bluuebunny/crossref_metadata_2025_split huggingface dataset:
Restoration of Dystrophin in Duchenne Muscular Dystrophy by Human iPS Cells Derived Skeletal Muscle Progenitor Cells
Authors: Xuan Wanling , Tang Yaoliang , Ashraf Muhammad 
Published: April 2020

Background and ObjectiveDuchenne muscular dystrophy (DMD) is caused by mutations of the gene that encodes the protein dystrophin. Loss of dystrophin leads to severe and progressive muscle‐wasting in both skeletal and heart muscles. Human induced pluripotent stem cells (hiPSCs) offer an important opportunity to treat a number of diseases. Here, we investigated whether givinostat, a histone deacetylase inhibitor (HDACi), could reprogram hiPSCs into muscle progenitor cells (MPCs) for DMD treatment.Methods and ResultshiPSCs were treated with CHIR99021(10 μM) for two days and followed by givinostat (100 nM) for 5 days in E6 medium (Fig.
1). After culture in E6 medium for additional seven days, cells were analyzed for myogenic makers (Pax7, desmin) expression. Reprogrammed cells (MPCs) from 4 human iPS cell lines expressed Pax7 and desmin. MPCs were differentiated into myotubes in N2 or DMEM medium with 2% horse serum expressing MF20. Additionally, we also generated MPCs from hiPSCs using CHIR99021 and FGF according to a previous study (Fig.
2) and referred these cells as control‐MPCs. We observed that givinostat induced MPCs exhibited superior proliferation and migration capacity using CCK‐8 assay, colony assay and migration assay compared to control‐MPCs. Moreover, we transplanted human myoblasts (1×105) and MPCs (1×105) labeled with GFP into tibialis anterior (TA) muscles of Mdx/scid mice, after injury with cardiotoxin (CTX) for one day prior to cell transplantation. One month after transplantation, dystrophin and GFP double positive fibers were analyzed. Givinostat induced MPCs showed higher engraftment capacity and restoration of dystrophin than treatment with control‐MPCs and human myoblasts. In addition, givinostat induced MPCs treated muscle contained significantly less inflammatory cells and showed limited muscle necrosis and fibrosis.ConclusionWe successfully generated highly expandable and integration free MPCs from multiple hiPS cell lines using CHIR99021 and givinostat. Givinostat induced MPCs showed marked and impressive regeneration and restored dystrophin in injured tibialis muscle compared to control MPCs. It is concluded that hiPSCs pharmacologically reprogrammed into MPCs with a small molecule, givinostat might be effective cellular source for treatment of muscle injury and restoration of dystrophin in DMD.Support or Funding InformationNational Institutes of Health grants R01 HL134354 & R01 AR070029 (M Ashraf, Y Tang, NL Weintraub)Schematic outline of generation of MPCs from hiPSCs using CHIR99021 and givinostat (Givi) small molecule combo.Figure 1Schematic outline generation of control MPCs from hiPSC.Figure 2
Peptide barcoding for high‐throughput functional evaluation of antibodies without immobilization
Authors: Aoki Wataru , Matsuzaki Yusei , Ohtani Yuta , Kajiwara Kaho , Miyamoto Kana , Miura Natsuko , Aburaya Shunsuke , Ueda Mitsuyoshi 
Published: April 2020

BackgroundDisplay technologies such as phage display and yeast display can evaluate a large number of antibodies in a high‐throughput manner, and have been used to isolate various antibody medicines. In display technologies, the immobilization of antibodies on the cell surface is essential to establish genotype–phenotype linkages, but this immobilization can affect functions of antibodies. Here, we developed a novel methodology, “peptide barcoding”, for high‐throughput quantitative measurement of binding properties of antibodies without immobilization. In the scheme of “peptide barcoding”, unique peptide barcodes are fused with each antibody, and they represent genotype–phenotype linkages. A mixture of different peptide‐barcoded antibodies is reacted with antigens, and peptide barcodes of functional antibodies are identified by mass spectrometry after cleavage by specific proteases. This methodology will enable high‐throughput development of antibodies for experimental reagents, diagnostics, and pharmaceuticals.MethodsUnique peptide barcodes were designed to have high detectability for mass spectrometry. Peptide‐barcoded nanobodies (anti‐CD4 or anti‐GFP) were produced by Pichia pastoris. A mixture of peptide‐barcoded nanobodies were reacted with antigen‐coated magnetic beads. Peptide barcodes of functional nanobodies were cleaved by a specific protease, enterokinase, and released peptides were identified by mass spectrometry. Precise dissociation constants of nanobodies were measured by BIACORE T200.Results & DiscussionWe designed peptide‐barcoded anti‐CD4 nanobody or anti‐GFP nanobody, and successfully produced them using P. pastoris. We measured the values of dissociation constants of the peptide‐barcoded nanobodies using surface plasmon resonance, and confirmed that addition of peptide barcodes didn’t affect affinities of these nanobodies. To demonstrate high‐throughput evaluation of free nanobodies, we produced peptide‐barcoded nanobody mutants, and mixed them with antigen‐coated magnetic beads. Released peptide barcodes from functional nanoboides were quantified by selected reaction monitoring, and we successfully ranked nanobody mutants in order of affinity. This methodology has potential scalability and could evaluate affinities of hundreds of antibodies in one shot [
1,
2].Support or Funding InformationThis research was supported by JST, CREST (grant number JPMJCR16G2), Japan.
Genome‐wide Association Study of Sperm Resistance to Freezing in Horses
Authors: Nikitkina Elena , Kudinov Andrey , Atroshchenko Mikhail , Dementeva Natalia , Pozovnikova Marina , Larkina Tatiana , Shcherbakov Yuri , Krutikova Anna , Musidray Artem 
Published: April 2020

