Cornelius , Woodham Emma F. , Machesky Laura M. , Bachg Sandra , Stange Richard , Jeong Hyun‐Woo , Adams Ralf H. , Bähler Martin , Hanley Peter J. 
Published: June 2021

Unconventional myosin‐X (Myo10) is a MyTH4‐FERM and PH domain containing actin‐based motor protein which is widely expressed among vertebrate tissues. MyTH4‐FERM myosins are associated with protrusions enriched in bundled actin filaments. Full‐length (fl) Myo10 is best known for its localization to the tips of filopodia and for its ability to induce and regulate the formation of these dynamic finger‐like structures. At the cellular level, Myo10 has been implicated in diverse functions, including oriented cell migration, axon guidance, melanosome transfer, spindle assembly and phagocytosis. However, the physiological functions at the organismal level of this filopodial cargo carrier are not clear. We studied the phenotypes of Myo10 reporter knockout mice (Myo10tm2), to elucidate Myo10 function in vivo. Using Western blot analysis, we showed that in homozygous knockout Myo10tm2/tm2 mice, the expression of motorized fl Myo10 was deleted, but the brain‐specific headless (hdl) isoform was not abolished. Macroscopic examination of Myo10‐deficient mice revealed ventral coat pigmentation defects (100% penetrance) and a limb malformation, syndactyly, with a high penetrance of >95%. 24% of homozygous KO embryos developed exencephaly, a severe neural tube closure defect that is lethal. Magnetic resonance imaging (MRI) of isolated brains revealed, in contrast to mice lacking the Myo10 cargo protein DCC, intact cerebral commissures. Furthermore, Myo10 KO mice consistently displayed bilateral persistence of the hyaloid vasculature, visualized by MRI and retinal whole mount preparations, whereas retinal vascularization, phagocytosis and chemotactic behavior were not impaired. Confocal spinning disk microscopy confirmed that the hdl isoform of Myo10 does not induce filopodia and we found out that hdl Myo10 strongly localizes to the plasma membrane in dependency of its PH domains, supporting the auto‐inhibition model in which head‐tail interactions of fl Myo10 abolishes inactive monomer binding to PIP3‐rich membranes. In conclusion, Myo10 is important for normal vertebrate development, both prenatal (neural tube closure and digit separation) and postnatal (hyaloid regression).Support or Funding InformationDeutsche Forschungsgemeinschaft (DFG)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Over‐expression of Mutant Rheb2 Proteins and mTOR Signaling
Authors: PARMAR NITIKA 
Published: June 2021

Activation of mTOR is regulated by the small G‐protein, Rheb. In mammalian systems there are two Rheb genes‐Rheb1 and Rheb2 (RhebL1). Based on a scan for key residues in Rheb1 that regulate its activity we created several mutants for Rheb2 using a site‐directed mutagenesis. These residues include Thr20, Ser44, Lys45, and Gln130. Wild type and mutant Rheb proteins were over‐expressed in HEK293 cells and impact on mTOR signaling was assessed via monitoring the phosphorylation of the S6 protein. Experiments were conducted under serum starved conditions to determine if over‐expression of wild type and/or mutant Rheb proteins offered a growth advantage to the cells. mTOR signaling was analyzed in cells grown in enriched, starved and starved/stimulated conditions. Our results indicate that over‐expression of the Q130S Rheb2 is able to rescue the starved phenotype observed, i.e. decrease in S6 phosphorylation; mutants S44A and K45A were unable to rescue this phenotype. Our results so far are unique and provide evidence that Rheb2 functions differently from Rheb1.Support or Funding InformationThis work was supported by a CI Faculty Development Grant and a Research Award to Nitika ParmarThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An exploration of the algal protein channelrhodospin and the origins of optogenetics
Authors: Smalley Daniel , Mackenzie Emerson , Meyer Mackenzie , Ouang Henry , Pandit Ayush , Parthasarathy Madhumitha , Rai Shubham , Seeley Lucas , Smuckler Hannah , Kessler Eric M 
Published: June 2021

