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https://www.readbyqxmd.com/read/28811500/human-microcephaly-protein-rttn-interacts-with-stil-and-is-required-to-build-full-length-centrioles
#1
Hsin-Yi Chen, Chien-Ting Wu, Chieh-Ju C Tang, Yi-Nan Lin, Won-Jing Wang, Tang K Tang
Mutations in many centriolar protein-encoding genes cause primary microcephaly. Using super-resolution and electron microscopy, we find that the human microcephaly protein, RTTN, is recruited to the proximal end of the procentriole at early S phase, and is located at the inner luminal walls of centrioles. Further studies demonstrate that RTTN directly interacts with STIL and acts downstream of STIL-mediated centriole assembly. CRISPR/Cas9-mediated RTTN gene knockout in p53-deficient cells induce amplification of primitive procentriole bodies that lack the distal-half centriolar proteins, POC5 and POC1B...
August 15, 2017: Nature Communications
https://www.readbyqxmd.com/read/28811362/nuclear-envelope-rupture-is-enhanced-by-loss-of-p53-or-rb
#2
Zhe Yang, John Maciejowski, Titia de Lange
The mammalian nuclear envelope (NE) forms a stable physical barrier between the nucleus and the cytoplasm, normally breaking down only during the cell cycle phase of mitosis. However, spontaneous transient NE rupture in interphase can occur when NE integrity is compromised such as when the nucleus experiences mechanical stress. For instance, deficiencies in the nuclear lamins and their associated proteins can cause NE rupture that is promoted by forces exerted by actin filaments. NE rupture can allow cytoplasmic nucleases to access chromatin, potentially compromising genome integrity...
August 15, 2017: Molecular Cancer Research: MCR
https://www.readbyqxmd.com/read/28810836/suppression-of-agr2-in-a-tgf-%C3%AE-induced-smad-regulatory-pathway-mediates-epithelial-mesenchymal-transition
#3
Lucia Sommerova, Eva Ondrouskova, Borivoj Vojtesek, Roman Hrstka
BACKGROUND: During cancer progression, epithelial cancer cells can be reprogrammed into mesenchymal-like cells with increased migratory potential through the process of epithelial-mesenchymal transition (EMT), representing an essential step of tumor progression towards metastatic state. AGR2 protein was shown to regulate several cancer-associated processes including cellular proliferation, survival and drug resistance. METHODS: The expression of AGR2 was analyzed in cancer cell lines exposed to TGF-β alone or to combined treatment with TGF-β and the Erk1/2 inhibitor PD98059 or the TGF-β receptor specific inhibitor SB431542...
August 15, 2017: BMC Cancer
https://www.readbyqxmd.com/read/28805828/mutations-in-keops-complex-genes-cause-nephrotic-syndrome-with-primary-microcephaly
#4
Daniela A Braun, Jia Rao, Geraldine Mollet, David Schapiro, Marie-Claire Daugeron, Weizhen Tan, Olivier Gribouval, Olivia Boyer, Patrick Revy, Tilman Jobst-Schwan, Johanna Magdalena Schmidt, Jennifer A Lawson, Denny Schanze, Shazia Ashraf, Jeremy F P Ullmann, Charlotte A Hoogstraten, Nathalie Boddaert, Bruno Collinet, Gaëlle Martin, Dominique Liger, Svjetlana Lovric, Monica Furlano, I Chiara Guerrera, Oraly Sanchez-Ferras, Jennifer F Hu, Anne-Claire Boschat, Sylvia Sanquer, Björn Menten, Sarah Vergult, Nina De Rocker, Merlin Airik, Tobias Hermle, Shirlee Shril, Eugen Widmeier, Heon Yung Gee, Won-Il Choi, Carolin E Sadowski, Werner L Pabst, Jillian K Warejko, Ankana Daga, Tamara Basta, Verena Matejas, Karin Scharmann, Sandra D Kienast, Babak Behnam, Brendan Beeson, Amber Begtrup, Malcolm Bruce, Gaik-Siew Ch'ng, Shuan-Pei Lin, Jui-Hsing Chang, Chao-Huei