The use of cryopreserved sperm has become a routine in horse breeding. Genetic factors affecting cryoresistance remain poorly understood. The aim of the study was to search for genome‐wide associations with a decreasing in sperm motility after freezing. Semen of 96 horses was frozen. Sperm motility was evaluated by computer‐assisted semen analysis (CASA)( ArgusSoft, Russia) before and after freezing. The evaluation was carried out using an Axio Imager microscope (Carl Zeiss Microscopy GmbH, Germany). DNA was isolated from sperm samples for genotyping on Affymetrix Equine HD array 96 SNP chips. Calculations were performed using the PLINK and EMMAX programs. The Genome‐Wide Association Studies (GWAS) was used for the analysis of genomic associations. As a result 45 significant associations were found. 14 are on the first chromosome in region 1: 166861063‐167517944 in the vicinity of the LOC11769038 gene. It is assumed that the polymorphisms found are associated with the formation of functional resistance of spermatozoa to cryopreservation. Studies have opened up the possibility of studying the changes in the cells after freezing. Found SNPs markers will be used to select males whose semen is suitable for cryopreservation.Support or Funding InformationThe study was supported by the Russian Science Foundation (project No. 18‐16‐00071)Manhattan plot of GWASFigure 1
Structure, Function and Mechanism of AztD, a Zinc Metallochaperone
Authors: Yukl Erik T. , Neupane Durga Prasad , Fullam Stephanie , Chacón Kelly 
Published: April 2020

Zinc is an essential element that is toxic in excess. Thus, zinc homeostasis is a critical function for all organisms. In bacteria, it is generally maintained by transcriptional regulation of zinc import and export proteins by zinc‐responsive transcriptional regulators. Disruption of zinc homeostasis in bacterial pathogens can result in dramatic attenuation of virulence, making these systems potential targets for the development of novel antibiotics. In particular, zinc importers of the ATP binding cassette (ABC) type are critical for the survival of many bacteria under zinc starvation conditions. Bacterial ABC transporters rely on a periplasmic or lipoprotein solute binding protein (SBP) to bind zinc with high affinity and specificity and deliver it to the membrane permease for import into the cytoplasm. Recently, the participation of other extracellular zinc‐binding proteins (e.g. metallochaperones) in metal import through ABC transporters has been demonstrated in some species. We are studying zinc homeostasis in Paracoccus denitrificans, an organism with two zinc‐specific ABC transporter operons znuABC and aztABCD. These are under transcriptional control of the zinc uptake regulator (Zur), are highly upregulated during zinc deprivation, and are required for normal growth under these conditions. Further, both operons are highly conserved in several human pathogens, including Klebsiella pneumoniae, a member of the carbapenem‐resistant Enterobacteriaceae (CRE) considered an “Urgent Threat” by the Centers for Disease Control. The aztABCD operon encodes a periplasmic metallochaperone (AztD) capable of transferring zinc to the associated SBP (AztC) through a specific, associative mechanism that can be followed in vitro using intrinsic tryptophan fluorescence. Crystal structures of AztD and AztC combined with kinetic data and mutational studies are beginning to reveal the function of AztD in zinc homeostasis and the molecular mechanism of zinc transfer. The observation that AztD is conserved in more than 500 genomes from diverse bacterial taxa suggests that this protein is an important component of zinc homeostasis and a newly identified family of zinc metallochaperones.Support or Funding InformationNational Institutes of Health 1R01GM122819‐01A1Docking model of the zinc transfer complex between the metallochaperone AztD (green) and the solute binding protein AztC (magenta)Figure 1
Deep Learning‐Based Point‐Scanning Super‐Resolution Imaging
Authors: Manor Uri , Fang Linjing , Howard Jeremy , Monroe Fred , Weiser Sammy , Kastner Kyle , Kirk Lyndsey , Harris Kristen , Pekkurnaz Gulcin , Yoon Blenda , Schiavon Cara , Zhang Tong 
Published: April 2020