Channelrhodopsin (ChR) is a light‐gated cation channel protein originally discovered in the algae Chlamydomonas reinhardtii. Composed of seven transmembrane helices (TM1‐7) covalently bound to all‐trans retinal (ATR), ChR permits cations to pass through cellular membranes when excited by light, functioning in algal photoreception. During excitation, ATR undergoes photoisomerization into the 13‐cis retinal form, triggering conformational changes that transfer protons between the protonated retinal Schiff base and its residue counterions. These protonation events control gating of the electronegative pore formed by TM1, 2, 3, and 7, allowing selective cation passage regulated by TM2 while excited, before thermally cycling back to ground state. The Blue Valley CAPS 2017–18 MSOE Center for BioMolecular Modeling SMART Team modeled channelrhodopsin chimaera C1C2 using JMol and 3D printing technology with the goal of exploring this protein's unique properties as utilized in the revolutionary field of optogenetics. Optogenetics uses light and genetic engineering to systematically target and manipulate cellular systems in living organisms, enabling unprecedented research and experimentation in disciplines such as neuroscience. Through Cre‐Lox site‐specific recombinase and adeno‐associated viral technology, researchers can express ChR in specific neurons, allowing light‐based experimental control of neural pathways and action potentials. Studies done with these techniques have shed new light onto neurological and psychiatric disorders, and could potentially hold the key to new effective treatment options.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CourseSource: Publishing Classroom Activities and Pedagogical Materials
Authors: Grover Neena , Fox Kristin , Bell J Ellis 
Published: June 2021

ASBMB developed a set of core concepts for undergraduates completing a major in biochemistry and molecular biology which have been published and are available in the form of learning goals and sample learning objectives on the ASBMB website. The goal of improving undergraduate education will be greatly enhanced if these developed core concepts are linked with our classroom implementation approaches. CourseSource (coursesource.org) is an online, open‐access journal that focuses on improving undergraduate biology education by publishing articles aligned with the learning goals created by various professional societies. The submitted articles and activities are peer‐reviewed and edited by members of the BMB community, including the three authors of this abstract. CourseSource is a robust and free resource that was developed using seed money from Howard Hughes Medical Institute and is currently funded by the National Science Foundation. Members of the BMB community can submit activities that they have developed in the classroom or during one of many workshops that were funded by NSF‐RCN grant. Materials used in classes are eligible for publication on CourseSource: from detailed case studies to short, 15‐minute in‐class activities that teach a single concept. There are different article types: “Science Behind the Lesson” summarizes scientific topics related to a submitted lesson, “Reviews” examine, evaluate, critique, and/or appraise topics, tools or materials that are of broad interest, and “Essays” cover topics or challenges that are of broad interest to the biology education community. CourseSource has created a ten‐minute interactive “course” that summarizes how to write a “Lesson” and provides templates for the types of articles it publishes. Detailed data on learning outcomes is not required for publication on CourseSource. In addition, as further assessment data are collected on the activities, they can be published in journals, such as, Biochemistry and Molecular Biology Education. CourseSource provides an opportunity to share our teaching resources with each other, contribute to the scholarship of teaching and learning, and improve access to peer‐reviewed resources for teaching.Support or Funding InformationSupported by NSF RCN‐UBE 0957205 and NSF‐DUE 1725130This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
2nd Grade Students Have PhUn Learning About Thermoregulation
Authors: Halpin Patricia A. 
Published: June 2021

Physiology Understanding (PhUn) week is a yearly event sponsored by the American Physiological Society (APS). As part of this annual event, physiologists visit K‐12 classrooms and teach students about physiology. While the event is usually held in November it can be held at different times of the year to accommodate students' and teachers' schedules. The different methods in which animals stay warm in the winter was the perfect topic to bring into the second grade classroom during the winter. Students learned the role of a physiologist and the definition of thermoregulation. The class was very interactive when describing the differences in how animals stay warm in the winter: hibernation, fur, huddling together, and warm winter jackets. The physiologist then focused the discussion on three animals: polar bear, human and bird. The degree to which the polar bear fur, human skin and bird's feathers provide insulation from the cold was discussed. To reinforce the knowledge learned, the students performed the Insulation Bag “hands‐in” activity. Three sets of hand mitts made from zipper lock bags were immersed into a bucket of ice water. One empty set of mitts mimicked human skin, a second set with bubble wrap in the mitts was used to mimic feathers and a third set had a layer of vegetable shortening to mimic the insulation of a polar bear. The students tested each type of bag and then determined in which mitt their hands were the warmest. They then met Phreezy bear (APS stuffed animal) and completing the activities in the APS Fun with Physiology activity book for grades 2–4. Each classroom was given an APS How do I Stay Warm poster to remember the lesson. This PhUn week event is sure to create a lasting memory for both students and physiologist alike.Support or Funding InformationAmerican Physiological SocietyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Newt‐derived extracellular vesicles drive mammalian neuron differentiation
Authors: Middleton Ryan C 
Published: June 2021