Chen, Megan T Cho, Patrick M Gaffney, Patrick E Gipson, Chyong-Hsin Hsu, Jameela A Kari, Yu-Yuan Ke, Cathy Kiraly-Borri, Wai-Ming Lai, Emmanuelle Lemyre, Rebecca Okashah Littlejohn, Amira Masri, Mastaneh Moghtaderi, Kazuyuki Nakamura, Fatih Ozaltin, Marleen Praet, Chitra Prasad, Agnieszka Prytula, Elizabeth R Roeder, Patrick Rump, Rhonda E Schnur, Takashi Shiihara, Manish D Sinha, Neveen A Soliman, Kenza Soulami, David A Sweetser, Wen-Hui Tsai, Jeng-Daw Tsai, Rezan Topaloglu, Udo Vester, David H Viskochil, Nithiwat Vatanavicharn, Jessica L Waxler, Klaas J Wierenga, Matthias T F Wolf, Sik-Nin Wong, Sebastian A Leidel, Gessica Truglio, Peter C Dedon, Annapurna Poduri, Shrikant Mane, Richard P Lifton, Maxime Bouchard, Peter Kannu, David Chitayat, Daniella Magen, Bert Callewaert, Herman van Tilbeurgh, Martin Zenker, Corinne Antignac, Friedhelm Hildebrandt
Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue...
August 14, 2017: Nature Genetics
https://www.readbyqxmd.com/read/28801621/efficient-generation-of-conditional-knockout-mice-via-sequential-introduction-of-lox-sites
#5
Takuro Horii, Sumiyo Morita, Mika Kimura, Naomi Terawaki, Mihiro Shibutani, Izuho Hatada
Conditional knockout using Cre/lox is essential for functional analysis of genes. CRISPR/Cas in combination with two sets of guide RNAs and a single-stranded oligonucleotide enables simultaneous insertion of two lox sequences. However, this method induces double-strand breaks at two sites on the same chromosome, which causes an undesirable chromosomal deletion and reduces the flanked lox (flox) rate. To solve this problem, we investigated a method that sequentially introduces each lox sequence at the 1-cell and 2-cell embryonic stages, respectively...
August 11, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28800587/highly-efficient-gene-inactivation-by-adenoviral-crispr-cas9-in-human-primary-cells
#6
Olaf Voets, Frans Tielen, Edo Elstak, Julian Benschop, Max Grimbergen, Jan Stallen, Richard Janssen, Andre van Marle, Christian Essrich
Phenotypic assays using human primary cells are highly valuable tools for target discovery and validation in drug discovery. Expression knockdown (KD) of such targets in these assays allows the investigation of their role in models of disease processes. Therefore, efficient and fast modes of protein KD in phenotypic assays are required. The CRISPR/Cas9 system has been shown to be a versatile and efficient means of gene inactivation in immortalized cell lines. Here we describe the use of adenoviral (AdV) CRISPR/Cas9 vectors for efficient gene inactivation in two human primary cell types, normal human lung fibroblasts and human bronchial epithelial cells...
2017: PloS One
https://www.readbyqxmd.com/read/28798030/ethanol-exposure-regulates-gabra1-expression-via-histone-deacetylation-at-the-promoter-in-cultured-cortical-neurons
#7
John Peyton Bohnsack, Vraj K Patel, A Leslie Morrow
GABAA-Rs mediate the majority of inhibitory neurotransmission in the adult brain. α1-containing GABAA-Rs are the most prominent subtype in the adult brain, and are important in both homeostatic function and several disease pathologies including alcohol dependence, epilepsy, and stress. Ethanol exposure causes a decrease of α1 transcription and peptide expression both in vivo and in vitro, but the mechanism that controls the transcriptional regulation is unknown. Since ethanol is known to activate epigenetic regulation of gene expression, we tested the hypothesis that ethanol regulates α1 expression through histone modifications in cerebral cortical cultured neurons...