Point scanning imaging systems (e.g. scanning electron or laser scanning confocal microscopes) are perhaps the most widely used tools for high resolution cellular and tissue imaging. Like all other imaging modalities, the resolution, speed, sample preservation, and signal‐to‐noise ratio (SNR) of point scanning systems are difficult to optimize simultaneously. In particular, point scanning systems are uniquely constrained by an inverse relationship between imaging speed and pixel resolution. Here we show these limitations can be mitigated via the use of deep learning‐based super‐sampling of undersampled images acquired on a point‐scanning system, which we termed point‐scanning super‐resolution (PSSR) imaging. Oversampled, high SNR ground truth images acquired on scanning electron or Airyscan laser scanning confocal microscopes were ‘crappified’ to generate semi‐synthetic training data for PSSR models that were then used to restore real‐world undersampled images. Remarkably, our EM PSSR model could restore undersampled images acquired with different optics, detectors, samples, or sample preparation methods in other labs. PSSR enabled previously unattainable 2 nm resolution images with our serial block face scanning electron microscope system. For fluorescence, we show that undersampled confocal images combined with a multiframe PSSR model trained on Airyscan timelapses facilitates Airyscan‐equivalent spatial resolution and SNR with ~100x lower laser dose and 16x higher frame rates than corresponding high‐resolution acquisitions. In conclusion, PSSR facilitates point‐scanning image acquisition with otherwise unattainable resolution, speed, and sensitivity.Linjing Fang1Fred Monroe2Sammy Weiser Novak1Lyndsey Kirk3Cara R. Schiavon1Seungyoon B. Yu4Tong Zhang1Melissa Wu1Kyle Kastner5Yoshiyuki Kubota6Zhao Zhang7Gulcin Pekkurnaz4John Mendenhall3Kristen Harris3Jeremy Howard8Uri Manor11. Waitt Advanced Biophotonics Center, Salk Institute for Biological Studies, La Jolla, CA, USA2. Wicklow AI Medical Research Initiative, San Francisco, CA, USA3. Department of Neuroscience, Center for Learning and Memory, Institute for Neuroscience, University of Texas at Austin, Austin, TX, USA4. Neurobiology Section, Division of Biological Sciences, University of California San Diego, La Jolla, CA, USA5. Montreal Institute for Learning Algorithms, Université de Montréal, Canada6. Division of Cerebral Circuitry, National Institute for Physiological Sciences, Okazaki, 444‐8787 Japan7. Texas Advanced Computing Center, University of Texas at Austin, Austin, TX, USA8. Fast.AI, University of San Francisco Data Institute, San Francisco, CA, USAFigure 1
WWOX possesses N‐terminal cell surface‐exposed epitopes WWOX7‐21 and WWOX7‐11 for signaling cancer growth suppression and prevention in vivo
Authors: Chang Nan-Shan , Wang Wan-Jen , Ho Pei-Chuan , Nagarajan Ganesan , Chen Yu-An , Kuo Hsiang-Ling 
Published: April 2020