Newts have the extraordinary ability to regenerate amputated limbs, as well as damaged heart, nerve and retinal tissue. This remarkable ability relies on the ability of the newt to generate a cluster of cells at site on injury, called a blastema, which is made up of progenitor cells from multiple tissue types. The blastema then directs the proliferation of cells to form new tissue and then the differentiation of those blastemal cells into their mature cell fates. The initial formation of the blastema requires rapid innervation of wound area.We have discovered that newt myogenic precursor cells (A1 cells) secrete extracellular vesicles (EVs) that, like all mammalian cell types, contain RNA and protein cargo that can modulate the morphology and gene expression patterns of the recipient cells that take up EVs. However, these newt EVs contain messenger RNA for multiple nerve differentiation factors. Cultured mammalian dorsal root ganglion (DRG) neurons, a type of peripheral sensory neuron, respond to treatments of newt EVs through increased axon growth and the up‐regulation of genes involved in neural cell differentiation and nerve cell fate determination. We hypothesize that the newt cells are responding to isolated culture conditions as though tissue injury has occurred and secrete EVs to promote nerve growth to facilitate blastema formation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Clinical Potential of Physical Microenvironment Stimulation for Wound Healing
Authors: Ko Ung Hyun , Choi Jongjin , Choung Jinseung , Moon Sunghwan , Shin Jennifer Hyunjong 
Published: June 2021

IntroductionSkin stretch by pressure contact, electric field by ion leakage are the most representative physical stimulus during skin wound process, and these physical factors are necessary for natural healing and wound homeostasis. However, when not adequately controlled of these stimulants, external physical cues can provide the mechanically harsh environment on skin cells like dermal fibroblasts and lead to promote excessive synthesis of matrix proteins by the activated fibroblasts, resulting in undesired scar formation. For severe wounds, however, it would be desired to maximize the matrix synthesis to promote faster regeneration, for which exogenously applied physical stimulations may be useful. In this study, we investigated suitable physical condition to develop highly functional fibroblasts and verified the potential of the physical stimulation cues at wound therapy using in vivo mouse model.MethodsNHDFs (Normal Human Dermal Fibroblasts, cell line) were cultured in DMEM supplementary with 10% fetal bovine serum, and 1% penicillin‐streptomycin. Electrospinning scaffolds were utilized to mimic cellular elongation by the wound stretching, and the electric field was applied through the cell culture medium by using DC power supply. Cells were expanded on electrospun fibers for two days, and cultured one day more without serum conditions. 0.25, 0.5, 0.75, 1 V/cm electric field were tested after the serum starvation. The electric field was given for 3 hours with periodic pattern (10 min stimulation, 20 min resting). NHDFs were expanded for two days more with TGF‐b1 treatment before being analyzed.ResultsqPCR analysis was done to quantify the gene expression changes by the physical cues. From the results, calponin, a‐SMA, FGF2, fibronectin genes were upregulated in all physical stimulation condition and showed a synergistic increase when elongation cues and electric field stimulation was both existed. These results were demonstrated in a‐SMA immunofluorescence images with the same aspect. In other words, each physical cues interactively facilitated the regenerative proteins by inducing the myofibroblasts differentiation. This increase of regeneration properties also observed in vivo mouse model and induced wound healing rates as well.DiscussionThe myofibroblasts are inadequate regenerative cells in the point of reversible wound healing. However, in the severe irreversible wound, excessive protein expression of myofibroblasts are more acceptable compared to normal fibroblasts. And, the physical microenvironment of fibroblasts can be one of the best tissue engineering options to increase the myofibroblasts differentiation.Support or Funding InformationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.