August 10, 2017: Journal of Pharmacology and Experimental Therapeutics
https://www.readbyqxmd.com/read/28794185/id-genes-are-essential-for-early-heart-formation
#8
Thomas J Cunningham, Michael S Yu, Wesley L McKeithan, Sean Spiering, Florent Carrette, Chun-Teng Huang, Paul J Bushway, Matthew Tierney, Sonia Albini, Mauro Giacca, Miguel Mano, Pier Lorenzo Puri, Alessandra Sacco, Pilar Ruiz-Lozano, Jean-Francois Riou, Muriel Umbhauer, Gregg Duester, Mark Mercola, Alexandre R Colas
Deciphering the fundamental mechanisms controlling cardiac specification is critical for our understanding of how heart formation is initiated during embryonic development and for applying stem cell biology to regenerative medicine and disease modeling. Using systematic and unbiased functional screening approaches, we discovered that the Id family of helix-loop-helix proteins is both necessary and sufficient to direct cardiac mesoderm formation in frog embryos and human embryonic stem cells. Mechanistically, Id proteins specify cardiac cell fate by repressing two inhibitors of cardiogenic mesoderm formation-Tcf3 and Foxa2-and activating inducers Evx1, Grrp1, and Mesp1...
August 9, 2017: Genes & Development
https://www.readbyqxmd.com/read/28794126/a-refined-method-to-study-gene-dosage-changes-in-vitro-using-crispr-cas9
#9
Teresa P Raposo, Henry O Ebili, Mohammad Ilyas
AIMS: Gene dosage can have a major impact on cell biology, although, hitherto, it has been difficult to study using in vitro models. We sought to refine and accelerate the development of 'gene dosage' models through using CRISPR/Cas9 (a gene editing technology) for sequential knockout of gene alleles. METHODS: Our method involved (1) using Cas9 nuclease mRNA rather than expression plasmids, (2) using a fluorescently labelled FAM-6 tracr complexed with guide RNA and (3) using high-resolution melting (HRM) analysis to screen for mutations...
August 9, 2017: Journal of Clinical Pathology
https://www.readbyqxmd.com/read/28793350/ifnar2-dependent-gene-expression-profile-induced-by-ifn-%C3%AE-in-pteropus-alecto-bat-cells-and-impact-of-ifnar2-knockout-on-virus-infection
#10
Qian Zhang, Lei-Ping Zeng, Peng Zhou, Aaron T Irving, Shang Li, Zheng-Li Shi, Lin-Fa Wang
Bats are important reservoirs of many viruses, which are capable of infecting the host without inducing obvious clinical diseases. Interferon and the downstream interferon regulated genes (IRGs) are known to act as the first line of defense against viral infections. Little is known about the transcriptional profile of genes being induced by interferon in bats and their role in controlling virus infection. In this study, we constructed IFNAR2 knockout bat cell lines using CRISPR technology and further characterized gene expression profiles induced by the most abundant IFN-α (IFN-α3)...
2017: PloS One
https://www.readbyqxmd.com/read/28783728/correction-of-a-pathogenic-gene-mutation-in-human-embryos
#11
Hong Ma, Nuria Marti-Gutierrez, Sang-Wook Park, Jun Wu, Yeonmi Lee, Keiichiro Suzuki, Amy Koski, Dongmei Ji, Tomonari Hayama, Riffat Ahmed, Hayley Darby, Crystal Van Dyken, Ying Li, Eunju Kang, A-Reum Park, Daesik Kim, Sang-Tae Kim, Jianhui Gong, Ying Gu, Xun Xu, David Battaglia, Sacha A Krieg, David M Lee, Diana H Wu, Don P Wolf, Stephen B Heitner, Juan Carlos Izpisua Belmonte, Paula Amato, Jin-Soo Kim, Sanjiv Kaul, Shoukhrat Mitalipov
Genome editing has potential for the targeted correction of germline mutations. Here we describe the correction of the heterozygous MYBPC3 mutation in human preimplantation embryos with precise CRISPR-Cas9-based targeting accuracy and high homology-directed repair efficiency by activating an endogenous, germline-specific DNA repair response. Induced double-strand breaks (DSBs) at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template...