Membrane hyaluronidase Hyal‐2 supports cancer cell growth. Inhibition of Hyal‐2 by specific antibody against Hyal‐2 or pY216‐Hyal‐2 leads to cancer growth suppression and prevention in vivo. By immunoelectron microscopy, tumor suppressor WWOX is shown to be anchored, in part, in the cell membrane by Hyal‐2. Alternatively, WWOX undergoes self‐polymerization and localizes in the cell membrane. Proapoptotic pY33‐WWOX binds Hyal‐2, and TGF‐β induces internalization of the pY33‐WWOX/Hyal‐2 complex to the nucleus for causing cell death. In contrast, when pY33 is downregulated and pS14 upregulated in WWOX, pS14‐WWOX supports cancer growth in vivo. Here, we investigated whether membrane WWOX receives extracellular signals via surface‐exposed epitopes, especially at the S14 area, that signals for cancer growth suppression and prevention. By using a simulated 3‐dimentional structure and generated specific antibodies, WWOX epitopes were determined at amino acid #7 to 21 and #286 to 299. Synthetic WWOX7‐21 peptide, or truncation to 5‐amino‐acid WWOX7‐11, significantly suppressed and prevented the growth and metastasis of melanoma and skin cancer cells in mice. Time‐lapse microscopy revealed that WWOX7‐21 peptide potently enhanced the explosion and death of 4T1 breast cancer stem cell spheres by ceritinib. This is due to rapid upregulation of proapoptotic pY33‐WWOX, downregulation of prosurvival pERK, prompt increases in Ca+2 influx, and disruption of the IκBα/WWOX/ERK prosurvival signaling. In contrast, pS14‐WWOX7‐21 peptide dramatically increased cancer growth in vivo and protected cancer cells from ceritinib‐mediated apoptosis in vitro, due to a prolonged ERK phosphorylation. Further, specific antibody against pS14‐WWOX significantly enhanced the ceritinib‐induced apoptosis. Together, the N‐terminal epitopes WWOX7‐21 and WWOX7‐11 are potent in blocking cancer growth in vivo. WWOX7‐21 and WWOX7‐11 peptides and pS14‐WWOX antibody are of therapeutic values in suppressing and preventing cancer growth in vivo.Support or Funding InformationMinistry of Science and Technology, Taiwan, National Health Research Institute, Taiwan, and Department of Defense, USAWWOX peptides regulate breast cancer cell sensitivity to ceritinib.Figure 1
Aspirin in Combination with Benznidazole During the Acute Phase of Chagas Disease Prevents Cardiovascular Dysfunction and Decrease Typical Cardiac Lesions in Mice Chronically Infected with Trypanosoma cruzi
Authors: Pinge-Filho Phileno , Pereira Rito Santo , Malvezi Aparecida Donizette , Lovo-Martins Maria Izabel , Lucchetti Bruno Fernando Cruz , Santos Jussevania Pereira , Tavares Eliandro Reis , Verri Waldiceu Aparecido , de Almeida Araújo Eduardo José , Lioni Lucy Megumi Yamauchi , Yamada-Ogatta Sueli Fumie , Martins-Pinge Marli Cardoso 
Published: April 2020

Chagas disease, caused by protozoan Trypanosoma cruzi, is one of the main causes of death due to cardiomyopathy and consequent heart failure in Latin American countries. The goal of treatment is to eliminate the parasite and decrease the probability of cardiomyopathy and interrupt the cycle of disease transmission. Benznidazole (BZ) and nifurtimox (NFX) are recognized by the World Health Organization as effective drugs for treatment of disease, but both are very toxic with limited efficacy, especially in the chronic phase. Studies have shown that low doses of aspirin (ASA) were protective in experimental T. cruzi Infection. We evaluated the efficacy of BZ in combination with ASA in low doses using a chronic infection model with T. cruzi Y strain. Our results show that ASA treatment prevented the typical cardiovascular dysfunction (hypertension and tachycardia) and cardiac lesions on chronic phase of disease. Moreover, mice treated with BZ+ASA had a smaller cardiac fibrotic area than those of BZ‐treated mice. These results were associated with an increase of eosinophils and reticulocytes and high levels of nitric oxide in plasma and cardiac tissue of animals treated with ASA compared to the controls. The protective effects of ASA or BZ+ASA on chronically infected mice disappeared when we used Boc‐2 (LXA4 receptor antagonist), indicating that the protector effect of ASA was mediated by aspirin‐triggered lipoxin. These results emphasize the importance of exploring new drug combinations in treatments that can be used in the acute phase of Chagas disease that are beneficial to chronic patients.Support or Funding InformationThe present study was supported by grants from Fundação Araucária ‐ chamada de projeto 09/2016 Programa Institucional de Pesquisa Básica e Aplicada ‐ Conv. 001/2017 ‐ protocolo 47.396 ‐ SIT. 31675, Conselho Nacional Desenvolvimento Científico e Tecnológico (CNPq), CAPES. PPF, MCMP, MILM, WAVJ, LMYL, SFYO are research fellows of CNPq. BFCL and ERT are research fellows of CAPES. RPS was supported by Ministério da Ciência e Tecnologia, Ensino Superior e Técnico Profissional de Mocambique.Graphical abstractFigure 1
The Role of κ‐Opioid Receptor‐Induced Autophagy in Synaptic Alterations
Authors: Georgoussi Zafiroula-Iro , Karoussiotis Christos , Sotiriou Aggeliki , Papavranoussi-Daponte Danae , Polissidis Alexia Victoria , Nikoletopoulou Vassiliki 
Published: April 2020