August 2, 2017: Nature
https://www.readbyqxmd.com/read/28782570/nanomedicine-and-advanced-technologies-for-burns-preventing-infection-and-facilitating-wound-healing
#12
Mirza Ali Mofazzal Jahromi, Parham Sahandi Zangabad, Seyed Masoud Moosavi Basri, Keyvan Sahandi Zangabad, Ameneh Ghamarypour, Amir R Aref, Mahdi Karimi, Michael R Hamblin
According to the latest report from the World Health Organization, an estimated 265,000 deaths still occur every year as a direct result of burn injuries. A widespread range of these deaths induced by burn wound happens in low- and middle-income countries, where survivors face a lifetime of morbidity. Most of the deaths occur due to infections when a high percentage of the external regions of the body area is affected. Microbial nutrient availability, skin barrier disruption, and vascular supply destruction in burn injuries as well as systemic immunosuppression are important parameters that cause burns to be susceptible to infections...
August 3, 2017: Advanced Drug Delivery Reviews
https://www.readbyqxmd.com/read/28781951/ifitm1-suppresses-expression-of-human-endogenous-retroviruses-in-human-embryonic-stem-cells
#13
Yudong Fu, Zhongcheng Zhou, Hua Wang, Peng Gong, Renpeng Guo, Jinmiao Wang, Xinyi Lu, Feng Qi, Lin Liu
Interferon-induced transmembrane protein 1 (IFITM1), a member of the IFITM protein family, is a component of a multimeric complex involved in the transduction of antiproliferation and cell adhesion signals. IFITM1 is thought to play a role in antiproliferation and immune surveillance, and has been shown to restrict infection by numerous viruses. It is highly expressed in human embryonic stem cells (hESCs) but its role in hESCs remains to be elucidated. In this study, knockout of IFITM1 mediated by CRISPR/Cas9 in hESCs did not affect self-renewal, pluripotency, telomerase activity or telomeres...
August 2017: FEBS Open Bio
https://www.readbyqxmd.com/read/28781079/rapid-generation-of-human-genetic-loss-of-function-ipsc-lines-by-simultaneous-reprogramming-and-gene-editing
#14
Andrew M Tidball, Louis T Dang, Trevor W Glenn, Emma G Kilbane, Daniel J Klarr, Joshua L Margolis, Michael D Uhler, Jack M Parent
Specifically ablating genes in human induced pluripotent stem cells (iPSCs) allows for studies of gene function as well as disease mechanisms in disorders caused by loss-of-function (LOF) mutations. While techniques exist for engineering such lines, we have developed and rigorously validated a method of simultaneous iPSC reprogramming while generating CRISPR/Cas9-dependent insertions/deletions (indels). This approach allows for the efficient and rapid formation of genetic LOF human disease cell models with isogenic controls...
July 24, 2017: Stem Cell Reports
https://www.readbyqxmd.com/read/28777946/engineered-epidermal-progenitor-cells-can-correct-diet-induced-obesity-and-diabetes
#15
Jiping Yue, Xuewen Gou, Yuanyuan Li, Barton Wicksteed, Xiaoyang Wu
Somatic gene therapy is a promising approach for treating otherwise terminal or debilitating diseases. The human skin is a promising conduit for genetic engineering, as it is the largest and most accessible organ, epidermal autografts and tissue-engineered skin equivalents have been successfully deployed in clinical applications, and skin epidermal stem/progenitor cells for generating such grafts are easy to obtain and expand in vitro. Here, we develop skin grafts from mouse and human epidermal progenitors that were engineered by CRISPR-mediated genome editing to controllably release GLP-1 (glucagon-like peptide 1), a critical incretin that regulates blood glucose homeostasis...
August 3, 2017: Cell Stem Cell
https://www.readbyqxmd.com/read/28777443/use-of-human-pluripotent-stem-cell-derived-cardiomyocytes-to-study-drug-induced-cardiotoxicity
#16
Agnes Maillet, Kim Peng Tan, Liam R Brunham
Drug-induced cardiotoxicity is the one of the most common causes of drug withdrawal from market. A major barrier in managing the risk of drug-induced cardiotoxicity has been the lack of relevant models to study cardiac safety. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have great potential in drug discovery and cardiotoxcity screens as they display many characteristics of the human myocardium and offer unlimited supply. This unit describes how to use pluripotent stem cells derived cardiomyocytes to study drug-induced cardiotoxicty using doxorubicin as an example...
August 4, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28769030/mir-30a-acts-as-a-tumor-suppressor-by-double-targeting-cox-2-and-bcl9-in-h-pylori-gastric-cancer-models
#17
Xuan Liu, Qing Ji, Chengcheng Zhang, Xiaowei Liu, Yanna Liu, Ningning Liu, Hua Sui, Lihong Zhou, Songpo Wang, Qi Li
Helicobacter pylori (H. pylori) is one of the most important factors that affect the development of gastric cancer, and its mechanism remains un-elucidated. Our present study found that, miR-30a is crucial for regulating the growth and migration of H. pylori infected gastric cancer in vitro by targeting COX-2 and BCL9. In details, double-stranded miR-30a precursor produced two single-stranded and matured miRNAs including miR-30a-3p and miR-30a-5p, which played significant biological functions in two different manners...
August 2, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28768826/mechanical-signals-activate-p38-mapk-pathway-dependent-reinforcement-of-actin-via-mechanosensitive-hspb1
#18
Laura Hoffman, Christopher C Jensen, Masaaki Yoshigi, Mary Beckerle
Despite the importance of a cell's ability to sense and respond to mechanical force, the molecular mechanisms by which physical cues are converted to cell-instructive chemical information to influence cell behaviors remain to be elucidated. Exposure of cultured fibroblasts to uniaxial cyclic stretch results in an actin stress fiber reinforcement response that stabilizes the actin cytoskeleton. p38 MAPK signaling is activated in response to stretch, and inhibition of p38 MAPK abrogates stretch-induced cytoskeletal reorganization...
August 2, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28767718/zygotic-vinculin-is-not-essential-for-embryonic-development-in-zebrafish
#19
Mitchell K L Han, Gerard N M van der Krogt, Johan de Rooij
The formation of multicellular tissues during development is governed by mechanical forces that drive cell shape and tissue architecture. Protein complexes at sites of adhesion to the extracellular matrix (ECM) and cell neighbors, not only transmit these mechanical forces, but also allow cells to respond to changes in force by inducing biochemical feedback pathways. Such force-induced signaling processes are termed mechanotransduction. Vinculin is a central protein in mechanotransduction that in both integrin-mediated cell-ECM and cadherin-mediated cell-cell adhesions mediates force-induced cytoskeletal remodeling and adhesion strengthening...
2017: PloS One
https://www.readbyqxmd.com/read/28765370/three-dimensional-biomimetic-vascular-model-reveals-a-rhoa-rac1-and-n-cadherin-balance-in-mural-cell-endothelial-cell-regulated-barrier-function
#20
Stella Alimperti, Teodelinda Mirabella, Varnica Bajaj, William Polacheck, Dana M Pirone, Jeremy Duffield, Jeroen Eyckmans, Richard K Assoian, Christopher S Chen
The integrity of the endothelial barrier between circulating blood and tissue is important for blood vessel function and, ultimately, for organ homeostasis. Here, we developed a vessel-on-a-chip with perfused endothelialized channels lined with human bone marrow stromal cells, which adopt a mural cell-like phenotype that recapitulates barrier function of the vasculature. In this model, barrier function is compromised upon exposure to inflammatory factors such as LPS, thrombin, and TNFα, as has been observed in vivo...
August 15, 2017: Proceedings of the National Academy of Sciences of the United States of America
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