Autophagy is a lysosomal degradation pathway that eliminates misfolded proteins and dysfunctional organelles to safeguard cellular homeostasis. In neurons, autophagy controls the quality of cytoplasmic proteins through degradation of important synaptic proteins and modulates synaptic organization and morphogenesis
(1). Emerging studies have shown that G protein coupled receptors (GPCRs) are direct sensors regulating the autophagic machinery
(2) and that opioid receptors regulate neurogenesis and neurotransmission with as yet unclarified mechanisms
(3,
4). In this regard, we have recently demonstrated that κ‐opioid receptor (κ‐OR) agonists induce autophagy in neuronal cells in vitro via a PTX‐sensitive G protein mechanism and identified the downstream components involved in this process
(5,
6). However, it is unknown whether κ‐OR agonists also mediate these effects in vivo and, specifically, whether opiodergic alterations mediated by selective opioid receptor agonists may control synaptic function and spine morphology through autophagy induction.We are now reporting by in vivo studies that brain extracts obtained from mice that have been treated with the κ‐OR selective agonist U‐50,488H display profound increases of specific autophagic markers. This κ‐OR induced autophagy increase is region‐specific and detected only in the hippocampal extracts. Moreover, isolated synaptosomes from saline‐ or U50,488H‐treated mice exhibited a profound decrease of a number of essential neuronal proteins enriched in dendritic spines, suggesting that these proteins are engulfed in the κ‐OR‐induced autophagic cargo. Our results demonstrate for the first time that κ‐OR‐induced autophagy plays a key role in synaptic function and dendritic spine density and may contribute to the mood disorders targeted by the dynorphin‐κ‐OR system.Support or Funding InformationThis work was supported by the GSRT Excellence II 3722 ‐”NO‐ALGOS” to Z.G; the SANITURA (MIS 5002514) grant, implemented under the Action for the Strategic Development on the Research and Technological Sectors & the OPENSCREEN‐GR “An Open‐Access Research Infrastructure of Chemical Biology and Target‐Based Screening Technologies for Human and Animal Health, Agriculture and the Environment” (MIS 5002691) funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020).
A cell permeant phosphopeptide mimetic of Niban restores endothelial function and inhibits p38 MAPK activation after vascular injury
Authors: Yim Tsz Wing , Perling Daniel , Polcz Monica , Komalavilas Padmini , Brophy Colleen , Cheung-Flynn Joyce 
Published: April 2020

Vascular injury leads to membrane disruption, ATP release, and endothelial dysfunction. Increases in the phosphorylation of p38 MAPK and decreases in the phosphorylation of Niban, a protein implicated in ER stress and apoptosis, are associated with vascular injury. A cell‐permeant phospho‐peptide mimetic of Niban (NiPp) was generated. The effects of NiPp in restoring endothelial function were determined ex vivo using intact rat aortic tissue (RA) after pharmacological activation of p38 MAPK and a clinically relevant stretch injury model. Anisomycin increased p38 MAPK phosphorylation and reduced endothelial‐dependent relaxation in RA. Treatment with NiPp prevented anisomycin‐induced reduction in endothelial function and increase in p38 MAPK phosphorylation (Figure
1). NiPp treatment also restored endothelial function after subfailure overstretch injury (Figure
2). Kinome screening experiments indicated that NiPp inhibits p38 MAPK. These data demonstrate that p38 MAPK and Niban signaling have a role in endothelial function, particularly in response to injury. Niban may represent an endogenous regulator of p38 MAPK activation. NiPp may serve as an experimental tool to further elucidate p38 MAPK regulation and as a potential therapeutic for endothelial dysfunction.Support or Funding InformationThis work was supported by the National Institutes of Health grants R01HL70715‐09 to CB and R01HL105731‐01 to JC.NiPp prevented anisomycin‐induced phosphorylation of p38 MAPK and endothelial dysfunction in rat aorta (RA).RA rings were treated with anisomycin (200μM) in the absence or presence of NiPp (500μM) pretreatment and endothelial function was determined in a muscle bath. A) Representative muscle bath tracings. B) Percent relaxation induced by 0.5μM CCH. C, D) Phosphorylation of p38 MAPK in RA after treatment as determined by Western blot analysis. n=10,*p<.05 in one‐way ANOVA with Tukey post‐test.Figure 1NiPp restored endothelial function and reduced p38MAPK phosphorylation in RA after subfailure stretch injury.RA was subjected to subfailure stretch and incubated in the absence or presence of NiPp (500μM) for 1 h. A) Endothelial‐dependent relaxation was determined in the muscle bath. n=5 rats; #p<.05, two‐way ANOVA. B, C) Phosphorylation of p38 MAPK in RA after stretch injury and NiPp treatment as determined by Western blot analysis n= 10. *p<.05, in one‐way ANOVA with Tukey post‐test.Figure 2